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three main ways.First, regarding evaluation, they challenged value-based pricing by allowing states to consider limited cost information in their evaluations.In particular, they allowed negotiation bodies in some countries (e.g.France and Italy) to revert back to production costs of drugs to motivate their decisions, with these costs no longer reflecting domestic investments as in period I, but rather the respective innovation efforts of the firm and public contributions to pharmaceutical R&D (I.1;OTMeds, 2021).Second, they reconfigured the topology of market actors.Even though the fragmentation of state agents between evaluation and negotiation agencies remained in place, many small state buyers essentially 'disappeared' from the pharmaceutical market and reappeared as interstate buying pools, with the BeNeLuxA creating its own evaluation and negotiation bodies (BeNeLuxA, 2018).Even negotiation agencies that did not merge were able to exchange price information through the EURIPID database, meaning that they were able to coordinate with each other and regain agency in their interactions with pharmaceutical corporations.Third, while jointly negotiated prices remain subject to confidentiality clauses, indications are that joint procurement succeeds in capping the vertiginous price spirals for highly innovative medicines (Vogler et al., 2021) -with one advocacy group even expressing confidence that 'the best [is] yet to come' in controlling price increases for new medicines through joint purchasing (EPHA, 2020). Pushing transparency further: transparency for democratic control.The vision of transparency for health system sustainability and access through inter-state collaboration and information sharing largely excluded communicating information to the public.While it was seen as a marked improvement over previous mobilizations of transparency by some activists (I.12)
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and policymakers (I.1), others remained sceptical as to the leverage these interstate initiatives really had (E.14): Voluntary collaboration can be a first step to increased transparency: it allows countries to exchange information on prices.But the tools do not lead to transparency automatically.We think it should be a specific requirement for new pharmaceutical initiatives to be [fully] transparent. . .(access to medicines activist) Here, the activist made an implicit distinction between transparency for coalitions of European states and creating visibility into pharmaceutical costs and prices for the broader public.At stake in these latest contestations is a further redefinition of transparency to reshape this market's politics, moving toward 'building a democratic control' over public expenditures (Barré-Sinoussi et al., 2019).This current situation begs the question of how the aftermath of Covid-19 will reshape the pharmaceutical market.Will we see the devising of another vision of transparency -transparency for democratic control -that could finally reorganize this market toward being more answerable to public concerns? Discussion This article asked: how do struggles around transparency reorganize markets?In addressing this question, it follows recent calls to deploy a constitutive perspective of transparency to study how actors claim, configure and contest the concept in different contexts (Heimstädt & Dobusch, 2020).We add to this literature in three crucial respects.First, we combine research on transparency with the literature on market devices to highlight the central role played by the transparency devices through which competing visions of transparency are enacted.We contend that transparency devices play a triple role in organizing markets, as they shape the evaluation of market
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objects, the topology of market actors, and the resulting prices.Second, drawing this device focus together with an investigation of the shifting political economies in which transparency mobilizations have been situated, we demonstrate how the enactment of different transparencies produces profound shifts in market power, including attempts to depoliticize transparency altogether and place it in a 'post-political' toolbox (this was particularly the case in our period II).Third and most important, leveraging insights from political economy for organization studies scholars, we investigate the struggles around transparency and its devices, showing how such struggles are instrumental to those seeking to challenge prevailing market orders and to reverse efforts to depoliticize market transparency.We discuss these contributions in turn. Devising transparency Building on a 'market politics' perspective that is sensitive to the struggles around concrete market organizations, we were able to show how the successive shifts in visions of transparency were mediated through a range of devices that together reorganized the European pharmaceutical market in various and often contentious ways.Rather than studying 'governance by transparency' in its normative sense (Fung, Graham, & Weil, 2007, p. 171), we traced how transparency was instrumented through these device assemblages and how this influenced market power and profit flows.While previous organization studies leveraged a Foucauldian perspective to study the governmental effects of transparency quests, these tended to revolve around the production and management of visibilities as a form of disciplinary control (Flyverbom, 2015).In broadening this perspective to the organization of markets, we find similar links between visions of transparency, their instrumentations, and the power of
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the agencies that promoted them, for instance when transparency devices in our period I enabled regulators to intervene in markets instead of using direct state aids. However, our account also painted a more complicated picture, where actors' own capacities to act, instrumented into the devices, also came to be questioned through them.The transparency devices we analysed shaped agencies -with 'states' and 'the pharmaceutical industry' themselves transforming into different actors depending on the transparency devices these actors deployed.But the actor topologies that were enacted through the devices were never settled for long, with struggles giving rise to further reshufflings.This was the case for instance with the European Reference Price system, a system favourable to pharmaceutical firms whose increasing complexity became a focal rallying cry for state activists contesting the market's organization.Thus, our study points toward a more complex engagement of agencies in devising transparency than previous studies may have suggested, and it opens fruitful avenues in studying the various 'misfires' that transparency devices may entail. Varieties of transparency, varieties of markets Our pragmatic approach allowed us to ground our definition of transparency devices in the practices of market actors themselves, tracing over time all those devices that actors mobilized in controversies around market transparency.As a result, the devices we encountered in our study substantially broaden the list of those previously studied, such as Hansen et al.'s (2015) three 'disclosure devices' (due diligence, rankings and big data analysis) or Hansen and Weiskopf's (2021) 'mediating technologies' (leagues, lists, social media and written testimonies).Our transparency devices included evaluation criteria, state
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committees, databases, coalition agreements, price grids, medication lists, accountancy rules and others.This array points to the multitude of organizational tools that can be deployed as transparency devices and, in different configurations, make specific political concerns knowable and governable (Flyverbom, 2015).We were able to highlight how actors themselves generate and fight over distinct visions and versions of transparency through this array of tools.Transparency, then, becomes a battlefield over technologies, where devices are mobilized and work together to change market power. It is the 'working together' that we wish to stress: our transparency devices did not act on their own, but only in the context of specific socio-material arrangements, as and when mobilized by market actors.Together with Hansen and Weiskopf (2021) and Mehrpouya and Salles-Djelic (2019), by being sensitive to this multiplicity our account demonstrates the extent to which the heterogeneity of the 'transparency' concept leads to a wide variety of social organizations.In our case, varieties of transparency existed not only in the objects of transparency (transparency of patents, costs, prices, funding) or in their devisings (transparency through lists, committees, timeframes, databases), but also and more crucially in who would ultimately benefit from it (transparency for states, corporations, state alliances, or the wider public).It is, importantly, in the different combinations of these categories -transparency of, through and for -that 'fuzzy transparency ideals' (Flyverbom, 2015, p. 174) give actors their power to act.Investigating the concrete and multiple configurations of transparency is all the more important in the context of markets as one type of organization whose decentralized and dispersed
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nature often conceals the 'structural advantage' that some actors enjoy over others (MacKenzie, 2019).Varieties of market transparency, we contend, generate varieties of market configurations -with each configuration ending up favouring specific kinds of actors and serving specific political interests.This makes it all the more important to study the relationship between definitions of transparency and the resulting organizational configurations, where the focus on the latter becomes an important entry point into organizational politics.We urge future research to deepen our knowledge of how struggles over transparency generate new forms of organizations, including considering the overlaps and entanglements between different transparency, accountancy and auditing devices in complex socio-material arrangements. The power of transparency struggles Finally, our study has significant implications for researchers interested in how the boundaries and overlaps between political and economic spheres are organized.While organizational scholars have made good inroads into studying the constitutive features of transparency -or how discourses and practices of transparency help build organizations -there is a dearth of work highlighting how the concept's economic and moral normativities are deployed and concealed.Our case encourages researchers to more fundamentally question the 'discursive and moral triumph' (Birchall, 2011, p. 66) of transparency as a pervasive organizational concept and an undisputed economic and social 'good' -in other words, to question the politics of the concept itself.Our study shows that what is organized in the name of transparency over time in fact represents highly selective politics -in our case, market politics.In his classic piece on the 'antipolitical economy', Barry (2002) argues that politics properly speaking can be fundamentally
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anti-political if contestations are shut off through technical arrangements, while conversely the politics of such organizational technologies is often vastly underestimated.This view chimes well with the analysis that we put forth in this paper, given how disputed the concept of transparency has been in the pharmaceutical market over the last decades and given the significance of what is at stake there. Taking a step back with its historical approach, our article helps better situate the specific organizational effects that concepts such as transparency have had in the rise of neoliberal market reforms.Similar to 'efficiency', 'accountability', or 'competition', transparency has held a central role among those post-political principles mobilized to organize economic life in the neoliberal society.While market transparency as a way of controlling firms and ensuring fair prices emerged as early as the 1970s, our article shows our period II ('transparency for corporations') from the 1990s as a crucial moment in history in which a specific vision of transparency, directly related to the implementation of New Public Management policies, was heralded as a universal one.Here, our work highlights dynamics similar to those detected by Mehrpouya and Salles-Djelic (2019), with the rise from the 1980s of what they call 'neoliberal' transparency.More crucially, however, we show how this moment was not the end of history for market transparency.The neoliberal, postpolitical definition of transparency has become increasingly contentious in recent years, with policymakers, pharmaceutical lobbyists and civil society activists arguing with strongly opposing views of the same principle.And this battlefield has extended well beyond pharma: during the recent wave
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of inflation, activists, trade unionists and policymakers called for 'transparency' over excessive profit-making by commodity firms, energy suppliers and property investors (AFP, 2022;Jones & Bruce, 2022).In our case, an unlikely coalition of NGOs and smaller European states has started to re-politicize the transparency principle by establishing struggles over its practical mobilizations in national health systems.We can draw parallels from these recent contestations to other post-political principles, for instance 'efficiency'.Once the monopoly of neoliberal policymakers, 'efficiency' has recently been reclaimed by critical activists to promote measures such as the nationalization of corporations (Mazzucato, Li, & Darzi, 2020) and the curbing of lobbying, which is deemed 'inefficient' (Transparency International, 2016).While it may be too early to tell whether we have entered the era of 'post-post-politics', our case clearly shows how contemporary contestations over such post-political concepts as transparency can re-enliven political debate.But, as our case demonstrated, such re-enlivening only becomes possible when civil society, state activists and researchers expose the socio-material arrangements behind these post-political concepts. Concluding Thoughts This paper is concerned with how struggles over transparency reshape the European pharmaceutical market.As Grossman et al. (2008, p. 108) quipped: 'the devil of transparency is mostly in the details' -and so, by extension, is its politics.Transparency has been a strategic concern in the organization of the European pharmaceutical market: a tool of marketization, corporate advancement and regulatory intervention.Highlighting the struggles around transparency in one public interest market is crucial not only for comprehending the concrete organizational effects of concepts that may often seem beyond contestation, but also in considering
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broader issues of how to transform market orders.As Wilson and Swyngedouw (2014) observe, the post-political age has brought to the fore how consensual principles such as transparency work to disempower critical voices and movements.But our article suggests that it has also opened new battlefields for state and civil society activists trying to disturb consensus politics.In our case, struggles over transparency have had a deep influence on the organization of the pharmaceutical market, leading to expanding corporate power and spiralling drug prices until the 2010s, but currently working in the opposite direction.Overall, as we have illustrated, the pharmaceutical market's failures are unlikely to be resolved by normative and overly general calls for more 'transparency', but only through a deep engagement with the specific, multiple and shifting orderings this concept entails. Table 1 . Archival sources used in this article. Table 4 . Transparency visions and transparency devices.
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Bridging Borders : Organizing Short-Term Agricultural Communication Exchange Programs This paper describes the efforts of the University of Florida (located in Gainesville, Florida) and the University of Guelph (located in Ontario, Canada, near Toronto) to jointly develop a unique pilot exchange program for undergraduate and graduate agricultural communication students. The program was designed to encourage students to think critically about agriculture, agricultural communications, and culture in each other's countries. During this exchange, nine University of Guelph students traveled to Florida for one week during their winter breaks and six University of Florida students traveled to Canada during their spring breaks. The exchanges took place in back-to-back weeks. Students met with university administrators and agriculture industry representatives, toured agricultural facilities, and visited cultural locations. This paper details the planning process that culminated in the two-week program, provides students' comments about what they learned during the study-abroad experience, and makes recommendations to faculty interested in organizing similar study-abroad programs. Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License. This professional development is available in Journal of Applied Communications: https://newprairiepress.org/jac/vol90/iss2/4 Professional Development Journal of Applied Communications, Vol. 90, No. 2, 2006, 17-27 ©ACE Bridging Borders: Organizing Short-Term Agricultural Communication Exchange Programs Abstract Emily Rhoades, Roslynn Brain, Ricky Telg, Tracy Irani, and Owen RobertsEmily Rhoades, Roslynn Brain, Ricky Telg, Tracy Irani, and Owen Roberts This paper describes the efforts of the University of Florida (located in Gainesville, Florida) and the University of Guelph (located in Ontario, Canada, near Toronto) to jointly develop a unique
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pilot exchange program for undergraduate and graduate agricultural communication students. The program was designed to encourage students to think critically about agriculture, agricultural communications, and culture in each other's countries. During this exchange, nine University of Guelph students traveled to Florida for one week during their winter breaks and six University of Florida students traveled to Canada during their spring breaks. The exchanges took place in back-to-back weeks . Students met with university administrators and agriculture industry representatives, toured agricultural facilities, and visited cultural locations . This paper details the planning process that culminated in the two-week program, provides students' comments about what they learned during the study-abroad experience, and makes recommendations to faculty interested in organizing similar study-abroad programs. In most businesses today, employers are looking for skills and competencies that extend beyond technical subject matter. Gorchels, Jambulingham, and Aurand (1999) note that cultural adaptability and work ethic are important traits for those being hired for international-related positions . Moreover, the authors write that adaptability is likely to increase with exposure to different cultures . Others have expressed the need to enhance American university curricula with international topics and to place more emphasis on "globalization" in general (Acker, 1999; Fugate & Jefferson, 2001; Moore & Woods, 2003; Redmann, Schupp, & Richardson, 1998; Tritz & Martin, 1997). One way to develop international competencies is through an international study tour. Two other skill sets that employers seek in new employees are effective communication abilities (Herman , 1995) and the ability to make decisions Journal of Applied Communications
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I 17 1 Rhoades et al.: Bridging Borders: Organizing Short-Term Agricultural Communicatio Published by New Prairie Press, 2017 and think critically. Professionals in agricultural communications have repeatedly voiced a need for graduates to possess strong communication skills in a variety of areas (Sitton, Cartmell, & Sargent, 2005; Sprecker & Rudd, 1998). The development of critical thinking skills in agricultural audiences has also been identified as an especially important need, based on findings that suggest potential deficiencies in students' abilities to think critically (Rudd, Baker, & Hoover, 2000). Research also has suggested a potentially important need to improve the critical thinking dispositions of agricultural communications students (Bisdorf-Rhoades, Ricketts, Irani, Lundy, & Telg, 2005; Telg & Irani, 2005). Pairing an international experience with these skill sets creates a desirable combination. But how can these important skills be brought together into an effective package? The University of Florida's Department of Agricultural Education and Communication and the University of Guelph's Ontario Agricultural College jointly developed a pilot exchange program that represented a first for each university. The purpose of the exchange was to expose undergraduate and graduate agricultural communication students to agriculture, agricultural communications, and culture in the other country, while enhancing critical thinking abilities. Nine University of Guelph (UoG) students traveled to the University of Florida (UF) for one week over their winter breaks, and six UF students traveled to Canada during their spring breaks. Students met with university administrators and agriculture industry representatives, toured agricultural facilities, and visited cultural locations. Students' critical thinking and perceived international competencies were
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assessed both before and after the exchange using both qualitative and quantitative instrumentation. This paper will explain how the University of Florida and the University of Guelph developed their study-abroad exchange program to teach college students about agriculture, culture, and communications in their respective countries. Furthermore, participating students' critical thinking skills and perceptions of their international experiences will be described, and recommendations for other universities that want to develop similar agricultural communication study-abroad tours will be provided . and think critically. Professionals in agricultural communications have repeatedly voiced a need for graduates to possess strong communication skills in a variety of areas (Sitton, Cartmell, & Sargent, 2005;Sprecker & Rudd, 1998). The development of critical thinking skills in agricultural audiences has also been identified as an especially important need, based on findings that suggest potential deficiencies in students' abilities to think critically (Rudd, Baker, & Hoover, 2000). Research also has suggested a potentially important need to improve the critical thinking dispositions of agricultural communications students (Bisdorf-Rhoades, Ricketts, Irani, Lundy, & Telg, 2005;Telg & Irani, 2005). Pairing an international experience with these skill sets creates a desirable combination. But how can these important skills be brought together into an effective package? The University of Florida's Department of Agricultural Education and Communication and the University of Guelph's Ontario Agricultural College jointly developed a pilot exchange program that represented a first for each university. The purpose of the exchange was to expose undergraduate and graduate agricultural communication students to agriculture, agricultural communications, and culture in the other country, while enhancing
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critical thinking abilities. Nine University of Guelph (UoG) students traveled to the University of Florida (UF) for one week over their winter breaks, and six UF students traveled to Canada during their spring breaks. Students met with university administrators and agriculture industry representatives, toured agricultural facilities, and visited cultural locations. Students' critical thinking and perceived international competencies were assessed both before and after the exchange using both qualitative and quantitative instrumentation. This paper will explain how the University of Florida and the University of Guelph developed their study-abroad exchange program to teach college students about agriculture, culture, and communications in their respective countries. Furthermore, participating students' critical thinking skills and perceptions of their international experiences will be described, and recommendations for other universities that want to develop similar agricultural communication study-abroad tours will be provided . Process The idea for such an exchange came about as a result of discussions at a professional conference in Kansas City, Missouri, in 2003 between the UF and UoG faculty advisers for the Agricultural Communicators of Tomorrow . The advisers believed both sets of students would be intrigued by the opportunity to travel to a different country-still in North America-and the opportunity to interact with fellow students twice: once in their own country and once in the other students' country. A study-abroad program also would be the stepping stone to begin a more formal agreement between the two universities for long-term study-abroad programs for students in any college major, notjust agriculture or agricultural communication . The advisers talked on the
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telephone and then face-to-face at the two professional conferences they attended in 2003 (the Association for Communication Excellence in Agriculture, Natural Resources, and Life and Human Sciences annual meeting and the National Agricultural Communicators of Tomorrow conference). As planning progressed, the target date was pushed back from 2004 to 2005. In 2005, UoG' s one-week winter break was the week immediately before UF's spring break respectively). Therefore, the exchange would occur in back-to-back weeks. As far as organizers could ascertain (after discussions in their universities' international programs offices), this exchange involving two consecutive weeks was a first for both universities. Each instructor secured the help of students to plan the weeks of activities. UF was responsible for all arrangements (except airfare) for the UoG students on their trip to Florida. UoG was responsible for all UF arrangements in Canada (again, excluding airfare). These arrangements included ground transportation, lodging, food, entertainment and activities, and educational excursions. Each working group consisted of three to five graduate and undergraduate students. Each student took one day and was in char ge of the overall arrangements for that day, arranging educational speakers and presenters, securing restaurant reservations, and planning entertainment and activities. The only aspect of the day not managed by students was transportation . Because they were immersed in the planning process for each day's activities, students learned first-hand about specialevent planning and coordination. During the early planning process, the UF and UoG planning groups met by telephone conference calls once every three months to discuss where they were in
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planning the events. About two months before the tour, the groups met about every other week to finalize plans. Each group wanted to give students from the other university a taste of agriculture and communications in their country, in addition to providing some specific cultural experiences. Each day had a different theme; themes included "Florida tourism," "Florida agriculture and communications," "Guelph agribusiness," and "Niagara region." The students learned about specific agricultural commodities and met with agricultural communicators and policymakers. While in Florida, Journal of Applied Communications I 19 3 students visited farms and companies representing the citrus, strawberry, equine, horticulture, and feeder cattle industries. While in Ontario, they experienced maple syrup production, Canadian horticulture, dairy cattle production, and wine production . These industry tours allowed students to learn about the indigenous agriculture of the province I state and hold open discussions with the farmers in those regions. Students also met with policymakers at the Florida Farm Bureau and with the Ontario Minister of Agriculture. These trips allowed students to learn about the policy issues facing both countries, such as urban sprawl, commodity prices, and free trade. To introduce students to the communications industry in both areas, tours were given of the Toronto Star and of the UF /Institute of Food and Agricultural Sciences Communications Services unit. Participants were also given a chance to experience life on the UoG and UF campuses through student-led tours. As for cultural and entertainment activities, both groups included a variety of experiences while trying to keep costs affordable. Activities held in
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Florida included a trip to Disney World's Magic Kingdom, a visit to an alligator farm, a day in St. Augustine (the nation's oldest city), attendance at a college basketball game, a taste of southern seafood, and a stroll along one of Florida's many beaches . In Canada, students enjoyed a "taste of Canada" dinner, a visit to a farmers' market, a trip to Niagara Falls, a day in Toronto, a semi-pro hockey game, and ice skating. Florida students visiting Canada were given the opportunity to see snow for the first time, while Canadian students got a taste of the humid Florida weather. Of the nine students from UoG, four were graduate students. Eight were students in Guelph's Ontario Agricultural College and one was a student in computer science. From Florida, six students traveled abroad; all were from the College of Agricultural and Life Sciences, with five from the Department of Agricultural Education and Communication and one from the Department of Family, Youth and Community Sciences. One UF student was a graduate student. The advisers also traveled with the student groups. Many of the students who traveled abroad also helped host the other group when they visited, which helped establish a feeling of camaraderie and friendship across the border. Both groups had a welcome reception on the first day of their respective study-abroad tours. Guelph students developed an online newsletter detailing their travels to Florida. Each day, one student was required to take photographs, write a short article about the day's events, and upload the story and
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photos. Three students from each university who engaged in the short-term study tour received academic credit. To earn the credit, they were required to research an aspect of the 20 I Journal of Applied Communications other country's agricultural industry and write a short paper about it. They also made a presentation about the industry they researched. As for travel expenses, each university developed a firm budget for the other university. The universities tried to keep expenses (excluding airfare) to around $475 to $500 (USD) per student. To avoid having to exchange money, funds were collected from one university's students and then used to pay for the other university's expenses. However, because UoG had three more students involved, the institution paid UF an additional amount of $1,425 ($475 x 3). Critical Thinking The tours and learning experiences provided opportunities to identify some ways to directly connect students' international study experience with their ability to think critically about what was discussed during the trips . The advisers involved wanted to examine whether an international experience would strengthen students' critical thinking dispositions . After receiving approval from the University of Florida Institutional Review Board, advisers developed a survey instrument based on previous research in critical thinking (Bisdorf-Rhoades, Ricketts, Irani, Lundy, & Telg, 2005) to gauge students' critical thinking dispositions. The researchers conducted tests both before and after the two-week exchange program to ascertain whether students' critical thinking dispositions had increased as a result of the studyabroad experience. Dispositions are trait-based, and would not necessarily be expected to change over
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a timeframe as short as a week; however, since this was an exploratory study, researchers felt it would be helpful to test. Fourteen students (six at UF and eight at UoG) completed the tests. The University of Florida Engagement, Maturity, and Innovativeness (UF-EMI) assessment was the survey instrument used to measure students' critical thinking dispositions. The instrument is a 33-item, 5-point Likert-type scale, with a demographics portion at the conclusion of the assessment. Results from the quantitative measurements of critical thinking dispositions were not found to be statistically significant when t-tests were run on the data, suggesting that critical thinking dispositions did not change during the two-week experience. Critical thinking skill-the expressed competency-was assessed qualitatively through content analysis of open-ended questions derived from the University of Florida Critical Thinking Skills (UF-CTS) instrument. The UF-CTS, a four-question, open-ended survey, was given to the students after the exchange, asking them for their respon _ ses about their experiences and thoughts during the program. Qualitative measures were appropriate since researchers theorized that critical thinking skill is contextually based. Some of the students' responses to the open-ended questions follow. It should be noted that during the two-week program, two major news stories involving U.S. I Canadian relations took place : the U.S. border remained closed to Canadian beef after it was supposed to reopen on March 7, and Canada opted out of the missile defense system for North America . Overall, students said this experience opened their eyes to life and agriculture across the U.S. I Canadian border. Canadian student
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responses There were many important messages garnered from this experience; however, I felt that the most important message was that the problems Florida agriculture is dealing with are very similar to those affecting Ontario agriculture. The major issues that crossed borders were urban sprawl and lack of public awareness. People tend to think that the problems they are faced with only affect them and are unaware that others elsewhere may be dealing with the same issues. This trip opened my eyes to this problem, and taught me to be less self-absorbed . The main conclusion that I brought back from this experience is that Florida has a thriving agriculture industry that ... faces many of the same issues facing Ontario agriculture. However, I do feel that Florida is more advanced in the education aspect of agriculture. They are already spreading the word, and I believe it would be a wise step to analyze in greater detail the successes and failures of their initiatives, so that Ontario can follow suit and bring some of their initiatives to our industry. One of the most unconvincing things that I saw in Florida is the way that the government is letting farmland be consumed by developers. I think they are thinking way too short-term and need to open their eyes to what Florida is going to look like in another 50 years. This is where I am so satisfied with the initiatives of the Ontario government and their more long-term vision. My beliefs and opinions have made a drastic shift
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since my return from Florida. Originally, I had the "Mickey Mouse" idea of Florida, with tourists and a lazy, simple lifestyle . Obviously, I know this was not the case everywhere, but I didn't realize how much of an impact agriculture has on Florida. I think the most meaningful message sent out was that as agricultural communication students, we must all work harder to increase public awareness about agricultural issues and the product our sector provides. U.S. student responses Throughout the week we visited with the Canadian students and were exposed to their perspectives on the border closure as well as other issues in agriculture. Although we may have differed in our opinions, we listened to each other's views. Being on the other side of the border when the cow trade market was yet again postponed greatly impacted my way of viewing the trade agreement. Being in another country where livelihoods were at stake caused me to look at the border closure for the global good and in the long run instead of looking at how it was affecting the U.S. cattle producers. The major conclusion that I have developed is that people do not know about agriculture . Although I already knew it, this trip has reinforced the fact that agricultural communicators are very important in getting the positive word out about agriculture to people who would possibly not know the facts. I also learned that there is not much difference between U.S. and Canadian agriculture . Both countries want to keep their people safe
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the best way they know how. The issues troubling farmers are also similar. My beliefs on Canadian agriculture have changed due to the fact that I was not aware of Canada's vast agricultural industry. I now see that Canadian farmers have the same types of problems that American farmers experience. Before this trip, I really only knew that Canada produced maple syrup and some cattle . I had no idea that Ontario had such good soil and the potential to grow such a wide variety of foods . Especially with regard to the wine industry, I learned that Ontario is producing some of the finest wines in the world. As far as culture goes, I had no idea that the "spirit of the cowboy" could exist anywhere else but America. However, I realized that being a farmer or rancher is about a way of life and an appreciation for the land that goes far beyond the Southern twang. The most important conclusion I developed was that good agricultural communications is critical to the survival of the ag industry both here and abroad. I realized how unaware the average American/ Canadian is about the needs of farmers and ranchers. 1. For a weeklong study-abroad program, try to keep all expenses (including airfare) to $1,000 or less. Much more than that will decrease students' interest in the program . 2. Identify someone in the country you plan to visit to help organize the activities in that country. Then develop a theme for each day of the visit. The
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organizers of this study tour found that a daily theme helped students understand what they were about to learn on a given day. 3. Start the planning process early. In this case, the two-week exchange took more than a year from initial discussion to final execution to plan. Study-abroad tours can be organized in a much shorter timeframe; however, for first-timers, it is recommended to build in plenty of planning time. Coordinate the planning. Meet frequently to make sure all issues are addressed before they become problems . 4. Enlist the help of students, recent graduates, and administrators. Students in the planning groups provided significant assistance to the overall program. They organized the days based around this question : "If I had never been to Florida I Ontario, what would I want to know I do?" They then planned the days accordingly. Administrators provided funding for the advisers to travel and meet with the touring groups. The recent graduates helped the planning groups arrange tours and provided meals free of charge. 5. Communicate the study-abroad experience to others. UoG students did an online newsletter while they traveled in Florida, and UF students made two presentations to students and faculty about their experiences in Canada. 6. Tie in many educational experiences and fun activities, but do not overdo it. Give students some "down" time. Build in activities and experiences that will engage students' critical thinking skills . A session conducted prior to the trip could allow instructors to introduce the concept of critical thinking, which could then
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be built upon through activities during the trip. 7. For logistical purposes, cap the number of students participating. Only 10 students were allowed to travel in the course described here. This made it possible for only one adviser to manage the students on the trip. The small size of the group also helped with tours; many agriculture locations will not give tours to groups larger than 15 or 20. This number is reached quickly once visiting students are added to students from the home institution. 8. If possible, tie in a research angle. Conclusions The students and faculty involved in this project believe it was an overwhelming success . It allowed students to see and experience life from a different perspective. Students developed lasting friendships, learned about agriculture in a different country, learned about international issues (trade, open borders), and learned communication skills and issues. They also applied their two weeks of U.S. I Canadian relations by analyzing, evaluating, and interpreting their experiences-all hallmarks of critical thinking. While the quantitative pretest and posttest for critical thinking disposition did not show significant differences, posttest qualitative answers did show that students were thinking critically about their experiences. It may be the case that a brief international experience alone may not stimulate critical thinking. Students were not introduced to the concept of critical thinking after the pretest and thus may not have increased their disposition significantly. More needs to be done with the students in fostering the development of the disposition . However, students' eyes were opened to the
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similar issues facing both countries and specific issues pertaining to each country, which is a crucial step in the critical thinking process . Students in both countries were somewhat stunned that policymakers (the Ontario Minister of Agriculture and Florida policymakers) would take time out of their busy schedules to talk with college students. They experienced agricultural issues and major news events-border closings, free trade, missile defense urban sprawl-firsthand from the other country's point of view . Students at both universities already want to do the exchange again . The advisers involved are looking into ways to make that happen. Also, at least three UoG students have inquired or formally applied to UF for master's or doctoral degrees, and one student is currently enrolled as a doctoral student. In addition, the advisers and faculty in both countries have already begun collaboration on joint U.S. I Canadian agricultural communication research grants. This synergy likely would not have come about without the study abroad experience . Probably the most telling component of the program is the friendships that were forged. Under the hot, humid Florida sun or in the frigid, snowy cold of the Canadian "maple sugar bush," students reached out to other students and became fast friends. They danced, laughed, and cried together. At the National Agricultural Communicators of Tomorrow conference, students from the UoG/UF exchange greeted each other like long-lost friends . Bringing together agricultural communicators from two cultures today established relationships that will continue when these students Journal of Applied Communications I 25
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Immunomodulatory Activity of a Traditional Sri Lankan Concoction of Coriandrum sativum L. and Coscinium fenestratum G. Objective To investigate the immunomodulatory activity of a traditional Sri Lankan concoction of Coriandrum sativum L. and Coscinium fenestratum (Gaertn.) Colebr., which is a Sri Lankan traditional medicine used to relieve inflammation and cold. Methods In vivo anti-inflammatory activity was tested using carrageenan-induced rat paw-edema model. Mechanism of anti-inflammatory activity was assessed by investigating the production of nitric oxide (NO), expression of iNOS enzyme, and reactive oxygen species (ROS) by rat peritoneal cells. The membrane stabilizing activity was also tested. The antibody response was determined by assessing the specific haemagglutination antibodies raised against sheep red blood cells. Results The three doses of freeze-dried concoction used ((human equivalent dose (HED)—183 mg/kg) 2 × HED and 1/2HED; n = 6 rats/group) showed significant inhibition of paw edema compared to water control at 3rd–5th hours (p < 0.05). Both HED and 1/2HED exhibited marked anti-inflammatory activity (72–83% inhibition at 4th-5th hours; p < 0.05). The HED of the concoction showed significant inhibition of NO (77.5 ± 0.73%, p < 0.001) and ROS production (26.9 ± 2.55%; p < 0.01) by rat peritoneal cells. Inhibition of NO production in the concoction treated rat peritoneal cells was confirmed by the lack of iNOS expression. The concoction also exhibited significant membrane stabilizing activity (IC50 = 0.0006 μg/ml; p = 0.001). HED resulted in a significantly high induction of specific antibody production against SRBC antigens as detected by SRBC haemagglutination assay (mean day 14 titers 253.3 compared
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to control: 66.7) (p < 0.01). Conclusions The traditional Sri Lankan concoction of C. sativum and C. fenestratum demonstrated potent in vivo anti-inflammatory activity, significant reduction of ROS, and NO production by rat peritoneal cells and the lack of iNOS expression confirmed the low NO production. The increased membrane stability also supports the anti-inflammatory activity of the concoction. Further, this concoction induced a significantly high antibody response reflecting its immunostimulatory activity. Together these results scientifically validate the therapeutic use of the concoction of C. sativum and C. fenestratum in Sri Lankan traditional medicinal system for immunomodulatory effects. Colebr. (family: Menispermaceae) is used as an immunomodulator for various types of ailments including relief of pain, inflammation, cold, and other viral infections for centuries [2]. Immunomodulatory and anti-inflammatory agents are therapeutically important since the pathogenesis of the common cold involves a complex interplay between replicating viruses and the host's inflammatory response [3]. A decoction is made using equal amounts of seeds of C. sativum (coriander; "Kottamalli" or "Kothamburu" in Sinhala and "Kottamalli" in Tamil) and stem of C. fenestratum (calumba wood or tree turmeric, "Veniwalgatta" in Sinhala; "Maramanjal" in Tamil) is a well-known home remedy in Sri Lanka for cold and inflammations, especially during the early stage of infection [2]. ese two ingredients are also constituents of the commercially available formulations called "Paspanguwa" along with three other plants parts, Zingiber officinale Roscoe., Oldenlandia corymbosa L., and Solanum surattense Burm.f.) and also in another commercial formulation called Samahan which is a combination of 14 ingredients including these two [4].
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e two plant parts, seeds of C. sativum and stem of C. fenestratum, are known to have a range of uses in traditional medicine and in Ayurveda. Coriander is used for treatment for anxiety, flatulence, loss of appetite, and convulsions [5]. Coriander seeds are used as carminative, diuretic, tonic, stimulant, stomachic, cooling agent, aphrodisiac, and analgesic [6]. Coriander has been attributed to have several medicinal uses, having antidiabetic, diuretic, cholesterol lowering, anticancer, anti-inflammatory, antifungal, antihelmintic, antioxidant, and antimicrobial effects [7][8][9][10][11]. Stem of C. fenestratum is thermogenic, ophthalmic, anti-inflammatory, vulnerary, depurative, stomachic, antiseptic, febrifuge, sudorific, and tonic [12,13]. Stem pieces of C. fenestratum are boiled and one cup is given for a fresh, deep cut, being the most common use against tetanus [2]. e root bark is used for dressing wounds and ulcers. C. fenestratum powder is mixed with ghee and used to apply for quick healing of ulcers. For snake bite poisoning, paste of C. fenestratum and turmeric is applied. C. fenestratum is reported to have anticancer, antimicrobial, antidiabetic, and antioxidant effect and is also used to treat cholera, gastroenteritis, and bleeding piles [14][15][16]. e seeds of C. sativum and stem of C. fenestratum have previously been shown to have antiinflammatory activity when tested alone and as ethanolic concoctions of individual ingredients [17,18]. Some immunostimulatory activity has also been reported with aqueous and ethanolic concoctions of C. sativum when used as a single ingredient [19][20][21][22]. e main objective of this study was to scientifically validate the traditional use of this concoction of seeds of C. sativum
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and stem of C. fenestratum as an immunomodulator. More specifically, we investigated its in vivo anti-inflammatory activity using the carrageenan-induced rat pawedema model and its effect on some of the immune cellular mechanisms including the production of nitric oxide (NO) and reactive oxygen species (ROS) and the expression of inducible nitric oxide synthase (iNOS) by rat peritoneal cells, membrane stabilizing activity of the concoction, and its immunostimulatory activity in enhancing antibody response. e reference drugs, aspirin, indomethacin, and prednisolone and also the syringes, needles, surgical blades, and cannulas (18G) were purchased from State Pharmaceuticals Corporation of Sri Lanka. Tissue culture plates (24 wells and 96 wells round and flat bottom), plates for Enzyme Linked Immunosorbant Assay (ELISA), round bottom tissue culture plates were purchased from Nunc, USA, and nitrocellulose filters (2 μM) and Whatman filter papers (No. 1) were obtained from Whatman Int. Ltd., UK. Reusable rat feeding needle was purchased from Orchid Scientifics, India. 2.2. Preparation of the Concoction of C. sativum and C. fenestratum. Seeds of C. sativum and stems of C. fenestratum were purchased from a reputed Ayurvedic store in Colombo, Sri Lanka, and authenticated by Dr. Chandima Wijesiriwardena at the Industrial Technology Institute, Colombo, Sri Lanka. Voucher specimens of C. sativum and C. fenestratum were deposited at the Institute of Biochemistry, Molecular Biology, and Biotechnology (IBMBB), University of Colombo, Sri Lanka. e concoction was made according to traditional Sri Lankan medicinal practice [2], by boiling 30 g each of C. sativum seeds and C. fenestratum stem in 1920 ml of water in
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a copper vessel till it reached approximately 240 ml. e concoction was filtered using Whatman No. 1 filter paper and freeze dried (Freezone 4.5-Labconco Corporation, USA). e human equivalent dose (HED) was calculated using the following formula [23]. weight of the concoction from 120 ml(mg) × weight of the rat(kg) × 6.2 Weight of a normal human(60 kg) , Rat metabolic rate � 6.2. (1) All experiments were conducted in accordance with the internationally accepted laboratory animal use and care, based on 3 Rs. Ethical clearance was obtained from the Research, Ethics and Higher Degrees committee of the IBMBB, University of Colombo. Animals were subjected to mild ether anesthesia for all procedures. Assessment of In Vivo Anti-Inflammatory Activity of the Concoction by Using Carrageenan-Induced Rat Paw-Edema Assay. ree doses of freeze-dried concoctionhuman equivalent dose (HED-183 mg/kg), high dose (2 × HED-366 mg/kg), and a low dose (1/2HED -92 mg/ kg), were orally administered to three groups of rats (n = 6/group). Indomethacin (5 mg/kg) was used as the reference drug (positive control) and water (2 ml) was administered to the control group. Paw volumes were measured hourly, after the carrageenan (0.1 ml of 1% carrageenan) injection on the left hind paw by using a digital Plethysmometer (Panlab sl., Barcelona, Spain) as described previously [24,25]. Assessment for Nitric Oxide Production by Rat Peritoneal Cells. Peritoneal cells were collected as described previously [26]. ree groups of rats were orally treated with HED (the optimum dose selected), prednisolone as reference drug (10 mg/kg), and water as control. One hour
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after the oral treatment, 1 ml of 0.1% carrageenan (1 mg/ml) was injected to the rat peritoneal cavity. Two hours after this, 40 ml of sterile phosphate buffered saline (PBS) was injected and approximately 35 ml of fluid was drained from peritoneal cavity. e drained peritoneal fluid was centrifuged at 500g for 10 minutes and resuspended in 1 ml of RPMI-1640 medium containing 1% bovine serum albumin (BSA) and total cell and differential cell counts were taken using a Neubauer's haemocytometer (Neubauer, Germany). Rat peritoneal cells as described above were used to evaluate the inhibitory effect of the HED against the production of nitric oxide. Cell suspension (200 μl of 1 × 10 6 /ml) was plated in 96 well tissue culture (TC) plates where 6 wells were maintained for each rat. e TC plate was incubated for 24 hours at 37°C in a 5% CO 2 incubator. After 24 hours, the supernatant was collected, centrifuged at 10, 000g for 10 minutes, and stored at −20°C for quantification of nitrite levels. Griess assay was used to quantify nitrite levels in rat peritoneal culture supernatants by mixing 100 μl of culture supernatant with equal volume of Griess solution (equal amounts of 1% Sulphanilamide and 0.1% N-(naphtyl) ethlenediamine hydrochloride) [27]. Optical density at 540 nm was measured 15 minutes after adding the Griess solution using an ELISA microplate reader (ELx 800-Universal Microplate Reader, Biotek Instruments, Canada). A dilution series of NaNO 2 standards from 100 to 0.781 μM were used to prepare nitrite standard curve. e amount of
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nitrites in μM was computed from the standard curve plotted for NaNO 2 . Assessment for the Expression of iNOS by Rat Peritoneal Cells. Total RNA was extracted from rat peritoneal phagocytic cells using the TRIzol reagent (1 ml of TRIzol to 1 × 10 6 cells) according to the manufacturer's instructions (Invitrogen, USA). Extracted RNA was quantified and then cDNA was synthesized using RNA (2 μg), dNTPs mixture (2 mM), random primers (500 ng), RNAsin (25 units), M-MLV reverse transcriptase enzyme (200 units), and the RT buffer (1X) and PCR was carried out for selected genes iNOS, eNOS, nNOS, and GAPDH independently using the same cDNA. Primers for rat iNOS were selected according to Linenluke et al. and thermal cycle parameters were initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1 min, and the final extension of 72°C for 10 min [28]. Primers for other constitutive forms of NOS, eNOs, and nNOS were selected as indicated in Liu et al. (annealing temperature for rat eNOS was 60°C, whereas the annealing temperature for rat nNOS was 62°C) [29]. Rat GAPDH gene which was used as the control or house-keeping gene was also amplified with the primers indicated in Wu et al. [30]. All amplified PCR products were resolved in 2% agarose gel and visualized by the UV transilluminator (Vilber-Laumart gel documentation system). Assessment of ROS Production in Rat Peritoneal Cells. Peritoneal cells collected as described in Section 2.5 were used to evaluate
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the effect of the HED against the production of ROS. Concentration of cell suspensions were adjusted to 4 × 10 5 cells/ml using complete RPMI containing 10% fetal bovine serum (cRPMI) and 8 × 10 4 cells in 200 μl of cell suspension were plated in 24-well culture plate and the final volume/well was increased to 400 μl with 200 μl of cRPMI added to each well. For each rat, 3 wells were maintained. Diphenyleneiodonium chloride (DPI) was used as in vitro positive control. For this, 200 μl of cell suspension obtained from a rat treated with water was plated with 200 μl of 10 μM DPI in cRPMI. Plate was incubated for 1 hour at 37°C with 5% CO 2 to allow the cell attachment. After one hour, 200 μl of supernatant was removed, 200 μl of 2 mg/ml of nitro blue tetrazolium (NBT) with 12 μg/ml phorbol 12-myristate 13acetate was added to each well and the plate was incubated for 30 minutes at 37°C with 5% CO 2 . After half an hour supernatant was removed and plate was washed twice with prewarmed (37°C) PBS. e plate was fixed using 70% methanol and allowed to dry and 120 μl of 2 M KOH and 140 μl of absolute dimethyl sulphoxide were added to each well and the plate was placed on a shaker for 10 minutes. Dissolved formazan (200 μl) was transferred into 96-well Evidence-Based Complementary and Alternative Medicine 3 ELISA plate and absorbance was read at 620 nm. e concentration of O −
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2 was calculated using standard NBT curve as described previously [31]. Assessment of Membrane Stabilizing Activity of Concoction. is assay was performed by heat-induced haemolysis of rat erythrocytes as described previously [26]. Tenfold dilution series of concoction was made using PBS for concentrations from 0.0001 to 1000 μg/ml. Dilutions of Aspirin was also made using PBS for the same concentrations and used as the standard drug. PBS was used as control. Rat erythrocytes washed and resuspended in PBS (20 μl) was added to each tube containing 980 μl of each concentration of test, aspirin and control samples. Samples were first incubated at 37°C for 15 min. Cell suspensions were centrifuged at 1500g for 3 min, the supernatants were removed and the cells were resuspended in 1 ml of PBS. Samples were then incubated at 54°C for 25 min to initiate heat-induced haemolysis and centrifuged at 1500g for 5 min. Supernatants (200 μl) were transferred into an ELISA plate and the optical density (OD) was measured at 540 nm. Percentage inhibition of haemolysis was calculated with respect to the controls and inhibitory concentration (IC 50 ) values were derived. Percent inhibition of haemolysis � [(OD control − OD sample/OD control] × 100. Assessment of the Effect of the Concoction on Rat Antibody Production and Detection of Antibodies by Haemagglutination Test. is experiment was designed to investigate the effect of oral treatment of rats with the concoction on their specific antibody production against SRBC antigens. e SRBC immunization was performed according to a modification of the previously described
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method [32][33][34]. Two groups of rats (n � 6/group) were orally treated with HED of concoction and water on days 1, 2, 3, 7, 8, and 9. A preparation of 0.5 × 10 9 cells of freeze-thawed SRBC was injected intraperitoneally on days 1 and 7. Serum collected on days 0, 7, and 14 was tested for anti-SRBC antibodies using SRBC haemagglutination assay [35]. Day 0 (preimmune) sera were used as the negative control, while days 7 and 14 sera were collected to ascertain the levels of antibodies after the exposure to SRBC antigen. Haemagglutination plates were incubated at 37°C for 16 hours. Statistical Analysis. Data were analyzed using the statistical package SPSS 17. Data were expressed as mean ± SD/SEM. One-way ANOVA was carried out; p ≤ 0.05 were considered as significant. e Mann-Whitney U test and independent t-test were carried out for small sampled tests. One-way ANOVA followed by post hoc Turkey was carried out to compare the inhibition of in vivo anti-inflammatory activity. Pearson corelation was calculated for dose dependency. In Vivo Anti-Inflammatory Activity of the Concoction. All three doses of the concoction showed significant antiinflammatory activity which was comparable to the positive control indomethacin (Figure 1). ere was a significant decrease in paw volumes in all three groups, compared to water control at 3 rd , 4 th , and 5 th hours (p < 0.05). e doses, 1/2HED and HED, overlapped at the first and the third hour and the ½HED showed the highest percentage inhibition in paw volumes at the
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fourth (83.5%) and the fifth (80.1%) hours followed by HED (72%). e percent inhibition of the double dose (2 × HED) increased gradually and reached its maximum at the 5 th hour (55.6%); however, its overall antiinflammatory effect was low compared to the other two doses (1/2HED and HED). is resulted in an inverse dosedependent activity at 4 th (r � −0.99; p � 0.03) and 5 th (r � −1.00; p � 0.001) hours. Since HED showed a significant level of anti-inflammatory activity in the first and second phases of inflammation, it was selected as the optimum dose for further assays. e concoction reported hereafter in the results section is the HED of the concoction. Figure 2, nitrite level was significantly inhibited by the concoction and percent inhibition was 77.5 ± 0.73% (p < 0.001). e reference drug and prednisolone had inhibited nitrite levels similarly by 91.5 ± 0.69% (p < 0.001). Inhibition of Nitric Oxide Production and iNOS Expression by Rat Peritoneal Cells. As shown in As shown in Figure 3, amplifications of GAPDH were observed in all three samples (RNA obtained from the rats injected with carrageenan and treated with the reference drug (prednisolone), water, and concoction). Amplification of iNOS was observed only in the control (water treated) and was absent in the concoction treated and the group treated with the reference drug. Amplification of nNOS was clearly observed for concoction treaded group, whereas amplification of both nNOS and eNOS was weak in the group treated with the reference drug. Inhibition of In
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Vivo ROS Production by Rat Peritoneal Cells. As shown in Figure 4, concoction had significantly inhibited the O 2 production and percent inhibition was 26.9 ± 2.55% (p � 0.002). e two positive controls, in vivo (prednisolone) and in vitro (DPI), had also inhibited the O − 2 production significantly and percent inhibitions of O − 2 production were 47.8 ± 1.78% and 48.5 ± 1.97%, respectively (p < 0.001). Figure 5, the concoction showed 88.34% of inhibition for heamolysis at 0.1 mg/ml and comparable to the reference drug aspirin (88.36%; p � 0.001) and IC 50 value of the concoction was 0.0006 μg/ml. Effect of Oral Administration of the Concoction on Rat Antibody Production. Effect of oral administration of the concoction on rat antibody production was evaluated by assessing the SRBC haemagglutination titers. e sera obtained on day 0 showed no antibodies against SRBC in both groups. On day 7, the SRBC haemagglutination titers in both concoction and control groups of rats were low and comparable (p > 0.05), but both groups showed agglutination ( Table 1). Titers of the concoction group on days 7 and 14 increased significantly from 100 (mean) to 253.33 (p � 0.03), whereas no significant change was observed in the control group. By day 14, the group of rats treated with the concoction showed a significant increase in their SRBC haemagglutination titers compared to day 14 sera of the control (mean value 253.3 and 66.7, respectively; p � 0.004). is showed that after two sets of 3-day oral treatment (and
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with two sets of antigen exposures), the concoction was able to induce a significant increase in the specific antibody response against SRBC. Discussion is study was designed to determine the immunomodulatory activity of the concoction of C. sativum and C. fenestratum. is was first ascertained by the in vivo antiinflammatory activity using the carrageenan-induced rat paw edema model and the results showed a significant antiinflammatory activity of the concoction. Since HED of concoction showed a significant level of anti-inflammatory activity at both first and second phases of inflammation, it was selected as the optimum dose for subsequent experiments to determine possible mechanisms of its anti-inflammatory activity. Further, this study demonstrated significant inhibition of nitric oxide and superoxide anion production by rat peritoneal cells and also in vitro membrane stabilizing capacity reflecting their possible contribution to the anti-inflammatory mechanisms. e specific Evidence-Based Complementary and Alternative Medicine inhibition of the expression of iNOS by the oral administration of the concoction confirmed the marked decrease of NO production by the rat peritoneal cells. e immunomodulatory activity was also shown by the enhancing effect on specific immune responses as evident by the high antibody titers raised against SRBC antigens following oral administration of concoction. is study reports for the first time the immunomodulatory activity of the concoction of C. sativum and C. fenestratum, including in vivo anti-inflammatory activity with mechanisms, that is, inhibition of ROS and RNS production and also the inhibition of iNOS gene expression by rat peritoneal cells and the immunomodulation to enhance antigen specific antibody response. Further,
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the findings of this study validate the use of the concoction (or decoction of C. sativum and C. fenestratum) for its traditional claims and use for treatment of cold and as a treatment at early stage of infections. In the carrageenan-induced paw-edema assay, the two doses (HED and 1/2HED) of the concoction showed significant anti-inflammatory effect during both early and late phases and there was marked inhibition during the late phase. e difference in the phases of the assay may occur due to different chemical and cellular components which come into action in the early, intermediate, and late phases of inflammation [36]. e inhibition in the first phase of inflammation is attributed to activities against serotonin and histamine, whereas inhibition during second phase is mainly due to activities against prostaglandins and suppression of mononuclear leukocyte migration [36]. In contrast to the biphasic pattern, the high inhibition during 2 nd hour (intermediate phase) shown by the HED of the concoction indicates that it may have inhibited the kinins which are known to play a role in between the first and second phases [37]. e inverse dose response observed in the present study with the 2 × HED exhibiting a lower anti-inflammatory activity is consistent with the results of a previous study where the potency of a high dose of an alcoholic preparation of coriander (500 mg/ml) was less compared to a low dose (200 mg/ml) [37]. In addition, two other studies have reported anti-inflammatory activity of ethanolic extract of C. sativum [17,38]. Ammar et al. have attributed
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the inhibitory effects of coriander fruits (one of the components in the concoction) to inhibition of all mediators released before the second phase as well as prostaglandin, the mediator in the second phase. Phytochemical studies on bioactive extracts of coriander have revealed the presence of unsaturated fatty acids, flavanoids, that is, quarcertin which may together produce an anti-inflammatory effect [37]. It is noteworthy that a comparative study with ethanolic extracts of Curcuma aromatica Salisb. and C. fenestratum have reported that C. fenestratum at a dose of 8 mg/kg exhibited in vivo anti-inflammatory to a lesser extent (34% inhibition at 3 hours). e anti-inflammatory activity was attributed to the presence of tannins and flavonoids [18]. Similarly, another comparison with ethanolic extracts of fruits of Coriandrum sativum leaves of Datura stramonium and Azadirachta indica at 200 mg/kg doses has shown that C. sativum had the less potent anti-inflammatory activity (41% inhibition at 3 hours) compared to A. indica which had the highest activity as reported in this study [17]. e present study used the traditional preparation of the concoction which has shown a much higher anti-inflammatory activity with half HED dose (92 mg/kg) having 84% inhibition. ese differences in anti-inflammatory activity may either be attributed to the different types of extraction types having different constituents with their intrinsic potencies. It may be possible that the aqueous extract used in the present study has more potent constituents or that when used as a combination they may show a synergistic effect. is antiinflammatory effects supports the widespread use of this
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combination in traditional medicinal practice in Sri Lanka and its use at the early stages of viral infections causing cold related symptoms. Recruitment of phagocytic cells (neutrophils and macrophages) are a characteristic feature of the late phase of inflammation [36]. In our previous studies on Ixora coccinea, we have shown that inhibition of the cell migration to the peritoneal cavity or to the site of inflammation as a mechanism that makes a significant contribution to the antiinflammatory effect [26]. In the early stage of action, polymorphonuclear cells predominate whereas mononuclear cells are more potent at the late stage [37]. In the present study, the HED of the concoction which exhibited the optimum anti-inflammatory activity was used to assess its effect on production of ROS (O − 2 ) and RNS (NO) by peritoneal cells. e significant decrease of both ROS and RNS reflects the cellular mechanisms that support the in vivo anti-inflammatory action shown in the paw-edema assay. Our previous studies on methnoloic leaf extract of I. coccinea and aqueous leaf extracts Vitex negundo have also shown similar inhibitory activity of NO and ROS production by rat peritoneal cells [26,48]. is study showed a considerable reduction in the nitrite levels in rat peritoneal cells obtained after the oral treatment of the concoction. e nitrite levels of the concoction treated rat cells are comparable to that of prednisolone treated group which has shown higher inhibition of NO production. Nitric oxide produced by inducible NOS plays an important role in inflammation and in regulation of the immune system
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[49]. Oral administration with the concoction showed a specific effect on expression of iNOS, whereas it had no effect on the expression of either nNOS or eNOS genes, the constitutive forms of NOS, which are necessary for the normal cell functions. In contrast, the carrageenaninduced and water treated control group was positive for iNOS gene expression. e reference drug used as the positive control has also shown a significant inhibitory effect on iNOS gene expression as well as the constitutive forms of NOS. e presence of GAPDH gene expression which is a house keeping gene [50] in all three groups was indicative of normal cell functions despite the specific effect on iNOS gene expression. ese findings confirm that the significantly reduced nitric oxide production in the concoction treated group was due to the specific inhibition of expression iNOS gene. It is also important to note that the concoction treatment has less or no inhibitory effects on the constitutive forms of NOS unlike the reference drug used as the positive control. e reduced or excess production of iNOS is known to leads to many immunological disorders. It is also responsible for the deleterious effects in inflammation [51,52]. Many plant components, such as flavonoids, sesquiterpene, and polyphenols, have been shown to inhibit the iNOS expression [53]. C. sativum and C. fenestratum both contain many flavonoids, alkoloids, and phenolics [14,54], which could have contributed to the low production of nitric oxide. However, no specific components have been shown to have the iNOS inhibitory activity from either C. sativum or
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C. fenestratum. One of the most immediate responses of monocytes to a variety of pathogenic stimuli is the production of the potent oxygen free radical, superoxide anion. e enzyme complex primarily responsible for the production of this highly reactive oxygen species is the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Superoxide anion is the first ROS produced and it can combine with nitric oxide to produce peroxinitrite which is highly deleterious to tissues [55]. Quantitative assessment of superoxide anion production by NBT assay showed the capability of concoction to inhibit superoxide anion production in rat peritoneal cells. us, the ability of the concoction to inhibit production of both NO and superoxide anion would contribute to preventing the formation of peroxinitrite. Our previous studies using the quantitative NBT assay have shown similar ROS inhibitory activity with methanolic leaf extracts of Ixora coccinea [25]. e concoction showed a significant membrane stabilizing activity, which indicates that it may be another mechanism that would contribute to the anti-inflammatory effects observed. At the onset of an inflammation, the cells undergo activation and release inflammatory mediators. Stabilization of cell and cell organelle membranes would prevent the release of inflammatory mediators. Membrane stabilization has been identified as a mechanism of antiinflammatory activity of other medicinal plants such as Solanum aethiopicum and Basella alba [56,57]. It is noteworthy that our findings on the concoction in the rat experimental system on enhanced antibody production are consistent with some of the previous studies when only the C. sativum extracts were used. Different preparations of aqueous and
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ethanolic extract of C. sativum had shown immunostimulant activity in mice [19], chickens [20], and fish [21,22]. It is possible that the immunostimulatory activity observed in the concoction of C. sativum or C. fenestratum is attributable to the effect from C. sativum. Alternately, it may be due to a combined effect of C. sativum or C. fenestratum since some of the immunostimulatory effects observed in the present study was comparatively higher compared to those observed when C. sativum was used by itself. Further studies are in progress to investigate on the exact active component(s) responsible of the Evidence-Based Complementary and Alternative Medicine increased antibody production and the specific mechanisms of activation of lymphocytes in producing an enhanced antibody response. Interestingly, the concoction or the combined extract used in the present study exhibited both anti-inflammatory as well as immunostimulatory effects showing a broad spectrum of immunomodulation. is shows the capacity of the concoction to modulate the innate immune response as well as induce an enhanced antibody response against specific antigens SRBC. ese immunomodulatory effects may have direct relevance to its traditional use in treatment of inflammation and cold where it could suppress inflammation relieving the cold symptoms, while the specific antibodies raised may prevent having antiviral effects preventing viral replication and further progression of the disease symptoms. According to traditional medical practitioners, the concoction is prescribed when an individual gets first symptoms of an infection meaning during or soon after an antigen exposure. It is also one of the home remedies practiced for years for treatment at
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early stage in infection [2]. It is noteworthy that rats were orally treated with the decoction just prior to the SRBC antigen exposure and in consecutive 2 days in both immunizations (day 1 and day 7). us, these rats were treated in a similar manner to prescribed ailment before checking their antibody levels and a marked elevation in antibody levels was observed within 14 days (for HED of concoction). is indicates the concoction in its traditionally prescribed dose (HED) has the capacity to boost the immune system to protect against an infection or by strengthening the immune system to combat the disease effectively by elevating antibodies against the specific antigen. is also further emphasizes an adjuvant effect by this concoction on antibody production against a specific antigen preparation. In a previous study, leaf extract of Vitex negundo and our previous studies have reported its adjuvant effect (to standard anti-inflammatory drugs) [48,58]. ese results emphasize further studies on identifying the bioactive components and other cellular mechanisms of lymphocyte activation such as the early and increased expression of costimulatory molecules that would enhance the immune response. Conclusions is study has shown the immunomodulatory activity of a concoction of C. sativum and C. fenestratum by possessing both anti-inflammatory (inhibition of inflammation, nitric oxide, superoxide anion production, and membrane stabilizing activity) and immunostimulatory (enhancement of antibody production) activities. e markedly reduced nitrite levels and superoxide anion and lack of iNOS gene expression in rat peritoneal cells and increased membrane stability may be the key immune cellular mechanisms which support this
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anti-inflammatory activity of the concoction. Furthermore, the significant elevation of antibody levels clearly supports the immunostimulatory activity which also shows long-term protection against specific antigens. erefore, this study for the first time scientifically validates the therapeutic claim of Sri Lankan traditional use of the concoction of coriander (C. sativum) and veniwalgata (C. fenestratum) for immunomodulatory effects. Data Availability e data used to support the findings of this study are available from the first author and corresponding author upon request. Additional Points (1) e concoction of Coriandrum sativum and Coscinium fenestratum shows a significant dose-dependent reduction of inflammation as shown in the rat paw edema model. (2) Several in vitro mechanisms were shown to contribute to the anti-inflammatory effect: (a) marked inhibition of NO and ROS production by rat peritoneal cells, (b) absence of iNOS expression in rat peritoneal cells confirm the inhibition of NO production, and (c) significant membrane stabilizing activity. (3) e concoction of C. sativum and C. fenestratum showed a significantly higher antibody response against specific antigens. Conflicts of Interest Shashika Dinethri Kothalawala is a Ph.D. Trainee, Institute for Veterinary Anatomy-Histology and Embryology, Justus Liebig University, Giessen, Germany. Jayamini C Harasgama is a PhD Trainee, Department of Marine Life Sciences and Fish Vaccine Research Center, Jeju National University, Republic of Korea. Galbada Arachchige Sirimal Premakumara is a Senior Lecturer, Department of Basic Sciences and Social Science, Faculty of Nursing, University of Colombo, Colombo 3. e other authors declare no conflicts of interest.
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Exercises in activating lymphatic system on fluid overload symptoms, abnormal weight gains, and physical functions among patients with heart failure: A randomized controlled trial Background Fluid overload remains a vexing problem in management of heart failure. The lymphatic system that plays the central role in fluid homeostasis has recently been explored as a potential target to counteract tissue fluid overload. The goal of the study was to evaluate the preliminary effects of exercises in activating lymphatic system on fluid overload symptoms, abnormal weight gains, and physical functions for patients with heart failure. Methods and results A pilot, pre- and post-test, randomized clinical trial was conducted to recruit a total of 66 patients who were randomized to receive either a 4-week The-Optimal-Lymph-Flow for Heart Failure (TOLF-HF) program or usual care alone. The primary outcome was the prevalence and burden of the fluid overload symptoms. Findings of the trial showed that the TOLF-HF intervention was effective in reducing the prevalence or burden of the majority of fluid overload symptoms. TOLF-HF intervention also demonstrated significant improvement in the outcomes of abnormal weight gains (MD: −0.82; 95% CI: −1.43 to −0.21; P = 0.010) and physical functions (F = 13.792, P < 0.001). Conclusions The TOLF-HF program focusing on activating lymphatic system through the performance of therapeutic lymphatic exercises holds the promise as an adjuvant therapy for patients with heart failure to manage fluid overload symptoms, reduce abnormal weight gains, and improve physical functions. Future larger-scale study with longer duration of follow-up is needed. Clinical Trial Registration http://www.chictr.org.cn/index.aspx, identifier ChiCTR2000039121.
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Introduction The increase in heart failure (HF) prevalence and considerable burden to healthcare systems have made HF a global pandemic (1,2). Fluid overload, the hallmark of HF, refers to a progressive body fluid retention or redistribution that impedes multiple body system functions (3). Many symptoms are associated with fluid overload symptoms, such as dyspnea, coughing, wheezing, edema, pain, or fatigue (3,4). These symptoms are highly prevalent and burdensome, leading to poor quality of life and decreased daily living functions (4). The experience of fluid overload symptoms not only is the most commonly cited reason for HF hospital admission or readmission but also creates substantial burdens on health system, patients, and families (5,6). Lymphatic system plays an essential role in body fluid regulation and pathogenesis of cardiovascular diseases (7)(8)(9). The lymphatic vascular system that lies parallel to the blood vascular system maintains fluid homeostasis between intravascular and interstitial spaces by collecting fluid from the interstitial space and draining it back into the venous circulation (10)(11)(12). It is estimated that up to 8 liters of interstitial fluid are scavenged each day by the lymphatic capillaries that line all organs and are transported by the unidirectional lymphatics to the blood circulation (10). Fluid overload occurs when fluid is not drained at the same rate as it leaks into the interstitial spaces (10,11). Activating lymphatic system holds a great promise for reducing fluid overload as the interstitial fluid is drained exclusively by lymphatic pumping (11,12). Exercise that stimulates muscle contraction and breathing can promote lymphatic flow, and potentially decreases fluid
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accumulation in tissues and interstitium (12,13). A three-to six-fold increase in the lymph clearance rates was observed using a scintigraphic device during human active exercise compared with resting levels (14). This phenomenon was also confirmed in an animal experiment using direct cannulation of a lymphatic duct in anaesthetized sheep (15). Therefore, therapeutic exercises focusing on optimizing lymph fluid flow can be promising to prevent and manage fluid overload symptoms in HF. This pilot study was conducted to examine the effects of exercises stimulating lymphatic system on fluid overload symptoms in HF. We hypothesized that lymphatic exercises that simulate lymphatic pumping and drainage could decrease fluid accumulation in tissues and interstitium in patients with HF, in turn, alleviate fluid overload symptoms and burden (12,13). We have developed and extensively tested a non-pharmacological The-Optimal-Lymph-Flow (TOLF) intervention that builds patients self-management skills to promote lymph fluid flow and results in reduced pain, swelling, lymph fluid level, reverse of mild lymphedema, and improved function and quality of life in cancer patients (16)(17)(18)(19)(20)(21)(22)(23)(24). TOLF includes strategies to promote lymph fluid flow: therapeutic lymphatic exercises, healthy diet, and proper sleep (16-24). The essential component of TOLF is 8-minute lymphatic exercises (i.e., muscle-tightening deep breathing, muscle-tightening pumping, and large muscle exercises) designed to stimulate lymphatic system by simulating lymphatic pumping to promote lymph fluid flow (16). We conducted a pilot single-blind two-group randomized controlled trial (RCT) to evaluate the preliminary effects of 4-week TOLF lymphatic exercise training in 66 adult patients with HF. Design and setting The-Optimal-Lymph-Flow for Heart Failure (TOLF-HF) trial utilized
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a prospective, single-center, two-arm RCT design. This trial was conducted in the Department of Cardiology of West China Hospital, the largest national center for the diagnosis and treatment of complicated and critical cardiovascular diseases in western China. The Ethics Committee of West China Hospital, Sichuan University approved this trial (No. 2019-202) and this trial was registered on Chinese Clinical Trial Registry (ChiCTR2000039121). The current study is reported following the CONSORT reporting guideline (25). Recruitment and participants Potential participants were identified from March 2019 to January 2020 via review of inpatient census lists and were introduced to the study two days before their discharge. A total of 66 patients met all eligibility criteria and consented to study participation after the recruiting researcher explained the study objectives, procedures, and possible risks/benefits. If patients agreed to participate in the trial, they were asked to sign a written informed consent and complete baseline assessment. Inclusion criteria for the trial included that patients were: (1) aged 18 to 80 years; (2) hospitalized with a primary diagnosis of HF; (3) classified as New York Heart Association (NYHA) functional class II or III; (4) willing to complete the home-based TOLF-HF program. The diagnosis of HF was made by an expert team of cardiologists in compliance with 2018 Chinese guidelines for the diagnosis and treatment of HF (26). We excluded patients who (1) had severe liver impairment (i.e., Child-Pugh score ≥10) or kidney insufficiency (i.e., an estimated glomerular filtration rate <30 ml/min/1.73 m 2 ) or malignant tumors; (2) had a terminal condition with
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a life expectancy of less than 6 months; (3) had received or were waiting for heart transplantation; (4) were undergoing respiratory muscle training or resistance training; (5) were participating in other research programs; or (6) were unable to read or understand Chinese. Randomization and masking Following baseline assessment, participants were randomly assigned to receive either a 4-week TOLF-HF program plus usual care or usual care alone with a 1:1 ratio. Randomization assignment was carried out by an independent research assistant using random numbers generated by the random number generator in the SPSS for Windows (Version 22.0; IBM Corp., Li et al. 10.3389/fcvm.2023.1094805 Frontiers in Cardiovascular Medicine Armonk, NY, USA) and individual allocations were concealed in sequentially numbered, opaque and sealed envelopes until interventions were assigned. Because of the nature of the exercise intervention, blinding participants and interventionist was not feasible. However, the outcome assessor and data analyst were blinded to the group allocation. Interventions Patients allocated to the control group only received usual care. In addition to guideline-directed medical therapy (26, 27), usual care included provision of a written discharge summary (including the diagnosis, disease course, inhospital treatment record and postdischarge medications, etc.) and oral instructions on appropriate lifestyle behaviors and medication management by the ward nurse upon discharge, and patients might be referred to post-discharge support as needed. All patients were routinely scheduled to visit the specialist clinic 4 weeks after discharge. No other structured educational or supportive postdischarge care was provided. Patients allocated to the intervention group received the 4-week TOLF-HF intervention in addition
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to usual care. TOLF-HF is a patient-centered behavioral program featuring self-care risk reduction strategies to promote lymph flow with the aim to improve fluid overload symptoms in patients with HF (16). Easyto-learn self-care strategies in activating lymphatic system consists of muscle-tightening deep breathing and muscletightening pumping exercises, as well as large muscle exercises. Specifically, muscle-tightening deep breathing activates lymphatic ducts and facilitates lymph fluid drain, muscle-tightening pumping helps lymph fluid flow and reduce fluid build-up, and large muscle exercises enhance lymph fluid flow and drain across the whole body (16). The self-care strategies along with their corresponding physiological rationales are presented in Table 1. TOLF-HF program was initiated as soon as possible after the baseline assessment and within 48 h before hospital discharge. A trained researcher (Q.M.) delivered the intervention in a 30-minute one-to-one meeting. First, a step-by-step video presentation showing how exercise should be done and how often it should be done was demonstrated to achieve unambiguous and standardized instruction. Next, patients were asked to practice on their own, and the researcher observed and corrected their wrong movements in a timely manner until they could perform all the movements themselves. The researcher made a video recording while patients were performing the lymphatic exercises. The video clip was provided to patients for them to review whenever necessary. Family members were involved throughout the procedure and asked to accompany and encourage patients' practices at home. After hospital discharge, subsequent WeChat contacts were made every week by the researcher to identify any associated barriers, offer pertinent advice, and
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motivate adherence to the TOLF-HF protocol. Outcomes We operationalized the primary outcome of the fluid overload symptoms as the prevalence and burden of symptoms associated with fluid retention (4, 28). Fluid overload symptoms were evaluated using the Chinese version of the Memorial Symptom Assessment Scale-Heart Failure (MSAS-HF) (29-31) to include symptoms of shortness of breath, difficulty breathing when lying flat, cough, lack of energy, swelling of legs or ankles, and waking up breathless at night (31, 32). Participants first reported whether they experienced each listed symptom for the past week; if so, then they reported the frequency on a scale ranging from 1 (rarely) to 4 (almost constantly) and severity on a scale from 1 (mild) to 4 (very severe) (29,30,33). Symptom burden scores are calculated as the mean of the frequency and severity of each symptom, with higher scores indicating greater symptom burden (33). The reliability of the MSAS-HF has been previously confirmed in the Chinese context with a Cronbach's α coefficient of 0.946 (31). The secondary outcomes included the number of abnormal weight gains and physical functions. An electronic scale and a structured daily log were provided to each participant to take home for recording body weight, and lymphatic exercise Muscle-tightening deep breathing exercises • The whole-body lymph fluid has to be drained through the lymphatic ducts above the heart. Muscle-tightening-deep-breathing stimulates lymphatic ducts and helps lymph fluid drain. • At least twice a day in the morning & at night before brushing teeth or as much as the patient wants throughout the day
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• Lymph fluid drains when muscles move. Muscle-tightening-deepbreathing creates the whole-body muscle movements that create muscle milking and pumping action and help to drain lymph fluid • Air-Travel: before take-off and after landing. • At least twice a day in the morning & at night before brushing teeth or as much as the patient wants throughout the day • Muscle-tightening pumping exercises build the muscles in upper and lower limbs that helps lymph fluid flow and drain. • Air-Travel: before take-off and after landing • Large muscle exercises create muscle milking and pumping to promote overall body lymph fluid flow and drain. • Air-Travel: get up and walk around for flight over 4 h. performance for participants in the TOLF-HF intervention group. All participants received the instructions on the use of the scale and self-monitor of weight daily at home. A rapid weight gain of greater than 1 kg in one day or greater than 2 kg in three days is considered as one occurrence of abnormal weight gain. Physical functions were measured using the physical subscale of the Minnesota Living with Heart Failure Questionnaire (MLHFQ) (34), which is one of the most commonly used disease-specific instruments that reflects how HF affects everyday life and functions of patients (35). The physical subscale of MLHFQ consists of 8 items rated on a 6-piont Likert scale from 0 (no impact) to 5 (very high impact). Summary scores range from 0 to 40; higher scores indicate poorer functioning. The Chinese version of the MLHFQ has been validated and the
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physical subscale demonstrated adequate reliability (α = 0.950) (36). Adverse events were defined as any discomfort or injury induced by the TOLF-HF program during the intervention period. Participants were asked to report to the research assistant immediately if an adverse event occurred due to the exercises. Data collection All participants completed the outcome measures at baseline and 4 weeks post-intervention under the guidance of an unbiased research assistant who was unaware of the study hypothesis and the group allocation, and were required to share their weight diary with the assistant via WeChat every week. Sociodemographic and clinical information were collected via patient self-report and review of electronic medical records. Sample size PASS software 15.0 (NCSS, LLC, Kaysville, UT, USA) was used to estimate the sample size based on the primary outcome measure informed by the results of our preliminary trial, which showed a 35% difference (40% vs. 75%) in the prevalence of shortness of breath (the most prevalent fluid overload symptom) between the groups. To detect this expected between-group difference at a two-sided significance level of 0.05 with 80% power, we required data from at least 28 participants per group. Accounting for an anticipated attrition rate of 15%, the final sample size needed for the study was 33 patients per group, a total of 66 patients to be randomized into control and intervention groups. Statistical analysis The baseline characteristics of the study participants were calculated using descriptive analyses stratified by treatment group. Data normality was verified using the Shapiro-Wilk test. Means with standard deviations (SDs) or
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medians with interquartile ranges (IQRs) were estimated for the continuous variables based on the normality of the data distribution. Differences between intervention and control groups were estimated using the independent sample t-test for normally distributed variables and the Mann-Whitney U test for variables that did not conform to normal distribution. Where appropriate, analysis of covariance (ANCOVA) was also calculated to adjust for baseline outcome measures and important demographics. Counts with percentages were calculated for the categorical variables. Pearson's χ 2 test with continuity correction or Fisher's exact probability test were used for between group differences. The modified Poisson regression models were built to estimate the adjusted risk ratios (RRs) with 95% confidence intervals (CIs) to estimate the intervention effect of TOLF-HF program on symptom prevalence while controlling for baseline covariates. All statistical analyses were conducted using SPSS 26.0 for Windows (IBM Corp., Armonk, NY, USA). A two-sided P value of less than 0.05 was set for significance level. Study participants The CONSORT flow diagram is shown in Figure 1. Eight-five patients were screened for eligibility; 66 patients met the inclusion criteria. Only six patients (9%) were not be able to complete the study, despite the final stage of the study being challenged by the COVID-19 outbreak. Thus, 60 patients completed both the baseline and follow-up assessments; data from the 60 patients were used for the analysis. Of the 60 patients, the mean age was 59.92 in years [standard deviation (SD) = 12.13] and 35% (n = 21) were female. The mean left ventricular ejection fraction (LVEF)
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for the participants was 34.63 (SD = 16.19); 48.3% of the participants were classified as NYHA class II and 51.7% class III. Detailed participant characteristics are presented in Table 2. Baseline characteristics between groups were similar, except for the number of co-morbidities: patients in TOLF-HF group suffered more co-morbidities as compared with control group (Z = 2.449, P = 0.014). 3.2. Primary outcome: fluid overload symptoms 3.2.1. The prevalence of fluid overload symptoms As shown in Table 3, no significant between-group differences at baseline were detected for symptom prevalence across the symptoms. At the study endpoint of week 4, the prevalence of shortness of breath (31.0% vs. 61.3%; P = 0.037) and swelling of arms or legs (13.8% vs. 41.9%; P = 0.033) were significantly lower in the TOLF-HF group than in the control group. The modified Poisson regression results shown in Figure 2 The number of abnormal weight gains A total of 39 times and 67 times of abnormal weight gains occurred during the study period in the intervention and control groups, respectively. As displayed in Figure 3, there was a significant between-group difference in favor of the TOLF-HF intervention group (MD: −0.82; 95% CI: −1.43 to −0.21; P = 0.010). Specifically, patients who received TOLF-HF intervention experienced 0.82 fewer times of rapid abnormal weight gain per person than those who only received care as usual. Physical functions As presented in Table 5, no significant group differences were found in the baseline values for physical functions. At the endpoint of the study, the MLHFQ physical
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subscale scores were significantly lower in the intervention group compared to the control group (F = 1,792, P < 0.001), indicating better physical functions for patients received the TOLF-HF intervention. Adverse events No exercise-related adverse events occurred throughout the course of the study. The TOLF-HF program was safe for patients who were discharged from the hospital with HF. Discussion The fundamental function of the lymphatic system in fluid homeostasis has long been overlooked, largely because of the difficulty in visualizing the transparent lymphatic vessels (8). This pilot clinical trial was the first to provide initial empirical evidence that the TOLF-HF program focusing on stimulating lymphatic system to promote fluid flow was effective in alleviating fluid overload symptoms of shortness of breath, cough, swelling of arms or legs and waking up breathless at night, decreasing abnormal weight gains, and improving physical functions for patients with HF. Recent research advances into the lymphatic network and the pathogenesis of cardiovascular diseases have made the lymphatic circulation an active target for the treatment of cardiovascular diseases (8,9). The innovative application of TOLF-HF lymphatic exercises in patients with HF may open new avenues for nonpharmaceutical interventions for prevention and management of fluid overload in HF. The accumulation of body fluid is an important driver of disease progression and symptom deterioration in HF (5). Worsening HF triggered by volume overload would manifest as multiple congestion symptoms including symptoms of pulmonary congestion and systemic venous congestion, causing patient distress (4,28). We found that the 4-week TOLF-HF lymphatic exercises in addition to standard patient
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care was beneficial in reducing either the prevalence or burden of the majority of the df, degree of freedom; SD, standard deviation; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; HF, heart failure; IQR, inter-quartile range; LVEF, left ventricular ejection fraction; NYHA, New York Heart Association (functional class); NT-proBNP, N-terminal pro-brain natriuretic peptide. *The data were compared by Pearson's χ 2 test with continuity correction. † The data were compared by Fisher's exact probability test. ‡ The data were expressed as median (IQR) and analyzed by the Mann-Whitney U test. Li et al. 10.3389/fcvm.2023.1094805 Frontiers in Cardiovascular Medicine fluid overload symptoms. Specifically, patients who received the intervention had 64.3% less likelihood of reporting waking up breathless at night and a trend towards less shortness of breath, cough, and swelling of arms or legs. Patients also benefited from the intervention by an alleviated symptom burden for shortness of breath, cough, and swelling of arms or legs. The symptomatic relief of trouble breathing (i.e., shortness of breath and waking up breathless at night) and coughing can be explained by the improved congestion in the pulmonary circulation while the relief of limb swelling can be explained by the improved congestion in the systemic circulation (37,38). The core TOLF-HF intervention consists of lymphatic exercises of muscle tightening, stretching and pumping movements coordinated with deep breathing to imitate the physiological process of lymph pumping and propulsion; in addition, lymphatic exercises also induce musculoskeletal contractions, arterial pulsations, skin tensions, postural changes, and breathing alterations. Such
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lymphatic exercises could produce a synergistic effect to accelerate fluid volume removal not only in the thoracic area but also throughout the whole body, thus achieving positive outcomes in fluid overload symptom relief (16). Our study found the TOLF-HF exercises did not reduce neither the prevalence nor the burden of difficulty breathing when lying flat or lack of energy. Difficulty breathing when lying flat represents the deteriorated cardiac health status and occurred more frequently in patients with more severe HF (39), while all patients recruited for this study were in chronic or stable phase and classified as NYHA class II or III. This might help explain the nonsignificant result for this symptom within our study. Lack of energy (also known as exercise intolerance or fatigue) in HF is a complex and multifactorial phenomenon; the exact pathogenesis of this Symptom Total prevalence TOLF-HF group (n = 29), n (%) Control group (n = 31), n (%) χ 2 P value* Forest plot showing estimates of RRs with 95% CIs of symptom prevalence between the TOLF-HF intervention and control groups after controlling for baseline prevalence values and the number of co-morbidities. RR, risk ratio; CI, confidence interval. 41). Therefore, it is reasonable that TOLF-HF exercises particularly targeting on the problem of fluid overload may not be as effective as expected in attenuating the perception of lack of energy. Weight fluctuation is an objective and measurable indicator of fluid volume status in HF (42). Our study suggested that the TOLF-HF intervention was effective in reducing the number of abnormal
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weight gains. Sudden weight gain commonly means that excess fluid is building up in the body and signals the worsening of cardiac pumping capacity (42). This finding supported our hypothesis that the TOLF-HF intervention, designed to stimulate the lymphatic system to promote fluid flow, was effective to decrease or offset the water retention in the body. The TOLF-HF intervention is safe and easy to perform while patients are sitting, standing, or lying; this makes easier for patients to adhere to the lymphatic exercises. Thus, the TOLF-HF intervention holds a great promise as an adjuvant intervention for patients with HF to maintain body weight within a stable range. Shortness of breath In addition, physical functions are important outcomes that reflects the impact of HF on patients' functional status (35). Findings of the trial demonstrated that the TOLF-HF Column bar graph showing the comparison of the number of abnormal weight gains between the TOLF-HF intervention and control groups. MD, mean difference; CI, confidence interval. (30,43) and the fact that the intervention prompted the patients to lead an active lifestyle. It is well-known that adopting a physically active lifestyle is one of the best ways that people can do to help prevent illness, preserve health, and enhance physical functions (44,45). To our knowledge, previous studies focusing on the effects of exercise intervention on fluid overload in HF were limited (46). This is a pioneer study that examined exercise-based intervention specifically targeting lymphatic system on alleviation of fluid overload symptoms by providing rigorous prospective randomized controlled data. Efficacious lymphatic transport
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entails the coordination of lymphatic pumping. Specifically, lymphatic contractions occur with a phase lag as the contractions propagated downstream from one lymphangion to the next generated the highest lymph flow (7, 10). The TOLF-HF exercises produced the desired effects as the intervention was designed to mimic the physiological process of lymphatic pumping (16). Apart from that, this intervention had a number of strengths. First, the TOLF-HF intervention is easy-to-learn and elderly-friendly, as such the intervention is especially appropriate for HF population. Second, the TOLF-HF intervention consists of a series of relatively lowintensity exercises and requires low physical demands. Even immobilized or bedridden patients can perform the most parts of the exercises. Finally, the TOLF-HF intervention has been shown to be feasible and acceptable to patients in this pilot trial as well as in previous experimental studies with breast cancer survivors (16-18, 24). Therefore, the TOLF-HF intervention is a pragmatic, effective, well-tolerated, and easily-integrated-into-daily-routine self-care program for the prevention and management of fluid overload and associated symptoms in patients with HF. We recognize that there are several limitations for the current study. There were no significant between-group differences in baseline characteristics except for the number of co-morbidities, some discrepancies still existed. Because of the relatively small sample size and the exploratory nature of this study, the possibility of chance findings cannot be excluded. Additional limitations include relatively short follow-up period, inclusion of the patients who had NYHA class II or III. Multicenter studies with a larger sample size, longer follow-up, and comprising of a wider spectrum of
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HF cases are needed. Patient participation and compliance with the exercise protocols are critical to fully elucidate the efficacy of the intervention. In the study, nearly 90% (26/29) patients reported performing the TOLF-HF exercises twice a day as prescribed. However, lack of real-time monitoring of the truly implemented exercise dose, limiting the study's ability to explore the dose-effectiveness of the intervention. Future technology innovation should focus on wearable device to monitor the adherence to TOLF lymphatic exercises. In conclusion, our study suggests that the TOLF-HF program focusing on promoting lymph fluid flow through the performance of therapeutic lymphatic exercises is an efficacious adjuvant therapy for patients with HF in the management of fluid overload symptoms, maintenance of stable body weight, and improvement of physical functions. Given its acceptability, practicality, safety, and effectiveness, the TOLF-HF program should be further tested as part of routine HF care. The current study represents an important initial step targeting the lymphatic system to counteract fluid accumulation and symptom exacerbations. Future research is to develop wearable devices for real-time monitor of the home-based TOLF-HF protocol, such innovation may allow detailed elucidation of the mechanisms of lymphatic system stimulation on fluid overload symptoms and body fluid level in HF. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Ethics statement The studies involving human participants were reviewed and approved by the Ethics Committee of West China Hospital of Sichuan University (No. 2019-202). The patients/participants provided their written informed consent to
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participate in this study.
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Hypoxic microenvironment in cancer: molecular mechanisms and therapeutic interventions Having a hypoxic microenvironment is a common and salient feature of most solid tumors. Hypoxia has a profound effect on the biological behavior and malignant phenotype of cancer cells, mediates the effects of cancer chemotherapy, radiotherapy, and immunotherapy through complex mechanisms, and is closely associated with poor prognosis in various cancer patients. Accumulating studies have demonstrated that through normalization of the tumor vasculature, nanoparticle carriers and biocarriers can effectively increase the oxygen concentration in the tumor microenvironment, improve drug delivery and the efficacy of radiotherapy. They also increase infiltration of innate and adaptive anti-tumor immune cells to enhance the efficacy of immunotherapy. Furthermore, drugs targeting key genes associated with hypoxia, including hypoxia tracers, hypoxia-activated prodrugs, and drugs targeting hypoxia-inducible factors and downstream targets, can be used for visualization and quantitative analysis of tumor hypoxia and antitumor activity. However, the relationship between hypoxia and cancer is an area of research that requires further exploration. Here, we investigated the potential factors in the development of hypoxia in cancer, changes in signaling pathways that occur in cancer cells to adapt to hypoxic environments, the mechanisms of hypoxia-induced cancer immune tolerance, chemotherapeutic tolerance, and enhanced radiation tolerance, as well as the insights and applications of hypoxia in cancer therapy. INTRODUCTION Cancer occurrence is markedly associated with increasing age, as patients aged over 60 years are more than twice as likely to develop invasive cancers than younger patients. The World Health Organization (WHO) estimates that by 2050, the proportion of the world
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population aged over 60 years will have increased from 12 to 22%, totaling over 2 billion individuals. 1 In the United States, cancer is the second leading cause of death after heart disease. In humans aged over 60 years, cancer is the leading cause of death. 2 Due to the aging of the global population, cancer has become a global public health issue, with a massive economic burden and complex treatment challenges. The distribution of oxygen partial pressure in tumor tissues is of particular interest to radiologists because the radiosensitivity of tumor tissues depends on the tissue O 2 tension. Tumor cells living under adequate hypoxic or hypoxic conditions are relatively resistant to radiation. 3 In 1953, Gray et al. found that well-oxygenated tumor cells responded trice as well to radiotherapy than hypoxic cells. 4 Tumor hypoxia was first proposed in 1955 by Thomlinson et al. in a study of tumor tissues from patients with lung cancer, and scientists have confirmed, through over 60 years of clinical and experimental evidence, that the hypoxic state is a widespread trait in a variety of solid tumors. The expression of key genes such as hypoxia-inducible factors (HIFs) and their various subunits, as well as the molecular regulatory mechanisms, were also explored under hypoxic conditions. [5][6][7][8][9][10][11][12][13][14] William Kaelin, Peter Ratcliffe, and Gregg Semenza have been awarded the 2019 Nobel Prize in Physiology or Medicine for the contributions of several scientists who have discovered how human and animal cells sense and adapt to oxygen supply (Fig. 1). 15 Hypoxia is present
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in 90% of solid tumors, which is considered a hallmark of cancer. 16,17 It is difficult to determine the hypoxic state in tumors due to variations in oxygen content between tissues, as well as differences in tumor size and measurement methods, and tissue oxygenation is highly variable, also within the same organ. 18 However, the available results indicate that the measurement of tumor partial pressure of oxygen (pO 2 ) in patients (polarographic technique) has demonstrated the presence of low values (<10 mmHg) in several different tumor types, including pancreatic cancer, head and neck tumors, breast cancer, cervical cancer, and melanoma. [19][20][21][22] Intra-tumor hypoxia is linked to decreased disease-free survival outcomes in several cancers including prostate, cervical cancer, and head and neck squamous cell carcinoma (HNSCC). [23][24][25][26] The hypoxic environment alters the expression levels of genes that modulate metabolism and other processes. Moreover, hypoxic signaling interacts with other cellular pathways to alter cancer cell malignant behaviors and is closely associated with cancer cell proliferation, migration, invasion and angiogenesis, and affects cancer treatment outcomes. 27 This article focuses on the unique hypoxic microenvironment in cancer, the possible mechanisms by which cells undergo transformation and malignancy, and the potential applications of these aspects. FACTORS CONTRIBUTING TO HYPOXIA IN CANCER The vasculature ensures the presence of tissue oxygen and energy substances. Vasculature endothelial cells (ECs) of cancer tissues or pre-neoplastic tissues are exposed to harmful substances, including various high-risk carcinogenic factors, such as drugs, carcinogens, pathogenic microorganisms, and the uniquely acidified tumor microenvironment (TME), which damage the shapes of
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ECs (for example, edema), resulting in dysregulated functions of cancer vasculature. 28 Cancer cells are characterized by high proliferative rates and active metabolism; they also consume high amounts of energy to support their increased cell proliferation and growth rates. 29 When metabolic oxygen demands exceed supply, the oxygen-deficient areas of cancer cells are exacerbated and they are change their metabolism. 30 Abnormal vascular structures and patterns that are due to dysregulated angiogenesis contribute highly to hypoxia. Although the expression of erythropoietin (EPO) and angiogenic factors are increased under hypoxic conditions, which promote the proliferation of vascular ECs, the arrangement is disorganized and non-functional vessels form. [31][32][33][34] Cancer rapidly grows such that the cancerous area is often deficient of vasculature and becomes hypoxic. 35 In the TME, chronic hypoxia occurs as the distance of cancer cells from blood vessels increases and O 2 diffusion decreases, which has been further validated in a mouse model of breast cancer. 36 Hypoxia can occur in tissues more than 100-200 µM from a functional blood supply, a phenomenon that is prevalent in solid tumors. 37 Additionally, using a model of rat brain gliom, Julien et al. found that the peritumor vasculature is vaso-responsive to hypoxia through alpha-smooth muscle actin, which can further exacerbate hypoxia in areas of tumor tissue with principle blood vessels. 38 Some blood vessels become abnormal and malfunction, resulting in hypoxia. 39 Non-cancer components and functions are greatly altered, including the activation and proliferation of stromal cells (for example, stellate cells and cancer-associated fibroblasts) and increased stromal components
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(for example, fibrin), 40,41 leading to remodeling of cancer morphology, such as vascular compression. This can result in impaired circulation and inadequate oxygen supply, further leading to thrombosis and increased tissue hypoxia. 42 Physiological effects of various factors, such as magnesium, change with the changing hypoxic environment. Under normoxic conditions, magnesium stimulates angiogenesis, whereas under hypoxic conditions, Mg inhibits angiogenesis. 43 The above factors can cause hypoxia in the TME (Fig. 2). Carcinogenic factors, such as drug, carcinogen, and microbiota dysbiosis, impair EC shape and function in the vascular system. TME is remodeled by tumor cells, stromal cells and stromal components (e.g., fibrin), resulting in vascular deformation due to pressure. High metabolism in cancer cells, such as increased nucleic acid synthesis and increased protein anabolism, leads to relative hypoxia. Dysregulated proliferation and alignment of vascular ECs result in the formation of non-functional blood vessels. With the increased distance between tumor cells and blood vessels, O 2 diffusion decreases and leads to hypoxia Hypoxic factors are prevalent in most solid tumors. Some tumors contain transient hypoxic cells, others contain chronic hypoxic cells, and others contain both components. This may include three different cell populations. The first type is chronically hypoxic cells. If left in situ, the cells die. These "doomed" cells survive alone after the necessary excision in the cell survival assay and does not affect the response of the tumor when left in situ, which is considered the main cause of cell necrosis in the central region of solid tumors. The second type is the chronically
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hypoxic cells that are viable if left in situ. These cancer cells are stimulated by hypoxia to promote proliferation, alter gene expression, and enhance cellular drug resistance and radioresistance. 16,[44][45][46][47][48][49] The third type is transient hypoxic cells, which are expressed closer to functional blood vessels, where the duration of hypoxia is short. Based on current evidence, tumor cells adapt to hypoxia by altering their signaling pathways. Hypoxia promotes malignant behavior of cancer cells, including proliferation, migration, infestation and epithelial-mesenchymal transition (EMT), and enhances immunotherapy, chemotherapy, and radiotherapy tolerance. HYPOXIA AND CARCINOGENESIS Hypoxia and genomic damage Hypoxia damages the cellular genome and drives carcinogenesis. In in vitro and in vivo hypoxic cancer models, gene mutation frequencies of hypoxic cancer cells were increased by 2-to 5-fold. [50][51][52] In tissue culture and animal models, gene amplification and mutations were associated with induction of DNA strand breaks during hypoxia, including DNA double-strand break (DSB) and single-strand break (SSB). [53][54][55] In addition, hypoxic conditions (<5%) could improve the efficiency of induced pluripotent stem cell (iPSC) generation from mouse and human somatic cells. 56 The hypoxic signaling microenvironment maintains stem cell selfrenewal by facilitating the reprogramming process. [57][58][59] The extrapolation of these iPSC studies provides insight into cancer stem cell (CSC), which can also exist in hypoxic ecological niches. 60 Hypoxia is known to promote and maintain CSC phenotypes. 61 CSCs are thought to have the potential to form tumor that will develop into cancer, especially when they metastasize with the cancer and will give rise to novel sources of cancer. 60
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Both mutation due to cellular genomic damage and the maintenance of CSCs are closely related to the production of large amounts of reactive oxygen species (ROS) under hypoxic conditions. 62,63 The aforementioned mutations and breaks stimulate expression of oncogenes, thereby inducing the formation of cancer cell variants that have the potential to metastasize and grow. 64 Hypoxia and genetic repair Hypoxia activates ataxia telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) checkpoints. ATM and ATR belong to the phosphatidylinositol 3-kinase-like protein kinase (PIKK) family and are the main members of the DNA damage checkpoints. They are activated by different types of DNA damage and regulate cell cycle checkpoints by phosphorylating their corresponding downstream proteins (CHK1 and CHK2). The specificities and functionalities of ATM and ATR during DNA damage differ, with ATM being mainly involved in DNA DSB repair. After hypoxia-mediated DNA double-strand breaks, the MRE11-RAD50-NBS1 (MRN) complex activates ATM and is autophosphorylated at serine 367 (ser367), serine 1893 (ser1893), serine 1981 (ser1981), and serine 2996 (ser2996), thereby inducing MRE11-RAD50-NBS1 (MRN) complexassociated recruitment of various complex phosphorylation cascades, such as p53 (cancer suppressor), CHK1, and CHK2, to the DNA DSB sites. These effects can be blocked by the inhibition of CDK2 activity. During G1/S or G2/M cell cycle progression, cells have more time to repair DNA damage before entering mitosis. ATR kinase phosphorylates p53 and CHK1 under extreme hypoxia (oxygen concentration <0.02%). 65 Once ATR is activated, it phosphorylates and inactivates CDC25 by phosphorylating CHK1 and CHK2, thereby resulting in failure to activate CDK2. These lesions can
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block G1/S or G2/M cell cycle progression by phosphorylating CDK1. [65][66][67] In glioma mice models, inactivation of the ATM/CHK2/p53 pathways promoted cancer formation. 68 Hypoxia cannot induce G2 arrest in CHK2-deficient cells, but these cells can undergo apoptosis under hypoxia. 67,69 Persistent hypoxia enhances DNA misreplication and DNA strand breaks, resulting in mutant cell phenotypes. 70 Non-homologous end joining (NHEJ) and homologous recombination (HR) regulate the repair of human DNA DSB. [71][72][73][74] DNA DSB are the most severe and extensive types of damage; HR can accurately repair these damages, especially in the S/G2 cell cycle stage. NHEJ is the simplest mechanism of DNA DSB repair and can act in all cell cycle phases, except for the M phase. In hypoxic conditions, HR repairs the lesions less frequently, whereas NHEJ activities are unaffected. 75,76 The mismatch repair (MMR) pathway can also be deregulated under hypoxic conditions. MMR is a DNA repair pathway that targets replicationrelated errors and primarily functions to correct the misintegration of nucleotides during DNA synthesis, thereby preventing permanent DNA damage in dividing cells. Hypoxia decreases the expression of MLH1 and MSH2, which induces mutations and dinucleotide repeat instability. These pathways may lead to sustained damage to intracellular RNA, resulting in cell transformation, including carcinogenesis. 77,78 HYPOXIA SIGNALING PATHWAYS AND CANCER CELLS HIFs and cancer cells To survive under hypoxic conditions, cancer cells reprogram their metabolism, protein synthesis, and cell cycle progression through the synergy of transcription factors. 79 One of the main reasons why cancer cells can survive in hypoxic environments is the activation
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of HIFs, which reprogram metabolism, protein synthesis, and cell cycle progression. 79,80 The HIF family has two distinct subunits: α (HIF-1α, HIF-2α, and HIF-3α) and β (HIF-1β). HIF-1α is widely expressed in all body tissues, whereas HIF-2α and HIF-3α only occur in specific tissues. 27 HIF-1α is an oxygen-unstable protein that becomes stable in response to hypoxia, iron chelators, and divalent cations. 81,82 In cell culture under hypoxic conditions, HIF-1α mRNA levels did not change, but HIF-1α protein levels increased. 83 HIF-1β is constitutively expressed in mammalian cells under normoxic conditions. 84 Owing to the presence of the oxygen-dependent proline hydroxylase family (PHD), under sufficient oxygen conditions, the HIF-α protein is hydroxylated and interacts with von Hippel-Lindau tumor suppressor protein (pVHL) to promote HIF-1α ubiquitin-proteasomal degradation. [85][86][87] Ectopic expression of PHD1 inhibited HIF-1α and suppressed tumor growth. 88 However, under hypoxic conditions, enzymatic activity of PHD is inhibited, preventing HIF-α hydroxylation and ubiquitin-mediated proteasomal degradation, leading to abnormal accumulation of HIF-α in cells. Significantly elevated mRNA of HIFs was detected after 1 h at O 2 concentrations <10%. 89 In addition, high expression of the CSN subunit CSN5 stabilizes HIF-1α aerobically by inhibiting HIF-1α prolyl-564 hydroxylation. 90 Also under normoxic conditions, elevated intracellular ROS induced sustained expression of HIF-1α protein. 91 The accumulated HIF-1α binds to HIF-1β and enters the nucleus to bind the hypoxia response element (HRE) in the promoter region of the target gene to reduce cellular oxygen consumption. 92,93 HIF-3α exerts the opposite effects in cancer cells by impairing angiogenesis, proliferation, and metabolism.
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94 Biologically, HIF is regulated by multiple signaling pathways, including the PI3K-mTOR signaling pathway; 95-98 JAK-STAT3 signaling pathway; 99 NF-κB pathway; 100,101 mitogen-activated protein kinase (MAPK) pathway; 102,103 Wnt/β-catenin pathway; 104 Notch pathway; 105 cancer suppressor gene deletion, such as p53, 106 phosphatase, and tensin homolog (PTEN); 107 and IDH1-R132H-FAT1-ROS-HIF-1α signaling pathway (Fig. 3). 108 Mechanistically, HIF regulates cancer cell growth by regulating genes encoding enzymes that hydrolyze sugars, angiogenic signaling genes, and apoptosis/stress response genes. 109 Hypoxia and cancer cell behaviors Among various cancers, patients whose primary cancers are hypoxic at diagnosis are more likely to have local recurrence and metastatic site recurrence, regardless of whether the initial treatment is surgery or radiation therapy. Cancer patients with conditions such as anemia or chronic obstructive pulmonary disease tend to have poorer prognostic outcomes and may have cancer-related hypoxia, at least in part, owing to increased propensity to develop metastatic disease. 26,110 Young et al. reported that exposure of cancer cells to a hypoxic environment in vitro or in vivo enhanced spontaneous metastasis. 111,112 Similar results were obtained in mouse models of fibrosarcoma and cervical cancer. 53,113 To an extent, hypoxic conditions eliminate tumor cells that are sensitive to hypoxia, and the surviving tumor cells adapt to hypoxia through their own molecular transformation. 114 Several early experiments have supported that hypoxiainduced increases in HIF-1α expression can drive the metastatic phenotype by upregulating genes involved in the metastatic cascade, such as urokinase-type plasminogen activator receptor (uPAR), matrix metalloproteinase 1 (MMP1), chemokine receptor 4 (CXCR4), osteopontin (OPN), known as
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secreted phosphoprotein 1 (SPP1), lysine oxidase (LOX), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF). 115-123 Hypoxia-exposed cancer cells are selective for the loss of p53 functions or increased expressions of the p53 negative regulator (MDM2), leading to increased resistance to apoptosis and increased metastasis. 124,125 Other experiments did not find correlation between hypoxia and p53. 126 HIF-1α binds and activates the MAX interactor-1 (MXI1), a repressor of the c-MYC transcriptional activity, and reduces the expressions of c-MYC, a factor that encodes mitochondrial DNA replication and promotes mitochondrial biogenesis, 127,128 which inhibits cancer cell mitochondrial biogenesis and cellular oxygen consumption, resulting in cancer growth and survival in a hypoxic environment. 129 Unfortunately, MYC overexpression occurs in 70% of human cancers. 130 HIF-2α promotes the expressions of the MYC and E2F target genes, which are involved in lipoprotein metabolism and ribosome biosynthesis. 131 Conversely, MYC maintains cancer stem cell self-renewal properties by selectively binding the promoter and activating the HIF-2α stemness pathway. 132 Both HIF-1α and MYC can enhance the glycolytic pathway and drive cancer proliferation and progression. 79,133 Under hypoxic conditions, HIF-1α directly and positively regulates ephrin A3 (EFNA3) expressions. Ephrin type-A receptor 2 (EphA2), a key functional mediator downstream of EFNA3, promotes sterol regulatory element binding protein (SREBP1) maturation, which drives the self-renewal, proliferation, and migration of hepatocellular carcinoma (HCC) cells. 35 Under the effects of HIF-1α, prostate intraepithelial neoplasia (PIN) cells highly express transglutaminase 2 (TGM2) and exhibit impaired androgen signaling, enhancing malignant progression. 23 In addition, some factors, such as Orai1; 134
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phospholipase D2 (PLD2); 135 annexin A3 (ANXA3); 136 CXCR4; 137 cysteine-rich protein 2 (CSRP2); 138 hematopoietic pre-B cell leukemia transcription factor-interacting protein (HPIP); 139 twist family bHLH transcription factor 2 (TWIST2); 140 and noncoding RNAs, such as long-stranded non-coding RNA (lncRNA) PVT1, 141 lncRNA-GAPLINC, 142 and LncRNA-MTA2TR; 143 and micro-RNAs, such as miR-525-5p, 144 miR-301a-3p, 145,146 and miR-141-3p, 147 play important roles in regulating hypoxia-induced cancer cell proliferation, migration, invasion, and angiogenesis in the TME under hypoxic conditions (Table 1). HYPOXIA AND CANCER CELL METABOLISM Hypoxia and glycolysis The majority of cancer cells increase glucose uptake and rely on glycolysis through a phenomenon known as the "Warburg effect". 30,148,149 The "Warburg effect," activated by MYC and HIF-1 in response to growth factors and hypoxia, is aerobic glycolysis Fig. 3 Biological changes in cancer cells adapt to hypoxia. Hypoxia promotes carcinogenesis by inducing DNA strand breaks, including DNA DSB and SSB, and by weakening DNA repair pathways, such as HR and MMR. HIF-1α is upgraded by PI3K-mTOR, JAK-STAT3, NF-κB, MAPK, Wnt/ β-catenin, and Notch pathway. Deletion of tumor suppressor genes, such as p53, PTEN, and ROS production, also contributes to the upregulation of HIF-1α. The loss of pVHL function under hypoxic conditions indirectly leads to HIF-1α accumulation. HIF-1α dimerizes with HIF-1β and enters the nucleus to bind to HRE, which regulates various downstream target genes (Table 1) to promote cancer cell proliferation, migration, invasion, EMT and angiogenesis with the purpose of meeting the nutrient and energy requirements for rapid genome replication. HIF-1α is the main transcription factor
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that promotes Warburg-like metabolism. 131,133,150 HIF-1α stimulates various enzymes such as glycolysis regulator phosphoglycerate mutase 1 (PGAM1), pyruvate kinase M (PKM), recombinant phosphoglycerate kinase 1 (PGK1), lactate dehydrogenase A (LDHA), lactate dehydrogenase C (LDHC), and lactate dehydrogenase-5 (LDH-5) to induce anaerobic metabolic shifts that lead to energy production. [151][152][153][154][155][156] HIF-2α promotes MYC target gene expression and enhances constitutive expression of LDHA. 131,157 In addition, HIF-1α inactivates pyruvate dehydrogenase (PDH) by activating pyruvate dehydrogenase kinase 1 (PDK1), which in turn fails to convert pyruvate to acetyl-CoA, preventing the entry of pyruvate into the tricarboxylic acid (TCA) cycle. This leads to lactate accumulation that increases intracellular adenosine triphosphate (ATP) levels and reduces hypoxic ROS production, thus rescuing these cells from hypoxia-induced apoptosis. [158][159][160] Lactate and H + generated by glycolysis cross cell membranes through monocarboxylate transport proteins (that is, MCT1/4), sodium hydrogen (Na + /H + ) exchanger (NHE) isoform 1 (NEH1), and carbonic anhydrase 9 (CAR9), contributing to cellular pH homeostasis and driving cancer proliferation and progression. 79,133,[161][162][163] Interestingly, cancer cells relying on MCT1 can autonomously consume lactate, predominating as a carbon source for the TCA cycle. 164 Pyruvate is converted from glycolysis to lactate, while lactate is used as a respiratory fuel to support the energy and synthetic functions of the TCA cycle, which is significant for the proliferation of cancer cells. 164 Hypoxia and lipid metabolism Lipid metabolism confers aggressiveness to malignant tumors by promoting membrane formation, energy storage, and production of signaling molecules, as well as by providing an important source of ATP production
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through fatty acid oxidation (FAO). 165 Lipids are composed of triglycerides and lipoids such as phospholipids, cholesterol, and cholesteryl esters. Lipid metabolism involves lipid synthesis, storage, and degradation. Fatty acid (FA) are essential for lipid biosynthesis and are dependent on the activities of fatty acid synthase (FASN), adenosine triphosphate citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturases (SCD). Endogenous FA biogenesis constitutes an oncogenic stimulus that drives malignant tumor progression. HIF-1 significantly upregulates sterol regulatory element-binding protein (SREBP)-1, a major transcriptional regulator of the FASN gene, which in turn promotes FASN expression. 166 Activation of FASN under hypoxic stress promotes de novo lipid synthesis and cell survival. 167,168 ACLY is the main enzyme responsible for the production of acetyl-CoA in the cytosol in most tissues, and its product, acetyl-CoA, is used to provide a variety of biosynthetic pathways, including lipid synthesis and cholesterol synthesis. ACLY is responsible for catalyzing the conversion of citrate and CoA to acetyl-CoA and oxaloacetate. 169 Studies have suggested that miR-27 and miR-195 are also upregulated in hypoxia-induced cardiomyocytes, suppressing the expression of ACLY and ACC. 170,171 The mechanism of ACLY regulation in tumor cells under hypoxic conditions is still unclear. ACC (including ACC1 and ACC2) is the rate-limiting enzyme in the FA synthesis pathway. 165 Under hypoxic conditions, the inhibition of ACC1 and ACLY increases the level of α-ketoglutarate by decreasing the level and activity of ETV4 to prevent hypoxia-induced apoptosis. 172 ACC2 is hydroxylated by PHD3, which inhibits FAO. Under hypoxic conditions, PHD3 loss reduces ACC2 hydroxylation and promotes
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FAO to provide energy. 173,174 SCD is a membrane protein of the endoplasmic reticulum that catalyzes the formation of monounsaturated FA (MUFA) from saturated FA, promotes tumor progression, and is associated with tumor recurrence and poor prognosis. [175][176][177] The main products of SCD are palmitoleic and oleic acids, providing key substrates for the production of complex lipids such as triglycerides, phospholipids, and cholesterol esters. Intermittently hypoxic mouse hepatocytes upregulate SCD1 mRNA and protein by increasing SREBP-1 and serum monounsaturated FAs. 178 HIF-1/2α promotes the progression of clear cell renal cell carcinoma by inducing SCD1 expression. 179,180 Glutamine is the most abundant amino acid in blood and has been identified as necessary to promote mitochondrial metabolism in rapidly dividing cancer cells. 181 HIF-1 activation leads to a significant decrease in the activity of the mitochondrial enzyme complex α-ketoglutarate dehydrogenase (αKGDH), which inhibits the oxidation of α-ketoglutarate (α-KG) as a product of the metabolic conversion of glutamine to succinate, its reductive carboxylation to isocitrate by isocitrate dehydrogenase (IDH), and then oxidation to citrate. 182,183 HIF promotes glutamine-derived citrate conversion to cytoplasmic acetyl-CoA, which increases FA synthesis. 184 Interestingly, the lipid catabolic metabolism also contributes to cancer metastasis. Under hypoxic conditions, the main enzymes associated with lipid catabolism in tumor cells are phospholipase A2 (PLA2), phospholipase D (PLD), and carnitine palmitoyltransferase 1 (CPT1). 165 PLA2 catalyzes the hydrolysis of glycerophospholipid (GPL) to produce lysophospholipid (Lyso-PL). 185 Lysophosphatidic acid stimulates PLA2 phosphorylation in a HIF-1α-dependent manner, promoting ovarian cancer cell metastasis in vivo. 186 PLD hydrolyzes phosphatidylcholine (PC) to produce
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phosphatidic acid (PA). 187 Activation of the PLD1/AKT pathway increases proliferation, migration, invasion, and epithelialmesenchymal transition (EMT) in HCC. 188 HIF-1α up-regulates the expression of PLD2 in colon cancer cells under hypoxic conditions. 135 In turn, PLD regulates HIF-1α at the translational level in a vHL non-dependent manner in renal cancer cells. 189,190 Prostate cancer cells with CPT1A overexpression showed enhanced proliferation, stemness, and tumor growth compared to controls under hypoxic conditions, mildly affecting the angiogenic response. 191 Breast cancer cells expressing CPT1C showed increased FAO, ATP production, and resistance to glucose deprivation or hypoxia. 192 However, experimental results have shown that HIF-1α and HIF-2α inhibit CPT1A expression in clear cell renal cell carcinoma, reduce FAs transport into mitochondria, and result in FAs being stored in lipid droplets. Compared with normal tissues, CPT1A expression and activity were reduced in clear cell renal cell carcinoma patient samples. 193 Similar results were observed in gastric cancer tissue. 194 Cancer cells optimize their requirements for rapid growth and aggressive progression by fine-tuning the lipid anabolic/catabolic switch. However, the dominant role of this fine-tuning mechanism remains unclear. Glucose and lipid metabolism in cancer cells under hypoxic conditions is showed in Fig. 4. HYPOXIA AND ANGIOGENESIS In the early 1970s, Folkman et al. popularized the concept of tumor angiogenesis, presenting that growing tumor cells must replenish their own blood supply to maintain oxygen and nutrients. 195 The accumulated experimental results have shown that hypoxia favors EC proliferation and migration. Deletion of p53 in cancer cells increases HIF-1α levels and enhances transcriptional
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activation of HIF-1-dependent VEGF and erythropoietin (EPO) in response to hypoxia, thus promoting EC proliferation, migration, and angiogenesis. 96,106,[196][197][198][199] Hypoxia-induced E74-like ETS transcription factor 3 (ELF3) mediates increased secretions of insulin like growth factor (IGF1) and VEGF to promote EC proliferation, migration, and angiogenesis. 200 HIF-1α mediates β-adrenergic receptor pro-angiogenesis. 39,[201][202][203] In addition, hypoxia stimulates the production of hyaluronic acid (a major component of the vascular basement membrane) and hyaluronidase activity, thus possibly promoting angiogenesis as a compensatory mechanism for hypoxia. 204 However, tumor angiogenesis is not necessarily equivalent to tumor blood supply, as the discontinuous basement membrane of immature neovascularization allows for plasma and protein extravasation, further elevation of intra-tumor interstitial pressure, persistent vascular collapse, and poor nutrient delivery. 205 In addition, some tumors exhibit an inability to maintain vascular survival, a condition that explains the well-formed, invasive peripheral and centrally necrotic hypoxic regions found in several highly angiogenic tumors. 206 Therefore, in most cancers, despite the high vascular density, the neointima that forms are typically twisted and dysregulated, not functional for blood transport, and less efficient in oxygen and nutrient transport as well as drug delivery. [31][32][33][34]203 Traditional anti-angiogenic strategies have attempted to reduce the vascular supply to the tumor, but their success has been limited by insufficient efficacy or the development of drug resistance. Normalization of vascular abnormalities may still be beneficial for tumor treatment with the available therapies. 31 HYPOXIA AND IMMUNE TOLERANCE Cancer immunotherapy has emerged as a promising strategy for the treatment of various cancers by stimulating the immune system
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of the patient. 207 Programmed death receptor 1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA4) blocking antibodies, which are drugs approved by the Food and Drug Administration (FDA), have shown promising results in clinical trials for the treatment of various cancers. For some cancers, including breast, pancreatic, colorectal, and prostate cancers, a high proportion of non-responders has been reported, one of the main causes of which is hypoxic stress. 208 Hypoxia-induced HIF-1α can promote programmed death ligand-1 (PD-L1) expression in cancer cells and suppress immune effects. 209 Moreover, hypoxia plays a central role in cancer progression and resistance to therapy by promoting various changes in the biology of stromal cells in the TME, including immune cells. The main mediators of transcriptional hypoxic responses are HIF-1α and HIF-2α, which induce gene transcription, leading to hypoxic responses, and are involved in the regulation of carcinogenesis as well as stromal responses. 210 HIF-1α has important functional roles in both innate and adaptive immune cells, including macrophages, 211 neutrophils, 212 dendritic cells (DCs), 213 and lymphocytes. 214 HIF-2α has been associated with macrophage NO homeostasis. 215 In addition, deletion of the aryl hydrocarbon nuclear translocator (ARNT)/HIF-1β gene in CD8 + T cells impairs the expression of cytolytic effector molecules, including perforins and granzymes. 216 Hypoxia and T cells A distinctive feature of T cells is the markedly increased glucose uptake through glucose transporter 1 (GLUT1) as they respond to immune challenges and differentiate into cytotoxic T lymphocytes (CTLs). 217,218 Activated lymphocytes generate energy largely through upregulation of aerobic glycolysis.
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217 Glycolysis requires T cells to activate and maintain the expression of glycolytic enzymes, for example, pyruvate kinase (PK) and lactate dehydrogenase (LDH), and also requires T cells to maintain high levels of glucose uptake by maintaining the expression of glucose transporters. Relatively high levels of exogenous glucose are required to maintain the CTL transcriptional program. 219,220 HIF-1α enhances the glycolytic activity of but not HIF-2α. 221 Elevated HIF-α expression of T cells in hypoxic environments is regulated by multiple pathways, including the PI3K/mTOR-dependent pathway, 98 protein kinase C (PKC) and Ca 2+ /calcineurin, 222 STAT3 dependence, 223 NF-κB, 100 MAPK pathways, 103 and T cell antigen receptors (TCRs) (Fig. 5). 98 Deletion of VHL impairs HIF-1α and HIF-2α degradation, which significantly elevates the expression of Glucose and lipid metabolism in cancer cells under hypoxic conditions. Glucose is taken up by cancer cells via GLUT1 and glycolysed to pyruvate via PKM, PGK1 and PGAM1. Hypoxic cancer cells promote pyruvate glycolysis to lactate by upregulating LDHA, LDHC and LDH-5, and the lactate produced is excreted outside the cell via MCT1/4. In addition, HIF-1α inactivates PDH by activating PDK1, which in turn fails to convert pyruvate to acetyl-CoA, preventing the entry of pyruvate into the TCA cycle. Cytoplasmic citrate is catalyzed by ALCY to acetyl-CoA, and acetyl-CoA catalyzed by ACC to malonyl-CoA. Acetyl-CoA and malonyl-CoA are catalyzed to FA via FASN upregulated by SREBP-1. SCD1 upregulated by SREBP-1 catalyzes the formation of MUFA from saturated FA. PHD3 loss reduces ACC2 hydroxylation and promotes FAO to provide energy. α-KG
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as a product of glutamine is reduced and carboxylated to isocitrate by IDH, and then oxidation to citrate. PLD hydrolyzes PC to produce PA. PLA2 catalyzes the hydrolysis of GPL to produce Lyso-PL various members of the CTL secretory granzyme family, including perforin and tumor necrosis factor α (TNF-α), thereby sustaining CTL effector functions. 224 In natural killer (NK) cells, APOBEC3G triggered site-specific editing of C-to-U RNA under hypoxia. These effects have been shown to occur independently of HIF-1α, suggesting the existence of other potential regulatory mechanisms. 225 HIF-α activities in CD8 + tumor-infiltrating lymphocytes (TILs) promote their accumulation and anticancer activities, 226 as well as the production of interferon-gamma (IFN-γ) by CD4 + T cells. 227 HIF-1α deficiency attenuates the ability of some CD8 + T cells to cross the endothelial barrier and reduces the abundance of infiltrating CD8 + T cells in the TME. This is because VEGF-A facilitates the recruitment of immune cells. 228 VEGF-A derived from T cells promotes VE-cadherin endocytosis in ECs, causing EC adhesion junction disruption and vascular homeostasis imbalances, resulting in higher expression of the adhesion molecule (VCAM-1), which mediates immune cell adhesion to the vascular endothelium. This finding suggests that T cell-derived VEGF-A affects T cell homing through the endothelial barrier. VEGF-A levels in cancers are inversely correlated with CD8 + T cell levels. 221 Loss of HIF-1α decreases the ratio of CD8 + to FOXP3 + cells in TILs. 229 After antigenic restimulation, the generation of effector cytokines from HIF-1α mutant T cells was suppressed. 221 Ectopic
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HIF-2α (but not HIF-1α) mediates the extensive changes in gene expression by altering the CD8 + T cell transcription factor network, regardless of VHL inhibition, including increased perforin, granzyme B, IL-2, integrins, and CXCR4. In addition, the increase in co-inhibitors such as lymphocyte activation gene-3 (LAG-3) and CTLA-4 and the decrease in IFN-γ expression is also mediated. Overall, these molecular expression changes enhance cytotoxic differentiation and lytic functions against cancer targets. 230 However, the antitumor effects of CTLs under hypoxic conditions remain unclear. It has been reported that hypoxia impairs T cell function by reducing the levels of IFN-γ, IL-2, and NK group 2 member D (NKG2D). 231,232 This is more in line with the actual tumor, where CD8 + and CD4 + T cells are significantly reduced in hypoxic areas of the tumor. T cell proliferations were strongly correlated with oxygen concentrations and were significantly suppressed under hypoxia (pO 2 = 1%), likely due to hypoxia-associated changes in Ca 2+ homeostasis in T cells. 233 HIF-1α inhibits IFN-γ, TNF-α, and granzyme B expressions in CD8 + , CD4 + T, and NK cells, which induces resistance to PD-1/PD-L1 blockade and suppresses T cell toxicity. 234 Hyperoxia treatment suppressed the expressions of adenosine, an immunosuppressive factor, 235 and reactive nitrogen production, 236 and increased IFN-γ levels and perforin granules. 237 Sustained hypoxic stimulation induces mitochondrial stress, resulting in the loss of mitochondrial functions, elevated ROS levels in T cells, T cell failure, 238 and impeded anti-PD-1 efficacy. 239 Because of the Warburg effect in cancer cells,
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which "ferments" glucose into lactic acid, lactic acid is transported outside the cell to prevent excess lactic acid accumulation. This stabilizes the intracellular pH of cancer cells while acidifying the extracellular environment. 30,133 HIF-1α induces high expression of tumor-associated carbonic anhydrase (CA), such as CA9 and CA12, which catalyze the reversible hydration of CO 2 to carbonic acid. 240 In hypoxic environments, the pH of the extracellular environment of cancer cells can be as low as 5.8-6.5, 208 which inhibits CTL activation, proliferation, and cytokine production, and also triggers T cell apoptosis. 241 Hypoxia induces cancer cells to express chemokines such as CC chemokine ligand 28 (CCL28) to recruit regulatory T cells (Tregs). 229 HIF-1α promotes Treg polarization and significantly contributes to colorectal cancer development and progression. This mechanism had no significant effect on the inhibition of Foxp3 expressions. Inhibiting Treg HIF-1α expression suppressed cancer growth. 242 However, the HIF-1α-dependent transcriptional program contributes to helper T cell (Th) 17 development by mediating glycolytic activities. Moreover, HIF-1α can activate RORvt and form a tertiary complex with retinoic acid-related orphan receptor γt (ROR-γt), as well as p300, and recruit it to the IL17 promoter. In addition, HIF-1α attenuates Treg development and differentiation by binding to Foxp3 and targeting it for proteasomal degradation. 223,243,244 Tregs express immunosuppressive factors, 245 immunosuppressive adenosine, 246 and cytokines such as IL-10, 247 IL-17, 248 and IL-35 247 to inhibit effector T cell toxicity; therefore, they are closely associated with poor cancer prognosis. 249 In addition, Tregs inhibit the expressions of nutrient transporters on
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CD8 + T cells, limiting nutrient uptake by T cells CTL increases glucose uptake via GLUT1, which is metabolized to lactate by glycolytic enzymes PK and LDH. Lactate acidifies the TME, which inhibits CTL activation during activation, thereby suppressing T cell proliferation. 250 Respiratory hyperoxia therapy may reduce the immunosuppressive effects of Tregs in the TME. 251 Amphiregulin (Areg) is an epidermal growth factor receptor (EGFR) ligand, and Areg-EGFR promotes HIF-1α levels, leading to cancer progression. 252 The lack of HIF-1α decreases the ratio of IFN-γ+ to FOXP3+ cells in CD4 + TILs, as HIF-1α deficiency reduces FOXP3 proteasomal degradation and cannot efficiently bind the IFN-γ promoter to drive Th1 responses. 227 Hypoxia and B cells The differentiation process of mammalian B cells includes pro-B, pre-B, immature B, and mature B cells. HIF-1α activity, which is regulated by miR-582, is high in human and murine bone marrow pro-B and pre-B cells, promoting cell proliferation, differentiation, apoptosis, and gene rearrangement. 253 However, reduced immature B cell stages and sustained high activities of HIF-1α reduced the abundance of surface B cell receptors (BCR), CD19, and B cell activator receptors and increased the expression of the proapoptotic factor (BIM), preventing normal B cell development. 254 Although hypoxia impairs B cell proliferation, it simultaneously alters B cell metabolism and promotes antigen-mediated differentiation. 255 HIF transcription factors suppress the activities of prolyl hydroxydioxygenase, which stabilizes HIF by hydroxylating HIF-1α and HIF-2α in conjunction with pVHL to disrupt HIF. Therefore, reducing antigen-specific B cells in germinal centers (GCs; produce long-lived plasma cells
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and memory B cells) disrupts high-affinity IgG production and shifts to IgG2c, early memory B cells, and recall antibody responses. In contrast, sustained hypoxia or HIF induction induced by VHL deficiency inhibits mTOR complex 1 (mTORC1) activity in B lymphoblastoid cells, which impairs B cell clonal expansion, activation-induced cytosine deaminase (AID) expression, and the ability to produce IgG2c and high-affinity antibodies. 256 HIF-1α is closely correlated with B-cell lymphoma (BCL-6). Upregulated BCL-6 promotes the immortalization of mouse embryonic fibroblasts and primary B cells by elevating cyclin D1 levels. 257 HIF-1α binds CXC chemokine receptor type 4 (CXCR4) to promote B-cell viability. 258 The immune effects of B cells on cancers have not been conclusively established. Large numbers of CD20+ B cell follicles and Foxp3+ low-infiltrating cells are associated with cancer survival and better recurrence-free survival outcomes in cancer patients, including gastric and pancreatic cancers. 259,260 Activation of HIF in B cells modulates immune responses by inducing VEGF to increase lymphatic and endothelial vessel formation and enhance DC maturation and antigen presentation. 261 Cancer-promoting effects of B cells have been reported. 262 Hypoxia activates HIF-1α and induces autocrine transforming growth factor-beta (TGF-β) signaling, promoting myofibroblast activation, CXCL13 induction, B lymphocyte recruitment, 263 and factor MYC secretion, which favors B cell proliferation and survival, 264 and drives cancer recurrence. 265 HIF-1α-dependent glycolysis facilitates CD1d hi CD5 + B cell expansion and promotes IL-10 expressions. 266 B cell-derived IL-35 is associated with attenuated macrophage as well as inflammatory T cell viabilities and inhibited functions of B cells as antigen
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presenting cells (APCs). 267 IL-10 and IL-35 promoted cancer cell metastasis, 268 and inhibited anticancer immune responses in mice. [269][270][271] In addition, one of the mechanisms of origin of extracellular immunosuppressive adenosine relies on CD73+ and CD19+ extracellular vesicles (EVs) from B, which is regulated by HIF-1α. 272,273 Regulatory B cells (Bregs) are a functional B cell subpopulation with the key function of secreting IL-10, thereby preventing the production of cytokines. 274 HIF-1α is a tumor suppressor in some cancers. 275 For instance, pancreaticspecific HIF-1α deficiency significantly accelerated Kras (G12D)driven pancreatic adenoma formation with a significant increase in intrapancreatic B lymphocytes. 276 Depletion of B cells increases exocrine tissue regeneration owing to a significant decrease in overall immune infiltrations and fibrosis. 277 Pancreatitis-induced tissue hypoxia and HIF-1α accumulation-induced B-cell depletion enhances pancreatic regeneration. B cell depletion in mice with pancreatitis significantly enhanced tissue regeneration and chemotherapeutic drug resistance. 278 Hypoxia and macrophages Hypoxia, HIF-1α, and tumor secretion of multiple chemokines facilitate cancer-associated macrophage (TAM) recruitment to the TME. [279][280][281] TAMs constitute a plastic and heterogeneous cell population and can account for up to 50% of certain solid tumors. 282 TAMs promote cancer progression by creating an immunosuppressive microenvironment, 283 and enhancing the progression and metastasis of various cancers. [284][285][286] Hypoxiainduced HIF-1α promotes the adaptation of TAMs to the hypoxic environment and is associated with cancer prognosis. 287 Lipopolysaccharides (LPS) promote HIF-1α expression by stimulating the expression of Toll-like receptors in macrophages. 288 Hypoxia (1.5% oxygen) leads to inadequate T-cell responses by increasing macrophage pro-inflammatory responses, including
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TGF-β, increased platelet-derived growth factor (PDGF), phagocytosis maintenance, and reduced antigen presentation. Hypoxia enhances fibrosis by promoting pro-fibrotic cytokine responses and isolating fibroblasts in the vicinity of granulomas. 289 The hypoxic environment leads to elevated HIF-1α levels in macrophages and promotes the expression of inducible nitric oxide synthase (iNOS), which rapidly blocks T cell proliferation through NO and subsequent peroxynitrite formation. 290-292 IL-15Rα+ TAMs downregulate CX3CL1 expression in cancer cells through the non-transcriptional activity of HIF-1α, reducing CD8 + T cells and/or increasing CD4 + T cells to reduce the ratio of CD8 + T cells to CD4 + T cells and thus weaken anti-cancer immunity. 293 In addition, macrophages express HIF-2α under hypoxic conditions and inactivate drug-induced chemoresistance to 5-FU through HIF-2α-mediated specific overexpression of dihydropyrimidine dehydrogenase (DPD). 294,295 Furthermore, acidic TME affect TAMs. Lactate, converted from pyruvate in cancer cells, plays a key signaling role by inducing HIF-1α-mediated expression of vascular endothelial growth factor in other processes. [296][297][298][299] In addition, M2-like TAM promotes cancer progression by remodeling the extracellular microenvironment. 282 Hypoxia and NK cells NK cells are bone marrow-derived and account for 10-18% of peripheral blood mononuclear lymphocytes. 300 NK cells can kill various cancer targets, including cancer cells with low expressions MHC-I. 301 Some cancer cells secrete chemokines to recruit NK cells. For instance, HCC cells are often in hypoxic microenvironments, an environment that helps CD103+ DCs to take up and clear cancer DNA, which is enhanced by blocking cell surface protein (CD47). By secreting IL-12 and CXCL9, activated CD103+ DCs induce
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NK cell recruitment; upregulate the expression of granzyme B, NKG2D, IFN-γ and TNF-α; and downregulate the expression of NKG2A, which in turn enhances antitumor effects. However, under hypoxic conditions, most HCC cells highly express the CD47 protein, which is associated with poor prognostic outcomes. 302,303 Expression of NKG2D receptors, intracellular perforin, and granzyme B in NK cells is severely impaired under hypoxic conditions, which suppresses NK cell immunotoxicity. 304 Hypoxia promotes cancer cell metalloproteinase ADAM10 expression through HIF-1α, leading to shedding of NK cell activation ligand (MICA) from cancer cell surfaces, thereby attenuating NK cell-mediated lysis. 291 In an inflammatory model, elevated NK cell HIF-1α levels stimulated the release of IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF), enhancing antimicrobial defenses while promoting M1 macrophage polarization, leading to slow angiogenesis and wound healing. 305 NK cell counts and cytotoxic effects were similarly suppressed in a chronic hypoxia mouse model, accompanied by inhibited IFN-γ secretion by NK cells. NK cells also secrete large amounts of MMP-9, which affects blood vessel remodeling. 306 Although the absence of NK cell HIF-1α inhibits tumor growth, it is associated with various challenges. For example, deletion of HIF-1α in NK cells suppressed the expression of the angiostatic soluble form of VEGF receptor 1 (sVEGFR1) in cancer, increasing VEGF bioavailability in the TME and leading to non-productive angiogenesis. The immature vascular phenotype promotes cancer cell intravasation and distant pulmonary metastases from melanoma in vivo. 202 Moreover, cancer cells influence the cytotoxic effects of NK cells. Upregulated HIF in the TME increases PD-L1 expression, decreases autophagic
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MHC-I expression, and inhibits NK cell recognition. 103,221 HIF-2α promotes IL-10 release by activating the STAT3 signaling pathway in HCC cells, thereby inhibiting the killing activities of NK cells, which in turn promotes HCC recurrence and metastasis. 307 HIF-2α stimulates ITPR1 expression in hypoxic cancer cells, activates autophagy in NK cells, inhibits granzyme B activity, and impairs the killing sensitivity of NK cells. 308 Activated autophagy in hypoxic cancer cells selectively degrades the pro-apoptotic NK-derived serine protease GZMB/granzyme B, leading to the suppression of NKmediated target cell apoptosis. 309 Hypoxia and neutrophils Neutrophils are the major players in the innate immune system. Neutrophil infiltration is uncommon in normal tissues. 310 Neutrophils are glycolytic cells that derive little ATP from oxidative phosphorylation and require gluconeogenesis to generate intracellular glycogen stores to kill bacteria. 311 In a hypoxic environment, neutrophils reduce glycogen recycling, leading to impaired functions, 311 and consume extracellular proteins to promote their own central carbon metabolism to maintain their functions. 312 Infiltrating neutrophils in the TME, known as tumor-associated neutrophils (TANs), promote cancer growth and is strongly associated with poor prognosis. 313 For instance, neutrophil-derived ROS damages hepatocyte DNA to drive HCC development. 314 Neutrophils can eliminate bacteria from the body, forming an immune barrier that allows cancers to start growing back; 315,316 therefore, they are closely correlated with prognostic outcomes of cancer patients. 310,317 Elimination of neutrophil infiltration leads to cancer regression and prolonged survival outcomes. 318 Sequencing of pancreatic single-cell transcriptome revealed that BHLHE40 is a key regulator of neutrophil polarization toward the
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TAN-1 phenotype, which has pro-cancer and immunosuppressive functions. However, the associated mechanism should be investigated. 319 Neutrophils have an extensive mitochondrial network that uses the glycolytic product (glycerol-3-phosphate) to maintain polarized mitochondria and produce ROS to regulate HIF-1α stability. 320 Neutrophil extracellular traps (NETs) promote cancer cell colonization by enhancing migration, invasion and cancer cell stemness, leading to poor cancer prognosis. Interestingly, NET formation is strongly associated with elevated HIF-1α levels in neutrophils. Downregulation of neutrophil HIF-1α can effectively inhibit NET-mediated circulating tumor cell (CTC) metastasis and prolong the median survival of mice with breast cancer lung metastasis. 321 However, some experiments have shown the anticancer effects of NK cells. Hypoxia favors neutrophil viability and function. 322 Hypoxia recruits polymorphonuclear neutrophils (PMNs), which are the main effector cells against endometrial adenocarcinoma growth, and induces cancer cell detachment from the basement membrane. 323 Upon relief of tumor hypoxia, recruitment of PMNs to the TME is significantly reduced; however, the recruited cells can efficiently kill cancer cells by releasing NADPH oxidase-associated MMP-9 and ROS. 324 Hypoxia and MDSCs Myeloid-derived suppressor cells (MDSCs) can suppress immune cell activity. Hypoxia and HIF-1α drive MDSCs recruitment to the TME. 280,281 This is because cancer cells in hypoxic areas express various cytokines, such as chemokine (C-C motif) ligand 26, G-CSF, and IL-6, to recruit MDSCs. 297,325,326 Functionally, MDSCs enhance the evasion of cancer cells from immune surveillance and promote cancer drug resistance. 327 Inhibiting the infiltration of MDSCs improves antitumor effects. 328 In an HCC model, Chiu et al. found that hypoxiainduced
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HIF-1α promotes the expression of ENTPD2/CD39L1, an indicator of poor HCC prognosis, which is thought to convert extracellular ATP to 5'-AMP, preventing the differentiation of MDSCs, but maintaining their survival. 329 In addition, under hypoxic conditions, MDSCs express multiple immunosuppressive factors. For instance, PD-L1 is secreted by hypoxia-induced HIF-1α, but not HIF-2α in MDSCs, 330 arginase and NO are promoted by HIF-1α-induced miR-210 expression, 331,332 surface ectonucleotidases CD39 and CD73, 333 TGF-β1, and exosomes, such as S100A9, RAR-related orphan receptor alpha (RORA), and PTEN, 281,334,335 promote cancer cell stemness and growth and inhibit CTL function. Hypoxia through HIF-1α dramatically alters the function of MDSCs in the TME and redirects their differentiation toward TAMs, although the macrophage subtype has not been clearly established. 331 These effects may be attributed to the ability of MDSCs to express SIRT1, 336 a factor that regulates the glycolytic activities of MDSCs and affects the functional differentiation of MDSCs, 337 and some miR-29a-containing exosomes. 338 Hypoxia and ILCs Innate lymphocytes (ILCs) consist of NK cells ILC1, ILC2, and ILC3, and are involved in the immune response to virus, bacteria, parasites, and transformed cells. 339,340 Their roles in cancer immunity and immunotherapy have not been established. 341,342 Specific changes in cancer cytokines alter ILC composition in cancers by inducing plasticity and altering ILC functions. 343,344 IL-15 promoted ILC1 granzyme A expressions and cytotoxicity, induced the apoptosis of murine leukemia stem cells (LSC), maintained anticancer immunity, and was positively correlated with survival outcomes. 345-347 IL33-activated ILC2 selectively expresses chemokine ligand 5 (CCL5), which recruits
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CD103+ DCs into the TME and activates CD8 + T cells to lyse cancer cells. 342 IL-33 results in massive amplifications of ILC2 and produces CXCR2 ligands from these cells, which enhances cancer cell-specific apoptosis through CXCR2. 348 Administration of hypoxic ILC2 resulted in a higher cancer volume, suggesting that hypoxiaexposed ILC2 enhanced the progression of pancreatic cancer cells. These outcomes were attributed to the conversion of ILC2 to ILCregs under hypoxic conditions, which inhibited T cell infiltration and IFN-γ expression by secreting IL-10. 349 IL-25 promotes intratumoral ILC2 infiltration and enhancing the ability of cancerinfiltrating MDSCs to suppress antitumor immunity and reduce survival outcomes in colorectal cancer (CRC) patients. 350 ILC3 have antitumor effects. Their intrinsic disruption in CRC drives dysfunctional adaptive immunity, cancer progression, and immunotherapeutic resistance. 351 The production of chemokines (CCL20) and pro-inflammatory cytokines (IL-1β) at the tumor site leads to ILC3 recruitment and activation. ILC3 secrete the chemokine CXCL10, which recruits CD4 + and CD8 + T cells and promotes antitumor immune responses. 352 IL-22, secreted by ILC3 cells, is required for initiation of DNA damage responses (DDR) after DNA damage. Stem cells that lose IL-22 signaling and are exposed to carcinogens escape DDR-controlled apoptosis, develop more mutations, and are more likely to cause colon cancer. 353 The interaction between immune cells and TME is showed in Fig. 6. HYPOXIA AND CHEMOTHERAPEUTIC RESISTANCE In vivo experiments have shown that hypoxia increases the tolerance of cancer cells to drug toxicity. [354][355][356] Hypoxia induces high expression of drug resistance genes in cancer cells,
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increases chemotherapeutic drug efflux, and reduces intracellular drug concentrations. Once they are combined with anticancer drugs, P-glycoprotein (PGP), known as multidrug resistance protein 1 (MDR1), and breast cancer resistance protein (BCRP) are ATPbinding cassette (ABC) transporters and energy-dependent drug excretion pumps that can pump drugs out of the cell by providing energy through ATP. 357 Therefore, the intracellular concentration of drugs continues to decrease, which weakens their cytotoxic effects until they are dissipated, and drug resistance occurs. ROS generated under hypoxic conditions induce PGP and BCRP expression through HIF-1α and increase cancer cell drug resistance. 358 HIF-1α induces multidrug resistance-associated protein 1 (MRP1) expressions and enhances drug resistance in colon cancer cells. 359 Moreover, HIF-1α enhances the inactivation of chemotherapeutic drugs and reduces their cytotoxic effects. Cytidine deaminase (CDD), an important metabolic enzyme involved in drug resistance development in cancer cells, is derived from cancer cells or bacteria within cancer cells. Elevated CDD levels promote the metabolism of gemcitabine to its inactive form. 360 Moreover, solute carrier (SLC) transport proteins are primarily involved in the uptake of small molecules into cells, and their absence affects the uptake of chemotherapeutic agents into cancer cells, leading to drug resistance. 361 The role of hypoxia in relation to CDD and SLC has not been fully established. Recently, alternative resistance pathways have been identified in hypoxic cancer cells. For instance, cancer and stromal cells in the TME, such as cancerassociated fibroblasts, secrete various cytokines, including IL-6, which induces HIF-1α expressions to regulate downstream chemotherapeutic resistance genes, such as olfactomedin 4
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(OLFM4), pyruvate kinase muscle 1 (PKM1) that enhances mitochondrial oxidative phosphorylation (OXPHOS), and expressions of non-coding genes, for example, miR-27a that increases PGP expression, to promote chemoresistance acquisition in cancer cells. [362][363][364][365][366][367] HIF-1α and cancer-associated fibroblast-secreted TGF-β signaling synergistically promote GLI family zinc finger 2 (GLI2) expressions through SMAD3, inducing CRC cell stemness and chemoresistance. 368 Upregulated HIF-2α promotes sorafenib resistance in hypoxic HCC cells by activating the TGF-α/EGFR and COX-2 pathways. 369,370 Under normoxic conditions, autophagy activation did not counteract cisplatin-induced stress, leading to cell death, whereas under hypoxic conditions, autophagy induction was enhanced, resolving cisplatin-induced stress and inhibiting the cisplatin-induced BCL2 interacting protein 3 (BNIP3) death pathway, allowing cell survival. 371 Downregulation of BNIP3 may contribute to the resistance of pancreatic cancer to hypoxiainduced cell death. 372 However, in non-small lung cancer samples, high expression of BNIP3 was significantly associated with HIF-1α and poorer overall survival was associated. 373 Due to fibrosis and formation of non-functional vessels in the TME, drug diffusion and delivery to cells away from functional vessels is decreased 374 , and this may trigger drug resistance in cancer cells. In addition, an altered pH gradient in the TME can attenuate drug action in cells (for example, alkylating agents and antimetabolites) to enhance drug resistance. 374 The mechanisms of hypoxia-enhanced tumor chemotherapy resistance is showed in Fig. 7. HYPOXIA AND RADIATION RESISTANCE Radiotherapy directly destroys macromolecule fixation and leads to DNA damage through the generation of free radicals, such as hydroxyl (OH•) and hydrogen (H•) radicals, by ionizing radiation (IR), a
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process known as "oxygen fixation theory". The damage caused by radiotherapy is permanent and irreversible. Hypoxia reduces the effectiveness of radiotherapy and free radicals become unstable, leading to limited extent of their damage, and most of the damage can be easily repaired. 375,376 Classical in vitro and in vivo experiments have shown that at O 2 (pO 2 ) partial pressures below 10 mmHg, tumor cells can acquire radiobiologic hypoxia and thus become relatively resistant to radiotherapy. For instance, at 1 mm Hg, cancer cells are three times more resistant to radiation than ordinary oxygen cells. 16 Several preclinical and clinical trials have demonstrated that the number of lethal DNA DSB formed under hypoxic conditions is also reduced by 2 to 3-fold and hypoxia can alter the expression and function of DNA DSB-associated genes. Under hypoxic conditions, additional DNA repair pathways can be Hypoxia induces neutrophils to produce ROS and NETs, which promote tumorigenesis and metastasis. HIF-1α induces MDSCs to secrete cytokines NO, arginase, PD-L1, RORA, PTEN, CD39, CD73 and S100A9, which inhibit immune response and promote the tumor cell stemness and growth. ILC1 secretes granzyme A and maintains antitumor efficacy. ILC2 enhances immune response through selective expression of CCL5, CXCR2. Hypoxia induces IL-10 expression in ILC2 and suppresses immunity. ILC3 secretes CXCL10 and IL-22, improving antitumor immune response activated. 16,[376][377][378][379] HIF-1α stimulates DNA-dependent protein kinase (DNA-PK) expression and repairs DNA DSB caused by ionizing radiation. 380 Radiation upregulates HIF-1 expression and enhances its activity in cancer cells. 381 Elevated HIF-1α levels confer radioresistance via various
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pro-cancer mechanisms. HIF-1α promotes ATP metabolism, and p53 activation and stimulates EC survival, thereby mediating the ultimate cancer radiation responses. 382,383 The serine peptidase inhibitor Kazal type 1 (SPINK1) is secreted in a HIF-dependent paracrine manner to reduce radiation-induced DNA damage and enhance radiation resistance in adjacent cancer cells through EGFR and nuclear factor erythroid 2-related factor 2 (NRF2). 384 Radiation-induced EC death leads to secondary tumor cell killing. 385 The vascular system also affects radiation therapy. Radiation-induced secretion of VEGF, FGF, and PDGF by cancers protects the cancer vasculature from radiation-mediated cytotoxicity and enhances the radioresistance of ECs. [386][387][388] Thus, VEGF, FGF, and PDGF inhibitors can significantly improve the efficiency of radiotherapy. 388,389 The radiosensitivity of hypoxic cancer cells was showed to be significantly increased after reoxygenation. 390 Elevated H 2 O 2 levels in cancer tissues exacerbate hypoxia-induced resistance to radiotherapy. Reducing oncological cellular H 2 O 2 or using it to generate oxygen through chemical reactions may improve radiotherapeutic outcomes. 391 However, these mechanisms require further evaluation (Fig. 8). HYPOXIA-MEDIATED THERAPY Targeted therapy Approximately 80-90% of deaths among cancer patients are directly or indirectly attributed to drug resistance. Progress in the development of new drugs is also hampered by drug resistance, which has become a considerable challenge in cancer treatment. 392 Hypoxia drives cancer development and progression. Therefore, it is essential to increase the oxygen concentration in TME. Oxygen delivery in the TME is increased by enhancing cancer vascular formation to elevate oxygen levels, however, the vessels formed in cancers are usually abnormal
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and non-functional. 39 Intravenous oxygen delivery to the cancer through blood is not feasible for reasons such as the potential risk of systemic ROS exposure and the low solubility of oxygen in blood. 393 Increasing oxygen delivery by increasing blood flow to the tumor through pharmacological vasodilatation methods has been proposed, but in practice it is difficult to improve or reduce blood flow to the tumor tissue. 394 To overcome this challenge, nano-and bio-based technologies have been used to carry oxygen generators to generate enough oxygen in the target cancer such as pancreatic ductal adenocarcinoma, colorectal cancer, fibrosarcoma, melanoma, etc., which aids in drug delivery and significantly improves the effectiveness of chemotherapy, radiotherapy as well as immunotherapy. 281,[395][396][397][398] Moreover, the reoxygenation process generates large amounts of ROS, which results in DNA damage, contributing to cancer cell death. 65,399 However, the damage this brings to the surrounding normal cells is a matter of concern. Second, inhibition of hypoxia-induced HIF and its downstream target genes is an effective strategy. 400 For instance, 32-134D, a low-molecular-weight compound that inhibits HIF-1/2-mediated gene expression in HCC cells, combined with anti-PD-1 increased HCC eradication rates in mice (from 25 to 67%). 401 HIF-1α inhibitors, such as topotecan, bortezomib (PS-341) as well as HIF-2α inhibitors, such as PT2399, PT2385 and PT2977, inhibit HIF-1/2α activities and their downstream target gene expressions. 402,403 These inhibitors reduce VEGF levels in circulating tumor cells (such as neuroblastoma, multiple myeloma, HCC and renal cell carcinoma) to inhibit tumor angiogenesis, [404][405][406][407][408] and enhances the effects of chemotherapeutic drugs, such
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