**1. Introduction**

The notion of a smart world, with the aid of smart devices, smartphones, smart cars, smart homes, and smart cities, the paradigm of smart everything, has been a vigorously researched topic for many years. This concept holds the view that people and the world itself will be overlaid with sensing and actuation, with the aid of the internet of things (IoT). Nowadays, IoT has been used in a large number of areas, such as government, industry, and academia [1], for different applications. For example, sensors are placed in buildings for attempting to save energy [2,3]; wireless sensor networks (WSNs) in vehicular communications trying to improve safety and transportation [4]; home automation [5];

industry [6]; or e-Health services which are relying on increased home sensing to support remote medicine and wellness [7].

Nowadays, more than half of the world's population lives in cities [8] with more than six devices per person connected to the Internet [9]. That means that billions of devices will be connected by 2020 to build the aforementioned smart city concept, which can range from end-user devices or wearables to vehicular communication systems, water and gas monitoring, smart lightning, structural monitoring, or smart healthcare systems, among others [10]. These solutions involve a high-density node environment, which in turn requires smaller outdoor and indoor cells leading to heterogeneous networks (HetNet).

Moreover, the advent of next generation 5G communication systems implies the use of ultra-dense small cells in order to increase coverage/capacity requirements inherent to the wide array of services to be offered [11]. In this context, multiple wireless communication systems can be employed in order to diversify service provision, leading to issues such as unpredictable interference sources, or idle cell interference which can greatly impact quality of service [12,13]. Interference analysis has also been considered as a potentially beneficial evaluation element that can be employed in order to enable covert communications in IoT scenarios [14].

The complexity in interference analysis is given by multiple factors, such as requested service types, heterogeneous service requirements, wireless system coordination, or user location and density. In this sense, HetNet architectures implement superimposed cell structures in order to provide adequate capacity requirements as a function of the requested service type. However, this has a serious impact in overall interference levels, requiring the use of cell coordination, such as almost blank subframe technique in order to minimize transmission times [15], employing game theory approaches to provide first order approximation to consider overall interference levels or a similar approach in order to analyze multi-user-multiple-input-multiple-output (MU-MIMO) user assignment in 5G systems [16]. Not only do user distribution and service type have an impact on interference, but also hardware constraints, such as sampling rate mismatch between end user devices and access points/base stations, also affect in the case of massive IoT deployments [17]. In this same line, interference levels can degrade operation of massively deployed transceivers, such as LoRa, owing to loss of ideal orthogonality and leading to increased packet loss [18]. In the future, these constraints can further be aggravated by the use of novel schemes, such as radio frequency (RF) energy harvesting, which in the case of IoT deployments, must consider coverage/capacity relationships in order to comply both with minimum harvested energy thresholds as well as with uplink/downlink signal to interference ratios [19]. Hence, interference analysis and control are one of the fundamental aspects to consider in the scenarios with variable quality of service (QoS) requirements and user density and location, intrinsic to IoT applications [20]. Different solutions have been recently proposed in order to control overall interference levels, such as content-aware cognitive control [21], resource split full duplex mechanisms [22], or machine learning techniques to provide adaptive transmit power control in WSNs [23].

For the successful implementation of the aforementioned dense node deployments, reliable and accurate channel models are necessary, addressing the different topologies and radio links to get reliable service, coverage, and capacity, as well as interference management. Furthermore, hot spots have a non-uniform traffic demand, so it is necessary to have three-dimensional (3D) realistic environments to achieve accurate models, which can lead to network performance improvement. The approaches followed in order to analyze interference in large areas with node density are usually based on statistical channel modelling and under certain model assumptions [13,24], providing certain consideration in relation with scenario characteristics, which can be eventually combined with measurement updates. Spatio-temporal techniques have also been proposed in order to analyze connection establishment phases in massive IoT deployment, based stochastic geometric models [25]. Measurement based interference characterization in IoT scenarios has also been proposed, assisted by supervised learning [26]. However, none of these approaches perform a complete analysis and system performance evaluation considering the whole morphology and topology of the considered scenario. Moreover, realistic wireless system operation exhibits a complex behavior, depending on conditions such as household/office environment, wireless systems under operation within the scenario under analysis or the density of transceivers considered [27].

The analysis on wireless node density and variations within wireless channel characteristics is relevant in functionalities related with applications such as wireless cooperative location systems [28] or in passive location systems [29], in which wireless channel characteristics (line of sight-non-line of sight channel conditions as well as multipath propagation) influence ranging estimations and hence, location performance. Network synchronization is another application in which node density impacts system operation of cooperative systems, influencing the value of the cooperative dilution intensity [30], which is influenced by wireless channel conditions.

Energy analysis is a relevant aspect in the operation of wireless communication systems and particularly in wireless sensor networks, with the existence of inherent limitations given by restrictive energy sources, processing capabilities, and compact form factor requirements. In this sense, wireless sensor network energy balance analysis is compulsory in order to implement efficient system level solutions, such as scheduling algorithms for sleep/active states in order to implement wireless sensor networks operating under partial coverage conditions [31]. By studying required coverage levels (given by receiver sensitivity thresholds, determined by transmission bit rates, adaptive modulation and coding schemes, and electronic device characteristics), transceivers can be dynamically set in sleep modes, resulting in effective energy reduction and hence, enhanced operation lifetime. In this context, estimation of wireless channel behavior, in terms of coverage estimation as well as in distribution of interference sources is relevant in order to analyze overall energy consumption impact, from physical layer as well as in access control and network layer.

In this work, we present a deterministic technique to model electromagnetic propagation in high node density scenarios, specifically an in-house 3D ray-launching (3D-RL) algorithm, based on geometrical optics (GO), geometrical theory of diffraction (GTD), and its extension the uniform theory of diffraction (UTD). With the aid of the 3D-RL simulation tool, the performance evaluation and interference characterization of a dense node density scenario has been performed in order to assess the key performance indicators of the network. The contributions of this work are aimed in providing a precise tool for coverage/capacity estimation, considering relevant multipath propagation phenomena in large complex scenarios. The proposed simulation methodology employs an optimized 3D-RL code with hybrid simulation (combining 3D-RL with neural network interpolators, the electromagnetic diffusion equation for diffraction estimation, and collaborative filtering of deep learning data base algorithms), enabling the possibility to simulate large, complex scenarios. A new simulation module has been implemented in order to perform estimation of error vector magnitude (EVM) for the complete simulation volume, hence enabling further quality of service analysis as a function of the employed modulation scheme.

The remaining parts of the paper are outlined as follow: The proposed simulation technique and the scenario description are explained in Section 2. Section 3 presents the simulation results in the high node density considered scenario, at ISM 2.4 GHz and 5.8 GHz frequency bands, with the received signal strength (RSS), signal to interference noise ratio (SINR), and performance analysis in terms of constellations plots and EVM characterization considering a ZigBee system (infrastructure nodes) as well as Bluetooth transceivers (high mobility devices/users), and coverage/capacity estimations examples. In Section 4, a campaign of measurements has been presented for the same considered scenario, achieving a good match between simulation and measurement results. In addition, the comparison between the scenario full of people and without people is presented in this section. Finally, conclusions and future work are summarized in Section 5.


**Biocultural Restoration of Traditional Agriculture: Cultural, Environmental, and Economic Outcomes of Lo'i Kalo Restoration in He'eia, O'ahu**

**Leah L. Bremer 1,2, Kim Falinski 2,3, Casey Ching 4, Christopher A. Wada 1, Kimberly M. Burnett 1, Kanekoa Kukea-Shultz 3,5, Nicholas Reppun 5, Gregory Chun 6,7, Kirsten L.L. Oleson 4 and Tamara Ticktin 8**

Received: 31 July 2018; Accepted: 25 November 2018; Published: 29 November 2018

**Abstract:** There are growing efforts around the world to restore biocultural systems that produce food while also providing additional cultural and ecological benefits. Yet, there are few examples of integrated assessments of these efforts, impeding understanding of how they can contribute to multi-level sustainability goals. In this study, we collaborated with a community-based non-profit in He'eia, O'ahu to evaluate future scenarios of traditional wetland and flooded field system agriculture (lo'i kalo; taro fields) restoration in terms of locally-relevant cultural, ecological, and economic outcomes as well as broader State of Hawai'i sustainability goals around food, energy, and water. Families participating in the biocultural restoration program described a suite of community and cultural benefits stemming from the process of restoration, including enhanced social connections, cultural (re)connections to place, and physical and mental well-being, which inspired their sustained participation. We also found benefits in terms of local food production that have the potential to provide economic returns and energy savings over time, particularly when carried out through a hybrid non-profit and family management model. These benefits were coupled with potential changes in sediment and nutrient retention with implications for water quality and the health of an important downstream fish pond (loko i'a) and coral reef social-ecological system. Compared with the current land cover (primarily invasive grasses), results suggest that full restoration of lo'i kalo would decrease sediment export by ~38%, but triple nitrogen export due to organic fertilizer additions. However, compared with an urban scenario, there were clear benefits of agricultural restoration in terms of reduced nitrogen and sediment runoff. In combination, our results demonstrate that a biocultural approach can support the social and financial sustainability of agricultural systems that provide multiple benefits valued by the local community and non-profit while also contributing to statewide sustainability goals.

**Keywords:** biocultural restoration; food energy water; ecosystem services; cultural services; sustainable agriculture; Hawai'i; taro; wetland agriculture; flooded field systems; lo'i kalo; sediment; nutrients

#### **1. Introduction**

Across the globe, multiple factors have stimulated a growing interest in biocultural approaches to ecological restoration [1–5], including the strengthening of indigenous cultural revitalization movements [6,7], increasing acknowledgement among conservation and restoration professionals of the importance of social-ecological linkages [8–11], and growing recognition that restoration success often depends on community engagement [12–14]. Biocultural approaches to restoration focus on both ecological outcomes, such as biodiversity restoration and erosion control, as well as cultural outcomes, such as restoration of culturally important species and the traditions associated with them and community (re)connection to place [15–17]. Building on broader theories of social-ecological systems that emphasize the links between humans and the environment [18,19], biocultural approaches place further emphasis on a place-based approach and explicitly recognize that cultural and biological outcomes are interlinked and mutually reinforcing [20]. For example, the restoration of indigenous food production systems can strengthen cultural identity and improve nutrition and food self-sufficiency, while also restoring habitat for native plants and animals [2]. Restored habitats can then reinforce other cultural traditions that are linked to them [20]

Whereas industrial monocultures are often framed in terms of tradeoffs among food production and other outcomes such as water quality [21], traditional agricultural systems are typically characterized as potentially sustainable systems with synergies among desired ecological, cultural, and economic outcomes [22–24]. A social-ecological systems or biocultural approach to restoring traditional agriculture explicitly focuses on the links between the ecological and socio-cultural processes that underpin these systems [3]. Garnering broad support for biocultural restoration can be challenging, however, as short-term revenue is typically lower than monoculture agriculture or alternative land uses like urbanization, and conservation efforts often prefer complete restoration of 'natural' systems to maximize ecological benefits. While a growing body of literature points to a wide array of cultural and ecological benefits of biocultural restoration that can contribute to multi-level sustainability goals [7,16,20], there are few studies that evaluate these benefits (as well as tradeoffs) in a holistic and inclusive way. A framework for evaluating synergies amongst and tradeoffs across objectives of biocultural restoration could facilitate inclusion of these approaches in multi-scale restoration planning and facilitate adaptive management [9].

There are several existing approaches that can be adapted to guide an evaluation framework for biocultural restoration projects. Considering the environmental dimensions, the food-energy-water (FEW) nexus has emerged as an important framework for illuminating hidden synergies and tradeoffs in agriculture of great relevance to local, regional, and international sustainability initiatives [25], such as the United Nations Sustainable Development Goals (SDGs). While the FEW nexus has the potential to shed light on biocultural restoration approaches, it has generally focused on large-scale agriculture and largely ignores cultural aspects that can play critical roles in long-term societal sustainability and adaptive management. As an important complement to address cultural dimensions, a growing theory and emerging examples of inclusive valuation of land-use futures within the ecosystem services or "nature's contributions to people" literature sheds light on strategies to bring together diverse methodologies to assess benefits and tradeoffs in terms of locally relevant and linked ecological, cultural, and socio-economic concerns [16,26,27]. This work has been furthered by indigenous and place-based perspectives on cultural ecosystem services, often framed as reciprocal human-environment relationships well-aligned with biocultural restoration approaches [17,20]. We propose that combining and adapting the FEW nexus and ecosystem services (including

place-based, indigenous cultural ecosystem services) frameworks has important potential to contribute to inclusive assessments of biocultural restoration.

Pacific islands can be considered as model systems to study biocultural restoration of traditional agriculture due to a combination of the socio-cultural significance of local food systems and their geographic isolation, which makes reduced dependence on imported food and energy sources an important part of building resilience. Pacific islands are also characterized by relatively small watersheds and tightly linked land-sea resources, making terrestrial agricultural practices and marine ecosystem health (also important for local food production) intricately linked [28,29]. In Hawai'i, elevated interest in the biocultural values of these traditional agricultural systems is demonstrated by a growing communities working to restore traditional terrestrial agriculture as well as nearshore aquaculture for linked cultural, economic, and environmental benefits [3,30]. The State of Hawai'i through the Aloha + challenge has also committed to interconnected sustainability goals around food, energy, and water (as well as links to local ecosystems and culture) and is recognized as an example of local implementation of the SDGs [31]. These commitments include: doubling food production in 20 years (Hawai'i currently imports nearly 90% of its food [32]); achieving 100% renewable energy by 2040; protecting watersheds and linked marine ecosystems; and facilitating re-connection to place and community-based management [31]. Achieving multiple sustainability goals simultaneously will require re-establishing community-based diversified agricultural systems with low energy requirements and few environmental tradeoffs. Yet, across the State, large tracts of agricultural land are currently fallow or used for high value export crops, largely due to the challenging economics of small-scale farming for local food production [33]. In this context, it is imperative to understand how biocultural approaches to traditional agricultural restoration can contribute towards achieving both local objectives and formal State of Hawai'i sustainability goals around food, energy, and water.

In order to contribute towards a better understanding of the multi-level outcomes of biocultural restoration projects, we collaborated with Kako'o ' ¯ Oiwi, a community-based non-profit in He'eia ¯ on O'ahu at the forefront of biocultural restoration of traditional agriculture, to evaluate the likely future benefits and tradeoffs of their vision around restoring a degraded and invaded wetland to lo'i kalo and native wetland plant communities. We specifically identified key ecological, cultural, and socio-economic outcomes of interest to Kako'o ' ¯ Oiwi and the local community who participate in ¯ the biocultural restoration efforts, and assessed those in combination with the broader State of Hawaii's sustainability goals around food, energy, and water [31]. For evaluating the various outcomes we used mixed methods, including participatory methods using an indigenous cultural ecosystem service process and framework [17]; sediment and nutrient retention ecosystem service modeling [34,35]; and food, energy, and water tradeoff analysis [25,36–39].

This study integrates diverse theories into a practical framework for evaluating biocultural restoration initiatives, with the aim of facilitating their on-the-ground planning, adaptive management, and assessment. Our research team built strong relationships with community members and non-profit staff to develop and apply the framework, ensuring that evaluation outcomes and approaches are reflective of local concerns while also linking to broader statewide sustainability objectives. Applying the framework collaboratively, we addressed the following research questions: (i) what are the locally-relevant cultural, environmental, and economic outcomes of biocultural restoration of lo'i kalo in He'eia, Hawai'i?; (ii) to what extent can biocultural restoration of lo'i kalo in He'eia, Hawai'i contribute to statewide sustainability goals around food, energy, and water?; and (iii) what are the synergies and tradeoffs among these outcomes and how can this inform design of biocultural restoration projects in He'eia and beyond?

#### **2. Methods**

#### *2.1. Site Description*

Our study site He'eia, O'ahu is an ahupua'a (traditional Hawaiian political-ecological boundary), where several NGOs are committed to a biocultural approach of restoring traditional social-ecological systems from mauka to makai (land-to-sea), which include wetland and marine fish ponds (loko i'a), upland agroforestry, and lo'i kalo. The area has recently been designated a Natural Estuarine Research Reserve, and the first of such reserves to explicitly focus on restoration for socio-cultural benefits [40]. The full He'eia watershed (11.5 km2) spans from the top of the Ko'olau mountain range to Paepae o He'eia (traditional fish pond) and then to Kane'ohe Bay, a 45 km ¯ 2 sheltered bay and highly valued conservation and subsistence and commercial fishing area. Average rainfall is 3020 mm at the summit and 1205 mm at the coast [41]. The watershed has two smaller basins, Ha'iku and 'Ioleka'a, that contribute to streamflow in He'eia stream (Figure 1).

We focus on a ~800,000 m2 wetland managed by the community-based non-profit Kako'o ' ¯ Oiwi ¯ who seek to restore the now primarily invasive vegetation to the lo'i kalo systems present until the 1930s. The mission of Kako'o ' ¯ Oiwi is to "perpetuate the cultural and spiritual practices of Native Hawaiians," ¯ of which restoring lo'i kalo through a biocultural approach is a central part. They have worked to restore lo'i kalo since 2008 and with the help of volunteers and a family program have successfully restored ~7,500 m2 of lo'i alongside other managed areas, and seek to restore another ~500,000 m2 to lo'i and other intercropped species (see below) in the next 20 years alongside a series of other restoration activities, including restoration of native wetland plant communities and agroforestry [42] (See SI Methods). They also aim to restore an additional ~100,000 m2 of the area as retention basins (including areas restored to loko i'a (fish ponds) and native wetland plant communities) with the remaining area including, streams, channels, access roads and small buildings, such as a commercial kitchen, poi (a traditional Hawaiian food made from mashed taro corms) mill, and community gathering place [42].

**Figure 1.** Location of He'eia wetland within the He'eia watershed, O'ahu. NERR = National Estuarine Research Reserve.

### *2.2. Outcomes Evaluated*

Emerging theory on biocultural restoration suggests that evaluations of biocultural projects should include place-based outcomes and indicators defined by and relevant to local actors [20,43]. At the same time, for biocultural approaches to gain traction in broader land-use planning initiatives, demonstrating their value to broader state, regional, and global sustainability goals and objectives is important [9,43] In the context of this study, we defined key outcomes of importance to Kako'o ¯ 'Oiwi's and the local community [ ¯ 42] as well to the achievement of statewide sustainability goals around food, energy, and water [31] (Table 1). An important *first* goal of Kako'o ' ¯ Oiwi is to build ¯ capacity of farmers and provide local families with the opportunity to learn to farm and care for lo'i kalo and (re)connect to the land. Among several initiatives to achieve this (see SI Methods), the organization began a family or 'ohana program where local families care for and maintain lo'i. There are currently 11 families participating in the project and scaling this program is an important part of the non-profit's growth strategy. *Second*, in line with the State's goal to double local food production, Kako'o ' ¯ Oiwi strives to produce traditional and diversified food crops. While financial ¯ return is not a central goal, the organization sees financial sustainability as an important part of their ability to sustain food production. *Third*, in line with the State of Hawai'i's goal to be 100% renewable by 2045, the organization seeks to understand how renewable energy could be incorporated into their long-term plans and how traditional agriculture can reduce energy inputs associated with food production and imports. *Fourth*, in line with the State's goal of protecting watersheds and marine ecosystems, there is a strong interest in understanding how lo'i kalo and broader wetland restoration influences the sediment and nutrient retention functions of the wetland given links to the downstream fish pond and critical coral reef habitat of Kane'ohe Bay, which both have high ecological, economic, ¯ and cultural value (Table 1).

**Table 1.** Outcomes of interest to Kako'o ' ¯ Oiwi and to the broader State of Hawai'i Aloha + Challenge ¯ sustainabilty goals [31].


*2.2. Air Quality Data*

The concentrations of six pollutants, SO2, NO2, CO, O3, PM2.5 and PM10, were monitored hourly over 22 cities across Sichuan Basin from January 2015 to December 2020. The data were made available by the China National Environmental Monitoring Center (http://www.cnemc.cn/ (accessed on 15 September 2021)).

The instruments for air quality monitoring were deployed according to the China Environmental Protection Standard HJ 664-2013. The equipment came from Shenzhen Aosen Purification Technology Co., Ltd., China. The gaseous pollutant and PM concentrations were measured following the Specifications and Test Procedures for Ambient Air Quality Continuous Automated Monitoring System HJ 654-2013 for SO2, NO2, O3 and CO, and HJ

653-2013 for PM2.5 and PM10, as stipulated in the National Environmental Protection Standards of the People's Republic of China. The air quality monitoring stations were located at least 50 m away from any notable stationary pollution sources, and the inlets for the instruments were placed at least 1 m higher than the roof of the building or wall [26]. Data quality assurance and quality control (QA/QC) were conducted following the technical guidelines on environmental monitoring quality management (HJ 630-2011) established in the National Environmental Protection Standards of the People's Republic of China. The validity of the data was checked following the national ambient air quality standards specified in the National Standards of the People's Republic of China (GB 3095-2012), as used in earlier studies [27,28]. The daily, monthly, and annual means of the data were calculated from the hourly concentrations (with ~80% of the available data to be considered as valid for calculating the mean).


**8. Conclusions**

was significantly higher in expanded cases compared with wild type individuals and also ALS-FTD patients with RE manifested cognitive behaviour before the onset of motor symptoms. In most cases the phenotype was compatible with a behavioural variant of FTD and frequently dominated by psychiatric symptoms, such as visual hallucination, paranoid behaviour with

Extrapyramidal and cerebellar signs were also observed in two patients, while a patients presented continuous lingual myclonus at disease onset. These cases suggested that clinical phenotype associated with RE in C9orf72 may be broader than originally thought, possibly involving extramotoneuronal structures such as the basal ganglia, cerebellum, brainstem

Sabatelli et al. and Chiò et al. [60, 61, 62] studied clinical phenotype of patients with repeat expansion in large population and also two Sardinian families with neurodegenerative diseases (FTD-ALS) in which mutations in different genes (TARDBP p.A382T mutation and repeat expansion GGGGCC C9orf72) co-existed as pathogenetic causes, giving varied pheno‐

Optineurin is an inhibitor protein that play an important role in the maintenance of the Golgi complex, in membrane trafficking and in exocytosis. Alternative splicing results in multiple

The human *OPTN* gene (Entrez Gene ID 10133) is located on chromosome 10p13, and it codes for a protein of 577 amino acids. In 2010 *OPTN* mutations have been described, for the first time, in ALS patients [63]. In the first paper about OPTN mutation three type of mutation have been found, two point mutation and one deletion. In 2011, a screening in the Caucasian population in SALS and FALS patients showed that *OPTN* mutations causing ALS are rare, especially in mainly Caucasian ALS subjects [64]. About Italian population, Del Bo and collaborators screened 274 ALS patients, 161 FALS and 113 SALS and the results showed six novel variants in both FALS and SALS patients, all occurring in an heterozygous state [13]. This data support the involvement of *OPTN* in ALS, especially in FALS patients, due to the

Del Bo et al. [13] suggested that ALS patients carrying OPTN mutations showed a prevalent lower-limb onset, with large variable age of onset (from 24 to 71 years of age) and progression (very aggressive forms with survival time < 1 year and very slow disease course over 10 years)

transcript variants encoding the same protein., three different isoforms are known.

persecutory delusions, aggressive behaviour and/or suicidal thoughts.

Reports in literature were in according [56-59].

86 Current Advances in Amyotrophic Lateral Sclerosis

nuclei.

types.

**7. Optineurin**

**7.1. Genotype**

**7.2. Phenotype**

1.2% cases found mutated [13].

Amyotrophic Lateral Sclerosis is a multifactorial and multigenic disease with still unknown aetiology and pathogenesis. We know many causative mutations in particular genes, both in familial and sporadic patients, and different clinical presentation of ALS. Genetic factors may play a role in determining the range of ALS phenotypes although in this moment no genes have been demonstrated to have a definite effect on phenotype (Chiò et al., 2011) [4]. Hetero‐ geneity between and among families and patients with same mutation suggests that environ‐ mental and other influences contribute to not only the rate of evolution and which signs predominate but also whether the disease will appear at all during life.

In this chapter we have identified cases in which connection between phenotype and genotype is possible and relevant.

We started from the ALS patients part of the recruitment of our Institute to define the possible clinical features that may be related to specific genes alteration.

For ALS patients with mutations in some genes, such as *SOD1*, there are an important clinical phenotypic heterogeneity at onset and during evolution of disease, different time of survival and velocity of progression with rapidly or slowly involvement of bulbar functions.

*TARDBP* is involved both in pathogenesis of frontotemporal dementia and ALS, but it's not sure that all patients with ALS will develop FTD and they don't demonstrated an homogenous clinical pattern.

In particular we will focus attention on connection between FUS mutations, and clinical presentation with upper limbs onset, developed weakness of the neck flexor/extensor muscles and bilateral scapular girdle and proximal muscle. In many cases this phenotype is correlated with rapidly bulbar evolution and frontotemporal behaviour alterations, with negative prognosis in short time. This focus is in particular due to the presence of FUS mutated patients in our cohort and the "poverty" of the literature about FUS and clinical features.

Another interesting suggestion is that sometimes mutated patients (i.e. SOD1) can have particular clinical course modulated by other causative or associated modified genes (ANG). It is an important issue that maybe indicates a central role of genotype in developing pheno‐ type.

For other genes it's difficult discovering association between genotype and phenotype for rarity of manifestation compared with more frequent mutations.

In conclusion, at this time, in front of a patient with ALS, a neurologist should has some "milestones" considering clinical phenotype:

**•** if patient's history suggests a familial form, it is important to perform a screening of four principal genes (SOD1, TARDBP, FUS, C9ORF) because they cover more than 50% of FALS;

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[10] Corrado L, Del Bo R, Castellotti B, Ratti A, Cereda C, Penco S, Sorarù G, Carlomagno Y, Ghezzi S, Pensato V, Colombrita C, Gagliardi S, Cozzi L, Orsetti V, Mancuso M, Siciliano G, Mazzini L, Comi GP, Gellera C, Ceroni M, D'Alfonso S, Silani V. Muta‐ tions of FUS gene in sporadic amyotrophic lateral sclerosis. J Med Genet. 2010;

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Our idea is that a specific mutation can cause a particular clinical onset, involvement and evolution of ALS, with a pathogenetic mechanism still unknown.

On the other side, now we have some ideas for type of disease correlated with particular mutations (i.e. FUS and TARDBP give a early onset, short duration of disease for early bulbar involvement) but it's impossible predict exactly what kind of phenotype can be developed by patient.

Different genes are involved in ALS disease, the importance of a good clinical characterization may help in choosing the genetic approach. We hypothesized that in the future, the symptoms observation may became more specific to indicate which gene is the most probably mutated. This idea may proceed in the same time of a better collaboration between clinicians and biologist to create a direct link from bed to bench.

This approach may be relevant for diagnostic use, so starting from neurological exam, that remains the essential element, and we can formulate diagnostic hypothesis that it can be surely confirmed by genetic test.

Acknowledgement

We are grateful to the patients and their families. We thank other members of General Neurology Department and Laboratory of Experimental Neurobiology for continuous working activity.


*Edited by Mark Schwartz*

The six chapters in this volume form a timely introduction to biofeedback research. The six authors describe how objective electrophysiological data provides useful data for assessment and therapy for physical therapists, psychologists and other healthcare professionals and their clients. The first chapter, "Biofeedback and neurofeedback in the treatment of migraine", is written by Ph.D. student Zivoder Ivana. The second chapter by Prof. Dumitrascu looks at pelvic floor rehabilitation and is titled "Factors predicting failure in anorectal biofeedback". The third chapter by Dr Liao Da-Vin is titled "Collaborative, socialnetworked posture training with posturing monitoring and biofeedback". It is a useful and timely description of the role of biofeedback to support rehabilitation therapy at home and describe how objective information of physiotherapy treatments could increase the quality of service and the consistency of treatment outcomes. The fourth chapter by Prof. Silva Hugo is titled "Bridging the clinic-home divide in muscular rehabilitation". It also covers the role of biofeedback to support rehabilitation therapy at home. The fifth chapter is written by Ph.D. student Grin-Yatsenko Vera and is titled "Effect of infra-low frequency neurofeedback on infra-slow-EEG-fluctuations". The sixth and final chapter in the volume, "Control systems of bionic limbs of the new generation and control systems with EMG signals of VR and games, toys" by M.A. Ivaniuk Natallia goes beyond the person and shows how biofeedback can be integrated in the behavior of a bionic hand.

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Biofeedback

Published in London, UK © 2018 IntechOpen © romankosolapov / iStock


**3. Results**

#### *3.1. Acute Carbogen Exposure Reduces Intraplaque Hypoxia*

To evaluate the e ffect of acute carbogen treatment on advanced atherosclerotic vein graft lesions, ApoE3\*Leiden mice that underwent vein graft surgery were exposed for 90 min to carbogen gas or normal breathing air. Mice exposed to carbogen gas (*n* = 8) showed a significant reduction of intraplaque (IP) hypoxia in the vein graft lesion compared to the air breathing group (*n* = 8) as shown by a 84% decrease in the immuno-area positive for pimonidazole in the lesions of carbogen treated mice compared to the control (*p*-value = 0.027) (Figure 1A–C).

**Figure 1.** Short term carbogen exposure drastically reduces intraplaque hypoxia. (**A**) Representative pictures of sections from vein graft lesions in ApoE3\* Leiden mice stained for pimonidazole in the control group (*n* = 8) and (**B**) one-time carbogen treated group (*n* = 8). (**C**) Quantification of pimonidazole positive area. Data are presented as mean ± SEM. \* *p* < 0.05; by two-sided Student's t test.

Regarding the aspect of vein graft patency, single 90-min carbogen exposure directly before sacrifice did not affect vein graft patency (Figure 2A), vessel wall area, lumen perimeter, lumen area or optimal lumen area (Figure 2B,C). Furthermore, weight nor cholesterol levels were changed (Figure S1).

The percentage of collagen present in the lesion was comparable between the two groups (Figure 2D) and at a cellular level, the percentage of macrophages (Figure 2E) and SMCs (Figure 2F) were not altered by the acute exposure to carbogen.

#### *3.2. Chronic Carbogen Exposure Does not Influence Intraplaque Hypoxia*

To evaluate the effect of hyperoxic carbogen treatment on plaque composition and remodeling we performed a chronic carbogen treatment on ApoE3\*Leiden mice with advanced atherosclerotic vein graft lesions. Mice were exposed for 90 min daily to carbogen gas (*n* = 13) or normal breathing air (*n* = 12) for 21 days. Neither weight or cholesterol levels were affected by the treatment (Figure S2).

Surprisingly chronic exposure to carbogen gas did not reduce intraplaque hypoxia in the treated group when compared to the air breathing group (Figure 3). In fact, the degree of pimonidazole staining in the vein graft area was not different between the two groups (Figure 3).

#### *3.3. Chronic Exposure to Carbogen Plays a Protective Role Against Occlusions*

Chronic carbogen treatment resulted in a beneficial effect on vein graft patency, increasing the rate of vein graft patency by 34.5% (Figure 4A). In fact, only 53% of the mice of the control group presented a patent vein graft (Figure 4A), while 87.5% of the mice exposed to carbogen gas had a patent vein graft.

**Figure 2.** Short term exposure to carbogen gas does not influence plaque size nor composition. (**A**) Quantitative measurement of vein graft patency in ApoE3\*Leiden mice from the control and one-time carbogen treated groups. Data are analyzed by Chi-square test (**B**) Representative pictures of MOVAT staining of vein graft sections from control (*n* = 8) and carbogen group (*n* = 8). (**C**) Quantification of vessel wall area, lumen perimeter, lumen area and optimal lumen area. Percentage of positive vessel wall area and representative pictures for (**D**) collagen (*n* = 8 for control and carbogen groups), (**E**) macrophages (*n* = 8 for control and carbogen groups) and (**F**) smooth muscle cells staining (*n* = 8 for control and *n* = 7 for carbogen groups). Data are presented as mean ± SEM.

**Figure 3.** Chronic carbogen treatment does not affect intraplaque hypoxia. Representative pictures of vein graft cross sections stained for pimonidazole in control (*n* = 8) and chronic-treated carbogen groups (*n* = 13) and quantitative measurement of percentage of vessel wall area positive for pimonidazole staining. Data are presented as mean ± SEM.

**Figure 4.** Chronic exposure to carbogen plays a protective role against vein graft occlusions. (**A**) Quantitative measurement of vein graft patency in ApoE3\*Leiden mice from the control and prolonged carbogen treated groups. Data are analyzed by Chi-square test. \* *p* < 0.05. Representative pictures of non-patent and patent vein grafts in ApoE3\* Leiden mice at day 28 after surgery in the right panel. (**B**) Quantitative measurements of vein graft thickening. In the right panel, representative pictures of MOVAT staining in vein grafts from control (*n* = 8) and long term carbogen treated mice (*n* = 13), with vessel wall area as the area between the two dotted lines. (**C**) Quantification of lumen perimeter and (**D**) optimal lumen area. Data are presented as mean ± SEM. \* *p* < 0.05; by two-sided Student's t test.

Vessel wall area thickening was not affected by exposure to carbogen gas since no differences could be detected between the two groups when taken only the patent grafts into account (Figure 4B). More importantly lumen size was affected by carbogen gas. In fact, carbogen treated mice presented a significant increase in the lumen perimeter when compared to control (Figure 4C,E, *p*-value = 0.048), and an increase in the optimal lumen area (Figure 4D, *p*-value = 0.067).

#### *3.4. Chronic Carbogen Treatment Does Not Have an E*ff*ect on Intraplaque Angiogenesis and Intraplaque Hemorrhage*

To see whether exposure to carbogen gas had an effect on the hypoxia triggered IP angiogenesis, the amount of CD31+ vessels in the vein graft lesions (white arrows in Figure 5A zoom in) was evaluated and no difference in the number of neovessels in the carbogen group was observed when compared to the control group (*p*-value > 0.99).

**Figure 5.** Chronic carbogen treatment does not affect intraplaque neovascularization. (**A**) Representative pictures of vein grafts lesions stained for DAPI (white), CD31 (green) and Ter119 (red). (**B**) Quantification of CD31 positive neovessels in the vessel wall area in the control group (*n* = 8) and in the carbogen treated group (*n* = 12). (**C**) Bar graphs representing the quantitative measurements for IPH in the control and long term carbogen treated groups. IPH was scored as not present, low, moderate or high. Total vessel wall gene expression of (**D**) Hif1a, (**E**) Cxcl12, (**F**) Vegfa and (**G**) Hif2a, relative to Hprt, was measured in the control and long term carbogen treated groups. Data are presented as mean ± SEM. \* *p* < 0.05; by two-sided Student's t test.

In addition, when corrected for intimal thickness no di fferences were observed between the groups (*p*-value = 0.91) (Figure 5A,B). As a measure of the quality of the IP angiogenesis the degree of intraplaque hemorrhage was analyzed (yellow stars in Figure 5A zoom-in) as the amount of Ter119+ cells found outside the neovessels and quantified as not present, low, moderate or high, and no di fferences could be seen when comparing the two groups (Figure 5C).

To determine the e ffects of hyperoxia on angiogenesis related genes the expression of Hif1a was analyzed. We surprisingly found a significant upregulation of Hif1a mRNA expression in the carbogen treated group when compared to the control (Figure 5D, *p*-value = 0.05), while mRNA expression of Cxcl12, Vegfa and Epas1 were not altered (Figure 5E–G). No di fferences between control and one-time carbogen treated group were found when analyzing gene expression in vein grafts from the acute carbogen treatment (Figure S3).

#### *3.5. Chronic Carbogen Treatment Induces Accumulation of Reactive Oxygen Species and Apoptosis*

Although an e ffect on Hif1a upregulation was observed, surprisingly no e ffect on angiogenesis could be seen. Therefore, we looked into other mechanisms that could possibly regulate Hif1a. We hypothesized that the mRNA upregulation of Hif1a in the carbogen treated group (Figure 5D) was caused by an accumulation of reactive oxygen species (ROS) induced by the carbogen treatment. ROS is known to be induced by prolonged hyperoxia [17] and to regulate the transcription of di fferent genes involved in hypoxia and in inflammation such as Hif1a and Il6.

Il6 mRNA expression was studied as a representative for ROS induced factors and quantification of its expression showed a trend towards increased expression in the carbogen group of the chronic exposure study when compared to the control group (Figure 6A, *p*-value = 0.09).

Next the presence of ROS was studied in the vein graft lesions by quantifying the amount of ROS–mediated DNA damage, analyzed by 8-hydroxy-2deoxy-guanosine (8OHdG) immunohistochemical staining. We determined the subcellular location of the staining of 8OHdG. As observed in Figure 6C, a strong 8OHdG positive staining was found in the nuclei of the cells, with an occasional staining outside of the nuclei in the mitochondria, seen as cytoplasmic staining (Figure 6C, right panel). This suggests that the main site of ROS induced DNA damage is nuclear, and not mitochondrial. 8OHdG positive staining could be seen as light blue staining in the nuclei of the cells as a results of co-localized DAPI and 8OHdG staining (Figure 6D zoom-in) and the quantification corrected for the vessel wall area resulted in an increase of DNA damage in the carbogen treated group when compared to the control group (Figure 6B,D), supporting the idea that ROS levels are increased.

ROS is known to induce apoptosis as a consequence of DNA damage. Therefore, the amount of cells positive for cleaved caspase 3 (CC3) in the atherosclerotic vein graft lesions was determined in the carbogen treated and in the control groups (Figure 6E). Due to their high oxygen consumption we hypothesized that macrophages could possibly be the main cell type a ffected by DNA damage induced by ROS and subsequent apoptosis. As shown in the bottom panel of Figure 6D, macrophages rich areas in the lesions of mice treated with carbogen were found to be strongly positive for CC3 when compared to control (Figure 6E bottom panel).

When looking at the total amount of cells positive for cleaved caspase 3 in the intimal area, an increase in apoptotic cells CC3+ in the lesions of mice exposed to carbogen gas was found compared to the air breathing group (Figure 6E, *p*-value = 0.06).

**Figure 6.** Chronic carbogen treatment induces accumulation of ROS. (**A**) Il6 gene expression relative to Hprt in the total vessel wall of control and chronic carbogen treated groups. (**B**) Quantification of the percentage of vessel wall area positive for 8OHdG. (**C**) Representative pictures of DAPI (in blue, left panel), 8OHdG (in green, central panel) and merged (right panel) staining in the vein graft lesions. (**D**) representative pictures of DAPI (blue) and 8OHdG (green) staining in vein graft lesions from control (*n* = 8) and carbogen treated (*n* = 12) mice. Light blue staining represents nuclei positive for 8OHdG and examples are indicated by white arrows. (**E**) In the top panels, representative pictures of DAPI (blue) and cleaved caspase 3 (CC3, in magenta) staining and in the bottom panels representative pictures of DAPI (blue), CC3 (magenta) and Mac3 (green) staining in control and carbogen groups respectively. In the right panel quantification of percentage of intimal area positive for cleaved caspase 3. Data are presented as mean ± SEM. \* *p* < 0.05 by two-sided Student's t test.

#### *3.6. Chronic Carbogen Exposure Reduces Inflammatory Cell Content*

The effects of chronic carbogen gas treatment on intraplaque inflammation were studied on macrophages since they produce high amounts of ROS, consume elevate amounts of O2 and are known to be hypoxic [6]. Interestingly, the group of mice exposed daily to carbogen for 21 days showed a significant reduction in macrophage content when compared to the control group breathing normal air (*p*-value = 0.0126).

When corrected for the differences in vein graft thickening, the relative percentage of macrophages was significantly decreased in the carbogen exposed group by the 15.2% (Figure 7A, *p*-value = 0.0044).

**Figure 7.** Chronic carbogen treatment reduces macrophages infiltration in the plaque. (**A**) Representative pictures of ApoE3\* Leiden mice vein grafts from control (*n* = 8) and chronic carbogen treated (*n* = 13) groups stained for Mac-3. In the right panel quantitative measurements of the percentage of vessel wall area positive for Mac-3. Data are presented as mean ± SEM. \*\* *p* < 0.01; by two-sided Student's t test. Total wall gene expression of (**B**) Ccl2 and (**C**) Tnf relative to Hprt. (**D**) Quantification of the number of cells positive for Mac-3 and Ki67 in the vessel wall area of control (*n* = 8) and carbogen treated group (*n* = 13) and representative pictures of the staining with DAPI presented in blue, Mac-3 in green and Ki67 in red. Data are presented as mean ± SEM. \*\* *p* < 0.01 by 2-sided Student t test.

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To study whether the decrease in macrophages was not due to a reduced infiltration of macrophages, nor a reduced proliferation of resident macrophages, local cytokines expression in the vein grafts was studied and the proliferation of macrophages was analyzed.

First, the mRNA expression levels of Ccl2 and Tnf in the vein graft atherosclerotic lesions were examined. The mRNA levels of Ccl2 and Tnf did not differ between the carbogen treated group and the control (Figure 7B,C).

Using a triple IHC staining for Mac-3, Ki-67 and DAPI the amount of proliferating macrophages was determined. As shown in Figure 7D and, there was no difference in the number of proliferative macrophages corrected for the vessel wall thickening (*p*-value = 0.16).

Thus, the data suggest that the reduction of plaque macrophages could be due to enhanced macrophage apoptosis.

#### *3.7. Chronic Carbogen Treatment Does Not A*ff*ect Plaque Size but Increases Plaque Stability*

To evaluate the effect of prolonged carbogen treatment and accumulation of ROS on plaque composition, the amount of collagen (positive collagen area in the total vessel wall) and smooth muscle cells (positive αSMA area in the total vessel wall) was analyzed, two main predictors of plaque stability.

The collagen content in the plaque was not affected by carbogen treatment, and was comparable between the two groups (Figure 8A). Similarly, SMCs content in the carbogen group was not different from the control group (Figure 8B). Interestingly, when calculating the plaque stability index, defined as the amount of collagen and SMCs divided by the vessel wall area, atherosclerotic plaques of the mice daily exposed to carbogen resulted to be more stable than the lesion of the control group (Figure 8C, *p*-value = 0.05).

**Figure 8.** Chronic carbogen treatment does not affect collagen nor smooth muscle cells content in the lesion but increases plaque stability. (**A**) Quantitative measurement vessel wall area positive for collagen in ApoE3\*Leiden mice from the control (*n* = 8) and carbogen treated (*n* = 13) groups. In the right panel representative pictures for collagen staining. (**B**) Quantification of percentage of vessel wall area positive for smooth muscle cell actin and representative pictures from the control (*n* = 8) and carbogen treated (*n* = 13) groups. (**C**) Quantification of plaque stability index. Data are presented as mean ± SEM.

#### *3.8. ROS Increases DNA Damage and Apoptosis in Bone Marrow Derived Macrophages In Vitro*

To unravel the molecular and cellular mechanism underlying the observed changes in macrophage content, in particular whether this could be due to hyperoxia induced ROS accumulation, we treated macrophages derived from bone marrow of APOE3\*Leiden mice with t-BHP, a known ROS mimic [19]. t-BHP treatment increased the occurrence of DNA damage in BMM as measured by 8-OHdG immunocytochemical staining (Figure 9A), confirming its activity as a ROS mimic and the induction of DNA damage by ROS.

**Figure 9.** ROS induces DNA damage and apoptosis on in vitro bone marrow macrophages (BMM) (**A**) Representative pictures of CTRL BMM and BMM treated with 200 or 400 μm t-BHP respectively are shown. Examples images stained for DAPI (blue), Mac3 (green) and 8OHdG (magenta) as well as a merged image are shown per each condition tested. (**B**) Quantification of 8OHdG expression as mean intensity is shown. (**C**) Total mRNA expression of Hif1a relative to Hprt. (**D**) Quantification of CC3 expression as mean intensity is shown. (**E**) Quantification of total amount of cells per condition tested expressed as total amount of positive DAPI nuclei. (**F**) Representative pictures of CTRL BMM and BMM treated with 200 or 400 μm t-BHP respectively. Examples images stained for DAPI (blue), Mac3 (green) and cleaved caspase 3 (CC3) (orange) as well as a merged image are shown per each condition tested. Data are presented as mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01; by two-sided Student's t test.

Quantification revealed a 2.2-fold increase in DNA damage in macrophages treated with 200 μm t-BHP (*p*-value = 0.006) and a two-fold increase in DNA damage in macrophages treated with 400 μm t-BHP (*p*-value = 0.006) when compared to control (Figure 9B).

We then evaluated the effect of the ROS mimic t-BHP on the expression of several genes. Similar to changes in expression in vivo, we found that t-BHP-induced ROS caused a significant increase of Hif1a mRNA expression (*p*-value = 0.007 and 0.02 respectively) when compared to control (Figure 9C). Interestingly, we also found that ROS caused an increase in the expression of pro-inflammatory genes Ccl2 and Tnf, but decreased Epas1 expression compared to control (Figure S4)

To assess if ROS ultimately causes apoptosis in cultured BMM, we examined the expression of CC3 and found a significant and dose dependent increase in CC3 expression, thus apoptosis, in t-BHP treated BMM when compared to control (Figure 9F). The group treated with 200 μm t-BHP showed a 10% increase (*p*-value = 0.03) and the group treated with 400 μm t-BHP a 27% increase (*p*-value = 0.01) in CC3 expression when compared to control (Figure 9D). Moreover, we observed a drastic reduction in the total number of cells by 72% and 70% in the groups treated with 200 and 400 μm t-BHP, respectively, when compared to control (Figure 9E, *p*-value = 0.01 for both groups). Combined these data demonstrate that ROS directly affects gene expression in macrophages and causes DNA damage and apoptosis.


**2. Effects of the SARS-CoV-2 virus on the cardiovascular system**

The COVID-19 pandemic greatly challenged clinicians, both due to the sheer number of patients, but also because of the lack of therapeutic consensus and incomplete understanding of disease pathogenesis. Most fatal cases of COVID-19 relate to a severe atypical pneumonia, accompanied by a sudden systemic deterioration, despite therapeutic intervention in the hospital setting.

The infection with the SARS-CoV-2 virus primarily affects the respiratory structures, but the involvement of the cardiovascular system is also frequent. Cardiovascular complications in addition to respiratory disease may develop in all phases of COVID-19, which can start with the dramatic picture of acute heart failure (ACF), acute coronary syndrome (ACS), pulmonary venous thromboembolism (VTE), or even sudden cardiac death, as shown in **Figure 1**. The pathophysiological mechanisms underlying these disproportionate effects of the SARS-CoV-2 infection on patients with cardiovascular comorbidities, however, remain incompletely understood [7]. Thromboembolic events, usually accompanied by violent, pulmonary, and/or systemic complications, have been described from early on, since the beginning of the pandemic, with infectious inflammatory response patterns rapidly shifting into a typical systemic inflammatory response syndrome (SIRS) or ARDS, which could potentially induce multi-organ failure (MOF) and, subsequently, death. As we enter the third year of the pandemic, COVID-19 pathophysiology is slowly unraveling as we begin to better comprehend the complex interplay between the direct cytotoxic effects of SARS-CoV-2 on pneumocytes and endothelial cells, the emerging local and systemic inflammatory response, and the ways in which these responses interact with hemostatic homeostasis, a mechanism which has been deemed as central and, at least to this extent, unprecedented [8].

#### **2.1 Cardiac tissue damage**

COVID-19 was initially considered to be solely a respiratory disease, yet clinical outcomes quickly revealed that, undeniably, this infection implies multi-organ involvement. Perhaps most notably, the heart has been shown to represents a target organ for SARS-CoV-2-related pathogenesis, with a high prevalence of cardiac injury following COVID-19, often diagnosed only through biomarker evaluation. Beyond subclinical myocardial damage, SARS-CoV-2 infection may also cause more aggressive, clinically apparent modifications, such as myocarditis, accompanied by a subsequent diastolic dysfunction or severe reduction of left ventricle ejection fraction, not to mention the fact that heart failure may represent a short−/long-term consequence of COVID-19-related inflammatory cardiomyopathy, with dramatic consequences regarding prognosis [9].

Regarding myocardial damage in COVID-19, although the full pathophysiology is still incompletely understood, multiple mechanisms are most likely incriminated (see **Figure 2**), which, globally, can be divided into two main groups: direct, specific modifications, related to the cytopathic effects of SARS-CoV-2 infection, and indirect, general modifications, commonly seen in other severe infections, as well [10].

### *2.1.1 Direct cytopathic myocardial injury*

The aforementioned ACE2, a type I transmembrane protein, highly expressed in different organs (heart, lungs, gut, and kidneys), mediates SARS-CoV-2 entry into

**Figure 1.** *Main COVID-19-associated cardiovascular complications and underlying pathophysiological mechanisms.*

the host cells, with different clinical implications, depending on the targeted organ, and represents the key molecular entity involved in the direct cytopathic effects of SARS-CoV-2 infection within the cardiac tissue. After entering the host cell through the host ACE2 receptor, SARS-CoV-2 utilizes the host's RNA-dependent RNA polymerase to replicate its own structural proteins, which are then assembled, and the newly formed virions are released from the infected cells, perpetuating the viral life cycle. Theoretically, as a consequence of this process, infected cells may become damaged/destroyed [11].

This idea is supported by a recent autopsy study, analyzing cardiac tissue from 39 consecutive patients who died as a consequence of COVID-19, which found viral genome in the myocardial tissue, yet in situ hybridization showed that the most likely localization of SARS-CoV-2 not to be in the cardiomyocytes, but rather in interstitial cells or macrophages invading the myocardial tissue [12]. Even so, in engineered heart tissue models of COVID-19 myocardial pathology, SARS-CoV-2 demonstrated

*Impairment of the Cardiovascular System during SARS-CoV-2 Infection DOI: http://dx.doi.org/10.5772/intechopen.103964*

#### **Figure 2.**

*Pathophysiology of COVID-19-related myocardial injury [15, 16].*

the ability to directly infect cardiomyocytes through ACE2, resulting in contractile deficits, cytokine production, sarcomere disassembly, and cell death [9].

Furthermore, ACE2 must not be viewed as a mere bystander in the pathophysiology of COVID-19 myocardial injury, seeing as, besides being the host cell receptor of SARS-CoV-2, ACE2 is an enzyme involved in the renin-angiotensin-aldosterone system (RAAS). Specifically, ACE2 cleaves angiotensin II, a very potent vasoconstrictor, into angiotensin 1–7, which manifests vasodilator and anti-inflammatory effects. ACE2 also demonstrates a weak affinity for angiotensin I (or proangiotensin, formed by the action of renin on angiotensinogen), competitively limiting angiotensin II synthesis by ACE. Angiotensin I is converted by ACE2 into the nonapeptide angiotensin 1–9, which will manifest vasodilator effects through subsequent angiotensin type 2 (AT2) receptor stimulation. Therefore, ACE2 can counteract the undesirable effects of angiotensin II, demonstrating vasodilator, antioxidant, and anti-fibrotic effects [13]. In the context of SARS-CoV-2 infection, after S protein binding is complete, the virus attaches ACE2 through membrane fusion and invagination, causing a downregulation of ACE2 enzymatic activity [13]. Additionally, ACE2 also demonstrates immunomodulatory properties, both directly, via its interactions with macrophages, and indirectly, as it reduces expression of angiotensin II, which stimulates inflammation [14]. Thus, ACE2 downregulation in the context of SARS-CoV-2 infection may increase angiotensin II levels, favoring AT1 receptor activity, with a subsequent vasoconstriction, fibrotic, proliferative, and pro-inflammatory effects [10].

#### *2.1.2 Indirect mechanisms of myocardial injury*

As is the case with all severe respiratory infections, COVID-19 has a general deleterious effect on the cardiovascular system, with fever and sympathetic activation causing tachycardia and implicitly increasing myocardial oxygen consumption [9, 10], while prolonged bed rest and systemic inflammation will favor coagulation disorders, as supported by clinical findings – both venous and atypical arterial thromboembolic events have been documented in COVID-19 patients (see subchapter 3.4. Thromboembolic events and bleeding risk). Hypoxemia, another hallmark of COVID-19, will determine enhanced oxidative stress and increased production of reactive oxygen species, with subsequent intracellular acidosis, mitochondrial damage, and cell death [7, 9].

Moreover, another series of indirect mechanisms for COVID-19-related myocardial damage appears as a result of the abnormal inflammatory response which may be elicited by SARS-CoV-2 infection (i.e. a pro-inflammatory surge, the so-called "cytokine storm," which may occur as early as 1 week after the initial exposure and infection) [15].

Indeed, individual immune response is the cardinal element behind SARS-CoV-2 infection progression. Upon viral genome expulsion into the host cytosol, SARS-CoV-2 viral replication begins, with aberrant RNA sequences, byproducts of replication, being, in turn, detected by intracellular receptors, which activate the cellular antiviral response, involving enhanced leukocyte chemotaxis and transcriptional induction of type I and III interferons (IFN-I/-III), followed by under-regulation of IFN-stimulated genes [16]. Lung cell damage incurred during replication will also activate the local immune response, resulting in monocyte/macrophage recruitment [16], while chemokines will induce specific leukocyte subset recruitment and coordination [16]. Circulating immune cell relocation in the pulmonary tissue will determine additional cytokine/chemokine production, while also creating multiple imbalances in immune cell populations – increased leukocyte count and neutrophillymphocyte ratio, with decreased lymphocytes (especially T cells [17]), thus setting the scene for immune response dysregulation [3].

In fact, the relationship between SARS-CoV-2 infection and extensive activation of inflammation signaling pathways has been well documented, representing the main immunopathological mechanism through which severe forms occur, in susceptible individuals. During the acute phase of the infection, a disproportionate response occurs between T helper cell populations (types 1 and 2), characterized by high circulating levels of interleukin (IL)-1β, IL-1RA, IL-2, IL-6, IL-7, IL-8, IL-9 IL-10, interferon gamma-induced protein-10 (CXCL10), monocyte chemoattractant protein-1 (CCL2), macrophage inflammatory protein 1α (CCL3) and 1β (CCL4), granulocyte colony-stimulating factor, vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF) α [16, 18, 19], which mediate widespread lung inflammation, in an attempt to eradicate the pathogen [3]. The resulting hyperinflammatory status, as well as the individual excessive levels of certain circulating cytokine species, have been independently associated with an unfavorable evolution and increased mortality [20]. This hyper-inflammatory state seems, at least intuitively, to be pivotal in the development of cardiac injury, seeing as positive correlations have been established between the increase in inflammatory markers and myocardial damage in COVID-19 [21, 22]. Indeed, this idea is additionally supported by previous studies, in other septic conditions, evidencing that the release of

pro-inflammatory cytokines such as TNFα and IL-1β, were responsible for myocardial cells depression through modulation of calcium channel activity and nitric oxide production [23].

It may also be the case that the cytokine storm following SARS-CoV-2 infection determines the AHF, recurrently seen in severe COVID-19, as the inflammatory activation and oxidative stress background are similarly expressed generally in heart failure, predisposing to a more severe clinical course [24].

Lastly, the aforementioned marked inflammatory changes will also take place in the endothelium, as shown in postmortem histological studies, evidencing lymphocytic endotheliitis with apoptotic bodies and viral inclusion in multiple organs [7, 25]. Endotheliitis can lead to disseminated intravascular coagulation, with small or large vessels thrombosis and infarction, and will determine significant new vessel growth through a mechanism of intussusceptive angiogenesis [25].

#### **2.2 Coagulation disturbances**

After becoming infected, roughly 20% of COVID-19 patients will be incapable of controlling/halting viral replication through their initial immune response, which may be aberrant/insufficient or overwhelmed by a high initial viral load, or both [26]. This subgroup of patients will thus progress to a more severe disease phenotype, with aggravating symptomatology secondary to uncontrolled viral replication, leading to host pneumocyte and endothelial cell apoptosis, which in turn will activate platelets, induce procoagulant factor expression (fibrinogen, factors V, VII, VIII, X, and von Willebrand), and increase inflammatory response, as the body tries and fails to keep the infection localized to the lungs [27]. This sequence of host responses will additionally damage the pulmonary parenchyma (through further destruction of pneumocytes, microangiopathy, and inflammatory microthrombi), causing even more severe symptoms and hindering oxygenation, thus imposing the need for an additional oxygen supply. Even so, at this point, a relative balance between procoagulant and anticoagulant (but also pro-inflammatory/anti-inflammatory) factors is still maintained. In only approximately 5% of symptomatic patients, the pro-inflammatory processes involved in the immune response to SARS-CoV-2 infection will derail into the so-called "cytokine storm," which will fuel pro-inflammatory and pro-coagulatory processes even further, resulting in systemic endotheliitis and capillary leakage, cellular dysfunction, organ dysfunction (including ARDS), and overt activation of the (systemic) coagulation cascade resulting in the need for critical organ support [28]. In fact, SARS-CoV-2 infection may trigger endothelial dysfunction not only through the direct cytopathic effect of invasion on vascular endothelial cells but also through indirect mechanisms, such as hypoxia and the induced inflammatory response [27]. Moreover, some patients have also manifested antiphospholipid antibodies [28].

Therefore, all factors of the classic Virchow triad are influenced during the course of COVID-19, and they contribute synergically to the risk of thromboembolic events: hemodynamic changes (increased blood viscosity due to elevated fibrinogen, but also venous stasis due to hospitalization and disease-related immobilization); hypercoagulability (due to an overwhelming inflammatory state, occurring early after infection); and endothelial injury/dysfunction (ACE2 receptor expression on endothelial cells allows viral entry and cytopathic effects – endotheliitis) [3].


Smoking - Prevention, Cessation and Health Effects

*Edited by Li Ping Wong and Victor Hoe*

Published in London, United Kingdom

### *Supporting open minds since 2005*

Smoking - Prevention, Cessation and Health Effects http://dx.doi.org/10.5772/intechopen.77769 Edited by Li Ping Wong and Victor Hoe

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Narendra Maddu, Azim Chowdhury, Linda Tang, Ignatio Madanhire, Charles Mbohwa, Xiu Liu

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