Three HO isoforms have been reported in humans: HO-1 (32 KDa), which is the inducible form, and two other isoforms with constitutive expression [HO-2 (36 KDa) and a putative HO-3 (33 KDa)] (Nitti et al., 2017). The inducible HO-1, is expressed at very small amounts in normal tissues, except the liver and spleen, where it plays a key role in the elimination process of damaged and aged red blood cells (Gottlieb et al., 2012). Protein expression of HO-1 can be enhanced by many stimuli, which can induce ROS production. Among the pharmacological and chemical stimuli known to induce HO-1, there is heme itself [a byproduct of HO-1 action), heavy metals (e.g., cobalt], inflammatory cytokines, ultraviolet radiation, bacterial membrane component lipopolysaccharide (LPS), hyperglycemia, H2O2, cell growth factors, NO, and CO (Ryter et al., 2006). On the other hand, the expression of HO-2 is not induced by the factors inducing HO-1. However, HO-2 is constitutively expressed in high levels in certain tissues such as the brain, testis, smooth muscle cells and endothelial cells (Chen et al., 2003). In addition to maintaining heme homeostasis, basal amounts of HO-2 also play a key role in anti-oxidant cellular responses by regulating the activation of extracellular SOD, Akt, and ASK-1, through the modulation of heme degradation rate into its biological byproducts. Furthermore, HO-2 plays a pivotal role in sensing levels of oxygen and in cellular response to hypoxia (Munoz-Sanchez and Chanez-Cardenas, 2014). The protective role of HO-2 in hypoxia was demonstrated in endothelial cells. HUVECs and aortic endothelial cells exposed to hypoxia were found to exhibit a stable HO-2 protein expression despite a significant decline in its mRNA expression and a decrease in general protein translation. An increased interaction of HO-2 transcript with polysomes was observed during hypoxic events, leading to a selective activation of HO-2 protein translation, which maintained protein expression levels of HO-2. The maintenance of steady levels of HO-2 protected endothelial cells from apoptosis during hypoxic episodes (He et al., 2010). Finally, HO-3 is encoded by a pseudogene derived

from the transcript of HO-2, but so far has only been identified in rats (McCoubrey et al., 1997).

The modulation of HO-1 expression involves a complex molecular interplay between different regulatory mechanisms. MAPK, phosphatidyl inositol 3-kinase (PI-3K)/Akt, protein kinases (PKA, PKC, and PKG) (Stocker and Perrella, 2006), NrF-2, activator protein (AP)-1, NF-κB, cyclic adenosine monophosphate-response element-binding (CREB) protein, and activation transcription factor (ATF)-2 are signaling pathways that have been found to play a role in gene regulation of HO-1 expression (Kim H.P. et al., 2011; Kim Y.M. et al., 2011). Of particular interest, in response to oxidative damage, NrF-2 controls the transcriptional induction of HO-1. NrF-2 is maintained in inactive form in the cytosol by interacting with protein Keap1, which prevents the nuclear translocation of NrF-2 and enhances proteasome-mediated degradation of NrF-2. Following induction of an oxidative stress state, NrF-2 is activated after its dissociation from Keap1; active NrF-2 translocates then to the nucleus and triggers the transcription of HO-1 gene. In support of the role of NrF-2 in controlling HO-1 expression, previous reports have found that fibroblasts from mice deficient in NrF-2 express low levels of HO-1 (Leung et al., 2003; Liu et al., 2007).

Several heavy metal protoporphyrins induce the expression of HO-1; however, Cobalt protoporphyrin (CoPP) is regarded as probably the most potent. CoPP indirectly modulates the production of two factors involved in regulating the expression of HO-1, NrF-2 and transcription regulator protein Bach1. Bach1 is a basic leucine zipper transcription factor that forms heterodimers with proteins from the Maf family and repress the transcription of the HO-1 gene. CoPP enhances the degradation of Bach1 protein. NrF-2 is also a member of the family of basic leucine zipper transcription factors, which is involved in protection against oxidative stress through its antioxidant response element (ARE)-directed induction of HO-1. CoPP also induces the overexpression of NrF-2 by decreasing its degradation, and being a basic leucine zipper transcription factor, the NrF-2 dimerizes with the Maf family transcription factors and together these heterodimers bind to ARE of the HO-1 promotor region resulting in enhancement in expression (Shan et al., 2006). Liu et al. (2013) also reported that CoPP increases the expression of the transcription factor forkhead box protein O1 (FOXO1), which results in transcriptional upregulation of HO-1 gene (Liu et al., 2013).

Besides this mRNA transcriptional activation of HO-1, it was found that HO-1 is also subjected to a post-transcriptional

regulatory process which aims at reducing the expression of HO-1 to prevent that its overexpression becomes constitutive. This negative regulation of HO-1 expression is under the control of two microRNA (miR) molecules, miR-217 and miR-377, which work together to reduce the mRNA levels of HO-1 and hence reduce its protein expression. Knocking down these two microRNA molecules abrogated the attenuation of HO-1 expression (Beckman et al., 2011). Lin P.H. et al. (2008) showed that HO-1 expression is also regulated by a post-translational mechanism under the action of the ubiquitin/proteasome system. These findings were further supported by the increased HO-1 expression in the presence of proteasome inhibitor, MG-132, in a model of heme-induced oxidative damage (Chen and Regan, 2005).

#### Role of HO-1 in Vascular Inflammation

It has been established that HO-1 is able to suppress inflammatory responses by the concomitant generation of CO and bilirubin, two anti-inflammatory molecules, and the removal of pro-inflammatory agent, heme. CO and bilirubin are reported to blunt both innate and adaptive immune responses by modulating the activation and functions of multiple cell types involved in inflammation such as immune cells, endothelial cells and platelets, which ultimately results in reduced recruitment and tissue infiltration of immune cells (Durante, 2011). The antiinflammatory role of HO-1 was determined in HO-1 knockout mice. In these mice, HO-1 deficiency led to higher release of pro-inflammatory molecules (Kapturczak et al., 2004). In patients undergoing a surgery, higher levels of HO-1 were associated with lower levels of pro-inflammatory cytokine IL-6 (Taha et al., 2010). HO-1 was found to lower leukocyte rolling, adhesion, and transmigration to the sub-intimal space, by blunting the expression and function of surface adhesion molecules. In contrast, the inhibition of HO-1 caused the overexpression and over activation of adhesion molecules, activating thus leukocyte recruitment (Belcher et al., 2006). Multiple preclinical and clinical studies have been performed in many inflammatory vascular diseases to understand the impact of HO-1 overexpression on the pathogenesis and progression of the inflammatory process. Much evidence shows that the induction of HO-1 in the vessel wall inhibited the transmigration of monocytes to the sub-intimal space facilitated by oxidized low-density lipoproteins (LDL), leading to reduced atherosclerotic lesion buildup in mice lacking LDL receptor, a widely used model for atherosclerosis (Ishikawa et al., 2001). Circulating amounts of bilirubin in the general public were found to negatively correlate with the incidence of ischemic accidents (Mayer, 2000). This is not surprising knowing that bilirubin was found to decrease endothelial activation and dysfunction (Kawamura et al., 2005).

#### Role of HO-1 in Angiogenesis

HO-1 and its gas byproduct CO have been recognized for their strong pro-angiogenic roles. Several pro-angiogenic molecules, including VEGF, were found to mediate their effects via the induction of HO-1. However, depending on the situation, the pro-angiogenic properties of HO-1 could be considered as beneficial or deleterious. For instance, favoring angiogenesis is critical for many physiological responses such as wound healing and neovascularization following an ischemic episode. However, activation of angiogenesis driven by HO-1 may be deleterious in other conditions such neoplastic states (Dulak et al., 2008).

In investigating the role of HO-1 in promoting angiogenesis, Zhao et al. (2011) found that partial deficiency of maternal HO-1 caused defects in the feto-maternal interface, structural defects in placental vasculature and deficiency in spiral artery remodeling. These alterations were not related to the fetal genetic background and were only dependent on the maternal genetic status of HO-1. Moreover, restoring blood flow and the reestablishment of optimal oxygen concentrations to an injury site are also achieved by a proper angiogenic process. The wound-healing sequence of events consists of multiple steps that all require an intact angiogenesis process (Bauer et al., 2005). Growth factors (e.g., VEGF), chemokines, and hypoxia-inducible factors (HIF) all contribute to the orchestration of the multi-step wound healing procedure (Tepper et al., 2005). The deletion of HO-1 in mice led to an impaired wound healing process compared to wildtype littermates. This was partly because of an impaired EPC mobilization capacity linked to capillary formation at the site of injury (Deshane et al., 2007). Furthermore, it has been noted that the natural activation of HO-1 expression in wounded skin areas was weaker and slower in diabetic mice compared to controls. In these animal models of diabetes, the local adenoviral-mediated delivery of HO-1, improved the wound healing process (Grochot-Przeczek et al., 2009). Furthermore, HO-1 was found to confer a cytoprotective role in ischemic cardiac tissue through the activation of protein expression of pro-angiogenic factors in infarcted areas of cardiac tissue (Zeng et al., 2010). Lin H.H. et al. (2008) observed that the transfer of the HO-1 gene to infarcted heart tissue confers protection, at least partly, through the activation of VEGF-A expression and the subsequent activation of angiogenesis. In addition, the overexpression of HO-1 in mesenchymal stem cells implanted in infarcted hearts, markedly reduced apoptosis and fibrosis in cardiac tissue and improved cardiac function and remodeling (Jeon et al., 2007). Recently, Maamoun et al. (2017) have reported that the incubation of HUVECs in intermittent high glucose conditions for 5 consecutive days caused the activation of ER stress response and an impaired capacity of cells to form tubelike structures on a 3-dimensional matrigel matrix. This effect was associated with a decrease in the mRNA and protein expression of VEGF-A, indicating reduced angiogenesis capacity in these cells. Authors found that the pharmacological induction of HO-1 with CoPP reduced ER stress response and prevented the effects of high glucose-mediated ER stress on angiogenic capacity of cells, further stressing the pro-angiogenic effects of HO-1 in endothelial cells (Maamoun et al., 2017).

It is crucial to also highlight that despite the evidence in literature in support of the beneficial role of HO-1 in angiogenesis control, some reports identified a negative impact of HO-1 in tumoral neovascularization. Some human tumors, such as renal cell carcinoma and prostate cancer, exhibit elevated expression levels of HO-1 (Goodman et al., 1997). HO-1 could therefore contribute to tumor cell growth, survival and progression and ultimately participate in the resistance to drug therapy

(Nowis et al., 2006). In these conditions, the inhibition of HO-1 reduced tumor development in murine cancer models indicating a potential therapeutic role for HO-1 inhibition in certain cancer types (Dulak et al., 2008). However, there are also some reports that show HO-1 may play an anti-angiogenic role in certain cancer situations. In prostatic cancer cells (PC3), Ferrando et al. (2011) found that the overexpression of HO-1 caused the downregulation of several pro-inflammatory and angiogenic factors (NF-κB, VEGF-A, VEGF-C). To model the in vivo angiogenic process, the authors intradermally inoculated PC3 cells, that had been transfected with a stable HO-1 DNA construct, into immunodeficient mice and found that these cells produced tumors that were less richly vascularized than controls (Ferrando et al., 2011). However, in other conditions driven by excessive angiogenesis such as diabetic retinopathy (Wilkinson-Berka, 2004) and nephropathy (Osterby and Nyberg, 1987), HO-1 induction was still reported to be protective, indicating the complexity of the physiological actions of HO-1. For instance, Fan et al. (2012) observed in streptozotocin-injected diabetic rats pre-injected with hemin, a HO-1 inducer, that retinal ganglion cells were less prone to apoptosis compared to controls (not pre-treated with hemin), suggesting that HO-1 induction in rat retinas protected against diabetes-mediated neurodegeneration. Furthermore, the expression levels of VEGF, a strong proangiogenic factor which contributes to exaggerated angiogenesis, was also attenuated in retinas from streptozotocin-injected rats treated with hemin (Fan et al., 2012).

Taken together, data outlined here identify HO-1 as a key modulator of the angiogenic process in different pathophysiological conditions. Therefore, HO-1 induction could be a potential therapeutic target for the inhibition of angiogenesis in carcinogenic conditions, as opposed to being a therapeutic target for the induction of angiogenesis in diabetic cardiovascular complications. These studies also highlight the complexity of signaling pathways underpinning the action of HO-1 in angiogenesis.

#### Role of HO-1 in Oxidative Stress

The most well-known properties of HO-1 are its anti-oxidant functions (Hayashi et al., 1999). Of particular note, the work conducted by Ishikawa et al. (2012) demonstrated that mice lacking the gene of HO exhibited a weaker anti-oxidant capacity and an extensive lipid peroxidation. The anti-oxidant capacity of HO-1 is due to its byproducts biliverdin and CO. Bilirubin strongly scavenges several oxygen free radicals including singlet oxygen, O2 −, ONOO− and RO2 radicals. Moreover, unlike GSH, bilirubin, because of its lipophilic nature, is closely associated with cell membranes, and hence protects them against lipid damage by peroxidation. Bilirubin was found to work better as an O2 − scavenger and inhibitor of ONOO−-mediated protein nitration than biliverdin (Jansen et al., 2010). In addition to its ROS scavenging properties, some studies proposed that the protective actions of bilirubin are also partly related to its capacity to inhibit inducible NOS (iNOS), which eventually leads to less production of the highly reactive and potent ONOO− free radical (Wang et al., 2004). CO also mediates part of the global anti-oxidant actions of HO-1 (Srisook et al., 2006). CO was reported to block ONOO−-mediated protein nitration in endothelial cells through heightened intracellular GSH levels (Li et al., 2007). The anti-oxidant role of HO-1 was further demonstrated recently in HUVECs treated with intermittent high glucose. Authors reported that the induction of HO-1 using CoPP prevented high glucose-mediated concomitant increase in superoxide production and reduction in NO release, indicating an improved NO bioavailability. These focal effects were associated with a decrease in ER stress response activation, reduced phosphorylation of NOX regulatory subunit p47phox at Ser345, and an enhanced phosphorylation of eNOS at its activatory site (Ser1177) (Maamoun et al., 2017).

There is accumulating evidence that oxidative stress contributes to the early stages as well as the maintenance and progression of diabetic cardiovascular complications. Therefore, HO-1, owing to its strong anti-oxidant properties, may be utilized as a viable and promising therapeutic target in these conditions. The protective actions of HO-1 induction have been demonstrated in animal models of diabetic cardiomyopathy and vasculopathy (Li et al., 2012). Moreover, in vitro studies have demonstrated the highly potent anti-oxidant effect of bilirubin with increased protection of vascular endothelial cells, where even concentrations of bilirubin in the nanomolar range protected cells against oxidative stress damage both through direct scavenging of ROS radicals and indirectly by blocking the activity of NOX complex (Abraham and Kappas, 2008).

### Role of HO-1 in Apoptosis

A high percentage of apoptotic endothelial cells was observed in animal models of diabetes-mediated vascular injury (Negi et al., 2011). Clinical data also suggest the involvement of apoptosis in diabetic vascular diseases where the expression of pro-apoptotic molecules was high while in contrast the expression of anti-apoptotic ones was low (Arya et al., 2011). HO-1 mediates its anti-apoptotic action through multiple molecular mechanisms including the inhibition of oxidative stress, ER stress and inflammation. NF-κB was shown to control cellular apoptosis in addition to its well-known role in inflammation. HO-1 induction by CoPP was found to reduce the expression of IL-6, a gene regulated by NFκB, and CHOP in response to stressful stimuli such as ischemia-reperfusion injury, with the consequent inhibition of apoptosis (Ben-Ari et al., 2013). Other mechanisms suggested for the anti-apoptotic effects of HO-1 include blunting the apoptotic signaling pathway mediated by the interaction of TNF-α with its receptor (Jaeschke and Woolbright, 2013). These anti-apoptotic actions of HO-1 may be driven by the release of CO, which was shown to protect against apoptosis in many cell types, including endothelial cells, fibroblasts, hepatocytes and pancreatic β-cells. Brouard et al. (2000) were able to show that HO-1 inhibits apoptosis, namely by generating CO. The inhibition of HO-1 activity by tin protoporphyrin (SnPPIX) failed to prevent endothelial cell apoptosis. The direct effect of CO on apoptosis was confirmed by showing that exposing endothelial cells to exogenous CO, while inhibiting HO-1 activity by SnPPIX, prevented apoptosis. Moreover, investigators have also explored

some of the signaling pathways behind the action of HO-1/CO, and showed that it is partially mediated by the activation of p38 MAPK signaling transduction pathway. This was further supported by the observation that the selective blockade of p38 MAPK by the over-expression of a dominant negative mutant resulted in the abolishment of the anti-apoptotic effect of HO-1 (Brouard et al., 2000). In addition, as previously discussed, in diabetic vascular disease, oxidative stress occurs in endothelial cells which subsequently leads to apoptosis. Hence, HO-1 by merely exerting its anti-oxidant effects is reported to protect these cells from apoptosis (Schmeichel et al., 2003). Other studies focused on the role of bilirubin in this pro-survival behavior of HO-1. It has been shown that bilirubin was found to mediate its anti-apoptotic role by upregulating extracellularsignal-regulated kinase (ERK) MAPK and PI-3K/Akt/eNOS pathways (Hung et al., 2010).

More recently, pharmacological induction of HO-1 using CoPP was reported to protect endothelial cells from high glucosemediated apoptosis through the alleviation of ER stress response. Maamoun et al. (2017) observed that the exposure of HUVECs to intermittent high glucose for a duration of 5 days led to cell death, while induction of HO-1 prevented this deleterious manifestation. The cytoprotective effects of HO-1 were associated with a reduction in the activation of PARP-1 and caspases 3 and 7. Furthermore, HO-1 induction in HUVECs prevented the activation of NF-κB and JNK pathways caused by high glucose treatment, indicating reduced cellular inflammation in the presence of HO-1 (Maamoun et al., 2017). While the amount of scientific evidence is still relatively small, some studies suggest HO-1 activation as a novel and promising strategy to improve survival of endothelial cells through HO-1 byproducts, bilirubin and CO, which may delay the development of various diabetic cardiovascular complications.

### CONCLUSION

Crosstalk between oxidative stress and ER stress significantly contributes to the onset and development of endothelial

#### REFERENCES

dysfunction and altered angiogenesis and therefore represent major therapeutic targets for alleviating micro- and macrovascular complications associated with this metabolic disturbance. HO-1 carries out, anti-oxidant, anti-inflammatory, anti-apoptotic and angiogenic actions, through its byproducts CO and bilirubin, and can thus affect therefore multiple cellular pathways involved in endothelial dysfunction, including oxidative stress and ER stress response. As a multitarget molecule affecting major aspects of cardiovascular dysfunction, HO-1 induction represents a potential therapeutic approach for cardiovascular complications associated with diabetes.

#### AUTHOR CONTRIBUTIONS

HM and AA wrote the manuscript and constructed the figures. TB, GP, and SM contributed to selected sections and critically revised the manuscript. All authors approved the final version for submission.

### FUNDING

This publication was made possible by a NPRP award (NPRP8-1750-3-360) from the Qatar National Research Fund (a member of Qatar Foundation) and a Qatar University high collaborative grant (QUCG-CPH-2018\2019-2) to AA. The publication of this article was funded by Qatar National Library.

#### ACKNOWLEDGMENTS

The figures in this article were partly produced by adapting freely available tools from Servier Medical Art, that are licensed under a Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licenses/by/3.0/. This manuscript was based on the doctoral thesis of HM.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Maamoun, Benameur, Pintus, Munusamy and Agouni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Plasma Heme Scavengers Alpha-1-Microglobulin and Hemopexin as Biomarkers in High-Risk Pregnancies

Grigorios Kalapotharakos1,2 \*, Katja Murtoniemi3,4, Bo Åkerström5 , Esa Hämäläinen6,7 , Eero Kajantie8,9,10, Katri Räikkönen11, Pia Villa12, Hannele Laivuori3,13,14,15 and Stefan R. Hansson1,2

1 Department of Clinical Sciences Lund, Skåne University Hospital, Lund, Sweden, 2 Department of Obstetrics and Gynecology, Lund University, Lund, Sweden, 3 Medical and Clinical Genetics, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 4 Department of Obstetrics and Gynecology, Turku University Hospital and University of Turku, Turku, Finland, 5 Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden, 6 HUSLAB, Helsinki University Hospital, Helsinki, Finland, 7 Department of Clinical Chemistry, University of Helsinki, Helsinki, Finland, 8 National Institute for Health and Welfare, Helsinki, Finland, 9 Children's Hospital, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 10 Research Unit for Pediatrics, Dermatology, Clinical Genetics, Obstetrics and Gynecology, Medical Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland, 11 Department of Psychology and Logopedics, Faculty of Medicine, University of Helsinki, Helsinki, Finland, 12 Department of Obstetrics and Gynecology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 13 Institute for Molecular Medicine Finland, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland, 14 Department of Obstetrics and Gynecology, Tampere University Hospital, Tampere, Finland, 15 Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland

#### Edited by:

Ana Claudia Zenclussen, Universitätsklinikum Magdeburg, Germany

#### Reviewed by:

Marcos Lopez, The University of Chicago, United States Deepesh Pandey, Johns Hopkins University, United States

#### \*Correspondence:

Grigorios Kalapotharakos<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Oxidant Physiology, a section of the journal Frontiers in Physiology

Received: 03 August 2018 Accepted: 06 March 2019 Published: 04 April 2019

#### Citation:

Kalapotharakos G, Murtoniemi K, Åkerström B, Hämäläinen E, Kajantie E, Räikkönen K, Villa P, Laivuori H and Hansson SR (2019) Plasma Heme Scavengers Alpha-1-Microglobulin and Hemopexin as Biomarkers in High-Risk Pregnancies. Front. Physiol. 10:300. doi: 10.3389/fphys.2019.00300 Women with established preeclampsia (PE) have increased plasma concentration of free fetal hemoglobin. We measured two hemoglobin scavenger system proteins, hemopexin (Hpx) and alpha-1-microglobulin (A1M) in maternal plasma using enzymelinked immunosorbent assay during the late second trimester of pregnancy in women with high and low risk of developing PE. In total 142 women were included in nested case-control study: 42 women diagnosed with PE and 100 controls (49 randomly selected high-risk and 51 low-risk controls). The concentration of plasma A1M in high-risk controls was higher compared to low-risk controls. Women with severe PE had higher plasma A1M levels compared to women with non-severe PE. In conclusion, the concentration of plasma A1M is increased in the late second trimester in high-risk controls, suggesting activation of endogenous protective system against oxidative stress.

Keywords: preeclampsia, heme, hemopexin, alpha-1 microglobulin, aspirin

### INTRODUCTION

Preeclampsia (PE) is a relatively common hypertensive disorder in pregnancy, affecting 4,6% of pregnancies worldwide (Abalos et al., 2013). The specific etiology of PE is, however, still not completely described. According to the most well-accepted model, PE is a two-stage disorder (Redman, 1991; Redman et al., 2014). The abnormal modification of the spiral arteries during

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placental development is thought to be the initial stage leading to reduced utero-placental perfusion and increased oxidative stress that in turn causes placental damage. Circulating toxic factors derived from the placenta cross the blood-placenta barrier and leak into the maternal circulation where they in turn trigger an inflammatory response and general endothelial damage. As a consequence of that, general organ damage develops, which leads to the typical manifestations of PE after 20th week of gestation, including hypertension, edema and proteinuria. Circulating syncytiotrophoblast microvesicles (Smarason et al., 1996), free fetal DNA (Zhong et al., 2002), cytokines (Hamai et al., 1997) and antiangiogenic factors (Powe et al., 2011) have been postulated as endothelial toxic factors derived from the fetus and the placenta. However, we still lack a full explanation on how placental damage leads to distinct maternal and fetal manifestations that occur either during early pregnancy or late pregnancy, so called early onset PE and late-onset PE. Early onset PE is linked to poor placentation while the late-onset is more determined by maternal risk factors such as obesity, diabetes mellitus and chronic hypertension, which are associated with a higher pre-pregnancy level of vascular inflammation (Roberts and Redman, 1993; Ness and Roberts, 1996).

Extracellular fetal hemoglobin (HbF) has been introduced in a series of earlier studies and suggested to have a crucial role in the etiology of PE (Hansson et al., 2013). Increased synthesis of HbF in the placenta was indicated by an up-regulation of the HbF genes and there was an accumulation of extracellular HbF in the vascular lumen of PE placenta (Centlow et al., 2008). Extracellular HbF induces oxidative stress by formation of reactive oxygen species resulting in damage to the blood-placenta barrier and leakage of extracellular HbF into the maternal circulation (May et al., 2011). As a consequence, plasma concentration of extracellular HbF has been shown to be increased in maternal plasma as early as the first trimester in women who later develop PE (Anderson et al., 2011). Increased plasma levels in the late third trimester has been shown to correlate to the maternal blood pressure (Anderson et al., 2018).

There are several defense mechanisms which protect against the harmful effects of extracellular hemoglobin (Hb). Haptoglobin (Hp) is the most important protective scavenger protein that binds extracellular Hb in plasma resulting in a complex that in turn is cleared via CD163 receptors on macrophages (Kristiansen et al., 2001; Schaer et al., 2006). Hemopexin (Hpx) has a complementary role to bind extracellular heme that is released as a metabolite when Hb is degraded by the rate-limiting enzyme heme-oxygenase (HO-1) (Nielsen et al., 2010; Tolosano et al., 2010). The resulting complex is cleared from the circulation by liver parenchymal cells via receptor-mediated endocytosis involving CD91/LRP1. Alpha-1-microglobulin (A1M) is another component of the heme scavenger system (Akerstrom and Gram, 2014). It is a lipocalin with heme-binding properties as well as being an antioxidant due to radical-scavenging and reductase properties. In a series of studies (Gram et al., 2015; Anderson et al., 2018), it has been shown that the plasma levels of Hp and Hpx are reduced, suggesting that in cases where the maternal endogenous protection system against extracellular HbF is overwhelmed, PE becomes clinically manifest. Cellular A1M expression of A1M is upregulated by increased oxidative stress and Hb/heme exposure (Olsson et al., 2007) and previous investigations have shown increased circulating plasma levels of A1M in women pregnant with PE (Olsson et al., 2010; Anderson et al., 2011) consistent with high circulating levels of Hb, heme and oxidants in this disease.

In the present study, we analyzed the plasma levels of A1M and Hpx in order to further understand the dynamics of these components of the Hb/heme scavenger system in the second trimester in women with high and low risk of developing PE. The cohort of patients were stratified according to known maternal risk factors, intervention with acetylsalicylic acid (ASA) as well as neonatal outcome.

### MATERIALS AND METHODS

#### Study Population

The present nested case-control study is a part of the multidisciplinary "Prediction and Prevention of Pre-eclampsia and Intrauterine Growth Restriction" (PREDO) project. Women with known risk factors for PE were prospectively recruited between September 2005 and June 2009 at ten participating maternity clinics in Finland. The ethics Committee at the Helsinki and Uusimaa Hospital District approved the study and written informed consent was obtained from all participants.

In total 142 women were included in this study: 42 women diagnosed with PE and 100 controls (49 randomly selected highrisk and 51 low-risk controls). Seven women with PE participated in the ASA trial (part of the PREDO project), as well, and were treated with low dose acetylsalicylic acid (mini-ASA 100mg/d) starting before 14th week of gestation. Three women who were taking mini-ASA and did not develop PE, were included as controls for this sub-group. The inclusion and exclusion criteria are described in **Supplementary Table S1**.

Preeclampsia was defined as a systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure ≥90 mmHg occurring after 20th weeks of gestation combined with a urinary 24-h protein excretion of ≥0.3 g or the dipstick equivalent in two consecutive measurements. Severe PE was defined as systolic blood pressure ≥160 mmHg and/or diastolic blood pressure ≥110 mmHg and/or proteinuria ≥5 g/24 h. Small for gestational age (SGA) was defined as a birthweight ≤minus 2 SDs.

All participants had their first visit at 12+0–14+0 weeks of gestation. Uterine artery blood flow was measured with Doppler ultrasound examination. Gestational age was confirmed by crown-rump length measurement. The first trimester mean arterial pressure (MAP) was calculated with the equation: MAP = diastolic blood pressure + (systolic blood pressure – diastolic blood pressure)/3.

Fasting blood samples were collected in all three trimesters. Plasma was separated within an hour by centrifugation and stored in −80 ◦ C until analysis. In the present study we determined serum A1M and Hpx concentrations from samples drawn at 26+0 to 28+0 weeks of gestation.

#### Hemopexin ELISA

fphys-10-00300 April 2, 2019 Time: 17:30 # 3

The Hpx concentrations were measured with a Human Hemopexin ELISA Kit from Genway Biotech Inc. The analysis was performed according to manufacturer's instructions and the absorbance was read at 450 nm using a Wallac 1420 Multilabel Counter.

#### A1M ELISA

The A1M concentrations were measured with an in-house A1M ELISA. Flat-bottom ninety six-well microtiter plates were coated with mouse monoclonal anti-A1M antibodies (clone 35.14) by incubation overnight at +4 ◦C under sealing film with 100 µl/well of a 5 µg/ml-solution in PBS. After washing three times with PBS + 0.05% tween-20, 100 µl of human urinary A1M reference standard samples (1.56 – 100 ng/ml in PBS + 0.05% tween-20) or unknown plasma samples (diluted 1000× with PBS + 0.05% tween-20) were added to the wells and incubated under sealing film for 1 h at room temperature, darkness and rotational shaking 250–500 rpm. After washing three times with PBS + 0.05% tween-20, 100 µl/well of the detection antibody solution was added (horse radish peroxidase-coupled mouse monoclonal anti-A1M antibody clone 57.10; 5 ng/ml in PBS + 0.05% tween-20) and incubated under sealing film for 1 h at room temperature, darkness and rotational shaking 250–300 rpm. After washing three times with PBS + 0.05% tween-20, 100 µl/well of TMB substrate (SureBlueTM TMB Microwell Peroxidase Substrate, KPL cat. no. 50-00-04) was added, sealed, and incubated 20 min without shaking, and the reaction was stopped by adding 100 µl/well of 1 M sulfuric acid. Absorbance at 450 nm was read in a Wallac 1420 Multilabel Counter (Perkin Elmer Life Sciences). The monoclonal anti-A1M antibodies were prepared against human urinary A1M by Agrisera AB (Vännäs, Sweden). Human urinary A1M was prepared in our lab as described (Akerstrom et al., 1995).

#### Statistical Analysis

Statistical analyses were performed using SPSS version 25.0 statistic software package. Normally distributed data were analyzed using one-way ANOVA followed by Tukey's post hoc tests. Kruskal-Wallis and Mann-Whitney test were used in case the data were not normally distributed and Bonferroni corrections were used in post hoc comparisons. Statistical significance was defined as p < 0.05.

#### RESULTS

Patient demographics and clinical characteristics are shown in **Table 1**, **2**. Women affected by PE had higher body mass index (29,3 kg/m2 ) compared to controls (23,7 kg/m2 ). There were less primiparas among women affected by PE compared to controls. There were less women with a previous pregnancy complicated by PE among controls compared to women affected by PE.

The distributions of plasma Hpx and A1M across the groups are shown in **Figures 1A,B**. Pairwise comparisons using significance values adjusted by the Bonferroni correction for multiple tests revealed that the concentration of Hpx in women TABLE 1 | Demographics of patients and controls.

abinary logistic regression, bmean, cmedian, d fisher's exact test; PE = preeclampsia, OR = odds ratio, CI = confidence interval, SD = standard deviation, BMI = body mass index. There was one type I diabetes mellitus and one Sjögren's syndrome in women who did not develop preeclampsia. There was not systemic lupus erythematosus in either group.

with PE was higher compared to low-risk normotensive women (median concentration 1.21 mg/ml versus 1.04 mg/ml, p = 0.014). There was no significant difference in Hpx concentration between women with PE and high-risk normotensive women (median concentration 1.11 mg/ml) or between high-risk and low-risk normotensive women. The concentration of A1M in high-risk controls was higher compared to low-risk controls (median concentration 16.08 µg/ml vs. 13.09 µg/ml, p = 0.002). There was no significant difference in A1M concentration between women with PE and high-risk controls or between women with PE and low-risk controls.

The concentrations of plasma Hpx and A1M in women with PE are shown in **Table 3**. We found no difference in Hpx or A1M concentrations between women with PE who gave birth to SGA infant and women with PE who gave birth to non-SGA infant. The concentration of A1M in women with severe PE was higher compared to women with non-severe PE. The distributions of Hpx and A1M across the subgroups of PE, high- and low-risk controls are shown in **Figures 1C,D**.

Continuous variables are presented as median values with interquartile range in parenthesis. n=number of cases, PE=preeclampsia.

Seven PE women received mini-ASA during the pregnancy and one of them gave birth to SGA infant. Thirty-five PE women did not receive mini-ASA and eight of them gave birth to SGA infant. Women who received ASA had higher Hpx concentration compared to women who did not receive ASA (median concentration 1,28 mg/ml versus 1,09 mg/ml, p = 0,025).

#### DISCUSSION

To the best of our knowledge, this is the first study evaluating the levels of maternal plasma Hpx and A1M in the late second trimester in PE women as well as in high- and low-risk controls without PE. The strength of the study is a carefully characterized cohort, where both women with predetermined risk factors for PE and a low-risk reference group were prospectively recruited (Girchenko et al., 2017).

Our analysis shows a significantly higher plasma A1M concentration in high-risk controls compared to lowrisk controls, while there was no significant difference in concentration of A1M between PE women and controls. In previous studies (Olsson et al., 2010; Anderson et al., 2011, 2016; Gram et al., 2015), the concentration of A1M in PE women was increased compared to controls when it was analyzed during the first trimester and third trimester, 24 h prior to delivery. In the aforementioned studies, normotensive pregnant women had been studied as a single undivided group, irrespective of risk factors for PE. However, it is reasonable to hypothesize that high-risk women are a distinct group characterized by increased oxidative stress compared to low-risk women.

In the present cohort, pre-pregnancy obesity was among the criteria conferring high-risk status to normotensive pregnant women. Obesity is associated with systemic oxidative stress (Furukawa et al., 2004). It can therefore be assumed that in high-risk controls, the endogenous protection system against oxidative stress is activated. The housekeeping protein A1M is an extravascular scavenger and tissue repair protein which has an important role in cleaning oxidative radicals and heme. It is upregulated during oxidative stress in general and by hemolysis specifically (Akerstrom and Gram, 2014). Thus, our results may suggest that increased oxidative stress is present in high-risk normotensive pregnant women compared to low-risk controls in late second trimester. A serious weakness with this argument, however, is that we have not included evaluation of oxidative stress markers. Evaluation of oxidative stress markers and reactive oxidative species measurements need to be undertaken before the association between A1M and oxidative stress in high-risk normotensive women is more clearly understood. Although A1M has not been shown to be an acute-phase protein, increased levels of A1M in high-risk normotensive women as a result of chronic inflammation related to obesity might be another explanation and needs to be clarified in further studies (Akerstrom and Gram, 2014).

Interestingly, the median plasma A1M concentration in high-risk normotensive women was increased compared to PE women, although the difference was not statistically significant. Higher concentration of A1M may confer its protective effect ameliorating the clinical impact of oxidative stress and preventing the development of PE in high-risk controls who remain asymptomatic. In line with this assumption, intravenous administration of a recombinant version of A1M in PE animal models has been successful in eliminating or at least significantly reducing the manifestations of preeclampsia (Sverrisson et al., 2014; Nääv et al., 2015; Gunnarsson et al., 2017). One can therefore speculate that the administration of exogenous A1M might have a similar effect in humans and PE women also.

Recent evidence suggests that extracellular HbF is elevated in the fetal circulation of pregnancies complicated by fetal growth restriction (Brook et al., 2018). We expect that among SGA infants in our cohort there are both constitutionally small infants and infants that have not reached their growth potential because of placental dysfunction. In the latter case, we expect that extracellular HbF in the fetal circulation is elevated causing, as we have previously shown (Centlow et al., 2008; May et al., 2011), damage to blood-placenta barrier and consequently leaking into maternal circulation, depleting Hpx. We hypothesized thus that women with PE and SGA infant would have lower levels of Hpx compared to women with PE and non-SGA infant, because Hpx binds and detoxifies heme and HbF that is released from the feto-placental unit. Although the levels of plasma Hpx was lower in women with PE who gave birth to SGA infant compared to women with PE and non-SGA infant, the difference was not significant. Type II error is possible due to a small sample size. One woman in the group of women with PE who gave birth to SGA infant had exceptionally high plasma Hpx and A1M values and this increased variability substantially. This woman differed from the other in that group by having a medical history of

Continuous variables are presented as median values with interquartile range in parenthesis. n=number of cases.

chronic hypertension and thus superimposed PE and she was the only woman who received mini-ASA in the group of women with PE who gave birth to SGA infant.

Recent studies (Ferrazzi et al., 2018; Tay et al., 2018) suggest that women with PE and SGA or growth restricted fetus have a distinct cardiovascular phenotype characterized by lower cardiac output and higher peripheral vascular resistance. It is tempting to speculate that there is a connection between the impaired cardiovascular function and the decreased levels of heme scavenger Hpx. A possible explanation may be leakage of extracellular HbF of fetoplacental origin in maternal circulation. As a consequence, heme is released from metabolized extracellular HbF and depleting Hpx as recently shown (Anderson et al., 2018). Imbalance in the scavenging capacity causes vasoconstriction by reducing nitric oxide availability and impaired cardiac function. In fact, heme has been shown to induce contractile dysfunction in human cardiomyocytes in vitro (Alvarado et al., 2015). Furthermore, Hpx has been shown to have cardio-protective effect and it preserves systolic function by limiting heme-driven oxidative stress in mice (Ingoglia et al., 2017). Further studies, where cardiac function is evaluated simultaneously with the concentration of heme in maternal circulation, are needed to further clarify whether this suggested mechanism has clinical relevance.

In contrast to previous studies, we found increased plasma concentration of Hpx in PE women compared to low-risk controls. After excluding controls and PE women treated with mini-ASA, there was no statistically significant difference in the concentrations of Hpx between PE women and controls. In previous studies, where women treated with mini-ASA were not included, the concentration of Hpx was decreased, albeit marginally, in early pregnancy in women who later developed PE (Anderson et al., 2016). The concentration of Hpx in PE women has been shown to be even more decreased compared to controls just before delivery (Anderson et al., 2018). Thus, when focusing on women not treated with mini-ASA, our results suggest that Hpx depletion, due to heme-Hpx binding, is a continuous process, which starts slowly in early pregnancy and intensifies in third trimester when PE is manifested clinically. Further studies with a significantly larger patient group are needed if we try to detect the very marginal differences of the early stages of this process in order to clarify the dynamics of this process.

As regards to mini-ASA prophylaxis, 3 high-risk controls and 7 PE women who had participated in ASA trial were included in this study. Overall, these 10 women had significantly higher plasma Hpx concentration compared to controls and PE women who did not receive mini-ASA prophylaxis. Mini-ASA could possibly have an impact on Hpx concentration by improving placental perfusion. We have previously hypothesized that the up-regulation of HbF gene expression in the preeclamptic placenta may be induced by hypoxia caused by insufficient placental perfusion (Gram et al., 2015). Mini-ASA may improve the placental perfusion through inhibition of the potent vasoconstrictor Thromboxane A2 (TXA2) or by preventing placental thrombosis formation improving the blood flow in the PE placenta. Improved placental perfusion could then possibly prevent up-regulation of the HbF gene expression thereby reducing the leakage of HbF and heme into the maternal circulation. As previously shown, small amount of heme administered intravenously in rhesus monkeys increases the Hpx levels by increasing the rate of Hpx synthesis (Foidart et al., 1982). In contrast, large amount of heme has been shown to reduces Hpx levels by increased Hpx catabolism. Further studies are needed to understand the role of mini-ASA on Hpx dynamics.

#### CONCLUSION

This study shows increased concentration of plasma A1M in high-risk normotensive pregnant women in late second trimester.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

SH, BÅ, and HL conceived and designed the analysis. GK and KM performed the analysis and wrote the manuscript. KM, PV, KR, EH, EK, and HL collected the data and contributed the data.

#### FUNDING

This project has been partly supported by Erasmus + Program of the European Union (Framework agreement number: 2013-0040). The PREDO project has been supported by EVO research funding (A special Finnish state subsidy for health science research), Academy of Finland, Signe and Ane Gyllenberg Foundation, Sigrid Juselius Foundation, University of Helsinki Research Funds, Finnish Medical Foundation, Juho Vainio Foundation, Novo Nordisk Foundation, Jane and Aatos Erkko Foundation, and Päivikki and Sakari Sohlberg Foundation.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2019.00300/full#supplementary-material

**Conflict of Interest Statement:** SH and BÅ holds patent related to diagnosis and treatment of preeclampsia and are co-founders of A1M Pharma and Preelumina Diagnostics (www.a1m.se). The pre-existing intellectual properties involve 4 patents owned by A1M Pharma;

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Kalapotharakos, Murtoniemi, Åkerström, Hämäläinen, Kajantie, Räikkönen, Villa, Laivuori and Hansson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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**2. ctDNA methodological approaches and technical considerations**

Circulating cell-free DNA (cfDNA) was first detected in 1948 by Mendel and Metais in the peripheral blood plasma of healthy and diseased individuals [8]. CfDNA levels can vary between 1 and 10 ng mL−1 in plasma and can be affected by physiological conditions such as exercise and acute inflammation [9]. In 1977, Leon et al. found that cfDNA was more elevated in cancer patients compared with healthy subjects, with higher levels correlating with higher burden of disease [10]. In 1989, Stroun et al. discovered that at least part of the plasma DNA in cancer patients originated specifically from cancer cells [11]. In the ensuing decades, knowledge and applications of tumour-derived cfDNA has rapidly evolved due to advances in molecular techniques, and also gave rise to the term, circulating tumour DNA (ctDNA).

**57**

*yield and purity [5].*

**Figure 2.**

*Current Utility and Future Applications of ctDNA in Colorectal Cancer*

clinical relevance, it is prudent to reflect on these biological variables.

Numerous inherent challenges have affected the development of ctDNA preanalytic and analytic methods. These include variable fragmentation, low abundance in plasma or serum volumes, tumour heterogeneity, and low stability as a

To minimise sample degradation and optimise stability, a number of preanalytical steps need to be carefully planned. Although there are currently no standardised methodology guidelines on ctDNA collection, storage and processing,

A variety of methods for detecting and characterising ctDNA have been reported. These can be broadly categorised as targeted and non-targeted

approaches. Differing performance characteristics, strengths and disadvantages may also facilitate complementary roles of these approaches in molecular analysis. **Table 1** lists examples of described methods. Applying any of these approaches in

*Pre-analytical components in ctDNA analysis. (1) Collection of blood samples (usually 5–10 mL) via phlebotomy. Currently, there is no guidance on the comparative impact of the source of blood draw (for example, peripheral venepuncture or intravascular ports) on ctDNA analysis [5]; (2) samples should be collected in tubes containing anticoagulants compatible with polymerase chain reaction methods, such as ethylenediaminetetraacetic acid (EDTA) [9]; (3) centrifugation of blood to separate cells should be performed promptly. The exact optimal time to centrifugation is not known and may depend on storage conditions and the presence of stabilising agents [16]. Current evidence suggests that plasma is preferred to serum samples, as in the latter case, cfDNA released during white blood cell lysis may lead to a dilutional effect [9]. (4) Processed plasma is then generally stored frozen, often in aliquots; (5) CfDNA is extracted using commercially available kits. There are multiple DNA purification strategies and modifications, which may variably impact on DNA*

A variety of tumour-specific molecular alterations may be identified by ctDNA including mutations, methylation variants, microsatellite alterations, copy number variations and structural changes [12]. Although the exact mechanisms are yet to be elucidated, ctDNA is thought to be released into the blood stream via biological processes such as apoptosis, necrosis, inefficient phagocytosis and active secretion [13, 14]. CtDNA has a short half-life of up to a few hours and accounts for generally only a small fraction of cfDNA, although concentration can vary widely from <0.01 to 90% [12]. The biological and tumoural determinants underlying ctDNA variations both between and within individuals are incompletely understood, but are likely affected by tumour burden, treatment response, circulatory elements, circadian rhythm, cellular turnover and clearance mechanisms [12, 15]. Somatic variants may also be found in healthy individuals, mostly commonly associated with clonal haematopoiesis [5]. Such variability, coupled with the often-low allele frequency of the molecular aberration of interest, demand sensitive and robust detection methods. As we interpret the results of ctDNA studies and consider their

*DOI: http://dx.doi.org/10.5772/intechopen.82316*

**2.1 Pre-analytic considerations**

**2.2 Detection methods**

result of the aforementioned biological factors [16].

the typical workflow is illustrated in **Figure 2**.


**5.1. Dietary fiber**

Dietary fiber can be classified in different ways: soluble versus insoluble based on water solubility; fermentable versus non-fermentable based on whether or not it can be fermented by the microbiota in the large intestine; and viscous versus non-viscous related to its viscosity [90]. Fruit, vegetables, and cereals are the major sources of dietary fiber. The analysis of 67 clinical trials on diets high in soluble fibers suggested that these fibers lower total cholesterol and LDL-C [91]. Water insoluble fibers remain unchanged during digestion and have no effect unless they displace foods supplying saturated fats and cholesterol [92]. Most of the available epidemiologic studies suggest that dietary fiber is inversely related to coronary artery disease [93]. Diet rich in dietary fiber is beneficial for the treatment of T2DM [94], as dietary fiber ameliorates postprandial hyperglycemia by delaying digestion and absorption of carbohydrates [95]. A recent systematic review of the literature reported that moderate amounts of fiber supplements (4–19 g/day) achieved little improvement in glycemic control or CVD risk factors [96]. It has been reported that increased intake of dietary fiber and low GI diet with legumes reduced blood pressure compared with wheat fiber diet in T2DM patients [95]. A cross-sectional study in adults men and women indicated that the highest total dietary fiber and insoluble dietary fiber intakes were associated with a significantly lower risk of overweight, high blood pressure, plasma apolipoprotein (apo) B, apo B, apo A–I, cholesterol, triacylglycerols, and homocysteine [97]. The fiber intake should, ideally, be 40 g/day (or 20 g/1000 kcal/day) or more and about half should be of the water-soluble type. People with T2DM are encouraged to choose ≥5 servings of fiber-rich vegetables or fruit and ≥4 servings of legumes per week to achieve the fiber intake goals set for the general population [98].

myocardial infarction, and breast cancer (EURAMIC) study found that the risk of MI was 60% lower for the highest quintile of adipose lycopene concentration compared to the lowest quintile, after adjustment for age, family history of CVD and cigarette smoking [109]. In a crosssectional study comparing Lithuanian and Swedish populations showing diverging mortality rates from CVD, lower blood lycopene levels were found to be associated with increased risk and mortality from CVD [110]. Many studies show that high consumption of tomato products can improve resistance to oxidation in people with T2DM [111]. Eating a lycopene-rich Mediterranean diet increases lycopene levels and can reduce the levels of hemoglobin A1c from 7.1 to 6.8% [112]. In a case-control study on serum β-carotene and the risk of T2DM, participants in the highest tertile of serum β-carotene levels had a 55% lower risk of developing T2DM [113]. In a quasi-experimental study, 32 T2DM patients received 200 g raw tomato daily for 8 weeks. There were significant decreases in systolic and diastolic blood pressure and also a significant increase in apoA-I compared with initial values, which suggests the beneficial role of tomato consumption in reducing cardiovascular risk associated with T2DM [114, 115].

Dietary Recommendations for Patients with Cardiovascular Disease and Diabetes

http://dx.doi.org/10.5772/intechopen.71391

41

N−3 fatty acids including α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) have a significant role in the prevention of CVD [116]. The evidence supports a dietary recommendation of ≈500 mg/day of eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) for CVD risk reduction [117]. A meta-analysis suggests that ALA consumption may also confer cardiovascular benefits, and each 1 g/d increment in ALA intake was associated with a 10% lower risk of CVD death [118]. Dietary sources of ALA include flaxseeds and flaxseed oil, walnuts and walnut oil, soybeans and soybean oil, pumpkin seeds, rapeseed oil, and olive oil [119]. In the GISSI Prevention Study, treatment with n-3 PUFA significantly lowered the risk of the primary endpoint (death, non-fatal MI, and stroke) [120]. Several mechanisms explaining the cardioprotective effect of the n-3 PUFA have been

Several groups are now beginning to use animal models of myocardial ischemia and reperfusion to explore whether certain nutrients, including ethanol and non-ethanolic components of wine, may have a specific protective effect on the myocardium, independently from the classical risk factors for coronary disease involved in vascular atherosclerosis and thrombosis [121]. Most epidemiological studies have suggested an inverse association between regular light to moderate drinking and the risks of CVD [122]. Researchers have wondered whether moderate alcohol consumption mediates some of its cardioprotective effects by stimulating NO, and conversely, whether binge drinking diminishes NO availability [123]. In a swine model of chronic ischemia, alcohol administration promoted angiogenesis, increased capillary and arteriolar density in non-ischemic myocardium [122]. Numerous studies indicate that moderate red wine consumption is associated with a protective effect on the cardiovascular system, which has largely been attributed to the rich content of phenolic compounds [124, 125]. Polyphenolic antioxidants scavenge the free radicals, inhibit lipid peroxidation (lipoproteins,

suggested including antiarrhythmic and antithrombotic roles [119].

**5.5. Ethanol and non-ethanolic components of wine**

**5.4. Fatty acids**

## **5.2. Polyphenols**

A number of antioxidants showed beneficial effect in experimental models of atherosclerosis and CVD [99, 100]. The main polyphenol dietary sources are fruit and beverages (fruit juice, wine, tea, coffee, chocolate, and beer), dry legumes, and cereals [101]. Dietary polyphenols have been shown to possess cardioprotective effects. Oleuropein inhibits the oxidation of LDL-C in vitro [102]. Dietary quercetin decreases lipid peroxidation and upregulates the expression of serum HDL-associated paraoxonase-1 (PON-1) in the liver [101]. PON-1 may mediate anti-atherogenic properties by protecting LDL-C from oxidation. Several studies have indicated that red wine polyphenolic compounds (RWPCs) were able to inhibit proliferation and migration of vascular cells. RWPCs induced NO-mediated endothelium-dependent relaxations in isolated arteries. The activation of eNOS led to an increase in [Ca2+]i and phosphorylation of eNOS by the PI3-kinase/Akt pathway [103]. RWPCs also increased endothelial prostacyclin release and inhibited the synthesis and the effects of endothelin-1 in endothelial cells [101].

#### **5.3. Lycopene**

Lycopene is a natural carotenoid found in tomatoes, which has biochemical functions as an antioxidant scavenger, hypolipidemic agent, and inhibitor of pro-inflammatory and prothrombotic factors [104]. Red fruits and vegetables, including tomatoes, watermelons, pink grapefruits, apricots, and pink guavas, contain lycopene. Processed tomato products are good dietary sources of lycopene [105]. Two major hypotheses have been proposed to explain the anti-atherogenic activities of lycopene. The non-oxidative action of lycopene results in an increase of gap-junction communication between cells and modulation of immune function [106] .The oxidative hypothesis supports the prevention of the oxidization of LDL-C as the initial step leading to its uptake by the macrophages inside the arterial wall and the formation of foam cells and atherosclerotic plaque [105]. A possible mechanism for the protective role of lycopene in CVD is via the inhibition of cellular 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis [107]. Results from the Harvard Medical School's Women's Health Study showed that women with the highest intake of tomato-based foods rich in lycopene had a reduced risk for CVD compared to women with a low intake of these foods [108]. The European multicenter case-control study on antioxidants, myocardial infarction, and breast cancer (EURAMIC) study found that the risk of MI was 60% lower for the highest quintile of adipose lycopene concentration compared to the lowest quintile, after adjustment for age, family history of CVD and cigarette smoking [109]. In a crosssectional study comparing Lithuanian and Swedish populations showing diverging mortality rates from CVD, lower blood lycopene levels were found to be associated with increased risk and mortality from CVD [110]. Many studies show that high consumption of tomato products can improve resistance to oxidation in people with T2DM [111]. Eating a lycopene-rich Mediterranean diet increases lycopene levels and can reduce the levels of hemoglobin A1c from 7.1 to 6.8% [112]. In a case-control study on serum β-carotene and the risk of T2DM, participants in the highest tertile of serum β-carotene levels had a 55% lower risk of developing T2DM [113]. In a quasi-experimental study, 32 T2DM patients received 200 g raw tomato daily for 8 weeks. There were significant decreases in systolic and diastolic blood pressure and also a significant increase in apoA-I compared with initial values, which suggests the beneficial role of tomato consumption in reducing cardiovascular risk associated with T2DM [114, 115].


*4.4. Limitations*

Despite our best attempts to maximize participants' motivation and attention during 1.5 h of cognitive testing, including the option to administer the battery over the course of two separate visits and flexibility in the number of breaks given, we cannot rule out the possibility that test performance was influenced by these factors or language comprehension skills. Another caveat is that alternative procedures for measuring a hypothesized skill could tap multiple domains or other domains that were not included in our analyses. Hence, our factor solution may not apply to other cognitive batteries. Studies that use a different combination of tests should examine the underlying structure of their battery rather than rely upon our cognitive domain classifications. Another limitation of our study is that classification of disease status, although conducted by teams with extensive experience working with this population, is an inherently imperfect process (as it is for diagnosis of prodromal AD in elderly adults with a neurotypical developmental history). Moreover, there was some circularity in the use of certain measures of the cognitive battery to aid in consensus diagnoses, which we believe was minimized by including the results of a physical exam and a variety of informant-based measures.

Lastly, none of the participants in our larger, multisite study with severe ID successfully completed all cognitive measures. Hence, the findings in the current study should not be generalized to those individuals, a significant minority of adults with DS. This points to the need to develop and evaluate procedures specifically targeting AD clinical progression in adults with more severe lifelong disabilities.