Vascular Cell Function Research Articles

Abstract 12715: Role of Novel Long Non-Coding RNA BAZ1A-AS1 in Neointima Formation in Human Saphenous Vein Graft Disease

Introduction: Saphenous veins (SVs) are frequently used in CABG surgery, but over 50% of the SV grafts fail in the first 10 years after surgery due to neointima formation. Long non-coding RNAs (lncRNAs) have emerged as attractive therapeutic targets and biomarkers for cardiovascular disease. However, studies on their role and mechanisms in humans are limited. This study aimed to unravel epigenetic mechanisms of neointima formation in human SVs and investigate the role of candidate lncRNAs in key vascular cell functions pertinent to neointima formation. Methods and Results: By using an ex vivo model of human SV graft disease wherein segments of human SV are incubated in tissue culture, we performed RNA-sequencing on human SV with and without neointima formation to interrogate the global transcriptomic changes, and determined 699 differentially expressed novel lncRNAs during neointimal formation. We then identified BAZ1A-antisense (AS)1 as a novel lncRNA highly upregulated during neointima formation. BAZ1A-AS1 overlaps antisense to its cis- regulatory gene, BAZ1A , which was reported to play a role in oxidative stress and DNA damage repair response. We found that one isoform of BAZ1A-AS1 primarily localizes to the nucleus of VSMCs. Interestingly, UV exposure of vascular smooth muscle cells (VSMCs) isolated from human SV induced significant upregulation of BAZ1A and BAZ1A-AS1, suggesting that BAZ1A-AS1 could be associated with chromatin repair processes activated during neointima formation. To investigate whether BAZ1A-AS1 can regulate VSMC functions pertinent to neointima formation, VSMCs were transfected with BAZ1A-AS1 antisense gapmers. Knockdown of BAZ1A-AS1 showed reduced cell proliferation and migration, and increased cell death and DNA damage in VSMCs. Furthermore, role of BAZ1A during neointima formation in vivo is currently under investigation in a carotid artery ligation model using BAZ1A knockout mice. Conclusions: We identified BAZ1A-AS1 as a novel lncRNA associated with neointima formation in human SV, and targeted inhibition of BAZ1A-AS1 or its cis-regulator gene BAZ1A could be a promising strategy for SV graft disease.

Differential endothelial senescence elicited by AQP1 regulation of epigenetic/metabolic responses

Abstract Introduction Accumulation of senescent endothelial cells with age is an important driver of vascular aging and related cardiovascular disease. However, little is known about the mechanisms and signaling pathways that underpin endothelial cell senescence. Aquaporin-1 (AQP1) has been implicated in sensing hydrogen peroxide (H2O2), which differentially modulates endothelial signal transduction. Nonetheless, the role of AQP1 in the regulation of metabolism and function in young and old vascular cells remains elusive. Purpose We sought to investigate whether 1) AQP1 differentially regulates vascular senescence and angiogenesis through epigenetic/metabolic response, senescence-associated secretory phenotype (SASP), and 2) AQP1 knockdown/inhibition rejuvenates senescent endothelial cells and restores angiogenesis. Methods We assessed the role of AQP1 in endothelial cell senescence and impaired angiogenesis through mitochondrial metabolic disorder, epigenetic alteration, and the SASP in human aortic endothelial cells and aortas from wild-type C57BL/6 mice. For these, we conducted AQP1 knockdown (with siRNA) or blockade of AQP1 (with Bacopaside-II, 10 μM) to inhibit H2O2 transport. Results Here we show the upregulation of AQP1 in the senescent endothelial cells (SEC, p.15) and aorta from old mice (>24 months) (p<0.01 and p<0.0001, respectively), facilitating outside-in H2O2 transport. Interestingly, AQP1 knockdown/inhibition reverses both replicative (increased SA-β-galactosidase positive cells) and stress-induced senescence (increased cell-cycle arrest markers p16, p19, and p21; and SASP markers IL1α, IL1β, and IL6) in SEC, whereas it triggers the senescence processes in the proliferating endothelial cells (PEC, p.5) (for each, p<0.05, n=6). Unlike in PEC or young aorta (3-month-old), AQP1 knockdown/inhibition restores angiogenesis in both SEC and old aortas. The interventions increased mitochondrial OXPHOS (represented by enhanced oxygen consumption rate (OCR)) in SEC, which is accompanied by marked increase in CaMKKβ-regulated AMPKT172 phosphorylation (for each, p<0.05, n=6) and in histone deacetylase-4 (HDAC4) phosphorylation at Ser632 (p<0.05) and further histone 3 (H3) acetylation (p<0.01). The HDAC4 phosphorylation leads to significant increase in Mef2A-mediated eNOS phosphorylation at Ser1177 (p<0.05). By contrast, AQP1 knockdown/inhibition induced a senescent-like state by a decrease in mitochondrial OCR and the phosphorylation of CaMKKβ-AMPK and HDAC4-Mef2A regulation of eNOS activity. Conclusion Our studies identify AQP1 as essential regulator of EC proliferation that differentially regulates angiogenesis via epigenetic/metabolic response and SASP modulation. Moreover, genetic or pharmacological inhibition of AQP1 reverses endothelial senescence and restores angiogenesis, representing a potential therapeutic target for vascular rejuvenation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)SwissLife Jubiläumsstiftung

The relationship between venous insufficiency and serum magnesium level

Magnesium is the second most common cation in the cell. In addition to its role as a cofactor in many enzymatic pathways in physiological processes, it is necessary for the regular functioning of vascular smooth muscle cells. Magnesium deficiency has been associated with exacerbation of inflammation, which plays a role in the aetiopathogenesis of many diseases. To investigate the potential relationship between serum magnesium level and the development of chronic venous insufficiency by comparison with healthy individuals. The study included 394 patients with venous insufficiency based on physical examination findings and colour Doppler ultrasonography, and 206 controls without venous insufficiency. Venous insufficiency was defined by colour Doppler as reflux lasting 0.5 seconds or more in superficial veins, and longer than one second in femoral and popliteal veins. Clinical, haematological and biochemical parameters, including serum magnesium level and indicators of inflammation, were compared between groups. A total of 600 participants were included. There was no significant difference between the groups in terms of age and gender. In total, 187 (47.46%) patients with chronic venous insufficiency and 105 (50.97%) of the control group were male (p=0.414). The median age of the patients with chronic venous insufficiency was 48 (min-max: 41-49), and the median age of the control group was 49.00 (min-max: 45.00-60.25) (p=0.064). Serum magnesium level was found to be significantly lower in the group with chronic venous insufficiency compared to the control group; 1.90 mg/dL (min-max: 1.82-2) versus 2.1 mg/dL (min-max: 2-2.2) (p<0.001), respectively. Low serum magnesium levels may pose a potential risk for the development of chronic venous insufficiency, which is common in the community.

A comprehensive analysis on the relationship between BDE-209 exposure and erectile dysfunction

Decabromodiphenyl ether (mainly BDE-209) is a commonly used brominated flame retardant in various industrial products. Although its damage to the reproduction system has been established, its effect on erectile function remains unclear. The present study investigated whether BDE-209 induced erectile dysfunction in male SD rats and the underlying mechanisms. Pubertal male rats were exposed to BDE-209 orally (0, 5, 50, and 500 mg/kg/day) for 28 days and the ICP (intracavernous pressure) and MAP (mean arterial pressure) were measured. After the rats were euthanized, the fibrosis and apoptosis levels were evaluated. Additionally, the endothelial function of the rat vascular endothelium cells and the human umbilical vein endothelial cells were impaired after treatment with 50 μM and 100 μM BDE-209. Moreover, the bioinformatics based on CTD database and ChIP-X Enrichment Analysis, version 3 (ChEA3) and molecular docking analysis demonstrated that 5 transcription factors (NFKB1, NR3C1, E2F5, REL, IRF4) might regulate endothelial function by affecting the expression of interactive genes (BCL-2, CAP3, CAT, TNF, MAPK1, and MAPK3). In summary, the present study demonstrated that BDE-209 might affect downstream interactive genes by binding to transcription factors, leading to corpus cavernosum endothelial dysfunction, thus contributing to erectile dysfunction in rats.

FOXO1 represses MCL1 transcription to regulate the function of vascular smooth muscle cells in intracranial aneurysm.

Intracranial aneurysm (IA) is a pathological dilation of the cerebral arteries. Vascular smooth muscle cell (VSMC) dysfunction assumes a role in IA development. In this context, this study probed the role of FOXO1 in human brain VSMC (HBVSMC) function via MCL1. FOXO1 and MCL1 expression in arterial wall tissues from IA patients and inflammatory cytokines (IL-1β, TNF-α, and IL-6) levels in the serum of IA patients were, respectively, detected with qRT-PCR and ELISA. Pearson's correlation analysis was utilized to analyze the correlation between FOXO1 and MCL1. After FOXO1 and/or MCL1 were overexpressed in HBVSMCs, caspase-3 and Cyt-c protein expression were examined by western blot, cell proliferation by CCK-8 and EdU assays, and cell apoptosis by flow cytometry. IL-1β, TNF-α, and IL-6 levels were assessed in the supernatant of HBVSMCs with ELISA. Dual-luciferase gene reporter and ChIP assays were conducted to evaluate the binding of FOXO1 to MCL1. FOXO1 expression was high and MCL expression was low in arterial wall tissues from IA patients, and IL-1β, TNF-α, and IL-6 levels were high in the serum of IA patients. There was an inverse correlation between FOXO1 and MCL1 mRNA levels. Moreover, FOXO1 bound to the MCL1 promoter to decrease MCL1 transcription. In addition, FOXO1 overexpression augmented cell apoptosis, caspase-3 and Cyt-c protein expression, and IL-1β, TNF-α, and IL-6 secretion, while reducing cell proliferation in HBVSMCs, which was abrogated by further MCL1 overexpression. FOXO1 impeded MCL1 transcription to curb HBVSMC proliferation and facilitate their apoptosis and inflammation.

The role of p53 in the alternation of vascular functions.

Ageing is a risk factor for many degenerative diseases. Cardiovascular diseases (CVDs) are usually big burdens for elderly, caregivers and the health system. During the aging process, normal functions of vascular cells and tissue progressively lost and eventually develop vascular diseases. Endothelial dysfunction, reduced bioavailability of endothelium-derived nitric oxide are usual phenomena observed in patients with cardiovascular diseases. Myriad of studies have been done to investigate to delay the vascular dysfunction or improve the vascular function to prolong the aging process. Tumor suppressor gene p53, also a transcription factor, act as a gatekeeper to regulate a number of genes to maintain normal cell function including but not limited to cell proliferation, cell apoptosis. p53 also crosstalk with other key transcription factors like hypoxia-inducible factor 1 alpha that contribute to the progression of cardiovascular diseases. Therefore, in recent three decades, p53 has drawn scientists’ attention on its effects in vascular function. Though the role of tumor suppressor gene p53 is still not clear in vascular function, it is found to play regulatory roles and may involve in vascular remodeling, atherosclerosis or pulmonary hypertension. p53 may have a divergent role in endothelial and vascular muscle cells in those conditions. In this review, we describe the different effects of p53 in cardiovascular physiology. Further studies on the effects of endothelial cell-specific p53 deficiency on atherosclerotic plaque formation in common animal models are required before the therapeutic potential can be realized.

Functions of Membrane Progesterone Receptors (mPRs, PAQRs) in Nonreproductive Tissues.

Gender differences in a wide variety of physiological parameters have implicated the ovarian hormones, estrogens and progesterone, in the regulation of numerous nonreproductive tissue functions. Rapid, nongenomic (nonclassical) progesterone actions mediated by membrane progesterone receptors (mPRs), which belong to the progestin and adipoQ receptor family, have been extensively investigated in reproductive and nonreproductive tissues since their discovery in fish ovaries 20 years ago. The 5 mPR subtypes (α, β, γ, δ, ε) are widely distributed in vertebrate tissues and are often expressed in the same cells as the nuclear progesterone receptor (PR) and progesterone receptor membrane component 1, thereby complicating investigations of mPR-specific functions. Nevertheless, mPR-mediated progesterone actions have been identified in a wide range of reproductive and nonreproductive tissues and distinguished from nuclear PR-mediated ones by knockdown of these receptors with siRNA in combination with a pharmacological approach using mPR- and PR-specific agonists. There are several recent reviews on the roles of the mPRs in vertebrate reproduction and cancer, but there have been no comprehensive assessments of mPR functions in nonreproductive tissues. Therefore, this article briefly reviews mPR functions in a broad range of nonreproductive tissues. The evidence that mPRs mediate progesterone and progestogen effects on neuroprotection, lordosis behavior, respiratory control of apnea, olfactory responses to pheromones, peripheral nerve regeneration, regulation of prolactin secretion in prolactinoma, immune functions, and protective functions in vascular endothelial and smooth muscle cells is critically reviewed. The ubiquitous expression of mPRs in vertebrate tissues suggests mPRs regulate many additional nonreproductive functions that remain to be identified.

Empagliflozin prevents neointima formation by impairing smooth muscle cell proliferation and accelerating endothelial regeneration.

BackgroundEmpagliflozin, an inhibitor of the sodium glucose co-transporter 2 (SGLT2) and developed as an anti-diabetic agent exerts additional beneficial effects on heart failure outcomes. However, the effect of empagliflozin on vascular cell function and vascular remodeling processes remains largely elusive.Methods/ResultsImmunocytochemistry and immunoblotting revealed SGLT2 to be expressed in human smooth muscle (SMC) and endothelial cells (EC) as well as in murine femoral arteries. In vitro, empagliflozin reduced serum-induced proliferation and migration of human diabetic and non-diabetic SMCs in a dose-dependent manner. In contrast, empagliflozin significantly increased the cell count and migration capacity of human diabetic ECs, but not of human non-diabetic ECs. In vivo, application of empagliflozin resulted in a reduced number of proliferating neointimal cells in response to femoral artery wire-injury in C57BL/6J mice and prevented neointima formation. Comparable effects were observed in a streptozocin-induced diabetic model of apolipoprotein E–/– mice. Conclusive to the in vitro-results, re-endothelialization was not significantly affected in C57BL/6 mice, but improved in diabetic mice after treatment with empagliflozin assessed by Evan’s Blue staining 3 days after electric denudation of the carotid artery. Ribonucleic acid (RNA) sequencing (RNA-seq) of human SMCs identified the vasoactive peptide apelin to be decisively regulated in response to empagliflozin treatment. Recombinant apelin mimicked the in vitro-effects of empagliflozin in ECs and SMCs.ConclusionEmpagliflozin significantly reduces serum-induced proliferation and migration of SMCs in vitro and prevents neointima formation in vivo, while augmenting EC proliferation in vitro and re-endothelialization in vivo after vascular injury. These data document the functional impact of empagliflozin on vascular human SMCs and ECs and vascular remodeling in mice for the first time.

A histological study of vascular wall resident stem cells in venous malformations.

Vascular wall resident stem cells (VW-SCs) play a key role in vascular formation and remodeling under both physiological and pathological situations. They not only serve as a reservoir to supply all types of vascular cells needed, but also regulate vascular homeostasis by paracrine effects. Venous malformations (VMs) are common congenital vascular malformations which are just characterized by the deficient quantity and abnormal function of vascular cells. However, the existence and role of VW-SCs in VMs is still unclear at present. In this study, the level and distribution of VW-SCs in 22 specimens of VMs were measured by immunochemistry, double-labeling immunofluorescence, and qPCR, followed by the Spearman rank correlation test. We found that both the protein and mRNA expression levels of CD34, vWF, VEGFR2, CD44, CD90, and CD105 were significantly downregulated in VMs compared with that in normal venules. VW-SCs were sporadically distributed or even absent within and outside the endothelium of VMs. The expression of the VW-SC-related markers was positively correlated with the density of both endothelial cells and perivascular cells. All those results and established evidence indicated that VW-SCs were more sporadically distributed with fewer amounts in VMs, which possibly contributing to the deficiency of vascular cells in VMs.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC.Graphical abstractmiR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow.

Background:Pericytes and vascular smooth muscle cells, collectively known as mural cells, are recruited through PDGFB (platelet-derived growth factor B)-PDGFRB (platelet-derived growth factor receptor beta) signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Here, we characterize the role of the transcription factor SRF (serum response factor) in MCs and study its function in developmental and pathological contexts.Methods:We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis using RNA-sequencing, immunohistology, in vivo live imaging, and in vitro techniques.Results:By postnatal day 6, pericytes lacking SRF were morphologically abnormal and failed to properly comigrate with angiogenic sprouts. As a consequence, pericyte-deficient vessels at the retinal sprouting front became dilated and leaky. By postnatal day 12, also the vascular smooth muscle cells had lost SRF, which coincided with the formation of pathological arteriovenous shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF (myocardin-related transcription factor) cofactors. We further show that MRTF-SRF signaling promotes pathological pericyte activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging, and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone.Conclusions:SRF is crucial for distinct functions in pericytes and vascular smooth muscle cells. SRF directs pericyte migration downstream of PDGFRB signaling and mediates pathological pericyte activation during ischemic retinopathy. In vascular smooth muscle cells, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of arteriovenous shunts. These essential roles in physiological and pathological contexts provide a rationale for novel therapeutic approaches through targeting SRF activity in MCs.

N-Methyl-D-Aspartic Acid Receptor-Mediated Vasodilation Is Attenuated in the Retinas of Diabetic Rats

Purpose Activation of N-methyl-d-aspartic acid (NMDA) receptors enhances nitric oxide (NO) production in retinal neuronal cells, and in turn, NO released from neuronal cells induces glial cell-mediated dilation of retinal arterioles in rats. The purpose of this study was to examine how neuronal cell-dependent, glial cell-mediated vasodilation is impacted in diabetic rat retinas. Methods Diabetes was induced in 6-week-old male Wistar rats by combining streptozotocin injection and D-glucose feeding. Two weeks later, the dilator function of retinal arterioles was assessed. Results Compared with non-diabetic rats, the dilator responses of retinal arterioles induced by intravitreal injection of NMDA and NOR3, an NO donor, were reduced in diabetic rats. Following the blockade of large-conductance Ca2+-activated K+ (BKCa) channels with iberiotoxin, no significant difference in the retinal vasodilator response to NOR3 was observed between non-diabetic and diabetic rats. Intravitreal injection of 14,15-epoxyeicosatrienoic acid, a vasodilatory factor released from glial cells, dilated retinal arterioles, and the response was diminished by diabetes. Conclusion These findings suggest that the impaired BKCa channel function in vascular cells is responsible for the diminished neuronal cell-dependent, glial cell-mediated dilation of retinal arterioles during the early stage of diabetes.

Generation of iPS-derived endothelial cells and use in organoids vascularizationin

The use of human pluripotent stem cells (iPS) for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types with an efficiency rate. The major challenge in the organoids formation is the vascularization of these. Here, we report the rapid and efficient differentiation of human iPS into mature vascular endothelial cells capable of forming vessels in collagen gels. iPS cells were committed to a mesodermal fate and subsequent exposure to VEGF resulted in the differentiation of endothelial cells. The protocol produced mature cells with efficiencies over 80% within six days. Upon purification to 99% via VECadherin as a surface marker, endothelial cells maintained their identity until passage 5, as assessed by marker gene expression and immunostainings (CD31, VECadherin, vWF). Functional tests have been carried out. The capability of endothelials cells to form capillaries in matrigel was tested and confirmed. IPS-derived endothelial cells are also able to capture oxidized LDL and form a tight monolayer that is destabilized by thrombin treatment. Tissue factor-induced thrombin generation in platelet free plasma is reduced in the presence of iPS-derived vascular endothelial cells showing an antithrombotic phenotype. Endothelial cells were cultivated in collagen gel during one week and form structured vessels with lumen. All together these results showed relevant in vitro functionality of iPS-derived vascular endothelial cells. Our results suggest that these endothelial cells could be used to vascularized organoids structure.

Abstract 507: Adipocyte-specific Cytokine Signaling Regulates Inflammation And Atherosclerosis

Adipose tissue inflammation has been implicated in various chronic inflammatory diseases and cancer. Perivascular adipose tissue (PVAT) surrounds the aorta as a fourth layer and was previously shown to regulate the function of endothelial and vascular smooth muscle cells. Moreover, PVAT contains various immune cells and therefore could contribute to the inflammatory environment in the aorta during atherosclerosis development via cross-talk with adventitia. We found that Western diet (WD) feeding increases the accumulation of immune cells in PVAT of atherosclerosis-prone Ldlr -/- mice and increases the expression of IL-1β. As IL-1 is essential for atherosclerosis development and its inhibition in patients in CANTOS trial reduced inflammation and cardiovascular events, we further evaluated the potential role of IL1R signaling in adipocytes of PVAT.We found that adipocyte-specific ablation of IL1R in Il1r fl/fl Ldlr -/- AdipoCre + mice fed with Western Diet for 16 weeks led to significant reduction in atherosclerosis when compared to Il1r fl/fl Ldlr -/- AdipoCre - controls. Gene expression analysis of isolated adipocytes revealed a significant reduction of expression of several chemokines, including Ccl2, Ccl5, Ccl7 as well as pro-atherogenic cytokines IL-6 and IL-1β . The analysis of immune cell composition by Flow cytometry and scRNA sequencing further demonstrated reduced representation of key pro-inflammatory cell subsets in PVAT of l1r fl/fl Ldlr -/- AdipoCre + which also reflected in their reduced accumulation in the aorta and atherosclerotic plaques. Our data suggest novel adipocyte-specific mechanisms controlling inflammation in atherosclerosis. IL1R signaling therefore directly regulates pro-inflammatory chemokines and cytokines in adipocytes thereby controlling immune cell recruitment to PVAT and aortic wall, which in turn contributes to the enhanced aortic inflammation and atherosclerosis.

Role of mechanosensitive channels/receptors in atherosclerosis.

Mechanical forces are critical physical cues that can affect numerous cellular processes regulating the development, tissue maintenance, and functionality of cells. The contribution of mechanical forces is especially crucial in the vascular system where it is required for embryogenesis and for maintenance of physiological function in vascular cells including aortic endothelial cells, resident macrophages, and smooth muscle cells. Emerging evidence has also identified a role of these mechanical cues in pathological conditions of the vascular system such as atherosclerosis and associated diseases like hypertension. Of the different mechanotransducers, mechanosensitive ion channels/receptors are gaining prominence due to their involvement in numerous physiological and pathological conditions. However, only a handful of potential mechanosensory ion channels/receptors have been shown to be involved in atherosclerosis, and their precise role in disease development and progression remains poorly understood. Here, we provide a comprehensive account of recent studies investigating the role of mechanosensitive ion channels/receptors in atherosclerosis. We discuss the different groups of mechanosensitive proteins and their specific roles in inflammation, endothelial dysfunction, macrophage foam cell formation, and lesion development, which are crucial for the development and progression of atherosclerosis. Results of the studies discussed here will help in developing an understanding of the current state of mechanobiology in vascular diseases, specifically in atherosclerosis, which may be important for the development of innovative and targeted therapeutics for this disease.

Vascular Permeability in Diseases.

Vascular permeability is a selective mechanism that maintains the exchange between vessels, tissues, and organs. The regulation was mostly studied during the nineteenth century by physiologists who defined physical laws and equations, taking blood, tissue interstitial, and oncotic pressure into account. During the last decades, a better knowledge of vascular cell functions and blood-vessel interactions opens a new area of vascular biology. Endothelial cell receptors vascular cell adhesion molecule (VCAM), intercellular cell adhesion molecule (ICAM), vascular endothelial growth factor receptor (VEGFR-2), receptor for advanced glycation end products (RAGE), and mediators were identified and their role in homeostasis and pathological situations was described. The molecular differences of endothelial cell junctions (tight, gap, and adherens junctions) and their role in vascular permeability were characterized in different organs. The main mediators of vasomotricity and permeability, such as prostaglandins, nitric oxide (NO), prostacyclin, vascular growth factor (VEGF), and cytokines, have been demonstrated to possess major functions in steady state and pathological situations. Leukocytes were shown to adhere to endothelium and migrate during inflammatory situations and infectious diseases. Increased vascular permeability is linked to endothelium integrity. Glycocalyx, when intact, may limit cancer cell metastasis. Biological modifications of blood and tissue constituents occurring in diabetes mellitus were responsible for increased permeability and, consequently, ocular and renal complications. Vascular pressure and fluidity are major determinants of pulmonary and cerebral edema. Beside the treatment of the infectious disease, of the blood circulation dysfunction and inflammatory condition, drugs (cyclooxygenase inhibitors) and specific antibodies anti-cytokine (anti-VEGF) have been demonstrated to reduce the severity and the mortality in diseases that exhibited enhanced vascular permeability.

The Potential Role of Peripheral Oxidative Stress on the Neurovascular Unit in Amyotrophic Lateral Sclerosis Pathogenesis: A Preliminary Report from Human and In Vitro Evaluations.

Oxidative stress, the alteration of mitochondrial function, and changes in the neurovascular unit (NVU) could play a role in Amyotrophic Lateral Sclerosis (ALS) pathogenesis. Our aim was to analyze the plasma redox system and nitric oxide (NO) in 25 ALS new-diagnosed patients and five healthy controls and the effects of plasma on the peroxidation/mitochondrial function in human umbilical cord-derived endothelial vascular cells (HUVEC) and astrocytes. In plasma, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), and nitric oxide (NO) were analyzed by using specific assays. In HUVEC/astrocytes, the effects of plasma on the release of mitochondrial reactive oxygen species (mitoROS) and NO, viability, and mitochondrial membrane potential were investigated. In the plasma of ALS patients, an increase in TBARS and a reduction in GSH and NO were found. In HUVEC/astrocytes treated with a plasma of ALS patients, mitoROS increased, whereas cell viability and mitochondrial membrane potential decreased. Our results show that oxidative stress and NVU play a central role in ALS and suggest that unknown plasma factors could be involved in the disease pathogenesis. Quantifiable changes in ALS plasma related to redox state alterations can possibly be used for early diagnosis.

Targeting Arginine in COVID-19-Induced Immunopathology and Vasculopathy.

Coronavirus disease 2019 (COVID-19) represents a major public health crisis that has caused the death of nearly six million people worldwide. Emerging data have identified a deficiency of circulating arginine in patients with COVID-19. Arginine is a semi-essential amino acid that serves as key regulator of immune and vascular cell function. Arginine is metabolized by nitric oxide (NO) synthase to NO which plays a pivotal role in host defense and vascular health, whereas the catabolism of arginine by arginase to ornithine contributes to immune suppression and vascular disease. Notably, arginase activity is upregulated in COVID-19 patients in a disease-dependent fashion, favoring the production of ornithine and its metabolites from arginine over the synthesis of NO. This rewiring of arginine metabolism in COVID-19 promotes immune and endothelial cell dysfunction, vascular smooth muscle cell proliferation and migration, inflammation, vasoconstriction, thrombosis, and arterial thickening, fibrosis, and stiffening, which can lead to vascular occlusion, muti-organ failure, and death. Strategies that restore the plasma concentration of arginine, inhibit arginase activity, and/or enhance the bioavailability and potency of NO represent promising therapeutic approaches that may preserve immune function and prevent the development of severe vascular disease in patients with COVID-19.

YAP and TAZ in Vascular Smooth Muscle Confer Protection Against Hypertensive Vasculopathy.

Background:Hypertension remains a major risk factor for cardiovascular diseases, but the underlying mechanisms are not well understood. We hypothesize that appropriate mechanotransduction and contractile function in vascular smooth muscle cells are crucial to maintain vascular wall integrity. The Hippo pathway effectors YAP (yes-associated protein 1) and TAZ (WW domain containing transcription regulator 1) have been identified as mechanosensitive transcriptional coactivators. However, their role in vascular smooth muscle cell mechanotransduction has not been investigated in vivo.Methods:We performed physiological and molecular analyses utilizing an inducible smooth muscle–specific YAP/TAZ knockout mouse model.Results:Arteries lacking YAP/TAZ have reduced agonist-mediated contraction, decreased myogenic response, and attenuated stretch-induced transcriptional regulation of smooth muscle markers. Moreover, in established hypertension, YAP/TAZ knockout results in severe vascular lesions in small mesenteric arteries characterized by neointimal hyperplasia, elastin degradation, and adventitial thickening.Conclusions:This study demonstrates a protective role of YAP/TAZ against hypertensive vasculopathy.

SPARC, a Novel Regulator of Vascular Cell Function in Pulmonary Hypertension.

Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature. After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach. C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-β1 (transforming growth factor β1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice. This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.