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The vascular endothelium plays a vital role during embryogenesis and aging and is a cell monolayer that lines the blood vessels. The immune system recognizes the endothelium as its own. Therefore, an abnormality of the endothelium exposes the tissues to the immune system and provokes inflammation and vascular diseases such as atherosclerosis. Its secretory role allows it to release vasoconstrictors and vasorelaxants as well as cardio-modulatory factors that maintain the proper functioning of the circulatory system. The sealing of the monolayer provided by adhesion molecules plays an important role in cardiovascular physiology and pathology.

Bone morphogenic protein receptor 2 (BMPR2) expression and signaling are impaired in pulmonary arterial hypertension (PAH). How BMPR2 signaling is decreased in PAH is poorly understood. Protein tyrosine phosphatases (PTPs) play important roles in vascular remodeling in PAH. To identify whether PTPs modify BMPR2 signaling, we used a siRNA-mediated high-throughput screening of 22,124 murine genes in mouse myoblastoma reporter cells using ID1 expression as readout for BMPR2 signaling. We further experimentally validated the top hit, PTPN1 (PTP1B), in healthy human pulmonary arterial endothelial cells (PAECs) either silenced by siRNA or exposed to hypoxia and confirmed its relevance to PAH by measuring PTPN1 levels in blood and PAECs collected from PAH patients. We identified PTPN1 as a novel regulator of BMPR2 signaling in PAECs, which is downregulated in the blood of PAH patients, and documented that downregulation of PTPN1 is linked to endothelial dysfunction in PAECs. These findings point to a potential involvement for PTPN1 in PAH and will aid in our understanding of the molecular mechanisms involved in the disease.

Medin, a small 50-amino acid peptide, is an internal cleaved product from the second discoidin domain of milk fat globule epidermal growth factor VIII (MFG-E8) protein. Medin has been reported as the most common amylogenic protein in the upper part of the arterial system, including aortic, temporal, and cerebral arterial walls in the elderly. Medin has a high affinity to elastic fibers and is closely associated with arterial degenerative inflammation, elastic fiber fragmentation, calcification, and amyloidosis. In vitro, treating with the medin peptide promotes the inflammatory phenotypic shift of both endothelial cells and vascular smooth muscle cells. In vitro, ex vivo, and in vivo studies demonstrate that medin enhances the abundance of reactive oxygen species and reactive nitrogen species produced by both endothelial cells and vascular smooth muscle cells and promotes vascular endothelial dysfunction and arterial stiffening. Immunostaining and immunoblotting analyses of human samples indicate that the levels of medin are increased in the pathogenesis of aortic aneurysm/dissection, temporal arteritis, and cerebrovascular dementia. Thus, medin peptide could be targeted as a biomarker diagnostic tool or as a potential molecular approach to curbing the arterial degenerative inflammatory remodeling that accompanies aging and disease.

Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Vascular endothelial dysfunction (VED) significantly results in catastrophic cardiovascular diseases with multiple aetiologies. Variations in vasoactive peptides, including angiotensin II and endothelin 1, and metabolic perturbations like hyperglycaemia, altered insulin signalling, and homocysteine levels result in pathogenic signalling cascades, which ultimately lead to VED. Endoplasmic reticulum (ER) stress reduces nitric oxide availability, causes aberrant angiogenesis, and enhances oxidative stress pathways, consequently promoting endothelial dysfunction. Moreover, the renin-angiotensin system (RAS) has widely been acknowledged to impact angiogenesis, endothelial repair and inflammation. Interestingly, experimental studies at the preclinical level indicate a possible pathological link between the two pathways in the development of VED. Furthermore, pharmacological modulation of ER stress ameliorates angiotensin-II mediated VED as well as RAS intervention either through inhibition of the pressor arm or enhancement of the depressor arm of RAS, mitigating ER stress-induced endothelial dysfunction and thus emphasizing a vital crosstalk., Conclusion: Deciphering the pathway overlap between RAS and ER stress may open potential therapeutic avenues to combat endothelial dysfunction and associated diseases. Several studies suggest that alteration in a component of RAS may induce ER stress or induction of ER stress may modulate the RAS components. In this review, we intend to elaborate on the crosstalk of ER stress and RAS in the pathophysiology of VED., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The large-conductance calcium-activated potassium (BK) channel is a critical regulator and potential therapeutic target of vascular tone and architecture, and abnormal expression or dysfunction of this channel is linked to many vascular diseases. Vascular remodelling is the early pathological basis of severe vascular diseases. Delaying the progression of vascular remodelling can reduce cardiovascular events, but the pathogenesis remains unclear. To clarify the role of BK channels in vascular remodelling, we use rats with BK channel α subunit knockout (BK α ‒/‒ ). The results show that BK α ‒/‒ rats have smaller inner and outer diameters, thickened aortic walls, increased fibrosis, and disordered elastic fibers of the aortas compared with WT rats. When the expression and function of BK α are inhibited in human umbilical arterial smooth muscle cells (HUASMCs), the expressions of matrix metalloproteinase 2 (MMP2), MMP9, and interleukin-6 are enhanced, while the expressions of smooth muscle cell contractile phenotype proteins are reduced. RNA sequencing, bioinformatics analysis and qPCR verification show that C1q/tumor necrosis factor-related protein 7 ( CTRP7 ) is the downstream target gene. Furthermore, except for that of MMPs, a similar pattern of IL-6, smooth muscle cell contractile phenotype proteins expression trend is observed after CTRP7 knockdown. Moreover, knockdown of both BK α and CTRP7 in HUASMCs activates PI3K/Akt signaling. Additionally, CTRP7 is expressed in vascular smooth muscle cells (VSMCs), and BK α deficiency activates the PI3K/Akt pathway by reducing CTRP7 level. Therefore, we first show that BK channel deficiency leads to vascular remodelling. The BK channel and CTRP7 may serve as potential targets for the treatment of cardiovascular diseases.

Lymphatic vessels, as the main tube network of fluid drainage and leukocyte transfer, are responsible for the maintenance of homeostasis and pathological repairment. Recently, by using genetic lineage tracing and single-cell RNA sequencing techniques, significant cognitive progress has been made about the impact of stem/progenitor cells during lymphangiogenesis. In the embryonic stage, the lymphatic network is primarily formed through self-proliferation and polarized-sprouting from the lymph sacs. However, the assembly of lymphatic stem/progenitor cells also guarantees the sustained growth of lymphvasculogenesis to obtain the entire function. In addition, there are abundant sources of stem/progenitor cells in postnatal tissues, including circulating progenitors, mesenchymal stem cells, and adipose tissue stem cells, which can directly differentiate into lymphatic endothelial cells and participate in lymphangiogenesis. Specifically, recent reports indicated a novel function of lymphangiogenesis in transplant arteriosclerosis and atherosclerosis. In the present review, we summarized the latest evidence about the diversity and incorporation of stem/progenitor cells in lymphatic vasculature during both the embryonic and postnatal stages, with emphasis on the impact of lymphangiogenesis in the development of vascular diseases to provide a rational guidance for future research.

Vascular diseases of the elderly are a topic of enormous interest in clinical practice, as they have great epidemiological significance and lead to ever-increasing healthcare expenditures. The mechanisms underlying these pathologies have been increasingly characterized over the years. It has emerged that endothelial dysfunction and chronic inflammation play a diriment role among the most relevant pathophysiological mechanisms. As one can easily imagine, various processes occur during aging, and several pathways undergo irreversible alterations that can promote the decline and aberrations that trigger the diseases above. Endothelial dysfunction and aging of circulating and resident cells are the main characteristics of the aged organism; they represent the framework within which an enormous array of molecular abnormalities occur and contribute to accelerating and perpetuating the decline of organs and tissues. Recognizing and detailing each of these dysfunctional pathways is helpful for therapeutic purposes, as it allows one to hypothesize the possibility of tailoring interventions to the damaged mechanism and hypothetically limiting the cascade of events that drive the onset of these diseases. With this paper, we have reviewed the scientific literature, analysing the pathophysiological basis of the vascular diseases of the elderly and pausing to reflect on attempts to interrupt the vicious cycle that connotes the diseases of aging, laying the groundwork for therapeutic reasoning and expanding the field of scientific research by moving from a solid foundation.

The antidiabetic effects of quercetin and metformin are well known. However, their synergistic effect in reversing the symptoms of diabetes-induced endothelial dysfunction remains unknown. In this study, we have investigated their synergistic effect in streptozotocin (STZ)-nicotinamide induced diabetic rats. Seventy-five rats were divided into five groups; normal control, diabetic control, treatment groups (10 mg/kg quercetin, 180 mg/kg metformin, and combined). The plasma glucose and lipid levels, liver enzymes, ex-vivo studies on aortic rings, histology of liver, kidney, pancreas, abdominal aorta and thoracic aorta, and immunohistochemical studies were carried out. The findings revealed that the combination of quercetin and metformin showed a greater antidiabetic effect than either drug, and rendered protection to the endothelium. The combination effectively reversed the hyperglycemia-induced endothelial dysfunction in diabetic rats. Furthermore, it also reversed the dysregulated expression of eNOS, 3-nitrotyrosine, VCAM-1, CD31 and SIRT-1. Overall, the present study's findings demonstrate that quercetin potentiates the activity of metformin to control the complications associated with diabetes., (© 2022. The Author(s).)

Purpose: The administration of anti-vascular endothelial growth factor agents is the standard firs-line therapy for ocular vascular diseases, but some patients still have poor outcomes and drug resistance. This study investigated the role of DCZ19903, a small molecule multitarget kinase inhibitor, in ocular angiogenesis., Methods: The toxicity of DCZ19903 was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays, flow cytometry, Calcein-AM/PI staining, and terminal uridine nick-end labeling staining. Oxygen-induced retinopathy and laser-induced choroidal neovascularization models were adopted to assess the antiangiogenic effects of DCZ19903 by Isolectin B4 (GS-IB4) and hematoxylin-eosin staining. EdU assays, transwell migration assays, tube formation, and choroid sprouting assays were performed to determine the antiangiogenic effects of DCZ19903. The antiangiogenic mechanism of DCZ19903 was determined using network pharmacology approach and western blots., Results: There was no obvious cytotoxicity or tissue toxicity after DCZ19903 treatment. DCZ19903 exerted the antiangiogenic effects in OIR model and choroidal neovascularization model. DCZ19903 inhibited the proliferation, tube formation, migration ability of endothelial cells, and choroidal explant sprouting. DCZ19903 plus ranibizumab achieved greater antiangiogenetic effects than DCZ19903 or ranibizumab alone. DCZ19903 exerted its antiangiogenic effects via affecting the activation of ERK1/2 and p38 signaling., Conclusions: DCZ19903 is a promising drug for antiangiogenic treatment in ocular vascular diseases., Translational Relevance: These findings suggest that DCZ19903 possesses great antiangiogenic potential for treating ocular vascular diseases.

Endothelium dysfunction produces peripheral vascular disease comorbidities in type 2 diabetes, including hypertension, and critical limb ischemia. In this study we aimed to test endothelial dysfunction, the vasodilator effects of a proteinase-activated receptor 2 (PAR2) agonist (2fLIGRLO), and thromboxane A 2 synthase inhibitor (ozagrel) on PAR2 vasodilation in hind limb arteries ex vivo, using Zucker Diabetic-Sprague Dawley (ZDSD) rats, a model of type 2 diabetes. Male Sprague Dawley rats (SD) and ZDSD were fed a high-fat content 'Western diet' from 16 to 20 weeks of age (wks) then fed a standard laboratory diet. We identified diabetic ZDSD rats by two consecutive blood glucose measurements > 12.5 mM, based on weekly monitoring. We used acetylcholine, 2fLIGRLO, and nitroprusside with wire-myograph methods to compare relaxations of femoral, and saphenous arteries from diabetic ZDSD (21-23 wks) to age-matched normoglycemic SD. All arteries showed evidence of endothelium dysfunction using acetylcholine (reduced maximum relaxations, reduced sensitivity), and higher sensitivities to 2fLIGRLO, and nitroprusside in ZDSD vs SD. Ozagrel treatment of ZDSD distal segments, and end-branches of saphenous arteries decreased their sensitivities to 2fLIGRLO. We tested aortas for altered expression of endothelium-specific gene targets using PCR array and qPCR. PAR2, and placental growth factor gene transcripts were 1.5, and 4-times higher in ZDSD than SD aortas. Hind limb arteries of ZDSD exhibit endothelium dysfunction having less GPCR agonist induced vasodilation by endothelial NO-release. Different expression of several endothelial genes in ZDSD vs SD aortas, including PAR2, suggests altered inflammatory, and angiogenesis signaling pathways in the endothelium of ZDSD., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Four of the Zucker Diabetic-Sprague Dawley rats used in this study were provided gratis by Charles River Canada to the investigators for evaluation., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Preeclampsia is a multi-system disease that can have severe, even fatal implications for the mother and fetus. Abnormal placentation can lead to ischaemic tissue injury and placental inflammation. In turn, the placenta releases anti-angiogenic factors into the maternal circulation. These systemically act to neutralise angiogenic factors causing endothelial dysfunction causing preeclampsia. Hydroxychloroquine is an immune modulating drug that is considered safe in pregnancy. There is epidemiological evidence suggesting it may reduce the risk of preeclampsia. Here, we examined the effects hydroxychloroquine on the production and secretion of sFlt-1, soluble endoglin (sENG), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) in primary human placenta, cytotrophoblasts and umbilical vein endothelial cells (endothelial cell model). Hydroxychloroquine treatment decreased mRNA expression of two sFlt-1 isoforms and its protein secretion. sENG was not reduced. Hydroxychloroquine treatment increased secretion of pro-angiogenic factor PIGF from endothelial cells. It did not significantly reduce the expression of the endothelial cell inflammation marker, ET-1, and inflammation induced expression of the adhesion molecule, VCAM. Hydroxychloroquine could not overcome leukocyte adhesion to endothelial cells. Hydroxychloroquine mitigates features of preeclampsia, but it does not reduce key markers of endothelial dysfunction., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Kadife et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Oxidative stress and inflammation damage play pivotal roles in vascular dementia (VaD). Trimethylamine N-oxide (TMAO), an intestinal microbiota-stemming metabolite, was reported to promote inflammation and oxidative stress, involved in the etiology of several diseases. Still, these effects have not been investigated in VaD. Here, we tested whether pre-existing, circulating, high levels of TMAO could affect VaD-induced cognitive decline. TMAO (120 mg/kg) was given to rats for a total of 8 weeks, and these rats underwent a sham operation or bilateral common carotid artery (2VO) surgery after 4 weeks of treatment. Four weeks after surgery, the 2VO rats exhibited hippocampal-dependent cognitive function declines and synaptic plasticity dysfunction, accompanied by an increase in oxidative stress, neuroinflammation, and apoptosis. TMAO administration, which increased plasma and hippocampal TMAO at 4 weeks postoperatively, further aggravated these effects, resulting in exaggerated cognitive and synaptic plasticity impairment, though not within the Sham group. Moreover, TMAO treatment activated the NLRP3 inflammasome and decreased SIRT1 protein expression within the hippocampus. However, these effects of TMAO were significantly attenuated by the overexpression of SIRT1. Our findings suggest that TMAO increases oxidative stress-induced neuroinflammation and apoptosis by inhibiting the SIRT1 pathway, thereby exacerbating cognitive dysfunction and neuropathological changes in VaD rats.

Vascular dysfunction: develops progressively with ageing; increases the risk of cardiovascular diseases (CVD); and is characterized by endothelial dysfunction and arterial stiffening, which are primarily mediated by superoxide-driven oxidative stress and consequently reduced nitric oxide (NO) bioavailability and arterial structural changes. Interventions initiated before vascular dysfunction manifests may have more promise for reducing CVD risk than interventions targeting established dysfunction. Gut microbiome-derived trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk, and can be suppressed by 3,3-dimethyl-1-butanol (DMB). We investigated whether DMB supplementation could prevent age-related vascular dysfunction in C57BL/6N mice when initiated prior to development of dysfunction. Mice received drinking water with 1% DMB or normal drinking water (control) from midlife (18 months) until being studied at 21, 24 or 27 months of age, and were compared to young adult (5 month) mice. Endothelial function [carotid artery endothelium-dependent dilatation (EDD) to acetylcholine; pressure myography] progressively declined with age in control mice, which was fully prevented by DMB via higher NO-mediated EDD and lower superoxide-related suppression of EDD (normalization of EDD with the superoxide dismutase mimetic TEMPOL). In vivo aortic stiffness (pulse wave velocity) increased progressively with age in controls, but DMB attenuated stiffening by ∼ 70%, probably due to preservation of endothelial function, as DMB did not affect aortic intrinsic mechanical (structural) stiffness (stress-strain testing) nor adventitial abundance of the arterial structural protein collagen. Our findings indicate that long-term DMB supplementation prevents/attenuates age-related vascular dysfunction, and therefore has potential for translation to humans for reducing CV risk with ageing. KEY POINTS: Vascular dysfunction, characterized by endothelial dysfunction and arterial stiffening, develops progressively with ageing and increases the risk of cardiovascular diseases (CVD). Interventions aimed at preventing the development of CV risk factors have more potential for preventing CVD relative to those aimed at reversing established dysfunction. The gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) induces vascular dysfunction, is associated with higher CV risk and can be suppressed by supplementation with 3,3-dimethyl-1-butanol (DMB). In mice, DMB prevented the development of endothelial dysfunction and delayed and attenuated in vivo arterial stiffening with ageing when supplementation was initiated in midlife, prior to the development of dysfunction. DMB supplementation or other TMAO-suppressing interventions have potential for translation to humans for reducing CV risk with ageing., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Placental extracellular vesicles (EVs) have increasingly been recognized as a major mediator of feto-maternal communication. However, the cellular and molecular mechanisms of the uptake of placental EVs by recipient cells are still not well-understood. We previously reported that placental EVs target a limited number of organs in vivo. In the current study, we investigated the mechanisms underlying the uptake of placental EVs into target cells. Placental EVs were derived from explant cultures of normal or preeclamptic placentae. The mechanisms underlying the uptake of placental EVs were elucidated, using the phagocytosis or endocytosis inhibitor, trypsin-treatment or integrin-blocking peptides. The endothelial cell activation was studied using the monocyte adhesion assay after the preeclamptic EVs exposure, with and/or without treatment with the integrin blocking peptide, YIGSR. The cellular mechanism of the uptake of the placental EVs was time, concentration and energy-dependent and both the phagocytosis and endocytosis were involved in this process. Additionally, proteins on the surface of the placental EVs, including integrins, were involved in the EV uptake process. Furthermore, inhibiting the uptake of preeclamptic EVs with YIGSR, reduced the endothelial cell activation. The interaction between the placental EVs and the recipient cells is mediated by integrins, and the cellular uptake is mediated by a combination of both phagocytosis and endocytosis.

Endothelial cells (ECs) line the inner surface of blood vessels and play a substantial role in vascular biology. Endothelial dysfunction (ED) is strongly correlated with the initiation and progression of many vascular diseases. Regulated cell death, such as ferroptosis, is one of the multiple mechanisms that lead to ED. Ferroptosis is an iron-dependent programmed cell death associated with various vascular diseases, such as cardiovascular, cerebrovascular, and pulmonary vascular diseases. This review summarized ferroptosis of ECs in vascular diseases and discussed potential therapeutic strategies for treating ferroptosis of ECs. In addition to lipid peroxidation inhibitors and iron chelators, a growing body of evidence showed that clinical drugs, natural products, and intervention of noncoding RNAs may also inhibit ferroptosis of ECs.

The endothelial glycocalyx (EGX) contributes to the permeability barrier of vessels and regulates the coagulation cascade. EGX damage, which occurs in numerous disease states, including sepsis and trauma, results in endotheliopathy. While influenza and other viral infections are known to cause endothelial dysfunction, their effect on the EGX has not been described. We hypothesized that the H1N1 influenza virus would cause EGX degradation. Human umbilical vein endothelial cells (HUVECs) were exposed to varying multiplicities of infection (MOI) of the H1N1 strain of influenza virus for 24 hours. A dose-dependent effect was examined by using an MOI of 5 (n = 541), 15 (n = 714), 30 (n = 596), and 60 (n = 653) and compared to a control (n = 607). Cells were fixed and stained with FITC-labelled wheat germ agglutinin to quantify EGX. There was no difference in EGX intensity after exposure to H1N1 at an MOI of 5 compared to control (6.20 vs. 6.56 Arbitrary Units (AU), p = 0.50). EGX intensity was decreased at an MOI of 15 compared to control (5.36 vs. 6.56 AU, p<0.001). The degree of EGX degradation was worse at higher doses of the H1N1 virus; however, the decrease in EGX intensity was maximized at an MOI of 30. Injury at MOI of 60 was not worse than MOI of 30. (4.17 vs. 4.47 AU, p = 0.13). The H1N1 virus induces endothelial dysfunction by causing EGX degradation in a dose-dependent fashion. Further studies are needed to characterize the role of this EGX damage in causing clinically significant lung injury during acute viral infection., Competing Interests: The authors have declared that no competing interests exist.

Nanotechnology has been developed to deliver cargos effectively to the vascular system. Nanomedicine is a novel and effective approach for targeted vascular disease treatment including atherosclerosis, coronary artery disease, strokes, peripheral arterial disease, and cancer. It has been well known for some time that vascular disease patients have a higher cancer risk than the general population. During atherogenesis, the endothelial cells are activated to increase the expression of adhesion molecules such as Intercellular Adhesion Molecule 1 (ICAM-1), Vascular cell adhesion protein 1 (VCAM-1), E-selectin, and P-selectin. This biological activation of endothelial cells gives a targetability clue for nanoparticle strategies. Nanoparticle formation has a passive targeting pathway due to the increased adhesion molecule expression on the cell surface as well as increased cell activation. In addition, the VCAM-1-targeting peptide has been widely used to target the inflamed endothelial cells. Biomimetic nanoparticles using platelet and leukocyte membrane fragment strategies have been promising techniques for targeted vascular disease treatment. Cyclodextrin, a natural oligosaccharide with a hydrophobic cavity, increase the solubility of cholesterol crystals at the atherosclerotic plaque site and has been used to deliver the hydrophobic drug statin as a therapeutic in a targeted manner. In summary, nanoparticles decorated with various targeting molecules will be an effective and promising strategy for targeted vascular disease treatment.

Rationale: Autopsy and biomarker studies suggest that endotheliopathy contributes to coronavirus disease (COVID-19)-associated acute respiratory distress syndrome. However, the effects of COVID-19 on the lung endothelium are not well defined. We hypothesized that the lung endotheliopathy of COVID-19 is caused by circulating host factors and direct endothelial infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objectives: We aimed to determine the effects of SARS-CoV-2 or sera from patients with COVID-19 on the permeability and inflammatory activation of lung microvascular endothelial cells. Methods: Human lung microvascular endothelial cells were treated with live SARS-CoV-2; inactivated viral particles; or sera from patients with COVID-19, patients without COVID-19, and healthy volunteers. Permeability was determined by measuring transendothelial resistance to electrical current flow, where decreased resistance signifies increased permeability. Inflammatory mediators were quantified in culture supernatants. Endothelial biomarkers were quantified in patient sera. Measurements and Main Results: Viral PCR confirmed that SARS-CoV-2 enters and replicates in endothelial cells. Live SARS-CoV-2, but not dead virus or spike protein, induces endothelial permeability and secretion of plasminogen activator inhibitor 1 and vascular endothelial growth factor. There was substantial variability in the effects of SARS-CoV-2 on endothelial cells from different donors. Sera from patients with COVID-19 induced endothelial permeability, which correlated with disease severity. Serum levels of endothelial activation and injury biomarkers were increased in patients with COVID-19 and correlated with severity of illness. Conclusions: SARS-CoV-2 infects and dysregulates endothelial cell functions. Circulating factors in patients with COVID-19 also induce endothelial cell dysfunction. Our data point to roles for both systemic factors acting on lung endothelial cells and viral infection of endothelial cells in COVID-19-associated endotheliopathy.

Synthetic phenotype switch of vascular smooth muscle cells (VSMCs) has been shown to play key roles in vascular diseases. Mounting evidence has shown that fatty acid metabolism is highly associated with vascular diseases. However, how fatty acids regulate VSMC phenotype is poorly understood. Hence, the effects of palmitic acid (PA) on VSMC phenotype were determined in this study. The effect of the PA on VSMCs was measured by live/dead and EdU assays, as well as flow cytometry. Migration ability of VSMCs was evaluated using transwell assay. The underlying targets of miR-22 were predicted using bioinformatics online tools, and confirmed by luciferase reporter assay. The RNA and protein expression of certain gene was detected by qRT-PCR or western blot. PA inhibited VSMC switch to synthetic phenotype, as manifested by inhibiting VSMC proliferation, migration, and synthesis. PA upregulated miR-22 in VSMCs, and miR-22 mimics exerted similar effects as PA treatment, inhibiting VSMC switch to synthetic phenotype. Inhibition of miR-22 using miR-22 inhibitor blocked the impacts of PA on VSMC phenotype modulation, suggesting that PA modulated VSMC phenotype through upregulation of miR-22 expression. We found that ecotropic virus integration site 1 protein homolog (EVI1) was the target of miR-22 in regulation of VSMC phenotype. Overexpression of miR-22 or/and PA treatment attenuated the inhibition of EVI1 on switch of VSMCs. These findings suggested that PA inhibits VSMC switch to synthetic phenotype through upregulation of miR-22 thereby inhibiting EVI1, and correcting the dysregulation of miR-22/EVI1 or PA metabolism is a potential treatment to vascular diseases.

Hypercholesterolaemia is a significant risk factor for developing vascular disease and fatty liver. Pineapple ( Ananas comosus ), a tropical fruit widely cultivated in Asia, is reported to exhibit antioxidant and cholesterol-lowering activity; however, the potential hypolipidaemic mechanisms of pineapple fruit remain unknown. Therefore, we aimed to identify the anti-hypercholesterolaemic mechanism of pineapple fruit and to study the effect of pineapple fruit intake on hypercholesterolaemia-induced vascular dysfunction and liver steatosis in a high-cholesterol diet (HCD)-fed rats. Male Sprague Dawley rats were fed with standard diet or HCD, and the pineapple fruit was orally administered to HCD-fed rats for 8 weeks. At the end of treatment, vascular reactivity and morphology of aortas, as well as serum nitrate/nitrite (NOx), were determined. Liver tissues were also examined for histology, lipid content, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) activity, and protein expression of cholesterol metabolism-related enzymes. Results showed that pineapple fruit reduced the levels of hepatic cholesterol and triglycerides, and improved histological characteristics of a fatty liver in HCD-fed rats. Pineapple fruit also increased serum NOx, restored endothelium-dependent vasorelaxation, and reduced structural alterations in aortas of rats fed the HCD. In addition, a reduction of HMGCR activity and the downregulation of hepatic expression of HMGCR and sterol-regulatory element-binding protein 2 (SREBP2), as well as the upregulation of hepatic expression of cholesterol 7α-hydroxylase (CYP7A1) and LDL receptor (LDLR) were found in pineapple fruit-treated hypercholesterolaemic rats. These results indicate that pineapple fruit consumption can restore fatty liver and protect vascular endothelium in diet-induced hypercholesterolaemia through an improvement of hepatic cholesterol metabolism.

Although α-synuclein (SNCA) is a well-known pathological molecule involved in synucleinopathy in neurons, its physiological roles remain largely unknown. We reported that serum SNCA levels have a close inverse correlation with blood pressure and age, which indicates the involvement of SNCA in age-related endothelial dysfunction. Therefore, this study aimed to elucidate the molecular functions of SNCA in the endothelium. We confirmed that SNCA was expressed in and secreted from endothelial cells (ECs). Exogenous treatment with recombinant SNCA (rSNCA) activated the Akt-eNOS axis and increased nitric oxide production in ECs. Treatment with rSNCA also suppressed TNF-α- and palmitic acid-induced NF-κB activation, leading to the suppression of VCAM-1 upregulation and restoration of eNOS downregulation in ECs. As for endogenous SNCA expression, replicative senescence resulted in the attenuation of SNCA expression in cultured ECs, similar to the effects of physiological aging on mice aortas. The siRNA-mediated silencing of SNCA consistently resulted in senescent phenotypes, such as eNOS downregulation, increased β-gal activity, decreased Sirt1 expression, and increased p53 expression, in ECs. Ex vivo assessment of endothelial functions using aortic rings revealed impaired endothelium-dependent acetylcholine-induced relaxation in SNCA knockout (KO) mice. Furthermore, SNCA KO mice, especially those on a high-fat diet, displayed elevated blood pressure compared with wild-type mice; this could be eNOS dysfunction-dependent because of the lower difference caused by L-NAME administration. These results indicate that exogenous and endogenous SNCA in ECs might physiologically maintain vascular integrity, and age-related endothelial dysfunction might be partially ascribed to loss-of-function of SNCA in ECs., (© 2022 Federation of American Societies for Experimental Biology.)

Background and Aims: Intervessel pit membranes (PMs) are important cell wall structures in the vessel system that may impact a plant's water transport and its susceptibility to vascular diseases. Functional roles of intervessel PMs largely depend on their structure and polysaccharide composition, which are the targets of this study., Methods: With grapevine used as a model plant, this study applied an immunogold-scanning electron microscopy technique to simultaneously analyse at high resolution intervessel PM structures and major pectic and hemicellulosic polysaccharides that make up intervessel PMs., Key Results: Intervessel PMs in functional xylem showed significant structural variation, with about 90 % of them being structurally intact with smooth or relatively smooth surfaces and the remaining 10 % with progressively degraded structures. The results also elucidated details of the removal process of cell wall materials from the intervessel PM surface toward its depth during its natural degradation. Four groups of pectic and hemicellulosic polysaccharides were immunolocalized in intervessel PMs and differed in their spatial distribution and abundance. Weakly methyl-esterified homogalacturonans (WMe-HGs, detected by JIM5) were abundant in the surface layer, heavily methyl-esterified homogalacturonans (HMe-HGs, detected by JIM7) and xylans detected by CCRC-M140 were mostly found in deeper layers, and fucosylated xyloglucans (F-XyGs, detected by CCRC-M1) were more uniformly distributed at different depths of the intervessel PM., Conclusions: Intervessel PMs displayed diverse structural variations in grapevine. They contained certain major groups of pectic and hemicellulosic polysaccharides with different spatial distributions and abundance. This information is crucial to reveal the polysaccharide profiling of the primary cell wall and to understand the roles of intervessel PMs in the regulation of water transport as well as in a plant's susceptibility to vascular diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In our study we investigated the relationships between adipocytokines in adipose tissue (AT) and cardiovascular disease (CVD) risk factors; (2) Methods: fat tissue biopsies were obtained from 134 patients with stable CAD undergoing coronary artery bypass grafting and 120 patients undergoing aortic or mitral valve replacement. Adipocytes were isolated from subcutaneous (SAT), epicardial (EAT), and perivascular AT (PVAT) samples, and cultured for 24 h, after which gene expression of adipocytokines in the culture medium was determined; (3) Results: men showed reduced ADIPOQ expression in EAT and PVAT, LEP expression in PVAT, and LEPR expression in SAT and PVAT compared to women. Men also exhibited higher SAT and lower PVAT IL6 than women. Meanwhile, dyslipidemia associated with decreased ADIPOQ expression in EAT and PVAT, LEPR in EAT, and IL6 in PVAT. Arterial hypertension (AH) associated with low EAT and PVAT ADIPOQ, and high EAT LEP, SAT, as well as PVAT LEPR, and IL6 in SAT and EAT. ADIPOQ expression decreased with increased AH duration over 20 years against an increased LEP background in ATs. Smoking increased ADIPOQ expression in all ATs and increased LEP in SAT and EAT, however, decreased LEPR in PVAT. Patients 51-59 years old exhibited the highest EAT and PVAT LEP, IL-6, and LEPR expression compared to other age groups; (4) Conclusions: decreased EAT ADIPOQ expression against an increased pro-inflammatory IL6 background may increase atherogenesis and contribute to CAD progression in combination with risk factors including male sex, dyslipidemia, and AH., Competing Interests: 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 © 2022 Gruzdeva, Dyleva, Belik, Sinitsky, Kozyrin and Barbarash.)

This study aimed to investigate the lipid profiles of aqueous humor from polypoidal choroidal vasculopathy (PCV) patients and identify potential biomarkers to increase the understanding of PCV pathomechanism. An ultra-high performance liquid chromatography-tandem mass spectrometry based untargeted lipidomic analysis was performed to acquire lipid profiles of aqueous humor of PCV patients and control subjects. Differentially expressed lipids were identified by univariate and multivariate analyses. A receiver operator characteristic curve (ROC) analysis was conducted to confirm the potential of identified lipids as biomarkers. Sixteen PCV patients and twenty-eight control subjects were enrolled in this study. In total, we identified 33 lipid classes and 639 lipid species in aqueous humor using the LipidSearch software. Of them, 50 differential lipids were obtained by combining univariate and multivariate statistical analyses (VIP>1 and P < 0.05), and 19 potential lipid biomarkers were identified by ROC analysis. In addition, significant alterations were found in several metabolic pathways, including glycerophospholipid, glycerolipid, and glycosylphosphatidylinositol-anchor biosynthesis. This study is the first to systematically characterize the alterations in lipid profiles in aqueous humor of PCV patients and screen for the potential lipid biomarkers for PCV diagnosis and treatment intervention. The results of this study are likely to broaden our understanding of the pathogenesis of PCV and contribute to improvements in the diagnosis and treatment of the disease., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The function of platelets - and thereby the balance between thrombosis and hemostasis - critically depends on their lipid composition. At the same time, platelets are capable of interacting with inflammatory cells by releasing lipids in a paracrine manner. Over the years, many studies have emphasized the importance of both, membrane and signaling lipids, in the signaling pathways underlying arterial thrombosis and chronic inflammation. In line with this, an imbalance of platelet lipid homeostasis is associated with thrombo-inflammatory diseases such as acute coronary syndrome. By establishing quantitative platelet lipidomic analysis, an opportunity has arisen to deepen our knowledge about platelet lipids regulating thrombo-inflammation and vice versa. Past and future investigations in this upcoming field are of great clinical importance since they will presumably pave the way for the identification of novel biomarkers. In addition, targeting specific regulators of the platelet lipid metabolism is a promising strategy to receive both anti-thrombotic and anti-inflammatory therapeutics and could be beneficial to a wide variety of patients with vascular thrombo-inflammatory diseases. This review summarizes the latest scientific findings in the field of platelet lipidomics research and does so by focusing on the metabolism of sphingolipids, oxylipins and phosphoinositides, which are affected by dynamic modifications in a pathophysiological manner. Further, this review elucidates the impact of these platelet lipids on thrombo-inflammatory cardiovascular diseases and highlights potential diagnostic and therapeutic targets., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

One of the major challenges of cardiovascular primary prevention approach is the absence of early biomarkers of endothelial dysfunction which may be useful for identifying at-risk subjects. Endothelial dysfunction is a systemic disorder in which traditional cardiovascular risk factors, such as aging, gender, hypertension, smoking, hyperglycemia, and dyslipidemia, as well as emerging risk determinants, such as fetal factors, gut microbiome alteration, clonal hematopoiesis, air pollution, and sleep disorders act synergistically to tip the endothelial balance in favor of vasoconstrictive, pro-inflammatory, and pro-thrombotic phenotypes. Endothelial dysfunction can start already in fetal life and may be regained once detrimental stimuli are removed. The hallmark of endothelial dysfunction is a marked reduction of nitric oxide (NO) bioavailability owing to epigenetic-sensitive dysregulation of the endothelial nitric oxide synthase (eNOS) gene and upregulation of reactive oxygen species (ROS) in endothelial cells (ECs). Advance in liquid-based assays and molecular biology tools are providing novel potential EC-specific biomarkers for prediction and diagnosis of endothelial dysfunction. Significant associations between clinically useful indexes of endothelial dysfunction, mainly brachial artery flow-mediated dilation (FMD), and increased number of endothelial microparticles (EMPs), increased levels of endoglin and endocan, as well as reduced levels of irisin were observed in subjects with one or more traditional risk factors. However, none entered in clinical practice yet. Smoking cessation, weight loss, physical exercise, and diet control are the milestones of cardiovascular primary prevention, and they may restore endothelial function via epigenetic-sensitive pathways able to reduce inflammation and oxidative stress and increase NO production . We briefly summarize well-known and novel molecular routes driving early endothelial dysfunction mainly in human ECs and related potential biomarkers which may add predictive or diagnostic value to the traditional non-invasive techniques. Also, we focus on clinical trials investigating lifestyle modifications and their impact on molecular routes involved in restoring endothelial function., (Copyright © 2022 Elsevier Inc. All rights reserved.)

A key aspect of cytokine-induced changes as observed in sepsis is the dysregulated activation of endothelial cells (ECs), initiating a cascade of inflammatory signaling leading to leukocyte adhesion/migration and organ damage. The therapeutic targeting of ECs has been hampered by concerns regarding organ-specific EC heterogeneity and their response to inflammation. Using in vitro and in silico analysis, we present a comprehensive analysis of the proteomic changes in mouse lung, liver and kidney ECs following exposure to a clinically relevant cocktail of proinflammatory cytokines. Mouse lung, liver and kidney ECs were incubated with TNF-α/IL-1β/IFN-γ for 4 or 24 h to model the cytokine-induced changes. Quantitative label-free global proteomics and bioinformatic analysis performed on the ECs provide a molecular framework for the EC response to inflammatory stimuli over time and organ-specific differences. Gene Ontology and PANTHER analysis suggest why some organs are more susceptible to inflammation early on, and show that, as inflammation progresses, some protein expression patterns become more uniform while additional organ-specific proteins are expressed. These findings provide an in-depth understanding of the molecular changes involved in the EC response to inflammation and can support the development of drugs targeting ECs within different organs. Data are available via ProteomeXchange (identifier PXD031804).

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia , a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca 2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca 2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling., Competing Interests: 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 © 2022 Batori, Chen, Bordan, Haigh, Su, Verin, Barman, Stepp, Chakraborty, Lucas and Fulton.)

Significance: Emerging evidence suggests that perivascular adipose tissue (PVAT) has a relevant role in the control of vascular tone in physiology and pathology. Healthy PVAT has anticontractile, anti-inflammatory, and antioxidative actions. Accumulating data from both human and experimental animal models indicate that PVAT dysfunction is conceivably coupled to cardiovascular diseases, and it is associated with vascular inflammation, oxidative stress, and arterial remodeling. Therefore, "healthy" PVAT may constitute a novel therapeutic target for the prevention and treatment of cardiovascular diseases. Recent Advances: Hydrogen sulfide (H 2 S) has been recognized as a vascular anti-contractile factor released from PVAT. The enzymes deputed to H 2 S biosynthesis are variously expressed in PVAT and strictly dependent on the vascular bed and species. Metabolic and cardiovascular diseases can alter the morphological and secretory characteristics of PVAT, influencing also the H 2 S signaling. Here, we discuss the role of PVAT-derived H 2 S in healthy conditions and its relevance in alterations occurring in vascular disorders. Critical Issues: We discuss how a better understanding may help in the prevention of vascular dysfunction related to alteration in PVAT-released H 2 S as well as the importance of the interplay between PVAT and H 2 S. Future Directions: We propose future directions to evaluate the contribution of each enzyme involved in H 2 S biosynthesis and their alteration/switch occurring in vascular disorders and the remaining challenges in investigating the role of H 2 S. Antioxid. Redox Signal . 37, 84-97.

Spotted fever group rickettsioses caused by Rickettsia ( R ) are devastating human infections, which mainly target microvascular endothelial cells (ECs) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial - infected ECs, namely R -ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R -ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R -ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R -ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation., Competing Interests: 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 © 2022 Zhou, Bei, Qiu, Chang, Nyong, Vasilakis, Yang, Krishnan, Khanipov, Jin, Fang, Gaitas and Gong.)

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45- cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

Background: Corneal transplantation is the only way to treat serious corneal diseases caused by corneal endothelial dysfunction. However, the shortage of donor corneal tissues and human corneal endothelial cells (HCECs) remains a worldwide challenge. We cultivated HCECs by the use of a conditioned medium from orbital adipose-derived stem cells (OASC-CM) in vitro. Then the HCECs were used to treat animal corneal endothelial dysfunction models via cell transplantation. The purpose of this study was to conduct a long-term observation and evaluation after cell transplantation., Methods: Orbital adipose-derived stem cells (OASCs) were isolated to prepare the conditioned medium (CM). HCECs were cultivated and expanded by the usage of the CM (CM-HCECs). Then, related corneal endothelial cell (CEC) markers were analyzed by immunofluorescence. The cell proliferation ability was also tested. CM-HCECs were then transplanted into monkey corneal endothelial dysfunction models by injection. We carried out a 24-month postoperative preclinical observation and verified the long-term effect by histological examination and transcriptome sequencing., Results: CM-HCECs strongly expressed CEC-related markers and maintained polygonal cell morphology even after 10 passages. At 24 months after cell transplantation, there was a CEC density of more than 2400 cells per square millimeter (range, 2408-2685) in the experimental group. A corneal thickness (CT) of less than 550 μm (range, 490-510) was attained. Gene sequencing showed that the gene expression pattern of CM-HCECs was similar to that of transplanted cells and HCECs., Conclusions: Transplantation of CM-HCECs into monkey corneal endothelial dysfunction models resulted in a transparent cornea after 24 months. This research provided a promising prospect of cell-based therapy for corneal endothelial diseases., (© 2022. The Author(s).)

The endothelium has a fundamental role in the cardiovascular complications of coronavirus disease 2019 (COVID-19). Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particularly affects endothelial cells. The virus binds to the angiotensin-converting enzyme 2 (ACE-2) receptor (present on type 2 alveolar cells, bronchial epithelial cells, and endothelial cells), and induces a cytokine storm. The cytokines tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 have particular effects on endothelial cells-leading to endothelial dysfunction, endothelial cell death, changes in tight junctions, and vascular hyperpermeability. Under normal conditions, apoptotic endothelial cells are removed into the bloodstream. During COVID-19, however, endothelial cells are detached more rapidly, and do not regenerate as effectively as usual. The loss of the endothelium on the luminal surface abolishes all of the vascular responses mediated by the endothelium and nitric oxide production in particular, which results in greater contractility. Moreover, circulating endothelial cells infected with SARS-CoV-2 act as vectors for viral dissemination by forming clusters that migrate into the circulation and reach distant organs. The cell clusters and the endothelial dysfunction might contribute to the various thromboembolic pathologies observed in COVID-19 by inducing the formation of intravascular microthrombi, as well as by triggering disseminated intravascular coagulation. Here, we review the contributions of endotheliopathy and endothelial-cell-derived extracellular vesicles to the pathogenesis of COVID-19, and discuss therapeutic strategies that target the endothelium in patients with COVID-19.

Elevated circulating levels of nutrient-derived trimethylamine N-oxide (TMAO) have been associated with the onset and progression of cardiovascular disease by promoting athero-thrombosis. However, in conditions like bariatric surgery (Roux-en-Y gastric bypass, RYGB), stable increases of plasma TMAO are associated with improved endothelial function and reduced cardiovascular morbidity and mortality, thus questioning whether a mechanistic relationship between TMAO and endothelial dysfunction exists. Herein, we translationally assessed the effects of acute TMAO exposure on endothelial dysfunction, thrombosis and stroke. After RYGB, fasting circulating levels of TMAO increased in patients and obese rats, in parallel with an improved gluco-lipid profile and higher circulating bile acids. The latter enhanced FXR-dependent signalling in rat livers, which may lead to higher TMAO synthesis post RYGB. In lean rats, acute TMAO injection (7 mg kg -1 ) 1.5-h before sacrifice and ex-vivo 30-min incubation of thoracic aortas with 10 -6  M TMAO did not impair vasodilation in response to acetylcholine (Ach), glucagon-like peptide 1, or insulin. Similarly, in lean WT mice (n = 5-6), TMAO injection prior to subjecting mice to ischemic stroke or arterial thrombosis did not increase its severity compared to vehicle treated mice. Endothelial nitric oxide synthase (eNOS) activity and intracellular stress-activated pathways remained unaltered in aorta of TMAO-injected rats, as assessed by Western Blot. Pre-incubation of human aortic endothelial cells with TMAO (10 -6  M) did not alter NO release in response to Ach. Our results indicate that increased plasmatic TMAO in the near-physiological range seems to be a neutral bystander to vascular function as translationally seen in patients after bariatric surgery or in healthy lean rodent models and in endothelial cells exposed acutely to TMAO., (© 2022. The Author(s).)

Endothelial dysfunction plays a critical role in many human angiogenesis-related diseases, including cancer and retinopathies. Small non-coding microRNAs (miRNAs) repress gene expression at the post-transcriptional level. They are critical for endothelial cell gene expression and function and are involved in many pathophysiological processes. The miR-181 family is one of the essential angiogenic regulators. This review summarizes the current state of knowledge of the role of miR-181 family members in endothelial cell dysfunction, with emphasis on their pathophysiological roles in aberrant angiogenesis. The actions of miR-181 members are summarized concerning their targets and associated major angiogenic signaling pathways in a cancer-specific context. Elucidating the underlying functional mechanisms of miR-181 family members that are dysregulated in endothelial cells or cancer cells is invaluable for developing miRNA-based therapeutics for angiogenesis-related diseases such as retinopathies, angiogenic tumors, and cancer. Finally, potential clinical applications of miR-181 family members in anti-angiogenic tumor therapy are discussed.

Background: Sarco/endoplasmic reticulum Ca 2+ -ATPase2 (SERCA2) is impaired in various organs in animal models of diabetes. The purpose of this study was to test the effects of an allosteric SERCA2 activator (CDN1163) on glucose intolerance, hepatosteatosis, skeletal muscle function, and endothelial dysfunction in diabetic ( db / db ) mice. Methods: Either CDN1163 or vehicle was injected intraperitoneally into 16-week-old male control and db / db mice for 5 consecutive days. Results: SERCA2 protein expression was decreased in the aorta of db / db mice. In isometric tension measurements of aortic rings from db / db mice treated with CDN1163, acetylcholine (ACh)-induced relaxation was improved. In vivo intraperitoneal administrations of CDN 1163 also increased ACh-induced relaxation. Moreover, CDN1163 significantly decreased blood glucose in db / db mice at 60 and 120 min during a glucose tolerance test; it also decreased serum insulin levels, hepatosteatosis, and oxygen consumption in skeletal muscle during the early period of exercise in db / db mice. Conclusions: CDN1163 directly improved aortic endothelial dysfunction in db / db mice. Moreover, CDN1163 improved hepatosteatosis, skeletal muscle function, and insulin resistance in db / db mice. The activation of SERCA2 might be a strategy for the all the tissue expressed SERCA2a improvement of endothelial dysfunction and the target for the organs related to insulin resistance.

Pulmonary hypertension in sickle cell disease is an independent predictor of mortality, yet the pathogenesis of pulmonary vascular disease in chronic hemolytic disorders remains incompletely understood and treatment options are limited primarily to supportive care. The release of extracellular hemoglobin has been implicated in the development of pulmonary hypertension, and in this study we explored the direct effects of hemin, the oxidized moiety of heme, on the pulmonary artery endothelium. We found that low dose hemin exposure leads to significantly increased endothelial cell proliferation, migration, and cytokine release as markers of endothelial dysfunction. Protein expression changes in our pulmonary artery endothelial cells showed upregulation of mesenchymal markers after hemin treatment in conjunction with a decrease in endothelial markers. Endothelial to mesenchymal transition (EndoMT) resulting from hemin exposure was further confirmed by showing upregulation of the transcription factors SNAI1 and SLUG, known to regulate EndoMT. Lastly, given the endothelial dysfunction and phenotypic transition observed, the endothelial cytoskeleton was considered a potential novel target. Inhibiting myosin light chain kinase, to prevent phosphorylation of myosin light chain and cytoskeletal contraction, attenuated hemin-induced endothelial hyper-proliferation, migration, and cytokine release. The findings in this study implicate hemin as a key inducer of endothelial dysfunction through EndoMT, which may play an important role in pulmonary vascular remodeling during the development of pulmonary hypertension in chronic hemolytic states.

We aimed to examine the effect of Honokiol (HKL) on endothelial dysfunction in type 2 diabetic rats and its possible mechanism. A high-fat diet and streptozotocin (STZ) were used to establish the type 2 diabetic model in rats. Part of these rats were intraperitoneally injected with HKL 10 mg/kg daily. Then the expression of Ser1177 phosphorylation of endothelial nitric oxide synthase (p-eNOS), eNOS, and CD31, vasodilation function, insulin signaling, indicators of oxidative stress and relative signaling pathway were measured. Human umbilical vein endothelial cells (HUVECs) were used to explore the underlying mechanism of the effect of HKL on high glucose-related endothelial injury in vitro. The data showed that HKL could reverse the decline of the expression of p-eNOS and CD31, endothelium-related vasodilation dysfunction, insulin resistance and activation of oxidative stress induced by type 2 diabetes in vivo. The similar results were obtained in vitro. In summary, our study demonstrates that HKL improves endothelial function and diminishes insulin resistance and oxidative stress, suggesting that HKL could be used as a treatment option for diabetes in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Sickle cell disease (SCD) patients experience chronic inflammation and recurrent vaso-occlusive episodes during their entire lifetime. Inflammation in SCD occurs with the overexpression of several inflammatory mediators, including transforming growth factor beta-1 (TGF-β1), a major immune regulator. In this study, we aimed to investigate the role played by TGF-β1 in vascular inflammation and vaso-occlusion in an animal model of SCD. Using intravital microscopy, we found that a daily dose of recombinant TGF-β1 administration for three consecutive days significantly reduced TNFα-induced leukocyte rolling, adhesion, and extravasation in the microcirculation of SCD mice. In contrast, immunological neutralization of TGF-β, in the absence of inflammatory stimulus, considerably increased these parameters. Our results indicate, for the first time, that TGF-β1 may play a significant ameliorative role in vascular SCD pathophysiology, modulating inflammation and vaso-occlusion. The mechanisms by which TGF-β1 exerts its anti-inflammatory effects in SCD, however, remains unclear. Our in vitro adhesion assays with TNFα-stimulated human neutrophils suggest that TGF-β1 can reduce the adhesive properties of these cells; however, direct effects of TGF-β1 on the endothelium cannot be ruled out. Further investigation of the wide range of the complex biology of this cytokine in SCD pathophysiology and its potential therapeutical use is needed.

Abstract: The abnormal proliferation of vascular smooth muscle cells (VSMCs) is a key pathological characteristic of vascular proliferative diseases. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that plays an important role in regulating cell growth, motility, proliferation, and survival, as well as gene expression in response to hypoxia, growth factors, and nutrients. Increasing evidence shows that mTOR also regulates VSMC proliferation in vascular proliferative diseases and that mTOR inhibitors, such as rapamycin, effectively restrain VSMC proliferation. However, the molecular mechanisms linking mTOR to vascular proliferative diseases remain elusive. In our review, we summarize the key roles of the mTOR and the recent discoveries in vascular proliferative diseases, focusing on the therapeutic potential of mTOR inhibitors to target the mTOR signaling pathway for the treatment of vascular proliferative diseases. In this study, we discuss mTOR inhibitors as promising candidates to prevent VSMC-associated vascular proliferative diseases., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Uremic toxins, such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS), contribute to endothelial dysfunction in chronic kidney disease (CKD). This process is mediated by several cellular pathways, but it is unclear whether cAMP-responsive element-binding protein (CREB) and activating transcription factor 1 (ATF1) participate in endothelial dysfunction in uremic conditions despite playing roles in inflammatory modulation. This study aimed to evaluate the expression, activation, and transcriptional activity of CREB/ATF1 in endothelial cells exposed to PCS, IS, and uremic serum (US). In vitro, ATF1 protein levels were increased by PCS and IS, whereas CREB levels were enhanced only by IS. Activation through CREB-Ser 133 and ATF1-Ser 63 phosphorylation was induced by PCS, IS, and US. We evaluated the CREB/ATF1 transcriptional activity by analyzing the expression of their target genes, including ICAM1, PTGS2, NOX1, and SLC22A6, which are related to endothelial dysfunction through their roles in vascular inflammation, oxidative stress, and cellular uptake of PCS and IS. The expression of ICAM1, PTGS2 and NOX1 genes was increased by PCS, IS, and US, whereas that of SLC22A6 was induced only by IS. KG-501, a CREB inhibitor, restored the inductive effects of PCS on ICAM1, PTGS2, and NOX1 expression; IS on ICAM1, PTGS2 and SLC22A6 expression; and US on NOX1 expression. The presence of CREB and ATF1 was observed in healthy arteries and in arteries of patients with CKD, which were structurally damaged. These findings suggest that CREB/ATF1 is activated by uremic toxins and may play a relevant role in endothelial dysfunction in CKD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pathologies of the vasculature including the microvasculature are often complex in nature, leading to loss of physiological homeostatic regulation of patency and adequate perfusion to match tissue metabolic demands. Microvascular dysfunction is a key underlying element in the majority of pathologies of failing organs and tissues. Contributing pathological factors to this dysfunction include oxidative stress, mitochondrial dysfunction, endoplasmic reticular (ER) stress, endothelial dysfunction, loss of angiogenic potential and vascular density, and greater senescence and apoptosis. In many clinical settings, current pharmacologic strategies use a single or narrow targeted approach to address symptoms of pathology rather than a comprehensive and multifaceted approach to address their root cause. To address this, efforts have been heavily focused on cellular therapies and cell-free therapies (e.g., exosomes) that can tackle the multifaceted etiology of vascular and microvascular dysfunction. In this review, we discuss 1 ) the state of the field in terms of common therapeutic cell population isolation techniques, their unique characteristics, and their advantages and disadvantages, 2 ) common molecular mechanisms of cell therapies to restore vascularization and/or vascular function, 3 ) arguments for and against allogeneic versus autologous applications of cell therapies, 4 ) emerging strategies to optimize and enhance cell therapies through priming and preconditioning, and, finally, 5 ) emerging strategies to bolster therapeutic effect. Relevant and recent clinical and animal studies using cellular therapies to restore vascular function or pathologic tissue health by way of improved vascularization are highlighted throughout these sections.

Cellular senescence is a complex, dynamic process consisting of the irreversible arrest of growth and gradual deterioration of cellular function. Endothelial senescence affects the cell's ability to repair itself, which is essential for maintaining vascular integrity and leads to the development of endothelial dysfunction, which has an important role in the pathogenesis of cardiovascular diseases. Senescent endothelial cells develop a particular, senescence-associated secretory phenotype (SASP) that detrimentally affects both surrounding and distant endothelial cells, thereby facilitating the ageing process and development of age-related disorders. Recent studies highlight the role of endothelial senescence and its dysfunction in the pathophysiology of several age-related diseases. MicroRNAs are small noncoding RNAs that have an important role in the regulation of gene expression at the posttranscriptional level. Recently, it has been discovered that miRNAs could importantly contribute to endothelial cell senescence. Overall, the research focus has been shifting to new potential mechanisms and targets to understand and prevent the structural and functional changes in ageing senescent endothelial cells in order to prevent the development and limit the progression of the wide spectrum of age-related diseases. The aim of this review is to provide some insight into the most important pathways involved in the modulation of endothelial senescence and to reveal the specific roles of several miRNAs involved in this complex process. Better understanding of miRNA's role in endothelial senescence could lead to new approaches for prevention and possibly also for the treatment of endothelial cells ageing and associated age-related diseases.

Small vessel disease (SVD) is one of the most frequent pathological conditions which lead to dementia. Biochemical and neuroimaging might help correctly identify the clinical diagnosis of this relevant brain disease. The microvascular alterations which underlie SVD have common origins, similar cognitive outcomes, and common vascular risk factors. Nevertheless, the arteriolosclerosis process, which underlines SVD development, is based on different mechanisms, not all completely understood, which start from a chronic hypoperfusion state and pass through a chronic brain inflammatory condition, inducing a significant endothelium activation and a consequent tissue remodeling action. In a recent review, we focused on the pathophysiology of SVD, which is complex, involving genetic conditions and different co-morbidities (i.e., diabetes, chronic hypoxia condition, and obesity). Currently, many points still remain unclear and discordant. In this paper, we wanted to focus on new biomarkers, which can be the expression of the endothelial dysfunction, or of the oxidative damage, which could be employed as markers of disease progression or for future targets of therapies. Therefore, we described the altered response to the endothelium-derived nitric oxide-vasodilators (ENOV), prostacyclin, C-reactive proteins, and endothelium-derived hyperpolarizing factors (EDHF). At the same time, due to the concomitant endothelial activation and chronic neuroinflammatory status, we described hypoxia-endothelial-related markers, such as HIF 1 alpha, VEGFR2, and neuroglobin, and MMPs. We also described blood-brain barrier disruption biomarkers and imaging techniques, which can also describe perivascular spaces enlargement and dysfunction. More studies should be necessary, in order to implement these results and give them a clinical benefit.

Cardiovascular disease (CVD) is a major cause of mortality worldwide. A better understanding of the mechanisms underlying CVD is key for better management or prevention. Oxidative stress has been strongly implicated in the pathogenesis of CVD. Indeed, several studies demonstrated that reactive oxygen species (ROS), via different mechanisms, can lead to endothelial cell (EC) dysfunction, a major player in the etiology of several CVDs. ROS appears to modulate a plethora of EC biological processes that are critical for the integrity of the endothelial function. This review seeks to dissect the role of oxidative stress-induced endothelial dysfunction in CVD development, with emphasis on the underlying mechanisms and pathways. Special attention is given to ROS-induced reduction of NO bioavailability, ROS-induced inflammation, and ROS-induced mitochondrial dysfunction. A better understanding and appraisal of these pathways may be essential to attenuate oxidative stress or reverse EC dysfunction, and hence, reduce CVD burden., Competing Interests: The authors declare no conflict of interest. GP (Gianfranco Pintus) is serving as one of the Editorial Board members of this journal. We declare that GP (Gianfranco Pintus) had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to GP (Giordano Pula)., (© 2022 The Author(s). Published by IMR Press.)

Endothelial cell senescence is involved in endothelial dysfunction and vascular diseases. However, the detailed mechanisms of endothelial senescence are not fully understood. Here, we demonstrated that deficiency of developmentally regulated GTP-binding protein 2 ( DRG2 ) induces senescence and dysfunction of endothelial cells. DRG2 knockout (KO) mice displayed reduced cerebral blood flow in the brain and lung blood vessel density. We also determined, by Matrigel plug assay, aorta ring assay, and in vitro tubule formation of primary lung endothelial cells, that deficiency in DRG2 reduced the angiogenic capability of endothelial cells. Endothelial cells from DRG2 KO mice showed a senescence phenotype with decreased cell growth and enhanced levels of p21 and phosphorylated p53, γH2AX, senescence-associated β-galactosidase (SA-β-gal) activity, and senescence-associated secretory phenotype (SASP) cytokines. DRG2 deficiency in endothelial cells upregulated arginase 2 ( Arg2 ) and generation of reactive oxygen species. Induction of SA-β-gal activity was prevented by the antioxidant N-acetyl cysteine in endothelial cells from DRG2 KO mice. In conclusion, our results suggest that DRG2 is a key regulator of endothelial senescence, and its downregulation is probably involved in vascular dysfunction and diseases.

Reactive intralymphovascular immunoblastic proliferations (ILVIPs) may mimic aggressive lymphomas and are rarely reported. Herein, we characterize the clinicopathologic features of 8 patients with ILVIPs. No patients had lymphadenopathy, hepatosplenomegaly, or other findings suggestive of lymphoma. The ILVIPs involved the small or large intestine (n=5) and appendix (n=3). Patients were evaluated for abdominal pain, suspected appendicitis, intestinal obstruction, diverticulitis, volvulus, or tumor resection. Histologic sections showed expanded lymphovascular spaces filled by intermediate to large immunoblasts, positive for CD38, CD43, CD45, CD79a, and MUM1/IRF4 in all cases tested. Five of 6 (83%) cases were positive for CD30. CD20 was weakly positive in a subset of cells in 2 (25%) cases, and PAX5 was weakly positive in 4 (50%) cases. The immunoblasts expressed polytypic light chains in all cases tested. In 1 case, a subset of immunoblasts expressed T-cell markers indicating the presence of a T-cell component. The immunoblasts were negative for ALK, BCL-2, BCL-6, CD10, CD56, CD138, and Epstein-Barr virus-encoded small RNA in all cases assessed. The proliferation index shown by Ki-67 was high with a median of 80%. In all 6 cases tested, the immunoblasts were shown within lymphatic channels highlighted by D2-40. In conclusion, ILVIPs can be rarely observed in patients with inflammatory or infectious conditions, especially in gastrointestinal tract surgical specimens. The immunoblasts are predominantly of B-lineage with a postgerminal center immunophenotype and are located within lymphatic channels. It is essential to distinguish reactive ILVIPs from aggressive lymphomas to avoid unnecessary therapy., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Cells in the tissues and organs of a living body are subjected to mechanical forces, such as pressure, friction, and tension from their surrounding environment. Cells are equipped with a mechanotransduction mechanism by which they perceive mechanical forces and transmit information into the cell interior, thereby causing physiological or pathogenetic mechano-responses. Endothelial cells (ECs) lining the inner surface of blood vessels are constantly exposed to shear stress caused by blood flow and a cyclic strain caused by intravascular pressure. A number of studies have shown that ECs are sensitive to changes in these hemodynamic forces and alter their morphology and function, sometimes by modifying gene expression. The mechanism of endothelial mechanotransduction has been elucidated, and the plasma membrane has recently been shown to act as a mechanosensor. The lipid order and cholesterol content of plasma membranes change immediately upon the exposure of ECs to hemodynamic forces, resulting in a change in membrane fluidity. These changes in a plasma membrane's physical properties affect the conformation and function of various ion channels, receptors, and microdomains (such as caveolae and primary cilia), thereby activating a wide variety of downstream signaling pathways. Such endothelial mechanotransduction works to maintain circulatory homeostasis; however, errors in endothelial mechanotransduction can cause abnormalities in vascular physiological function, leading to the initiation and progression of various vascular diseases, such as hypertension, thrombosis, aneurysms, and atherosclerosis. Recent advances in detailed imaging technology and computational fluid dynamics analysis have enabled us to evaluate the hemodynamic forces acting on vascular tissue accurately, contributing greatly to our understanding of vascular mechanotransduction and the pathogenesis of vascular diseases, as well as the development of new therapies for vascular diseases.