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The endothelium, a cellular monolayer lining the blood vessel wall, plays a critical role in maintaining multiorgan health and homeostasis. Endothelial functions in health include dynamic maintenance of vascular tone, angiogenesis, hemostasis, and the provision of an antioxidant, anti-inflammatory, and antithrombotic interface. Dysfunction of the vascular endothelium presents with impaired endothelium-dependent vasodilation, heightened oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Recent studies have implicated altered endothelial cell metabolism and endothelial-to-mesenchymal transition as new features of endothelial dysfunction. Endothelial dysfunction is regarded as a hallmark of many diverse human panvascular diseases, including atherosclerosis, hypertension, and diabetes. Endothelial dysfunction has also been implicated in severe coronavirus disease 2019. Many clinically used pharmacotherapies, ranging from traditional lipid-lowering drugs, antihypertensive drugs, and antidiabetic drugs to proprotein convertase subtilisin/kexin type 9 inhibitors and interleukin 1β monoclonal antibodies, counter endothelial dysfunction as part of their clinical benefits. The regulation of endothelial dysfunction by noncoding RNAs has provided novel insights into these newly described regulators of endothelial dysfunction, thus yielding potential new therapeutic approaches. Altogether, a better understanding of the versatile (dys)functions of endothelial cells will not only deepen our comprehension of human diseases but also accelerate effective therapeutic drug discovery. In this review, we provide a timely overview of the multiple layers of endothelial function, describe the consequences and mechanisms of endothelial dysfunction, and identify pathways to effective targeted therapies. SIGNIFICANCE STATEMENT: The endothelium was initially considered to be a semipermeable biomechanical barrier and gatekeeper of vascular health. In recent decades, a deepened understanding of the biological functions of the endothelium has led to its recognition as a ubiquitous tissue regulating vascular tone, cell behavior, innate immunity, cell-cell interactions, and cell metabolism in the vessel wall. Endothelial dysfunction is the hallmark of cardiovascular, metabolic, and emerging infectious diseases. Pharmacotherapies targeting endothelial dysfunction have potential for treatment of cardiovascular and many other diseases.

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1β and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its mechanism being incompletely understood. Emerging evidence has demonstrated that the endothelium represents the Achilles' heel in COVID-19 patients and that endothelial dysfunction precipitates COVID-19 and accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. Primary human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) were treated with serum from control subjects or COVID-19 patients. Downstream monocyte adhesion and associated gene/protein expression was evaluated in endothelial cells exposed to COVID-19 patient serum in the presence of KLF2 activator (Atorvastatin) or KLF2 overexpression by an adenoviral vector. Here, we demonstrate that the expression of KLF2 was significantly reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1β and TNF-α, two cytokines observed in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis and reduced angiogenesis). Treatment of HPMECs with atorvastatin or KLF2 adenovirus ameliorate COVID-19 serum-induced increase in endothelial inflammation and monocyte adhesion by increasing KLF2 expression. Altogether, the present study demonstrates that genetic and pharmacological activation of KLF2 represses COVID-19 associated endothelial dysfunction, heralding a potentially new direction to treat endothelialitis accompanying COVID-19.

Atherosclerosis, the principal cause of cardiovascular death worldwide, is a pathological disease characterized by fibro-proliferation, chronic inflammation, lipid accumulation, and immune disorder in the vessel wall. As the atheromatous plaques develop into advanced stage, the vulnerable plaques are prone to rupture, which causes acute cardiovascular events, including ischemic stroke and myocardial infarction. Emerging evidence has suggested that atherosclerosis is also an epigenetic disease with the interplay of multiple epigenetic mechanisms. The epigenetic basis of atherosclerosis has transformed our knowledge of epigenetics from an important biological phenomenon to a burgeoning field of cardiovascular research. Here, we provide a systematic and up-to-date overview of the current knowledge of three distinct but interrelated epigenetic processes (including DNA methylation, histone methylation/acetylation, and non-coding RNAs), in atherosclerotic plaque development and instability. Mechanistic and conceptual advances in understanding the biological roles of various epigenetic modifiers in regulating gene expression and functions of endothelial cells (vascular homeostasis, leukocyte adhesion, endothelial-mesenchymal transition, angiogenesis, and mechanotransduction), smooth muscle cells (proliferation, migration, inflammation, hypertrophy, and phenotypic switch), and macrophages (differentiation, inflammation, foam cell formation, and polarization) are discussed. The inherently dynamic nature and reversibility of epigenetic regulation, enables the possibility of epigenetic therapy by targeting epigenetic “writers”, “readers”, and “erasers”. Several Food Drug Administration-approved small-molecule epigenetic drugs show promise in pre-clinical studies for the treatment of atherosclerosis. Finally, we discuss potential therapeutic implications and challenges for future research involving cardiovascular epigenetics, with an aim to provide a translational perspective for identifying novel biomarkers of atherosclerosis, and transforming precision cardiovascular research and disease therapy in modern era of epigenetics.

Endothelial cells line the innermost layer of a blood vessel and regulate multiple functional aspects of vascular homeostasis, such as vascular tone, inflammation, vessel permeability, and angiogenesis. Endothelial cells possess multiple specialized mechanosensing molecules or microstructures (termed “mechanosensors”) that sense different patterns of blood flow (in a concerted manner). Mechanosensing is a prerequisite step for integrating the mechanosignal into endothelial cells and leads to different patterns of endothelial gene expression. Emerging evidence using genetically engineered mice and pharmacological modulators has shown that these mechanosensors are critical regulators of endothelial function and probably the pathogenesis of atherosclerosis-related cardiovascular diseases. In this chapter, we delineate the specific 2role of each identified mechanosensor in vascular endothelial cell biology and atherosclerosis and highlight the possibility of targeting these mechanosensors to treat atherosclerosis.

Angiogenesis, the formation of new blood vessels, is tightly regulated by gene transcriptional programs. Yin Ying 1 (YY1) is a ubiquitously distributed transcription factor with diverse and complex biological functions; however, little is known about the cell-type-specific role of YY1 in vascular development and angiogenesis. Here we report that endothelial cell (EC)-specific YY1 deletion in mice led to embryonic lethality as a result of abnormal angiogenesis and vascular defects. Tamoxifen-inducible EC-specific YY1 knockout (YY1(iΔEC)) mice exhibited a scarcity of retinal sprouting angiogenesis with fewer endothelial tip cells. YY1(iΔEC) mice also displayed severe impairment of retinal vessel maturation. In an ex vivo mouse aortic ring assay and a human EC culture system, YY1 depletion impaired endothelial sprouting and migration. Mechanistically, YY1 functions as a repressor protein of Notch signaling that controls EC tip-stalk fate determination. YY1 deficiency enhanced Notch-dependent gene expression and reduced tip cell formation. Specifically, YY1 bound to the N-terminal domain of RBPJ (recombination signal binding protein for Ig Kappa J region) and competed with the Notch coactivator MAML1 (mastermind-like protein 1) for binding to RBPJ, thereby impairing the NICD (intracellular domain of the Notch protein)/MAML1/RBPJ complex formation. Our study reveals an essential role of endothelial YY1 in controlling sprouting angiogenesis through directly interacting with RBPJ and forming a YY1-RBPJ nuclear repression complex.

Endothelial dysfunction is the common molecular basis of multiple human diseases, such as atherosclerosis, diabetes, hypertension, and acute lung injury. Therefore, primary isolation of high-purity endothelial cells (ECs) is crucial to study the mechanisms of endothelial function and disease pathogenesis. Mouse lung ECs (MLECs) are widely used in vascular biology and lung cell biology studies such as pulmonary inflammation, angiogenesis, vessel permeability, leukocyte/EC interaction, nitric oxide production, and mechanotransduction. Thus, in this paper, we describe a simple, and reproducible protocol for the isolation and culture of MLECs from adult mice using collagenase I-based enzymatic digestion, followed by sequential sorting with PECAM1 (also known as CD31)- and ICAM2 (also known as CD102)-coated microbeads. The morphology of isolated MLECs were observed with phase contrast microscope. MLECs were authenticated by CD31 immunoblotting, and immunofluorescent staining of established EC markers VE-cadherin and von Willebrand factor (vWF). Cultured MLECs also showed functional characteristics of ECs, evidenced by DiI-oxLDL uptake assay and THP-1 monocyte adhesion assay. Finally, we used MLECs from endothelium-specific enhancer of zeste homolog 2 (EZH2) knockout mice to show the general applicability of our protocol. To conclude, we describe here a simple and reproducible protocol to isolate highly pure and functional ECs from adult mouse lungs. Isolation of ECs from genetically engineered mice is important for downstream phenotypic, genetic, or proteomic studies.

Angiogenesis is essential for growth and repair, and irregularity of angiogenesis contributes to numerous malignant, inflammatory and ischemic disorders. Transcription factors that regulate angiogenic gene expression are critical for proper vascular development and homeostasis. Here we show that YingYang1( YY1) is essential for vascular development both in embryonic and postnatal angiogenesis. We first found that endothelium-specific deletion of YY1 leads to embryonic lethality due to vascular defects. We further found inducible endothelial YY1 knockout (YY1 iΔEC ) mice exhibited abnormality of sprouting angiogenesis with a blunted-end, aneurysm-like structure with fewer and dysmorphic filopodia in EC tip cells compared with those of wild-type mice. Similar results are also identified in vitro by using cultured HUVEC cells. Moreover, we found that endothelial deletion of YY1 also leads to severe impairments in retinal vessel remodeling and maturation. Mechanistically, we show that YY1 is a robust endogenous inhibitor of Notch1 signaling that controls explicitly endothelial cell fate. YY1 deficiency remarkably enhances Hey1 expression, resulting in impaired retinal sprouting angiogenesis, which can be rescued by the treatment of the Notch inhibitor. Furthermore, YY1 regulates Hey1 expression by binding to RBP-J via a highly conserved ankyrin (ANK) repeat domain, then inhibits the NICD-MAML1- RBPJ complex formation by competing with MAML1 binding to RBP-J, which attenuates expression of downstream target genes Hey1. Lastly, we found that tumor growth and angiogenesis is reduced significantly in YY1 iΔEC mice, suggesting that endothelial YY1 is required for pathological angiogenesis in mouse tumor models. Altogether, our study reveals a new role for YY1 as a central mediator of Notch signaling and an essential regulator in both physiologic and pathologic angiogenesis.

Objective: We have previously shown that docking protein Grb2-associated binder 1 (Gab1) is a mechano-effector protein in response to fluid shear stress and regulates postnatal angiogenesis. The aim of this study was to determine the in vivo role of endothelial Gab1 in flow-mediated vascular remodeling and atherosclerosis and explore the underlying mechanisms. Methods and Results: To determine the role of endothelial Gab1 in disturbed flow-induced vascular remodeling in vivo, we performed partial carotid artery ligation in Gab1 endothelium-restricted knockout (Gab1-ecKO) mice and wild-type (WT) littermates, and we observed that Gab1-ecKO mice resulted in increased intima-media thickness. To examine the role of endothelial Gab1 in atherosclerosis, we next crossed Gab1-ecKO mice with ApoE KO mice. After partial ligation, Gab1-ecKO;ApoE KO mice under high fat diet showed increased atherosclerotic lesion size compared to Gab1-WT;ApoE KO mice. The levels of proatherogenic genes intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and leukocyte infiltration were all increased in Gab1-ecKO;ApoE KO mice. Using loss- and gain-of-function studies in cultured human endothelial cells (ECs), we found that Gab1 depletion by siRNA augmented monocyte adhesion to ECs by increasing ICAM-1 and VCAM-1 expression in response to the proinflammatory cytokine TNF-α. Conversely, adenoviral overexpression of Gab1 inhibited TNFα-induced monocyte adhesion to ECs and upregulation of ICAM-1 and VCAM-1 in ECs. Conclusions: These results demonstrate that endothelial Gab1 represses disturbed flow-induced vascular remodeling and atherogenesis through inhibition of vascular inflammation. Our findings suggest that Gab1 activation might represent novel approaches for the treatment of vascular diseases, including intimal hyperplasia and atherosclerosis.