Contribution of the Arterial Cells to Atherosclerosis and Plaque Formation.

Atherosclerosis is commonly known as an inflammatory disease that is characterized by lipid deposition in the arterial wall, causing gradual restriction or complete blockade of blood flow, which can cause complications such as myocardial infarction, stroke, or peripheral artery disease. Several factors contribute to initiation and progression of atherosclerotic plaque formation. The role of macrophages and leukocytes in atherosclerosis have been well explored. Here, we provide an overview of what has been reported on the role and impact of the arterial cells on plaque formation, and vice versa. The atherogenic environment can trigger transformation and dedifferentiation of the endothelial cells, smooth muscle cells, and fibroblasts whereby they can either directly contribute to plaque formation, or influence its composition. Recent studies have demonstrated the plasticity in the identity of the arterial cells, formation of intermediate cell types that share the characteristics of multiple cell types, and have revealed novel roles and functions for these cells in atherosclerosis. The potential for all vascular cells to cross-transdifferentiate, and detection of cells with mosaic characteristics in the atherosclerotic plaques reveal that the plaque environment is a complex and dynamic environment that could regulate the disease progression independent from the circulating lipid levels. We will also provide an overview on the interplay between sex and atherosclerosis, which has remained an underexplored area.

GPR126 is a specifier of blood-brain barrier formation in the mouse central nervous system.

The blood-brain barrier (BBB) acquires unique properties to regulate neuronal function during development. The formation of the BBB, which occurs in tandem with angiogenesis, is directed by the Wnt/ß-catenin signaling pathway. Yet the exact molecular interplay remains elusive. Our study reveals the G protein-coupled receptor GPR126 as a critical target of canonical Wnt signaling, essential for the development of the BBB's distinctive vascular characteristics and its functional integrity. Endothelial cell-specific deletion of the Gpr126 gene in mice induced aberrant vascular morphogenesis, resulting in disrupted BBB organization. Simultaneously, heightened transcytosis in vitro compromised barrier integrity, resulting in enhanced vascular permeability. Mechanistically, GPR126 enhanced endothelial cell migration, pivotal for angiogenesis, acting through an interaction between LRP1 and ß1 integrin, thereby balancing the levels of ß1 integrin activation and recycling. Overall, we identified GPR126 as a specifier of an organotypic vascular structure, which sustained angiogenesis and guaranteed the acquisition of the BBB properties during development.

Inflammatory breast cancer microenvironment repertoire based on DNA methylation data deconvolution reveals actionable targets to enhance the treatment efficacy.

BACKGROUND: Although the clinical signs of inflammatory breast cancer (IBC) resemble acute inflammation, the role played by infiltrating immune and stromal cells in this aggressive disease is uncharted. The tumor microenvironment (TME) presents molecular alterations, such as epimutations, prior to morphological abnormalities. These changes affect the distribution and the intricate communication between the TME components related to cancer prognosis and therapy response. Herein, we explored the global DNA methylation profile of IBC and surrounding tissues to estimate the microenvironment cellular composition and identify epigenetically dysregulated markers. METHODS: We used the HiTIMED algorithm to deconvolve the bulk DNA methylation data of 24 IBC and six surrounding non-tumoral tissues (SNT) (GSE238092) and determine their cellular composition. The prognostic relevance of cell types infiltrating IBC and their relationship with clinicopathological variables were investigated. CD34 (endothelial cell marker) and CD68 (macrophage marker) immunofluorescence staining was evaluated in an independent set of 17 IBC and 16 non-IBC samples. RESULTS: We found lower infiltration of endothelial, stromal, memory B, dendritic, and natural killer cells in IBC than in SNT samples. Higher endothelial cell (EC) and stromal cell content were related to better overall survival. EC proportions positively correlated with memory B and memory CD8+ T infiltration in IBC. Immune and EC markers exhibited distinct DNA methylation profiles between IBC and SNT samples, revealing hypermethylated regions mapped to six genes (CD40, CD34, EMCN, HLA-G, PDPN, and TEK). We identified significantly higher CD34 and CD68 protein expression in IBC compared to non-IBC. CONCLUSIONS: Our findings underscored cell subsets that distinguished patients with better survival and dysregulated markers potentially actionable through combinations of immunotherapy and epigenetic drugs.

Dopaminylation of endothelial TPI1 suppresses ferroptotic angiocrine signals to promote lung regeneration over fibrosis.

Lungs can undergo facultative regeneration, but handicapped regeneration often leads to fibrosis. How microenvironmental cues coordinate lung regeneration via modulating cell death remains unknown. Here, we reveal that the neurotransmitter dopamine modifies the endothelial niche to suppress ferroptosis, promoting lung regeneration over fibrosis. A chemoproteomic approach shows that dopamine blocks ferroptosis in endothelial cells (ECs) via dopaminylating triosephosphate isomerase 1 (TPI1). Suppressing TPI1 dopaminylation in ECs triggers ferroptotic angiocrine signaling to aberrantly activate fibroblasts, leading to a transition from lung regeneration to fibrosis. Mechanistically, dopaminylation of glutamine (Q) 65 residue in TPI1 directionally enhances TPI1's activity to convert dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP), directing ether phospholipid synthesis to glucose metabolism in regenerating lung ECs. This metabolic shift attenuates lipid peroxidation and blocks ferroptosis. Restoring TPI1 Q65 dopaminylation in an injured endothelial niche overturns ferroptosis to normalize pro-regenerative angiocrine function and alleviate lung fibrosis. Overall, dopaminylation of TPI1 balances lipid/glucose metabolism and suppresses pro-fibrotic ferroptosis in regenerating lungs.

Elucidating of the metabolic impact of risperidone on brain microvascular endothelial cells using untargeted metabolomics-based LC-MS.

Risperidone is useful for the treatment of schizophrenia symptoms; however, it also has side effects, and an overdose can be harmful. The metabolic effects of risperidone at high therapeutic doses and its metabolites have not been elucidated. Endogenous cellular metabolites may be comprehensively analyzed using untargeted metabolomics-based liquid chromatography-mass spectrometry (LC-MS), which can reveal changes in cell regulation and metabolic pathways. By identifying the metabolites and pathway changes using a nontargeted metabolomics-based LC-MS approach, we aimed to shed light on the potential toxicological effects of high-dose risperidone on brain microvascular endothelial cells (MVECs) associated with the human blood brain barrier. A total of 42 metabolites were selected as significant putative metabolites of the toxicological response of high-dose risperidone in MVECs. Six highly correlated pathways were identified, including those involving diacylglycerol, fatty acid, ceramide, glycerophospholipid, amino acid, and tricarboxylic acid metabolism. We demonstrated that methods focused on metabolomics are useful for identifying metabolites that may be used to clarify the mechanism of drug-induced toxicity.

Nuclear factor of activated T-cells 5 is indispensable for a balanced adaptive transcriptional response of lung endothelial cells to hypoxia.

AIMS: Chronic hypoxia causes detrimental structural alterations in the lung, which may cause pulmonary hypertension and are partially mediated by the endothelium. While its relevance for the development of hypoxia-associated lung diseases is well known, determinants controlling the initial adaptation of the lung endothelium to hypoxia remain largely unexplored. METHODS AND RESULTS: We revealed that hypoxia activates the transcription factor nuclear factor of activated T-cells 5 (NFAT5) and studied its regulatory function in murine lung endothelial cells (MLECs). EC-specific knockout of Nfat5 (Nfat5(EC)-/-) in mice exposed to normobaric hypoxia (10% O2) for 21 days promoted vascular fibrosis and aggravated the increase in pulmonary right ventricular systolic pressure as well as right ventricular dysfunction as compared with control mice. Microarray- and single-cell RNA-sequencing-based analyses revealed an impaired growth factor-, energy-, and protein-metabolism-associated gene expression in Nfat5-deficient MLEC after exposure to hypoxia for 7 days. Specifically, loss of NFAT5 boosted the expression and release of platelet-derived growth factor B (Pdgfb)-a hypoxia-inducible factor 1 alpha (HIF1α)-regulated driver of vascular smooth muscle cell (VSMC) growth-in capillary MLEC of hypoxia-exposed Nfat5(EC)-/- mice, which was accompanied by intensified VSMC coverage of distal pulmonary arteries. CONCLUSION: Collectively, our study shows that early and transient subpopulation-specific responses of MLEC to hypoxia may determine the degree of organ dysfunction in later stages. In this context, NFAT5 acts as a protective transcription factor required to rapidly adjust the endothelial transcriptome to cope with hypoxia. Specifically, NFAT5 restricts HIF1α-mediated Pdgfb expression and consequently limits muscularization and resistance of the pulmonary vasculature.

Akt is a mediator of artery specification during zebrafish development.

The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. While vascular endothelial growth factor a (VEGF-A) drives DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B, Akt, in zebrafish by generating a quadruple mutant (aktΔ/Δ), where expression and activity of all akt genes-akt 1, 2, 3a, and 3b are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to reestablish normal arterial specification in aktΔ/Δ. The Akt-loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.

Human heart-on-a-chip microphysiological system comprising endothelial cells, fibroblasts, and iPSC-derived cardiomyocytes.

In recent years, research on organ-on-a-chip technology has been flourishing, particularly for drug screening and disease model development. Fibroblasts and vascular endothelial cells engage in crosstalk through paracrine signaling and direct cell-cell contact, which is essential for the normal development and function of the heart. Therefore, to faithfully recapitulate cardiac function, it is imperative to incorporate fibroblasts and vascular endothelial cells into a heart-on-a-chip model. Here, we report the development of a human heart-on-a-chip composed of induced pluripotent stem cell (iPSC)-derived cardiomyocytes, fibroblasts, and vascular endothelial cells. Vascular endothelial cells cultured on microfluidic channels responded to the flow of culture medium mimicking blood flow by orienting themselves parallel to the flow direction, akin to in vivo vascular alignment in response to blood flow. Furthermore, the flow of culture medium promoted integrity among vascular endothelial cells, as evidenced by CD31 staining and lower apparent permeability. The tri-culture condition of iPSC-derived cardiomyocytes, fibroblasts, and vascular endothelial cells resulted in higher expression of the ventricular cardiomyocyte marker IRX4 and increased contractility compared to the bi-culture condition with iPSC-derived cardiomyocytes and fibroblasts alone. Such tri-culture-derived cardiac tissues exhibited cardiac responses similar to in vivo hearts, including an increase in heart rate upon noradrenaline administration. In summary, we have achieved the development of a heart-on-a-chip composed of cardiomyocytes, fibroblasts, and vascular endothelial cells that mimics in vivo cardiac behavior.

miRNA-Driven Regulation of Endothelial-to-Mesenchymal Transition Differs among Thoracic Aortic Aneurysms.

Thoracic aortic aneurysms (TAAs) represent a serious health concern, as they are associated with early aortic dissection and rupture. TAA formation is triggered by genetic conditions, in particular Marfan syndrome (MFS) and bicuspid aortic valve (BAV). During the aneurysmatic process, aortic endothelial cells can undergo endothelial-to-mesenchymal transition (End-MT) with consequent phenotypic and functional alterations. We previously documented that MFS TAA is characterized by miR-632-driven End-MT exacerbation, whereas in BAV aortopathy, the occurrence of this process remains still controversial. We investigated the End-MT process and the underlined regulatory mechanisms in BAV, TAV and MFS TAA tissues. Gene expression and immunohistochemical analysis were performed in order to analyze some important miRNAs and genes characterizing End-MT. We documented that BAV endothelium maintains the expression of the endothelial homeostasis markers, such as ERG, CD31 and miR-126-5p, while it shows lower levels of miR-632 and mesenchymal markers compared with MFS. Interestingly, we also found higher levels of miR-632 in MFS patients' blood. Our findings definitively demonstrate that the End-MT process does not characterize BAV that, among the other TAAs, better maintains the endothelial features. In addition, our results suggest miR-632 as a promising diagnostic/prognostic factor in MFS aortopathy.

Dynamics of Endothelial Cell Diversity and Plasticity in Health and Disease.

Endothelial cells (ECs) are vital structural units of the cardiovascular system possessing two principal distinctive properties: heterogeneity and plasticity. Endothelial heterogeneity is defined by differences in tissue-specific endothelial phenotypes and their high predisposition to modification along the length of the vascular bed. This aspect of heterogeneity is closely associated with plasticity, the ability of ECs to adapt to environmental cues through the mobilization of genetic, molecular, and structural alterations. The specific endothelial cytoarchitectonics facilitate a quick structural cell reorganization and, furthermore, easy adaptation to the extrinsic and intrinsic environmental stimuli, known as the epigenetic landscape. ECs, as universally distributed and ubiquitous cells of the human body, play a role that extends far beyond their structural function in the cardiovascular system. They play a crucial role in terms of barrier function, cell-to-cell communication, and a myriad of physiological and pathologic processes. These include development, ontogenesis, disease initiation, and progression, as well as growth, regeneration, and repair. Despite substantial progress in the understanding of endothelial cell biology, the role of ECs in healthy conditions and pathologies remains a fascinating area of exploration. This review aims to summarize knowledge and concepts in endothelial biology. It focuses on the development and functional characteristics of endothelial cells in health and pathological conditions, with a particular emphasis on endothelial phenotypic and functional heterogeneity.

HMGB1 released from pyroptotic vascular endothelial cells promotes immune disorders in exertional heatstroke.

BACKGROUND AND OBJECTIVE: Exertional heatstroke (EHS) mainly occurs in healthy young people with rapid onset and high mortality. EHS immune disorders can cause systemic inflammatory responses and multiple organ failure; however, the underlying mechanisms remain unclear. As high mobility group box 1 (HMGB1) is a prototypical alarmin that activates inflammatory and immune responses, this study aimed to investigate the effect and mechanism of HMGB1 in the pathogenesis of EHS. METHODS: Peripheral blood mononuclear cell (PBMC) transcriptome sequencing of healthy volunteers, classical heatstroke patients, and EHS patients was performed. A mouse model of EHS was established and murine tissue damage was evaluated by H&E staining. HMGB1 localization and release were visualized using immunofluorescence staining. Human umbilical vein endothelial cells (HUVECs) and THP-1 cells were co-cultured to study the effects of HMGB1 on macrophages. A neutralizing anti-HMGB1 antibody was used to evaluate the efficacy of EHS treatment in mice. RESULTS: Plasma and serum HMGB1 levels were significantly increased in EHS patients or mice. EHS-induced endothelial cell pyroptosis promoted HMGB1 release in mice. HMGB1 derived from endothelial cell pyroptosis enhanced macrophage pyroptosis, resulting in immune disorders under EHS conditions. Administration of anti-HMGB1 markedly alleviated tissue injury and systemic inflammatory responses after EHS. CONCLUSIONS: The release of HMGB1 from pyroptotic endothelial cells after EHS promotes pyroptosis of macrophages and systemic inflammatory response, and HMGB1-neutralizing antibody therapy has good application prospects for EHS.

Endothelial Cell-Derived Extracellular Vesicles Promote Aberrant Neutrophil Trafficking and Subsequent Remote Lung Injury.

The development of acute respiratory distress syndrome (ARDS) in sepsis is associated with substantial morbidity and mortality. However, the molecular pathogenesis underlying sepsis-induced ARDS remains elusive. Neutrophil heterogeneity and dysfunction contribute to uncontrolled inflammation in patients with ARDS. A specific subset of neutrophils undergoing reverse transendothelial migration (rTEM), which is characterized by an activated phenotype, is implicated in the systemic dissemination of inflammation. Using single-cell RNA sequencing (scRNA-seq), it identified functionally activated neutrophils exhibiting the rTEM phenotype in the lung of a sepsis mouse model using cecal ligation and puncture. The prevalence of neutrophils with the rTEM phenotype is elevated in the blood of patients with sepsis-associated ARDS and is positively correlated with disease severity. Mechanically, scRNA-seq and proteomic analys revealed that inflamed endothelial cell (EC) released extracellular vesicles (EVs) enriched in karyopherin subunit beta-1 (KPNB1), promoting abluminal-to-luminal neutrophil rTEM. Additionally, EC-derived EVs are elevated and positively correlated with the proportion of rTEM neutrophils in clinical sepsis. Collectively, EC-derived EV is identified as a critical regulator of neutrophil rTEM, providing insights into the contribution of rTEM neutrophils to sepsis-associated lung injury.

Increased vascular deposition of oxidized LDL in untreated juvenile dermatomyositis.

BACKGROUND: Juvenile dermatomyositis (JDM) is a systemic vasculopathy associated with metabolic derangements and possible increased risk for premature atherosclerosis. Oxidation of low-density lipoprotein (LDL) in the endothelium is an early step in atherosclerotic plaque formation. It is not known if oxidized LDL is altered in children with untreated JDM. The deposition of oxidized LDL in the vasculature of muscle biopsies (MBx) from patients with untreated JDM and pediatric controls was assessed. FINDINGS: Frozen tissue sections of MRI-directed MBx from 20 female children with untreated JDM and 5 female controls were stained with DAPI and fluorescently labeled antibodies against von Willebrand factor (vWF) and LDL oxidized by copper (oxLDL). Blood vessels were identified by positive vWF staining, and total fluorescence of oxLDL within the vessel walls was measured. Children with untreated JDM had increased deposition of oxLDL in the walls of muscle vasculature compared to healthy children (difference in means ± SEM = 19.86 ± 8.195, p = 0.03). Within the JDM cohort, there was a trend towards increased oxLDL deposition with longer duration of untreated disease (r = 0.43, p = 0.06). There was no significant correlation found between oxLDL deposition and markers of acute JDM disease activity including disease activity scores or muscle enzymes. CONCLUSIONS: This study found increased deposition of oxLDL within blood vessels of children with untreated JDM supporting the concern that these children are at increased risk for premature atherosclerosis from chronic exposure to vascular oxLDL. This study highlights the importance of early diagnosis and treatment initiation to ameliorate cardiovascular damage.

FLI-1-driven regulation of endothelial cells in human diseases.

Endothelial cells (ECs) are widely distributed in the human body and play crucial roles in the circulatory and immune systems. ECs dysfunction contributes to the progression of various chronic cardiovascular, renal, and metabolic diseases. As a key transcription factor in ECs, FLI-1 is involved in the differentiation, migration, proliferation, angiogenesis and blood coagulation of ECs. Imbalanced FLI-1 expression in ECs can lead to various diseases. Low FLI-1 expression leads to systemic sclerosis by promoting fibrosis and vascular lesions, to pulmonary arterial hypertension by promoting a local inflammatory state and vascular lesions, and to tumour metastasis by promoting the EndMT process. High FLI-1 expression leads to lupus nephritis by promoting a local inflammatory state. Therefore, FLI-1 in ECs may be a good target for the treatment of the abovementioned diseases. This comprehensive review provides the first overview of FLI-1-mediated regulation of ECs processes, with a focus on its influence on the abovementioned diseases and existing FLI-1-targeted drugs. A better understanding of the role of FLI-1 in ECs may facilitate the design of more effective targeted therapies for clinical applications, particularly for tumour treatment.

Cell Death in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by increased pulmonary vascular resistance because of vascular remodeling and vasoconstriction. Subsequently, PAH leads to right ventricular hypertrophy and heart failure. Cell death mechanisms play a significant role in development and tissue homeostasis, and regulate the balance between cell proliferation and differentiation. Several basic and clinical studies have demonstrated that multiple mechanisms of cell death, including pyroptosis, apoptosis, autophagy, ferroptosis, anoikis, parthanatos, and senescence, are closely linked with the pathogenesis of PAH. This review summarizes different cell death mechanisms involved in the death of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs), the primary target cells in PAH. This review summarizes the role of these cell death mechanisms, associated signaling pathways, unique effector molecules, and various pro-survival or reprogramming mechanisms. The aim of this review is to summarize the currently known molecular mechanisms underlying PAH. Further investigations of the cell death mechanisms may unravel new avenues for the prevention and treatment of PAH.

Exosomes Isolation, Purification, and Characterization.

Exosomes are double-layered lipid membranous nanovesicles that are endosomal in origin and secreted by almost all cells. They are 30-130 nm in size and contain various molecular signatures such as miRNAs, mRNAs, DNA, lipids, and proteins. Due to their highly heterogeneous content, exosomes have a major role in influencing cellular physiology and pathology. Although exosome research has been in progress for a long time, its biomedical applications have recently been expanding due to its bio-friendly nature. However, the most challenging part is its isolation to obtain quality exosomes with good yield. Therefore, in this chapter, we have described appropriate protocols for exosome isolation and characterization along with alternative purification methods.

Generation of Directly Reprogrammed Human Endothelial Cells.

Direct reprogramming provides a novel breakthrough for generating functional endothelial cells (ECs) without the need for intermediate stem or progenitor states, offering a promising resource for cardiovascular research and treatment. ETV2 is a key transcription factor that has been identified as a pioneering factor for specifying endothelial lineage. Achieving precise ETV2 induction is essential for effective endothelial reprogramming, and maintaining the reprogrammed cellular phenotype relies on a specific combination of growth factors and small molecules. Thus, we hereby provide a straightforward and comprehensive protocol for generating two distinct types of reprogrammed ECs (rECs) from human dermal fibroblasts (HDFs). Early rECs demonstrate a robust neovascularization property but lack the mature EC phenotype, while late rECs exhibit phenotypical similarity to human postnatal ECs and have a neovascularization capacity similar to early rECs. Both cell types can be derived from human somatic source cells, making them suitable for personalized disease investigations, drug discovery, and disease therapy.

CBX7 promotes choroidal neovascularization by activating the HIF-1α/VEGF pathway in choroidal vascular endothelial cells.

Vascular endothelial growth factor (VEGF) signaling is crucial for choroidal neovascularization (CNV), a major pathological feature of neovascular age-related macular degeneration (nAMD). Gene transcription of VEGF is mainly regulated by hypoxia-inducible factor 1-alpha (HIF-1α). The chromobox (CBX) family polycomb protein (Pc) subgroup includes CBX2, CBX4, CBX6, CBX7, and CBX8. CBX4 enhances hypoxia-induced VEGF expression and angiogenesis in hepatocellular carcinoma (HCC) cells by increasing HIF-1α's transcriptional activity. The objective of the study was to examine the functions of members of the CBX family Pc subgroup in choroidal vascular endothelial cells (CVECs) during CNV. CBX4 and CBX7 expression was up-regulated in hypoxic human choroidal vascular endothelial cells (HCVECs). In HCVECs, CBX7 facilitated HIF-1α transcription and expression, while CBX4 did not. In HCVECs, CBX7 stimulated HIF-1α's nuclear translocation and transcriptional activity, which in turn stimulated VEGF transcription and expression. The CBX7/HIF-1α/VEGF pathway promoted the migration, proliferation, and tube formation of HCVECs. The CBX7/HIF-1α/VEGF pathway was up-regulated in CVECs and in the mouse model with laser-induced CNV. Mouse CNV was lessened by the blockade of CBX7 through the down-regulation of HIF-1α/VEGF. In conclusion, CBX7 enhanced pro-angiogenic behaviors of hypoxic CVECs by up-regulating the HIF-1α/VEGF pathway, which contributing to the formation of mouse laser-induced CNV.

VEGF-A165a and angiopoietin-2 differently affect the barrier formed by retinal endothelial cells.

Exposure to VEGF-A165a over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A. In view of the clinical relevance of anti-VEGF therapy, we also studied whether blocking VEGF-A-driven signaling is sufficient to prevent barrier dysfunction induced by a combination of both growth factors. Although angiopoietin-2 stimulated proliferation of iBREC, the formed barrier was not weakened at a concentration of 3 nM: Cell index values remained high and expression or subcellular localization of claudin-1 and vascular endothelial cadherin, respectively, were not affected. Angiopoietin-2 enhanced the changes induced by VEGF-A165a and this was more pronounced at lower concentrations of VEGF-A165a. Specific inhibition of the VEGF receptors with tivozanib as well as interfering with binding of VEGF-A to its receptors with bevacizumab prevented the detrimental effects of the growth factors; dual binding of angiopoietin-2 and VEGF-A by faricimab was marginally more efficient. Uptake of extracellular angiopoietin-2 by iBREC can be efficiently prevented by addition of faricimab which is also internalized by the cells. Exposure of the cells to faricimab over several days stabilized their barrier, confirming that inhibition of VEGF-A signaling is not harmful to this cell type. Taken together, our results confirm the dominant role of VEGF-A165a in processes resulting in increased permeability of retinal endothelial cells in which angiopoietin-2 might play a minor modulating role.

Notch transcriptional target tmtc1 maintains vascular homeostasis.

Proper lung function requires the maintenance of a tight endothelial barrier while simultaneously permitting the exchange of macromolecules and fluids to underlying tissue. Disruption of this barrier results in an increased vascular permeability in the lungs, leading to acute lung injury. In this study, we set out to determine whether transcriptional targets of Notch signaling function to preserve vascular integrity. We tested the in vivo requirement for Notch transcriptional signaling in maintaining the pulmonary endothelial barrier by using two complementary endothelial-specific Notch loss-of-function murine transgenic models. Notch signaling was blocked using endothelial-specific activation of an inhibitor of Notch transcriptional activation, Dominant Negative Mastermindlike (DNMAML; CDH5CreERT2), or endothelial-specific loss of Notch1 (Notch1f/f; CDH5CreERT2). Both Notch mutants increased vascular permeability with pan-Notch inhibition by DNMAML showing a more severe phenotype in the lungs and in purified endothelial cells. RNA sequencing of primary lung endothelial cells (ECs) identified novel Notch targets, one of which was transmembrane O-mannosyltransferase targeting cadherins 1 (tmtc1). We show that tmtc1 interacts with vascular endothelial cadherin (VE-cadherin) and regulates VE-cadherin egress from the endoplasmic reticulum through direct interaction. Our findings demonstrate that Notch signaling maintains endothelial adherens junctions and vascular homeostasis by a transcriptional mechanism that drives expression of critical factors important for processing and transport of VE-cadherin.