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Background Disturbed flow generates oscillatory shear stress (OSS), which in turn leads to endothelial inflammation and atherosclerosis. Piezo1, a biomechanical force sensor, plays a crucial role in the cardiovascular system. However, the specific role of Piezo1 in atherosclerosis remains to be fully elucidated. Methods and Results We detected the expression of Piezo1 in atherosclerotic mice and endothelial cells from regions with disturbed blood flow. The pharmacological inhibitor Piezo1 inhibitor (GsMTx4) was used to evaluate the impact of Piezo1 on plaque progression and endothelial inflammation. We examined Piezo1's direct response to OSS in vitro and its effects on endothelial inflammation. Furthermore, mechanistic studies were conducted to explore the potential molecular cascade through which Piezo1 mediates endothelial inflammation in response to OSS. Our findings revealed the upregulation of Piezo1 in apoE−/− (apolipoprotein E) atherosclerotic mice, which is associated with disturbed flow. Treatment with GsMTx4 not only delayed plaque progression but also mitigated endothelial inflammation in both chronic and disturbed flow‐induced atherosclerosis. Piezo1 was shown to facilitate calcium ions (Ca2+) influx in response to OSS, thereby activating endothelial inflammation. This inflammatory response was attenuated in the absence of Piezo1. Additionally, we identified that under OSS, Piezo1 activates the Ca2+/CaM/CaMKII (calmodulin/calmodulin‐dependent protein kinases Ⅱ) pathways, which subsequently stimulate downstream kinases FAK (focal adhesion kinase) and Src. This leads to the activation of the OSS‐sensitive YAP (yes‐associated protein), ultimately triggering endothelial inflammation. Conclusions Our study highlights the key role of Piezo1 in atherosclerotic endothelial inflammation, proposing the Piezo1–Ca2+/CaM/CaMKII‐FAK/Src‐YAP axis as a previously unknown endothelial mechanotransduction pathway. Piezo1 is expected to become a potential therapeutic target for atherosclerosis and cardiovascular diseases.

Abstract Introduction Systemic sclerosis (SSc) is a complex autoimmune disease characterized by fibrosis, vascular damage, and immune dysregulation. Fractalkine or chemokine (C‐X3‐C motif) ligand 1 (CX3CL1), a chemokine and adhesion molecule, along with its receptor CX3CR1, have been implicated in the inflammatory processes of SSc. CX3CL1 functions as both a chemoattractant and an adhesion molecule, guiding immune cell trafficking. This systematic review examines the role of CX3CL1 and its receptor CX3CR1 in the pathogenesis of SSc, with a focus on pulmonary and vascular complications. Methods A systematic literature search was conducted across databases including PubMed, Scopus, and Web of Science from inception to November 2020. The search focused on studies investigating the CX3CL1/CX3CR1 axis in the context of SSc. Results The review identified elevated CX3CL1 expression in SSc patients, particularly in the skin and lungs, where CX3CR1 is expressed on infiltrating immune cells. Higher levels of CX3CL1 were correlated with the severity of interstitial lung disease in SSc patients, indicating a potential predictive marker for disease progression. CX3CR1‐positive monocytes and NK cells were recruited to inflamed tissues, contributing to fibrosis and tissue damage. Animal studies showed that inhibition of the CX3CL1/CX3CR1 axis reduced fibrosis and improved vascular function. Conclusion The CX3CL1/CX3CR1 axis plays a key role in immune cell recruitment and fibrosis in SSc. Elevated CX3CL1 levels are associated with lung and vascular complications, making it a potential biomarker for disease progression and a promising therapeutic target.

Abstract Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, areas exposed to high shear stress (HSS) are protected from plaque development. Endothelial extracellular vesicles (EVs) have been shown to regulate inflammation and senescence, and therefore play a crucial role in vascular homeostasis. Whilst previous studies have shown links between hemodynamic forces and EV release, the effects of shear stress on the release and uptake of endothelial EVs remains elusive. We aim to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent HUVECs were exposed to LSS or HSS for 24 h. Large and small EVs were isolated from conditioned medium by centrifugation and size exclusion chromatography. They were characterised by TEM, Western blot, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently‐labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions. Additionally, endothelial LSS‐EVs have a greater affinity for HUVECs than HSS‐EVs or EVs derived from platelets, erythrocytes and leukocytes. Proteomic analysis revealed that LSS‐EVs were enriched in adhesion proteins (PECAM1, MCAM), participating in EV uptake by endothelial cells. LSS‐EVs also carried mitochondrial material, which may be implicated in elevating ROS levels in recipient cells. These findings suggest that shear stress influences EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction.

Abstract Angiogenesis is crucial for successful bone defect repair. Co‐transplanting Bone Marrow Stromal Cells (BMSCs) and Endothelial Cells (ECs) has shown promise for vascular augmentation, but it face challenges in hostile tissue microenvironments, including poor cell survival and limited efficacy. In this study, the mitochondria of human BMSCs are isolated and transplanted to BMSCs from the same batch and passage number (BMSCsmito). The transplanted mitochondria significantly boosted the ability of BMSCsmito‐ECs to promote angiogenesis, as assessed by in vitro tube formation and spheroid sprouting assays, as well as in vivo transplantation experiments in balb/c mouse and SD rat models. The Dll4‐Notch1 signaling pathway is found to play a key role in BMSCsmito‐induced endothelial tube formation. Co‐transplanting BMSCsmito with ECs in a rat cranial bone defect significantly improves functional vascular network formation, and improve bone repair outcomes. These findings thus highlight that mitochondrial transplantation, by acting through the DLL4‐Notch1 signaling pathway, represents a promising therapeutic strategy for enhancing angiogenesis and improving bone repair. Hence, mitochondrial transplantation to BMSCS as a therapeutic approach for promoting angiogenesis offers valuable insights and holds much promise for innovative regenerative medicine therapies.

Abstract 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.

Abstract Oligodendrocyte precursor cells (OPCs) migrate extensively using blood vessels as physical scaffolds in the developing central nervous system. Although the association of OPCs with the vasculature is critical for migration, the regulatory mechanisms important for OPCs proliferative and oligodendrocyte development are unknown. Here, a correlation is demonstrated between the developing vasculature and OPCs response during brain development. Deletion of endothelial stimulator of interferon genes (STING) disrupts angiogenesis by inhibiting farnesyl‐diphosphate farnesyltransferase 1 (FDFT1) and thereby reducing cholesterol synthesis. Furthermore, the perturbation of metabolic homeostasis in endothelial cells increases interleukin 17D production which mediates the signal transduction from endothelial cells to OPCs, which inhibits oligodendrocyte development and myelination and causes behavioral abnormalities in adult mice. Overall, these findings indicate how the endothelial STING maintains metabolic homeostasis and contributes to oligodendrocyte precursor cells response in the developing neocortex.

Angiogenesis is a central component of vital biological processes such as wound healing, tissue nourishment, and development. Therefore, angiogenic activities are precisely maintained with secreted factors such as angiopoietin‐1 (Ang1), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF). As an element of intracellular communication, extracellular vesicles (EVs)—particularly EVs of vascular origin—could have key functions in maintaining angiogenesis. However, the functions of EVs in the control of angiogenesis have not been fully studied. In this study, human umbilical vein endothelial cell line (HUVEC)‐derived small EVs (

Abstract Studying acute changes in vascular endothelial cells in humans is challenging. We studied ten African American women and used the J‐wire technique to isolate vein endothelial cells before and after a four‐hour lipid and heparin infusion. Dynamic changes in lipid‐induced oxidative stress and inflammatory markers were measured with fluorescence‐activated cell sorting. We used the surface markers CD31 and CD144 to identify human endothelial cells. Peripheral blood mononuclear cells isolated from blood were used as a negative control. The participants received galantamine (16 mg/day) for 3 months. We previously demonstrated that galantamine treatment effectively suppresses lipid‐induced oxidative stress and inflammation. In this study, we infused lipids to evaluate its potential to increase the activation of endothelial cells, as assessed by the levels of CD54+ endothelial cells and expression of Growth arrest‐specific 6 compared to the baseline sample. Further, we aimed to investigate whether lipid infusion led to increased expression of the oxidative stress markers IsoLGs and nitrotyrosine in endothelial cells. This approach will expedite the in vivo identification of novel pathways linked with endothelial cell dysfunction induced by oxidative stress and inflammatory cytokines. This study describes an innovative method to harvest and study human endothelial cells and demonstrates the dynamic changes in oxidative stress and inflammatory markers release induced by lipid infusion.

Extracellular vesicles (EV), important messengers in intercellular communication, can load and transport various bioactive components and participate in different biological processes. We previously isolated glioma human endothelial cells (GhECs) and found that GhECs, rather than normal human brain endothelial cells (NhECs), exhibit specific enrichment of MYO1C into EVs and promote the migration of glioma cells. In this study, we explored the mechanism by which MYO1C is secreted into EVs. We report that such secretion is dependent on RAB31, RAB27B, and FAS. When expression of RAB31 increases, MYO1C is enriched in secretory EVs. Finally, we identified an EV export mechanism for MYO1C that promotes glioma cell invasion and is dependent on RAB31 in GhECs. In summary, our data indicate that the knockdown of RAB31 can reduce enrichment of MYO1C in extracellular vesicles, thereby attenuating the promotion of glioma cell invasion by GhEC‐EVs.

Abstract Ferroptosis has been confirmed to be associated with various diseases, but the relationship between ferroptosis and atherosclerosis (AS) remains unclear. Our research detailly clarified the roles of ferroptosis in three continuous and main pathological stages of AS respectively (injury of endothelial cells [ECs], adhesion of monocytes, and formation of foam cells). We confirmed that oxidized low‐density lipoprotein (ox‐LDL), the key factor in the pathogenesis of AS, strongly induced ferroptosis in ECs. Inhibition of ferroptosis repressed the adhesion of monocytes to ECs by inhibiting inflammation of ECs. Ferroptosis also participated in the formation of foam cells and lipids by regulating the cholesterol efflux of macrophages. Further research confirmed that ox‐LDL repressedthe activity of glutathione peroxidase 4 (GPX4), the classic lipid peroxide scavenger. Treatment of a high‐fat diet significantly induced ferroptosis in murine aortas and aortic sinuses, which was accompanied by AS lesions and hyperlipidemia. Treatment with ferroptosis inhibitors significantly reduced ferroptosis, hyperlipidemia, and AS lesion development. In conclusion, our research determined that ox‐LDL induced ferroptosis by repressing the activity of GPX4. Antiferroptosis treatment showed promising treatment effects in vivo. Ferroptosis‐associated indexes also showed promising diagnostic potential in AS patients.

Abstract Choroidal atrophy is a common fundus pathological change closely related to the development of age‐related macular degeneration (AMD), retinitis pigmentosa, and pathological myopia. Studies suggest that choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first cells lost in choroidal atrophy. It is found that endothelial cells derived from human pluripotent stem cells (hPSC‐ECs) through the MESP1+ mesodermal progenitor stage express CECs‐specific markers and can integrate into choriocapillaris. Single‐cell RNA‐seq (scRNA‐seq) studies show that hPSC‐ECs upregulate angiogenesis and immune‐modulatory and neural protective genes after interacting with ex vivo ischemic choroid. In a rat model of choroidal ischemia (CI), transplantation of hPSC‐ECs into the suprachoroidal space increases choroid thickness and vasculature density. Close‐up examination shows that engrafted hPSC‐ECs integrate with all layers of rat choroidal vessels and last 90 days. Remarkably, EC transplantation improves the visual function of CI rats. The work demonstrates that hPSC‐ECs can be used to repair choroidal ischemia in the animal model, which may lead to a new therapy to alleviate choroidal atrophy implicated in dry AMD, pathological myopia, and other ocular diseases.

Abstract Long‐term, intense endurance exercise training can occasionally induce endothelial micro‐damage and cardiac fibrosis. The underlying mechanisms are incompletely understood. Twenty healthy, well‐trained male participants (10 runners and 10 cyclists) performed a strenuous high‐intensity interval training (HIIT) session matched by age, height, weight and maximal oxygen consumption. We assessed the acute exercise response of novel cardiac biomarkers of fibrosis [e.g., galectin‐3 (Gal‐3) and soluble suppression of tumorigenicity 2 (sST2)] per exercise modality and their relationship with haemodynamic contributors, such as preload, afterload and cardiac contractility index (CTi), in addition to endothelial damage by sustained activation and shedding of endothelial cells (ECs). Serum Gal‐3 and sST2 concentrations were investigated by enzyme‐linked immunosorbent assays; haemodynamics were analysed via impedance plethysmography and circulating ECs by flow cytometry. The Gal‐3 and sST2 concentrations and ECs were elevated after exercise (P

Abstract Background The plasticity of endothelial cells (ECs) is crucial for tissue response to injury. Myocardial infarction can profoundly affect EC function, leading to a shift toward mesenchymal differentiation. Methods We utilized human single‐nucleus RNA sequencing data to investigate the dynamic changes and cellular interactions between normal and post‐infarction ECs. Results We identified two distinct subpopulations of ECs: A transient subpopulation characterized by short‐term mesenchymal gene expression and a long‐term subpopulation characterized by myocardial gene expression. Trajectory analysis revealed the differentiation pathways and potential roles over time and space. Furthermore, we uncovered the proliferation, differentiation, hypoxic, and inflammatory responses of ECs to injury. Conclusions Our study provides a comprehensive and detailed characterization of endothelial cell states, highlighting the role of activated endothelial cell subpopulations in promoting inflammation and tissue repair after infarction.

Abstract Background Tumor‐associated angiogenesis mediates the growth and metastasis of most solid cancers. Targeted therapies of the VEGF pathways can effectively block these processes but often fail to provide lasting benefits due to acquired resistance and complications. Results Recently, we discovered βIV‐spectrin as a powerful regulator of angiogenesis and potential new target. We previously reported that βIV‐spectrin is dynamically expressed in endothelial cells (EC) to induce VEGFR2 protein turnover during development. Here, we explored how βIV‐spectrin influences the tumor vasculature using the murine B16 melanoma model and determined that loss of EC‐specific βIV‐spectrin dramatically promotes tumor growth and metastasis. Intraperitoneally injected B16 cells formed larger tumors with increased tumor vessel density and greater propensity for metastatic spread particularly to the chest cavity and lung compared to control mice. These results support βIV‐spectrin as a key regulator of tumor angiogenesis and a viable vascular target in cancer.

Abstract Pivotal roles of extracellular vesicles (EVs) in the pathogenesis of central nervous system (CNS) disorders including acute brain injury are increasingly acknowledged. Through the analysis of EVs packaged miRNAs in plasma samples from patients with intracerebral hemorrhage (ICH), it is discovered that the level of EVs packaged miR‐143‐3p (EVs‐miR‐143‐3p) correlates closely with perihematomal edema and neurological outcomes. Further study reveals that, upon ICH, EVs‐miR‐143‐3p is robustly secreted by astrocytes and can shuttle into brain microvascular endothelial cells (BMECs). Heightened levels of miR‐143‐3p in BMECs induce the up‐regulated expression of cell adhesion molecules (CAMs) that bind to circulating neutrophils and facilitate their transendothelial cell migration (TEM) into brain. Mechanism‐wise, miR‐143‐3p directly targets ATP6V1A, resulting in impaired lysosomal hydrolysis ability and reduced autophagic degradation of CAMs. Importantly, a VCAM‐1–targeting EVs system to selectively deliver miR‐143‐3p inhibitor to pathological BMECs is created, which shows satisfactory therapeutic effects in both ICH and traumatic brain injury (TBI) mouse models. In conclusion, the study highlights the causal role of EVs‐miR‐143‐3p in BMECs’ dysfunction in acute brain injury and demonstrates a proof of concept that engineered EVs can be devised as a potentially applicable nucleotide drug delivery system for the treatment of CNS disorders.

Abstract Intercellular communication is critical to the formation and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via the transmission of cell‐generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, mechanically defined, synthetic electrospun fibrous matrices are utilized in conjunction with a microfabrication‐based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during their assembly into interconnected multicellular networks. It is found that cell force‐mediated matrix displacements in deformable fibrous matrices underly directional extension and migration of neighboring ECs toward each other prior to the formation of stable cell‐cell connections enriched with vascular endothelial cadherin (VE‐cadherin). A critical role is also identified for calcium signaling mediated by focal adhesion kinase and mechanosensitive ion channels in MIC that extends to multicellular assembly of 3D vessel‐like networks when ECs are embedded within fibrin hydrogels. These results illustrate a role for cell‐generated forces and ECM mechanical properties in multicellular assembly of capillary‐like EC networks and motivates the design of biomaterials that promote MIC for vascular tissue engineering.

Abstract Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear‐stressed endothelial cells demonstrated that the non‐degradative ubiquitination of several GTPases is regulated by mechano‐signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non‐degradative ubiquitination fine‐tunes the function of GTPases by modifying their interacting network. Specifically, WWP2‐mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress‐induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial‐specific Wwp2‐knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non‐degradative ubiquitination of GTPases in endothelial mechano‐activation.

Abstract Prolactin (PRL) is a polypeptide hormone that is mainly synthesized and secreted by the lactotroph cells of the pituitary. There are two main isoforms of PRL: 23‐kDa PRL (named full‐length PRL) and vasoinhibins (including 5.6–18 kDa fragments). Both act as circulating hormones and cytokines to stimulate or inhibit vascular formation at different stages and neovascularization, including endothelial cell proliferation and migration, protease production, and apoptosis. However, their effects on vascular function and cardiovascular diseases are different or even contrary. In addition to the structure, secretion regulation, and signal transduction of PRL/vasoinhibins, this review focuses on the pathological mechanism and clinical significance of PRL/vasoinhibins in cardiovascular diseases.

Abstract Objective Amiodarone (AM) is a drug commonly used in patients with ventricular arrhythmias. It can damage vascular endothelial cells and easily cause phlebitis. At present, the prevention and treatment of phlebitis induced by the use of AM is not clear due to the lack of corresponding primary research. Isoliquiritigenin (ISL) has an anti‐inflammatory effect, but until now, has not been explored much in the field of research in primary care nursing. The purpose of this study is to investigate the efficacy and mechanism of action of ISL in treating phlebitis induced by AM. Methods In our study, we used human umbilical vein endothelial cells (HUVECs) that were divided into three groups: the NC group (normal), the AM group (AM 30 μmol/L for 24 h), and the ISL pretreatment group (isoliquiritigenin 10 μmol/L after 1 h of pretreatment with amiodarone for 24 h). We used CCK‐8 to detect cell proliferation, cell scratch assay to detect the migration capability of cells, flow cytometry to measure apoptosis, angiogenesis assay to check the total length and total branches of angiogenesis, and PCR and WB to detect the expression of PCNA, casepase‐3, and VEGFA. WB was used to detect NF‐κBp65 and p‐NF‐κBp65 expression. Results Compared with the AM group, the ISL pretreatment promoted cell proliferation and migration, inhibited cell apoptosis, increased the total length and total branches of angiogenesis, and downregulated p‐NF‐κBp65 expression. Conclusion ISL shows promise in the prevention and treatment of clinical phlebitis and can be used as a potential therapeutic drug to prevent phlebitis.

Abstract Diabetes is a known risk factor for various cardiovascular complications, mediated by endothelial dysfunction. Despite the high prevalence of this metabolic disorder, there is a lack of in vitro models that recapitulate the complexity of genetic and environmental factors associated with diabetic endothelial dysfunction. Here, we utilized human induced pluripotent stem cell (iPSC)‐derived endothelial cells (ECs) to develop in vitro models of diabetic endothelial dysfunction. We found that the diabetic phenotype was recapitulated in diabetic patient‐derived iPSC‐ECs, even in the absence of a diabetogenic environment. Subsequent exposure to culture conditions that mimic the diabetic clinical chemistry induced a diabetic phenotype in healthy iPSC‐ECs but did not affect the already dysfunctional diabetic iPSC‐ECs. RNA‐seq analysis revealed extensive transcriptome‐wide differences between cells derived from healthy individuals and diabetic patients. The in vitro disease models were used as a screening platform which identified angiotensin receptor blockers (ARBs) that improved endothelial function in vitro for each patient. In summary, we present in vitro models of diabetic endothelial dysfunction using iPSC technology, taking into account the complexity of genetic and environmental factors in the metabolic disorder. Our study provides novel insights into the pathophysiology of diabetic endothelial dysfunction and highlights the potential of iPSC‐based models for drug discovery and personalized medicine.

Abstract The involvement of endothelial barrier function in abdominal aortic aneurysm (AAA) and its upstream regulators remains unknown. Single‐cell RNA sequencing shows that disrupted endothelial focal junction is an early (3 days) and persistent (28 days) event during Angiotensin II (Ang II)‐induced AAA progression. Consistently, mRNA sequencing on human aortic dissection tissues confirmed downregulated expression of endothelial barrier‐related genes. Aldehyde dehydrogenase 2 (ALDH2), a negative regulator of AAA, is found to be upregulated in the intimal media of AAA samples, leading to testing its role in early‐stage AAA. ALDH2 knockdown/knockout specifically in endothelial cells (ECs) significantly increases expression of EC barrier markers related to focal adhesion and tight junction, restores endothelial barrier integrity, and suppresses early aortic dilation of AAA (7 and 14 days post‐Ang II). Mechanically, ELK3 acts as an ALDH2 downstream regulator for endothelial barrier function preservation. At the molecular level, ALDH2 directly binds to LIN28B, a regulator of ELK3 mRNA stability, hindering LIN28B binding to ELK3 mRNA, thereby depressing ELK3 expression and impairing endothelial barrier function. Therefore, preserving vascular endothelial barrier integrity via ALDH2‐specific knockdown in ECs holds therapeutic potential in the early management of AAAs.

Abstract Background Aggressive brain tumours, whether primary gliomas or secondary metastases, are characterised by hypervascularisation and are fatal. Recent research has emphasised the crucial involvement of endothelial cells (ECs) in all brain tumour genesis and development events, with various patterns and underlying mechanisms identified. Main body Here, we highlight recent advances in knowledge about the contributions of ECs to brain tumour development, providing a comprehensive summary including descriptions of interactions between ECs and tumour cells, the heterogeneity of ECs and new models for research on ECs in brain malignancies. We also discuss prospects for EC targeting in novel therapeutic approaches. Conclusion Interventions targeting ECs, as an adjunct to other therapies (e.g. immunotherapies, molecular‐targeted therapies), have shown promising clinical efficacy due to the high degree of vascularisation in brain tumours. Developing precise strategies to target tumour‐associated vessels based on the heterogeneity of ECs is expected to improve anti‐vascular efficacy.

Background Stroke is a leading global cause of human death and disability, with advanced aging associated with elevated incidences of stroke. Despite high mortality and morbidity of stroke, the mechanisms leading to blood‐brain barrier dysfunction and development of stroke with age are poorly understood. In the vasculature of brain, endothelial cells (ECs) constitute the core component of the blood‐brain barrier and provide a physical barrier composed of tight junctions, adherens junctions, and basement membrane. Methods and Results We show, in mice, the incidents of intracerebral bleeding increases with age. After isolating an enriched population of cerebral ECs from murine brains at 2, 6, 12, 18, and 24 months, we studied age‐associated changes in gene expression. The study reveals age‐dependent dysregulation of 1388 genes, including many involved in the maintenance of the blood‐brain barrier and vascular integrity. We also investigated age‐dependent changes on the levels of CpG methylation and accessible chromatin in cerebral ECs. Our study reveals correlations between age‐dependent changes in chromatin structure and gene expression, whereas the dynamics of DNA methylation changes are different. Conclusions We find significant age‐dependent downregulation of the Aplnr gene along with age‐dependent reduction in chromatin accessibility of promoter region of the Aplnr gene in cerebral ECs. Aplnr is associated with positive regulation of vasodilation and is implicated in vascular health. Altogether, our data suggest a potential role of the apelinergic axis involving the ligand apelin and its receptor to be critical in maintenance of the blood‐brain barrier and vascular integrity.

Abstract Skeletal muscle capillarization is proportional to muscle fibre mitochondrial content and oxidative capacity. Skeletal muscle cells secrete many factors that regulate neighbouring capillary endothelial cells (ECs), including extracellular vesicles (SkM‐EVs). Peroxisome proliferator‐activated receptor γ coactivator 1‐α (PGC‐1α) regulates mitochondrial biogenesis and the oxidative phenotype in skeletal muscle. Skeletal muscle PGC‐1α also regulates secretion of multiple angiogenic factors, but it is unknown whether PGC‐1α regulates SkM‐EV release, contents and angiogenic signalling potential. PGC‐1α was overexpressed via adenovirus in primary human myotubes. EVs were collected from PGC‐1α‐overexpressing myotubes (PGC‐EVs) as well as from green fluorescent protein‐overexpressing myotubes (GFP‐EVs), and from untreated myotubes. EV release and select mRNA contents were measured from EVs. Additionally, ECs were treated with EVs to measure angiogenic potential of EVs in normal conditions and following an oxidative stress challenge. PGC‐1α overexpression did not impact EV release but did elevate EV content of mRNAs for several antioxidant proteins (nuclear factor erythroid 2‐related factor 2, superoxide dismutase 2, glutathione peroxidase). PGC‐EV treatment of cultured human umbilical vein endothelial cells (HUVECs) increased their proliferation (+36.6%), tube formation (length: +28.1%; number: +25.7%) and cellular viability (+52.9%), and reduced reactive oxygen species levels (−41%) compared to GFP‐EVs. Additionally, PGC‐EV treatment protected against tube formation impairments and induction of cellular senescence following acute oxidative stress. Overexpression of PGC‐1α in human myotubes increases the angiogenic potential of SkM‐EVs. These angiogenic benefits coincided with increased anti‐oxidative capacity of recipient HUVECs. High PGC‐1α expression in skeletal muscle may prompt the release of SkM‐EVs that support vascular redox homeostasis and angiogenesis.

ABSTRACT Kawasaki disease (KD) is a systemic vasculitis of unknown etiology, which tends to involve coronary arteries and can lead to acquired heart disease in children. The immuno‐inflammatory response and vascular endothelial dysfunction are important causes of coronary artery disease in patients with KD. Multisystem inflammatory syndrome in children (MIS‐C) is a rare inflammatory disease in children identified in recent years, which is caused by severe acute respiratory syndrome coronavirus 2 infection; this disease overlaps with KD. This review examines research progress concerning the immuno‐inflammatory response and vascular endothelial dysfunction associated with KD, as well as differences between KD and MIS‐C.

Hypoxia‐induced endothelial dysfunction is known to be involved in the pathogenesis of several vascular diseases. However, it remains unclear whether the pentose phosphate pathway (PPP) is involved in regulating the response of endothelial cells to hypoxia. Here, we established an in vitro model by treating EA.hy926 (a hybrid human umbilical vein cell line) with cobalt chloride (CoCl2; a chemical mimic that stabilizes HIF‐1α, thereby leading to the development of hypoxia), and used this to investigate the involvement of PPP by examining expression of its key enzyme, glucose‐6‐phosphate dehydrogenase (G6PD). We report that CoCl2 induces the accumulation of HIF‐1α, leading to endothelial cell dysfunction characterized by reduced cell viability, proliferation, tube formation, and activation of cytokine production, accompanied with a significant decrease in G6PD expression and activity. The addition of 6‐aminonicotinamide (6‐AN) to inhibit PPP directly causes endothelial dysfunction. Additionally, N‐Acetylcysteine (NAC), a precursor of glutathione, was further evaluated for its protective effects; NAC displayed a protective effect against CoCl2‐induced cell damage by enhancing G6PD activity, and this was abrogated by 6‐AN. The effects of CoCl2 and the involvement of G6PD in endothelial dysfunction have been confirmed in primary human aortic endothelial cells. In summary, G6PD was identified as a novel target of CoCl2‐induced damage, which highlighted the involvement of PPP in regulating the response of endothelial cell CoCl2. Treatment with NAC may be a potential strategy to treat hypoxia or ischemia, which are widely observed in vascular diseases.

Expanding on previous demonstrations of the therapeutic effects of adeno‐associated virus (AAV) carrying small‐hairpin RNA (shRNA) in downregulating the mechanistic target of rapamycin (mTOR) in in vivo retinal vascular disorders, vascular endothelial growth factor (VEGF)‐stimulated endothelial cells were treated with AAV2‐shmTOR to examine the role of mTOR inhibition in retinal angiogenesis. AAV2‐shmTOR exposure significantly reduced mTOR expression in human umbilical vein endothelial cells (HUVECs) and decreased downstream signaling cascades of mTOR complex 1 (mTORC1) and mTORC2 under VEGF treatment. Moreover, the angiogenic potential of VEGF was significantly inhibited by AAV2‐shmTOR, which preserved endothelial integrity by maintaining tight junctions between HUVECs. These data thus support previous in vivo studies and provide evidence that AAV2‐shmTOR induces therapeutic effects by inhibiting the neovascularization of endothelial cells.

Abstract In pulmonary artery hypertension (PAH), emerging evidence suggests that metabolic abnormalities may be contributing to cellular dysfunction in PAH. Metabolic abnormalities such as glycolytic shift have been observed intracellularly in several cell types in PAH, including microvacular endothelial cells (MVECs). Concurrently, metabolomics of human PAH samples has also revealed a variety of metabolic abnormalities; however the relationship between the intracellular metabolic abnormalities and the serum metabolome in PAH remains under investigation. In this study, we utilize the sugen/hypoxia (SuHx) rodent model of PAH to examine the RV, LV and MVEC intracellular metabolome (using targeted metabolomics) in normoxic and SuHx rats. We additionally validate key findings from our metabolomics experiments with data obtained from cell culture of normoxic and SuHx MVECs, as well as metabolomics of human serum samples from two different PAH patient cohorts. Taken together, our data, spanning rat serum, human serum and primary isolated rat MVECs reveal that: (1) key classes of amino acids (specifically, branched chain amino acids—BCAA) are lower in the pre‐capillary (i.e., RV) serum of SuHx rats (and humans); (2) intracellular amino acid levels (in particular BCAAs) are increased in SuHx‐MVECs; (3) there may be secretion rather than utilization of amino acids across the pulmonary microvasculature in PAH and (4) an oxidized glutathione gradient is present across the pulmonary vasculature, suggesting a novel fate for increased glutamine uptake (i.e., as a source of glutathione). in MVECs in PAH. In summary, these data reveal new insight into the shifts in amino acid metabolism occurring across the pulmonary circulation in PAH.

Background Homeostasis of the vessel wall is cooperatively maintained by endothelial cells (ECs), smooth muscle cells, and adventitial fibroblasts. The genetic deletion of fibulin‐4 (Fbln4) in smooth muscle cells (SMKO) leads to the formation of thoracic aortic aneurysms with the disruption of elastic fibers. Although Fbln4 is expressed in the entire vessel wall, its function in ECs and relevance to the maintenance of valvulo‐arterial integrity are not fully understood. Methods and Results Gene silencing of FBLN4 was conducted on human aortic ECs to evaluate morphological changes and gene expression profile. Fbln4 double knockout (DKO) mice in ECs and smooth muscle cells were generated and subjected to histological analysis, echocardiography, Western blotting, RNA sequencing, and immunostaining. An evaluation of the thoracic aortic aneurysm phenotype and screening of altered signaling pathways were performed. Knockdown of FBLN4 in human aortic ECs induced mesenchymal cell–like changes with the upregulation of mesenchymal genes, including TAGLN and MYL9. DKO mice showed the exacerbation of thoracic aortic aneurysms when compared with those of SMKO and upregulated Thbs1, a mechanical stress–responsive molecule, throughout the aorta. DKO mice also showed progressive aortic valve thickening with collagen deposition from postnatal day 14, as well as turbulent flow in the ascending aorta. Furthermore, RNA sequencing and immunostaining of the aortic valve revealed the upregulation of genes involved in endothelial‐to‐mesenchymal transition, inflammatory response, and tissue fibrosis in DKO valves and the presence of activated valve interstitial cells. Conclusions The current study uncovers the pivotal role of endothelial fibulin‐4 in the maintenance of valvulo‐arterial integrity, which influences thoracic aortic aneurysm progression.

Abstract Background Venous thromboembolism (VTE) is one of the leading complications in glioma patients. Neutrophil extracellular traps (NETs) have been reported to play a critical role in the physiopathology of cancer. We aimed to investigate the presence and potential role of NETs in the hypercoagulable state in glioma patients. Moreover, we evaluated the interaction between NETs and endothelial cells (ECs) in glioma patients. Methods The plasma levels of NETs were detected by enzyme‐linked immunosorbent assay. The NET procoagulant activity was performed based on fibrin formation assays. The NET generation and NET‐treated ECs in vitro were observed by confocal microscopy. Activated platelets (PLTs) and PLT‐neutrophil aggregates were detected by flow cytometry. Results Plasma NET markers were significantly higher in stage III/IV glioma patients than in stage I/II glioma patients and healthy subjects. PLTs from glioma patients tended to induce NET formation than those from healthy subjects. NETs contributed to the hypercoagulable state in glioma patients. After ECs were incubated with NETs isolated from stage III/IV glioma patients, they lost their intercellular connections and were converted into procoagulant phenotypes. Combining DNase I and activated protein C markedly decreased endothelial dysfunction. Conclusions Our results showed the interaction between NETs and hypercoagulability in glioma patients. Targeting NETs may be a potential therapeutic and prevention direction for thrombotic complications in glioma patients.

Abstract Endothelial dysfunction in atherosclerotic cardiovascular diseases has become one of the main characteristics in patients with diabetes mellitus, which is usually caused by abnormal inflammation and oxidative stress response. Presently, we focused on the role of Notoginsenoside R1 (NR1), a major component isolated from Panax notoginseng, in endothelial dysfunction caused by high glucose (HG). Human umbilical vein endothelial cells (HUVECs) were treated with HG and then dealt with NR1. Cell counting kit‐8 assay and 5‐bromo‐2′‐dexoyuridine assay were conducted to examine cell proliferation and viability. Flow cytometry was used to measure apoptosis. The angiogenesis of HUVECs was determined by tube formation assay. Moreover, the expressions of miR‐147a, inflammatory cytokines (TNF‐α, IL‐6, and IL‐10) and oxidative stress markers malondialdehyde, superoxide dismutase, and glutathione peroxidase were measured. The protein levels of MyD88/TRAF6/NF‐κB axis, Bax, Bcl2, and Caspase3 were detected by Western blot. Furthermore, gain and loss of functional assays of miR‐147a were performed to verify the role of miR‐147a in NR1‐mediated effects. Our data confirmed that NR1 (at 10–40 μM) reduces HG‐induced HUVECs proliferation and viability inhibition, mitigates apoptosis, and enhances tube formation ability. Meanwhile, NR1 inhibited oxidative stress and inflammatory response and blocked the activation of the MyD88/TRAF6/NF‐κB pathway induced by HG. In addition, NR1 promoted the expression of miR‐147a, which targeted MyD88. Overexpression of miR‐147a markedly inactivated MyD88/TRAF6/NF‐κB pathway, while the miR‐147a inhibitors reversed NR1‐mediated protective effect in HG‐induced HUVECs through activating MyD88/TRAF6/NF‐κB pathway. In conclusion, NR1 relieves HG‐induced endothelial cell injury by downregulating the MyD88/TRAF6/NF‐κB pathway via upregulating miR‐147a.

Abstract Reorganization of host red blood cells by the malaria parasite Plasmodium falciparum enables their sequestration via attachment to the microvasculature. This artificially increases the dwelling time of the infected red blood cells within inner organs such as the brain, which can lead to cerebral malaria. Cerebral malaria is the deadliest complication patients infected with P. falciparum can experience and still remains a major public health concern despite effective antimalarial therapies. Here, the current understanding of the effect of P. falciparum cytoadherence and their secreted proteins on structural features of the human blood‐brain barrier and their involvement in the pathogenesis of cerebral malaria are highlighted. Advanced 2D and 3D in vitro models are further assessed to study this devastating interaction between parasite and host. A better understanding of the molecular mechanisms leading to neuronal and cognitive deficits in cerebral malaria will be pivotal in devising new strategies to treat and prevent blood‐brain barrier dysfunction and subsequent neurological damage in patients with cerebral malaria.

Background Lipoprotein lipase (LPL)‐derived fatty acid is a major source of energy for cardiac contraction. Synthesized in cardiomyocytes, LPL requires translocation to the vascular lumen for hydrolysis of lipoprotein triglyceride, an action mediated by endothelial cell (EC) release of heparanase. We determined whether flow‐mediated biophysical forces can cause ECs to secrete heparanase and thus regulate cardiac metabolism. Methods and Results Isolated hearts were retrogradely perfused. Confluent rat aortic ECs were exposed to laminar flow using an orbital shaker. Cathepsin L activity was determined using gelatin‐zymography. Diabetes was induced in rats with streptozotocin. Despite the abundance of enzymatically active heparanase in the heart, it was the enzymatically inactive, latent heparanase that was exceptionally responsive to flow‐induced release. EC exposed to orbital rotation exhibited a similar pattern of heparanase secretion, an effect that was reproduced by activation of the mechanosensor, Piezo1. The laminar flow‐mediated release of heparanase from EC required activation of both the purinergic receptor and protein kinase D, a kinase that assists in vesicular transport of proteins. Heparanase influenced cardiac metabolism by increasing cardiomyocyte LPL displacement along with subsequent replenishment. The flow‐induced heparanase secretion was augmented following diabetes and could explain the increased heparin‐releasable pool of LPL at the coronary lumen in these diabetic hearts. Conclusions ECs sense fluid shear‐stress and communicate this information to subjacent cardiomyocytes with the help of heparanase. This flow‐induced mechanosensing and its dynamic control of cardiac metabolism to generate ATP, using LPL‐derived fatty acid, is exquisitely adapted to respond to disease conditions, like diabetes.

Background: The development of coronary vessels in embryonic mouse heart involves various progenitor populations, including sinus venosus (SV), endocardium, and proepicardium. ELA/APJ signaling is known to regulate coronary growth from the SV, whereas VEGF-A/VEGF-R2 signaling controls growth from the endocardium. Previous studies suggest hypoxia might regulate coronary growth, but its specific downstream pathways are unclear. In this study, we further investigated the role of hypoxia and have identified SOX17- and VEGF-R2-mediated signaling as the potential downstream pathways in its regulation of developmental coronary angiogenesis., Results: HIF-1α stabilization by knocking out von Hippel Lindau (VHL) protein in the myocardium (cKO) disrupted normal coronary angiogenesis in embryonic mouse hearts, resembling patterns of accelerated coronary growth. VEGF-R2 expression was increased in coronary endothelial cells under hypoxia in vitro and in VHL cKO hearts in vivo. Similarly, SOX17 expression was increased in the VHL cKO hearts, while its knockout in the endocardium disrupted normal coronary growth., Conclusion: These findings provide further evidence that hypoxia regulates developmental coronary growth potentially through VEGF-R2 and SOX17 pathways, shedding light on mechanisms of coronary vessel development., (© 2024 American Association for Anatomy.)

Atherosclerotic lesions mainly form in arterial areas exposed to low shear stress (LSS), where endothelial cells express a senescent and inflammatory phenotype. Conversely, areas exposed to high shear stress (HSS) are protected from plaque development. Endothelial extracellular vesicles (EVs) have been shown to regulate inflammation and senescence, and therefore play a crucial role in vascular homeostasis. Whilst previous studies have shown links between hemodynamic forces and EV release, the effects of shear stress on the release and uptake of endothelial EVs remains elusive. We aim to decipher the interplay between these processes in endothelial cells exposed to atheroprone or atheroprotective shear stress. Confluent HUVECs were exposed to LSS or HSS for 24 h. Large and small EVs were isolated from conditioned medium by centrifugation and size exclusion chromatography. They were characterised by TEM, Western blot, tunable resistive pulse sensing, flow cytometry and proteomics. Uptake experiments were performed using fluorescently-labelled EVs and differences between groups were assessed by flow cytometry and confocal microscopy. We found that levels of large and small EVs in conditioned media were fifty and five times higher in HSS than in LSS conditions, respectively. In vivo and in vitro uptake experiments revealed greater EV incorporation by cells exposed to LSS conditions. Additionally, endothelial LSS-EVs have a greater affinity for HUVECs than HSS-EVs or EVs derived from platelets, erythrocytes and leukocytes. Proteomic analysis revealed that LSS-EVs were enriched in adhesion proteins (PECAM1, MCAM), participating in EV uptake by endothelial cells. LSS-EVs also carried mitochondrial material, which may be implicated in elevating ROS levels in recipient cells. These findings suggest that shear stress influences EV biogenesis and uptake. Given the major role of EVs and shear stress in vascular health, deciphering the relation between these processes may yield innovative strategies for the early detection and treatment of endothelial dysfunction., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Cold-induced vasoconstriction is a significant contributor that leads to chilblains and hypothermia in humans. However, current animal models have limitations in replicating cold-induced acral injury due to their low sensitivity to cold. Moreover, existing in vitro vascular chips composed of endothelial cells and perfusion systems lack temperature responsiveness, failing to simulate the vasoconstriction observed under cold stress. This study presents a novel approach where a microfluidic bioreactor of vessel-on-a-chip was developed by grafting the inner microchannel surface of polydimethylsiloxane with a thermosensitive hydrogel skin composed of N-isopropyl acrylamide and gelatin methacrylamide. With a lower critical solution temperature set at 30°C, the gel layer exhibited swelling at low temperatures, reducing the flow rate inside the channel by 10% when the temperature dropped from 37°C to 4°C. This well mimicked the blood stasis observed in capillary vessels in vivo. The vessel-on-a-chip was further constructed by culturing endothelial cells on the surface of the thermosensitive hydrogel layer, and a perfused medium was introduced to the cells to provide a physiological shear stress. Notably, cold stimulation of the vessel-on-a-chip led to cell necrosis, mitochondrial membrane potential (ΔΨ m ) collapse, cytoskeleton disaggregation, and increased levels of reactive oxygen species. In contrast, the static culture of endothelial cells showed limited response to cold exposure. By faithfully replicating cold-induced endothelial injury, this groundbreaking thermosensitive vessel-on-a-chip technology offers promising advancements in the study of cold-induced cardiovascular diseases, including pathogenesis and therapeutic drug screening., (© 2024 Wiley Periodicals LLC.)

Abstract Background Convallotoxin (CNT), present in lily of the valley (Convallaria majalis), is a toxin that causes food poisoning among humans and companion animals. Although various symptoms of CNT poisoning have been well described, hypercoagulability owing to CNT is only empirically known among some veterinarians, and the underlying mechanism remains to be elucidated. CNT exerts cytotoxic effects on endothelial cells. Objectives This study aimed to determine whether CNT induces the expression of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade, in endothelial cells and leads to a hypercoagulable state. Methods Human umbilical vein endothelial cells (HUVECs) were used for in vitro experiments. HUVECs were treated with or without CNT (50 and 100 nM) for 4 h. Phosphate‐buffered saline was used as a control. Cell viability was determined using the WST‐8 assay. Quantitative real‐time polymerase chain reaction was performed to determine TF mRNA expression. TF protein expression was observed using a laser scanning confocal microscope. Results The viability of HUVECs significantly reduced after CNT treatment compared with that of non‐treated cells (p

Abstract Background Severe carotid stenosis is a common cause of stroke. In addition, previous clinical studies revealed that patients symptomatic of carotid stenosis suffer from increased episodes of stroke compared with their asymptomatic counterparts. However, the mechanism underlying these differences in the recurrence of stroke remains unclear. Objective The present study aimed to evaluate the levels of neutrophil extracellular traps (NETs) in the plasma of patients with severe carotid stenosis and investigate whether NETs induced procoagulant activity (PCA) in severe carotid stenosis. The study also sought to investigate the interactions between platelets or endothelial cells (ECs) and NETs. Methods The levels of NETs in plasma were quantified using enzyme‐linked immunosorbent assay (ELISA). In addition, NETting neutrophils and neutrophil‐platelet aggregates were detected through flow cytometry. On the other hand, the morphology of NETs formation and endothelial cells were analyzed through confocal microscopy. Finally, the procoagulant activity (PCA) of NETs and endothelial cells were assessed through ELISA and fibrin formation. Results Patients with symptomatic carotid stenosis patients had significantly higher levels of NETs markers compared with their asymptomatic counterparts and healthy subjects. In addition, increased levels of neutrophil‐platelet aggregates induced the generation of NETs in patients with symptomatic carotid stenosis. Moreover, NETs contributed to PCA through tissue factor (TF), in patients with carotid stenosis. Furthermore, NETs disrupted the endothelial barrier and converted endothelial cells (ECs) into PCA to enhance the PCA in patients with carotid stenosis. Conclusions The current study revealed differences in the levels of NETs in the plasma of symptomatic and asymptomatic patients suffering from carotid stenosis. The study also uncovered the interaction between NETs and thrombogenicity in carotid stenosis. Therefore, inhibiting NETs may be a potential biomarker and therapeutic target for recurring stroke in severe carotid stenosis.

Abstract Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by elevated pulmonary arterial pressure and organized thrombi within pulmonary arteries. Riociguat is a soluble guanylate cyclase stimulator and is approved for patients with inoperable CTEPH or residual pulmonary hypertension after pulmonary endarterectomy (PEA). Previous work suggested that riociguat treatment is associated with an increased risk of bleeding, although the mechanism is unclear. The aim of this study is to assess how riociguat affects primary hemostasis by studying its effect on the interaction between platelets and endothelial cells derived from CTEPH patients. Pulmonary artery endothelial cells (PAECs) were isolated from thrombus‐free regions of PEA material. Purified PAECs were cultured in flow chambers and were stimulated with 0.1 and 1 µM riociguat for 24 h before flow experiments. After stimulation with histamine, PAECs were exposed to platelets under shear stress. Platelet adhesion and expression of von Willebrand Factor (VWF) were evaluated to assess the role of riociguat in hemostasis. Under dynamic conditions, 0.1 and 1.0 µM of riociguat suppressed platelet adhesion on the surface of PAECs. Although riociguat did not affect intracellular expression and secretion of VWF, PAECs stimulated with riociguat produced fewer VWF strings than unstimulated PAECs. Flow cytometry suggested that decreased VWF string formation upon riociguat treatment may be associated with suppressed cell surface expression of P‐selectin, a protein that stabilizes VWF anchoring on the endothelial surface. In conclusion, Riociguat inhibits VWF string elongation and platelet adhesion on the surface of CTEPH‐PAECs, possibly by reduced P‐selectin cell surface expression.

Abstract It is unknown whether biological sex influences phenotypes of commercially available human pulmonary artery endothelial cells (HPAECs). Ten lots of commercial HPAECs were used (Lonza Biologics; PromoCell). Five (50%) were confirmed to be genotypically male (SRY+) and five (50%) were confirmed to be female (SRY−). Experiments were conducted between passages five and eight. HPAEC phenotype was confirmed with a panel of cell expression markers. Standard assays for proliferation, migration and tube formation were performed in triplicate with technical replicates, under three treatment conditions (EndoGRO; Sigma‐Aldrich). Apoptosis was assessed by exposing cells treated with complete media or low serum media to hypoxic (1% oxygen) or normoxic (20% oxygen) conditions. Laboratory staff was blinded. The median (range) age of male and female donors from whom the HPAECs were derived was 58 (48–60) and 56 (33–67), respectively. Our results suggest decreased proliferation in genotypically female cells compared with male cells (p = 0.09). With increasing donor age, female cells were less proliferative and male cells were more proliferative (p = 0.001). Female cells were significantly more apoptotic than male cells by condition (p = 0.001). Female cells were significantly more migratory than male cells in complete media but less migratory than male cells under vascular endothelial growth factor enriched conditions (p = 0.001). There are subtle sex‐based differences in the behavior of HPAECs that depend on donor sex and, less so, age. These differences may undermine rigor and reproducibility. Future studies should define whether biological sex is an important regulator of HPAEC function in health and disease.

Abstract Modulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. Here we sought to better understand how β‐arrestins, intracellular proteins that regulate agonist‐mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress. We performed both in vitro studies with primary endothelial cells subjected to β‐arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of β‐arrestin 1 and β‐arrestin 2. We found that β‐arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of β‐arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. β‐arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, β‐arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR‐II), whose disruption similarly led to an impaired endothelial shear response. β‐arrestins also regulated Smad transcription factor phosphorylation by BMPR‐II. Mice with endothelial specific deletion of β‐arrestin 1 and β‐arrestin 2 were found to have impaired retinal angiogenesis. In conclusion, we have identified a novel role for endothelial β‐arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR‐II and contribute to vascular development.

Abstract Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4‐thiouracil (4TUc) into 4‐thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin‐affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CMUPRT mice) and tested our hypothesis that CM‐derived miRNAs (CMmiRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PBsEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PBsEV of CMUPRT mice 6 h after 4TUc injection. In PBsEV, 4TUd was incorporated into CM‐specific/enriched miRs including miR‐208a, but not into miRs with other organ or tissue‐type specificities. 4TUd‐labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM‐derived miR‐208a (CMmiR‐208a) levels peaked 12 h after experimentally induced MI in PBsEV and 24 h after MI in the lung. Notably, miR‐208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PBsEV from mice that underwent MI (MI‐PBsEV) or sham surgery (Sham‐PBsEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR‐208a and cooperatively regulate inflammation via the NF‐κB pathway, was lower in the lungs of MI‐PBsEV–treated animals than the lungs of animals treated with Sham‐PBsEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro‐inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR‐208a antagomirs prior to MI surgery. Thus, CMUPRT mice enables us to track PBsEV‐mediated transport of CMmiRs and identify an miR‐208a‐mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.

Endothelial cell function and metabolism are closely linked to differential use of energy substrate sources and combustion. While endothelial cell migration is promoted by 2‐phosphofructokinase‐6/fructose‐2,6‐bisphosphatase (PFKFB3)‐driven glycolysis, proliferation also depends on fatty acid oxidation for dNTP synthesis. We show that inflammatory activation of human umbilical vein endothelial cells (HUVECs) by interleukin‐1β (IL‐1β), despite inhibiting proliferation, promotes a shift toward more metabolically active phenotype. This was reflected in increased cellular glucose uptake and consumption, which was preceded by an increase in PFKFB3 mRNA and protein expression. However, despite a modest increase in extracellular acidification rates, the increase in glycolysis did not correlate with extracellular lactate accumulation. Accordingly, IL‐1β stimulation also increased oxygen consumption rate, but without a concomitant rise in fatty acid oxidation. Together, this suggests that the IL‐1β‐stimulated energy shift is driven by shunting of glucose‐derived pyruvate into mitochondria to maintain elevated oxygen consumption in HUVECs. We also revealed a marked donor‐dependent variation in the amplitude of the metabolic response to IL‐1β and postulate that the donor‐specific response should be taken into account when considering targeting dysregulated endothelial cell metabolism.

Background The small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA‐ROCK (rho associated coiled‐coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)‐mediated angiogenesis, whereas other work suggests VEGF‐antagonistic effects of the RhoA‐ROCK axis. Methods and Results To elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant‐negative (T19N), or wild‐type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune‐incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild‐type RhoA but not dominant‐negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell‐related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF‐induced angiogenic sprouting in control‐treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA‐related effects, but increased angiogenic sprouting and migration of control‐treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer–based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR‐2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression. Conclusions Our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well‐characterized downstream effectors ROCK and LIMK.

ABSTRACT The transmembrane protein heart of glass1 (HEG1) directly binds to and recruits Krev interaction trapped protein 1 (KRIT1) to endothelial junctions to form the HEG1–KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel‐like factors 4 and 2 (KLF4 and KLF2), which are implicated in endothelial vascular homeostasis; however, the effect of acute inhibition of the HEG1–KRIT1 interaction remains incompletely understood. Here, we report a high‐throughput screening assay and molecular design of a small‐molecule HEG1–KRIT1 inhibitor to uncover acute changes in signaling pathways downstream of the HEG1–KRIT1 protein complex disruption. The small‐molecule HEG1–KRIT1 inhibitor 2 (HKi2) was demonstrated to be a bona fide inhibitor of the interaction between HEG1 and KRIT1 proteins, by competing orthosterically with HEG1 through covalent reversible interactions with the FERM (4.1, ezrin, radixin, and moesin) domain of KRIT1. The crystal structure of HKi2 bound to KRIT1 FERM revealed that it occupies the same binding pocket on KRIT1 as the HEG1 cytoplasmic tail. In human endothelial cells (ECs), acute inhibition of the HEG1–KRIT1 interaction by HKi2 increased KLF4 and KLF2 mRNA and protein levels, whereas a structurally similar inactive compound failed to do so. In zebrafish, HKi2 induced expression of klf2a in arterial and venous endothelium. Furthermore, genome‐wide RNA transcriptome analysis of HKi2‐treated ECs under static conditions revealed that, in addition to elevating KLF4 and KLF2 expression, inhibition of the HEG1–KRIT1 interaction mimics many of the transcriptional effects of laminar blood flow. Furthermore, HKi2‐treated ECs also triggered Akt signaling in a phosphoinositide 3‐kinase (PI3K)‐dependent manner, as blocking PI3K activity blunted the Akt phosphorylation induced by HKi2. Finally, using an in vitro colocalization assay, we show that HKi6, an improved derivative of HKi2 with higher affinity for KRIT1, significantly impedes recruitment of KRIT1 to mitochondria‐localized HEG1 in CHO cells, indicating a direct inhibition of the HEG1–KRIT1 interaction. Thus, our results demonstrate that early events of the acute inhibition of HEG1–KRIT1 interaction with HKi small‐molecule inhibitors lead to: (i) elevated KLF4 and KLF2 gene expression; and (ii) increased Akt phosphorylation. Thus, HKi’s provide new pharmacologic tools to study acute inhibition of the HEG1–KRIT1 protein complex and may provide insights to dissect early signaling events that regulate vascular homeostasis.

Abstract Acute respiratory distress syndrome (ARDS) is a major clinical problem without available therapies. Known risks for ARDS include severe sepsis, SARS‐CoV‐2, gram‐negative bacteria, trauma, pancreatitis, and blood transfusion. During ARDS, blood fluids and inflammatory cells enter the alveoli, preventing oxygen exchange from air into blood vessels. Reduced pulmonary endothelial barrier function, resulting in leakage of plasma from blood vessels, is one of the major determinants in ARDS. It is, however, unknown why systemic inflammation particularly targets the pulmonary endothelium, as endothelial cells (ECs) line all vessels in the vascular system of the body. In this study, we examined ECs of pulmonary, umbilical, renal, pancreatic, and cardiac origin for upregulation of adhesion molecules, ability to facilitate neutrophil (PMN) trans‐endothelial migration (TEM) and for endothelial barrier function, in response to the gram‐negative bacterial endotoxin LPS. Interestingly, we found that upon LPS stimulation, pulmonary ECs showed increased levels of adhesion molecules, facilitated more PMN‐TEM and significantly perturbed the endothelial barrier, compared to other types of ECs. These observations could partly be explained by a higher expression of the adhesion molecule ICAM‐1 on the pulmonary endothelial surface compared to other ECs. Moreover, we identified an increased expression of Cadherin‐13 in pulmonary ECs, for which we demonstrated that it aids PMN‐TEM in pulmonary ECs stimulated with LPS. We conclude that pulmonary ECs are uniquely sensitive to LPS, and intrinsically different, compared to ECs from other vascular beds. This may add to our understanding of the development of ARDS upon systemic inflammation.

Abstract Pulmonary arterial hypertension (PAH) is associated with significant morbidity and mortality. PAH is characterized by pulmonary artery remodeling, elevated right ventricular pressure (RVP) and, ultimately, cardiac failure. Pulmonary endothelial cells can sense danger or damage caused by mechanical injury or pathogens through alarmin cytokines. These cytokines can signal proliferation to restore barrier integrity or aberrant hyperproliferation and remodeling. We hypothesized that IL‐33 signals pulmonary artery endothelial cells to proliferate under hypertensive conditions during the remodeling response and rise in RVP. To test this hypothesis, pulmonary hypertension (PH) was induced in C57Bl/6J, IL‐33 receptor gene deleted (ST2−/−) and MYD88 gene deleted (MYD88−/−) mice by exposure to 10% O2 and SU5416 injections (SUHX). RVP, arterial wall thickness, endothelial cell proliferation and IL‐33 levels and signaling were evaluated. In response to SUHX. RVP increased in C57Bl/6J mice in response to SUHX (49% male and 70% female; p

Insufficient vessel maintenance adversely impacts patients in terms of tissue reperfusion following stroke or myocardial infarction, as well as during wound healing. Angiogenesis impairment is a feature typical of metabolic disorders acting at the cardiovascular level, such as diabetes. Therapeutic angiogenesis regulation offers promising clinical implications, and natural compounds as pro-angiogenic nutraceuticals hold valuable applications in regenerative medicine. By using cultured endothelial cells from human umbilical veins (HUVEC) we studied functional and molecular responses following exposure to erucin, a natural isothiocyanate derived from Brassicaceae plants and extracted from the seeds of rocket. Erucin (at nanomolar concentrations) promotes cell migration and tube formation, similar to vascular endothelial growth factor (VEGF), through mobilizing paxillin at endothelial edges. At the molecular level, erucin induces signaling pathways typical of angiogenesis activation, namely Ras, PI3K/AKT, and ERK1/2, leading to VEGF expression and triggering its autocrine production, as pharmacological inhibition of soluble VEGF and VEGFR2 dampens endothelial functions. Furthermore, erucin, alone and together with VEGF, preserves endothelial angiogenic functions under pathological conditions, such as those induced in HUVEC by high glucose (HG) exposure. Erucin emerges as a compelling candidate for therapeutic revascularization applications, showcasing promising prospects for natural compounds in regenerative medicine, particularly in addressing angiogenesis-related disorders., (© 2024 John Wiley & Sons Ltd.)

Background Cardiac hypertrophy and fibrosis are common adaptive responses to injury and stress, eventually leading to heart failure. Hypoxia signaling is important to the (patho)physiological process of cardiac remodeling. However, the role of endothelial PHD2 (prolyl‐4 hydroxylase 2)/hypoxia inducible factor (HIF) signaling in the pathogenesis of cardiac hypertrophy and heart failure remains elusive. Methods and Results Mice with Egln1Tie2Cre (Tie2‐Cre‐mediated deletion of Egln1 [encoding PHD2]) exhibited left ventricular hypertrophy evident by increased thickness of anterior and posterior wall and left ventricular mass, as well as cardiac fibrosis. Tamoxifen‐induced endothelial Egln1 deletion in adult mice also induced left ventricular hypertrophy and fibrosis. Additionally, we observed a marked decrease of PHD2 expression in heart tissues and cardiovascular endothelial cells from patients with cardiomyopathy. Moreover, genetic ablation of Hif2a but not Hif1a in Egln1Tie2Cre mice normalized cardiac size and function. RNA sequencing analysis also demonstrated HIF‐2α as a critical mediator of signaling related to cardiac hypertrophy and fibrosis. Pharmacological inhibition of HIF‐2α attenuated cardiac hypertrophy and fibrosis in Egln1Tie2Cre mice. Conclusions The present study defines for the first time an unexpected role of endothelial PHD2 deficiency in inducing cardiac hypertrophy and fibrosis in an HIF‐2α–dependent manner. PHD2 was markedly decreased in cardiovascular endothelial cells in patients with cardiomyopathy. Thus, targeting PHD2/HIF‐2α signaling may represent a novel therapeutic approach for the treatment of pathological cardiac hypertrophy and failure.

Abstract Endotheliopathy causes COVID‐19 conflicting complications.