Endothelial Cell Dysfunction Research Articles

The HIF2α-dependent Upregulation of SETDB1 Facilitates Hypoxia-induced Functional and Phenotypical Changes of Pulmonary Microvascular Endothelial Cells.

Background: Emerging studies have reported the vital role of histone modification in the dysfunction of pulmonary vascular endothelial cells, which acts as the key reason to drive the hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension (PH). This study aims to investigate the role of a histone 3 lysine 9 (H3K9) methyltransferase, SET domain bifurcated 1 (SETDB1), in hypoxia-induced functional and phenotypical changes of pulmonary vascular endothelial cells. Methods: Primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as cell model. Specific knockdown and overexpression strategies were used to systematically determine the molecular regulation and function of SETDB1 in PMVECs. Results: SETDB1 is highly expressed and significantly upregulated in the pulmonary vascular endothelium of lung tissue isolated from SU5416/hypoxia-induced PH (SuHx-PH) rats, and also in pulmonary arterial endothelial cells (PAECs) from idiopathic pulmonary arterial hypertension (IPAH) patients, comparing to their respective controls. In primarily cultured rat PMVECs, treatment of hypoxia or CoCl2 induces significant upregulation of HIF2α, SETDB1 and H3K9me3. Specific knockdown and overexpression strategies indicate the hypoxia- or CoCl2-induced upregulation of SETDB1 is mediated through a HIF2α-dependent mechanism. Knockdown of SETDB1 significantly inhibits the hypoxia- or CoCl2-induced apoptosis, senescence and endothelial to mesenchymal transition (EndoMT) in rat PMVECs. Moreover, treatment of the specific inhibitor of histone methyltransferase, Chaetocin, effectively attenuates the disease pathogenesis of SuHx-PH in rat. Conclusions: Our results suggest that the HIF2α-dependent upregulation of SETDB1 facilitates hypoxia-induced functional and phenotypical changes of PMVECs, potentially contributing to the hypoxia-induced pulmonary vascular remodeling and PH.

Super-Enhancer-Driven Syndecan-4 Regulates Intercellular Communication in Hypoxic Pulmonary Hypertension.

Unveiling pro-proliferation genes involved in crosstalk between pulmonary artery endothelial cells and pulmonary artery smooth muscle cells (PASMCs) are important to improving the therapeutic outcome of pulmonary hypertension (PH). Although growing studies have shown that super-enhancers (SEs) play a pivotal role in pathological and physiological processes, the SE-associated genes in PH and their impact on PASMC proliferation remain largely unexplored. We used serotype 5 adenovirus-associated virus to interfere with syndecan-4 and constructed an SU5416 combined with hypoxia-PH model. Chromatin immunoprecipitation sequencing analysis, chromatin immunoprecipitation quantitative polymerase chain reaction, and bioinformatics were used to confirm early growth response 1 was involved in regulating syndecan-4-associated SE in PASMCs. The effects of syndecan-4 and its underlying mechanisms were subsequently elucidated using Western blot, coimmunoprecipitation, and cell coculture assays. Herein, we identified a novel SE-associated gene, syndecan-4, in hypoxia-exposed PASMCs. Syndecan-4 was transcriptionally driven by early growth response 1 via an SE and was significantly overexpressed in hypoxic PASMCs and plasma from patients with PH. Mechanism studies revealed that syndecan-4 induces PASMC proliferation by interacting and regulating protein kinase C α ubiquitination. In addition, syndecan-4 was enriched in exosomes secreted from hypoxic PASMCs, which subsequently transported and led to pulmonary artery endothelial cell dysfunction. Syndecan-4 inhibition in hypoxia by serotype 5 adenovirus-associated virus treatment attenuated the pulmonary artery remodeling and development of PH invivo. Taken together, our results demonstrate that an SE-driven syndecan-4 modulates crosstalk of PASMCs and pulmonary artery endothelial cells and promotes vascular remodeling via the protein kinase C α and exosome pathway, thus providing potential targets for the early diagnosis and treatment of hypoxic PH.

Redox Imbalance and Cardiovascular Pathogenesis: Exploring the Therapeutic Potential of Phytochemicals

Abstract: The intriguing role of the oxidation system in cardiovascular disease lies in its contribution to chronic and acute increases in intracellular reactive oxygen species (ROS), driving the progression of cardiovascular diseases (CVDs). ROS, produced as by-products of oxidative physiological and metabolic events, act as mediators in various signaling pathways contributing to cardiovascular pathology. The delicate equilibrium between the production of free radicals and antioxidant defense shifts in favor of the former, resulting in redox imbalance and extensive cellular damage. Among CVDs, coronary artery disease (CAD) remains as the leading cause of death globally. Understanding the significance of oxidative damage in the dysfunction of endothelial cells, atherosclerosis, and other pathogenic events and pathways is crucial for preventing and managing CVD. Consequently, it is imperative to comprehend the mechanism/s underlying the pathogenic alterations of CVD due to oxidative damage to develop effective prevention strategies. Many studies have reported bioactive phytochemical/s as potential therapies against CVDs, modulating ROS generation, controlling the CVD-related inflammatory mediators and protecting the vascular system. Therefore, this review provides an update for understanding how the phytoconstituents exhibit preventive roles in oxidative stress-related CVD, thus improving the quality of life of people. This study conducted a thorough literature search on CVD, oxidative imbalance, and phytoconstituents. The search was performed using multiple search engines and the main keywords, and only English publications until June 2023 were included. However, there is a need for more research and clinical trials to fully elucidate the efficacy and safety of these phytochemicals for managing the disease.

PKM2-Driven Lactate Overproduction Triggers Endothelial-To-Mesenchymal Transition in Ischemic Flap via Mediating TWIST1 Lactylation.

The accumulation of lactate is a rising risk factor for patients after flap transplantation. Endothelial-to-mesenchymal transition (EndoMT) plays a critical role in skin fibrosis. Nevertheless, whether lactate overproduction directly contributes to flap necrosis and its mechanism remain unknown. The current study reveals that skin flap mice exhibit enhanced PKM2 and fibrotic response. Endothelial-specific deletion of PKM2 attenuates flap necrosis and ameliorates flap fibrosis in mice. Administration of lactate or overexpressing PKM2 promotes dysfunction of endothelial cells and stimulates mesenchymal-like phenotype following hypoxia. Mechanistically, glycolytic-lactate induces a correlation between Twist1 and p300/CBP, leading to lactylation of Twist1 lysine 150 (K150la). The increase in K150la promotes Twist1 phosphorylation and nuclear translocation and further regulates the transcription of TGFB1, hence inducing fibrosis phenotype. Genetically deletion of endothelial-specific PKM2 in mice diminishes lactate accumulation and Twist1 lactylation, then attenuates EndoMT-associated fibrosis following flap ischemia. The serum lactate levels of flap transplantation patients are elevated and exhibit predictive value for prognosis. This findings suggested a novel role of PKM2-derived lactate in mediating Twist1 lactylation and exacerbates flap fibrosis and ischemia. Inhibition of glycolytic-lactate and Twist1 lactylation reduces flap necrosis and fibrotic response might become a potential therapeutic strategy for flap ischemia.

A Co-Culture System for Studying Cellular Interactions in Vascular Disease

Cardiovascular diseases (CVDs) are leading causes of morbidity and mortality globally, characterized by complications such as heart failure, atherosclerosis, and coronary artery disease. The vascular endothelium, forming the inner lining of blood vessels, plays a pivotal role in maintaining vascular homeostasis. The dysfunction of endothelial cells contributes significantly to the progression of CVDs, particularly through impaired cellular communication and paracrine signaling with other cell types, such as smooth muscle cells and macrophages. In recent years, co-culture systems have emerged as advanced in vitro models for investigating these interactions and mimicking the pathological environment of CVDs. This review provides an in-depth analysis of co-culture models that explore endothelial cell dysfunction and the role of cellular interactions in the development of vascular diseases. It summarizes recent advancements in multicellular co-culture models, their physiological and therapeutic relevance, and the insights they provide into the molecular mechanisms underlying CVDs. Additionally, we evaluate the advantages and limitations of these models, offering perspectives on how they can be utilized for the development of novel therapeutic strategies and drug testing in cardiovascular research.

Loss of Y-chromosomal genes in macrophages induces paracrine phenotypic alterations in cardiac cells

Abstract Background At the intersection of gender medicine and ageing, mosaic loss of the Y chromosome (mLoY) in hematopoietic cells has emerged as a novel cardiovascular risk factor. As the most prevalent chromosomal aberration in men, mLoY is age-associated and characterised by interindividual varying frequencies of LoY cells in blood. It is associated with an increased risk for cardiovascular disease and mortality. A murine model of mLoY demonstrates myocardial fibrosis and reduced lifespan. However, the interactions between LoY hematopoietic and cardiac cells remain elusive. It is uncertain, whether the loss of single genes or combinations of multiple genes drive the observed effects, impeding the development of targeted therapies. Purpose We aimed to examine the impact of individual Y-chromosomal genes in macrophages concerning their paracrine effects on cardiac cells types. Methods and results To identify target genes, we analysed two single-nuclei RNA sequencing datasets of patient-derived PBMCs and THP-1 monocytic cells for their expression of Y-chromosomal genes. We identified nine genes that exhibited robust and concordant expression in both datasets. Seven were protein coding (RPS4Y, DDX3Y, EIF1AY, ZFY, UTY, USP9Y and KDM5D) and two were non-coding (TTTY14 and LINC00278). Using siRNA pools, we achieved >50% reductions in mRNA content in THP-1 M0 macrophages for all genes (p<0.05) and collected conditioned media (CM). As previous data suggested increased myocardial fibrosis in mLoY, we assessed the impact of CM on primary human cardiac fibroblasts. Notably, treatment with CM from ZFY, TTTY14 or LINC00278 silenced macrophages induced collagen 1A1 protein expression by 1.44-, 1.30- and 1.86-fold, respectively (all p<0.05), suggesting that loss of these genes might contribute to myocardial fibrosis. To explore potential effects on cardiomyocytes, neonatal rat cardiomyocytes were exposed to CM. We observed reduced beating frequencies upon treatment with CM from ZFY, TTTY14 or UTY silenced macrophages (relative fold-changes 0.45, 0.40, 0.53, respectively; all p<0.05). While suggestive of cardiomyocyte stress, we found no changes in the expression of the stress markers Nppa or Nppb, possibly indicating direct effects on ion transporters. Given the strong association of cardiovascular disease with endothelial cell dysfunction, we investigated the effects of CM on human umbilical vein endothelial cells (HUVEC). CM from ZFY and KDM5D silenced macrophages induced ICAM protein expression in HUVEC by 1.9- and 1.6-fold, respectively (both p<0.05). Conclusion Loss of individual Y-chromosomal genes in monocytic cells elicits distinct paracrine effects on various cardiac cell types. Particularly, silencing of ZFY in macrophages demonstrated profound effects on all investigated cell types, warranting further investigation. Additionally, two long non-coding RNAs, previously unexplored in cardiovascular disease, influenced paracrine activity of macrophages.

MIR181A1HG is a novel long noncoding RNA regulating vascular inflammation and atherosclerosis

Abstract Background Endothelial cell (EC) dysfunction and vascular inflammation are critical in the initiation and progression of atherosclerosis. Long noncoding RNAs (lncRNAs) plays a critical role in vascular pathology, but relatively little is known about their involvement in controlling vascular inflammation. MIR181A1HG is a lncRNA locating juxta-position with miR-181a1 and miR-181b1, both involved in vascular inflammation. However, the role of MIR181A1HG in vascular inflammation is still unknow. Purpose The study aims to investigate the role of MIR181A1HG in regulating vascular inflammation and atherosclerosis. Methods We examined expression of MIR181A1HG in both human and mouse atherosclerotic lesions. Loss-of-function and gain-of-function studies, multiple RNA–protein interaction assays luciferase reporter and chromatin immunoprecipitation assays were used to investigate the role and molecular mechanisms of MIR181A1HG in the vascular inflammation and atherosclerosis. The single-cell RNA-sequencing were used to analyze atherosclerotic phenotypes of MIR181A1HG-/-ApoE-/- mice. Results MIR181A1HG was abundant in ECs and significantly increased in both humans and mouse atherosclerotic lesions. Deficiency of MIR181A1HG effectively suppressed NLRP3 inflammasome, ECs activation, and atherosclerotic lesion formation. In contrast, EC-specific MIR181A1HG overexpression promoted NLRP3 inflammasome, ECs activation, and atherosclerotic lesion formation, which were reversed by pharmacological inhibition of NLRP3 (MCC950). MIR181A1HG functioned by regulating NLRP3 inflammation, ECs activation though decoying Foxp1 away from the promoters of targets genes, which is independently of miR181a1/b1 cluster. EC-specific Foxp1 silencing reversed in vivo anti-atherosclerotic effect mediated by MIR181A1HG-deletion.

Impact of plasma from patients with severe aortic stenosis on valvular endothelial cells

Abstract Background The natural history of aortic stenosis (AS) encompasses a spectrum of pathophysiological stages, including endothelial dysfunction, inflammation, fibrosis and calcification. Accumulating data underscore the detrimental impact of the haemostatic system on AS, contributing to disease progression and the development of a prothrombotic phenotype within the valve. Understanding these mechanisms is pivotal for the exploration of novel therapeutic avenues, particularly in the context of transcatheter aortic valve replacement (TAVR). Following TAVR, the native diseased aortic valve persists, maintaining its pathological activity and thrombogenic effects on the bioprosthetic valve, potentially influencing its durability and patient outcomes. The precise effects of plasma and its various components on valvular endothelial cells (VECs) dysfunction remains inadequately explored. Purpose To evaluate the influence of plasma derived from individuals with severe AS on aortic VECs. Methods Plasma samples obtained from patients with severe AS undergoing TAVR (n=100) were collected immediately before the procedure, alongside plasma samples from healthy individuals. Factor (F)Xa activity within the plasma samples was quantified using fluorometer-based method. Porcine VECs were then exposed to either plasma (at 10% concentration) or isolated FXa (1 nM), with or without different pharmacological modulators. Protein expression levels were assessed by Western blot analyses. The level of reactive oxygen species (ROS) was evaluated by dihydroethidium staining. Results Exposure of VECs to plasma from severe AS patients promoted ROS formation compared to plasma from healthy individuals (1402 ± 438 AU vs 732 ± 137 AU, p=0.001) (Figure 1). The heightened oxidative stress elicited by plasma from severe AS patients was attenuated by empagliflozin, perindoprilat, losartan, neutralizing antibodies directed against IL-1, IL-6 and TNF-α. Plasma from severe AS patients showed elevated FXa activity compared to plasma from healthy individuals (179.4 ± 101.3 ng/mL vs 63.2 ± 87.2 ng/mL, p=0.02). Stimulation of VECs with isolated FXa resulted in a sustained formation of ROS, which was blunted by empagliflozin, perindoprilat, losartan, rivaroxaban, dabigatran and PAR-1, 2 and 4 antagonists. Quantification of protein expression in VECs stimulated by isolated FXa revealed an endothelial dysfunction phenotype characterized by increased expression of SGLT2, VCAM-1 and COX-1, and decreased expression of eNOS. Conclusions Plasma from patients with severe AS exerts a detrimental effect on aortic VECs, characterized by a pronounced oxidative stress response. This adverse impact is mediated by multiple mechanisms, including activation of the AT1R/NADPH oxidase/SGLT2 pathway, upregulation of pro-inflammatory cytokines, and activation of PAR. FXa plays a pivotal role by promoting oxidative stress in VECs and contributing to valvular endothelial dysfunction.Pro-oxidant state in VECs.

Genetically Engineered Clostridium Prevents Perivascular Adipose Tissue and Endothelial Senescence and Dysfunction in Aging

Abstract Introduction (Peri)vascular senescence is a bona fide risk for several aging-associated diseases that threatens healthspan. Gut microbiome may be one of triggers that promotes vascular diseases as host ages. However, the interplay between vascular senescence and gut microbiome is largely unknown. Our recent studies revealed an increase in gut-derived phenylacetic acid (PAA) in the aging population (TwinsUK Aging Cohort, n=7,303) and aged mice. Our preliminary shotgun metagenomics also showed that ppfor-harboring Clostridium sp. ASF356 age-dependently generates PAA, which induces perivascular adipose tissue (PVAT) and endothelial cell (EC) senescence and dysfunction in old mice. Yet, it remains unclear whether and how genetic engineering of Clostridium sp. ASF356 (by depleting ppfor gene) can prevent aortic PVAT-EC senescence and restore vascular function in aging. Methods For bacterial genetic engineering, we depleted ppfor gene in Clostridium ASF356 by ClosTron technique and further mono-colonized aged mice (24-months old) and germ-free mice, as control, to reduce plasma PAA and thereby prevent aortic PVAT-EC senescence and improve endothelial dysfunction. Results As a proof of the concept, our studies exhibited that mono-colonization of germ-free mice with wild-type Clostridium sp. ASF356 markedly elevates plasma PAA levels. It was accompanied by hallmarks of aortic PVAT-EC senescence, including increased DNA damage (↑ γ-H2A.X phosphorylation), heightened SASP (↑ IL-6 and VCAM1), and proliferative arrest (↑ p16INK4a and p21WAF1/Cip1). Additionally, our findings revealed upreglation of Notch1 in PVAT, contributing to reduced energy supply (↓ UCP1) and augmented senescence-messaging secretome (↑ IL-6) towards ECs. Our further studies revealed that mono-colonization of aged mice (pre-treated with antibiotics) with Δppfor Clostridium ASF356 mutants intriguingly resulted in decreased plasma PAA levels. This intervention led to the rescues of cellular senescence in both aortic PVAT and ECs, consequently improving vascular function. Specifically, mice subjected to this treatment exhibited a significant reduction in SA-β-galactosidase+ cells, as well as decreased levels of γ-H2A.X phosphorylation, p16INK4a, p21WAF1/Cip1, and IL-6 in their aortic PVAT and ECs compared to wild-type aged mice. Conclusions The current study suggests that genetically engineered Δppfor Clostridium ASF356 mutants reverse cellular senescence and restore function in aortic PVAT and endothelial cells, highlighting the potential for targeted gut microbiome manipulation as an innovative senotherapy to combat vascular aging.

Coronary microvascular dysfunction in postmenopausal minipigs with multiple risk factors

Abstract Background Coronary microvascular angina occurs in both men and women, however, the majority of patients are postmenopausal women with multiple cardiovascular risk factors, such as diabetes mellitus (DM), chronic kidney disease (CKD) and dyslipidemia. No treatment is available for coronary microvascular dysfunction, requiring more in depth knowledge on the mechanisms underlying this syndrome in this expanding group of patients. Purpose Here we studied microvascular function in vivo and and in vitro in isolated small coronary arteries, in post-menopausal miniswine with multiple cardiovascular risk factors. Methods DM (streptozotocin), hypercholesterolemia (HC, high fat, high sugar diet), CKD (renal artery embolization) and menopause (ovariectomy, OVX) were induced in 5 adult female minipigs (1.5-2 years old, ~40kg, DM+HC+CKD+OVX) for 6 months, while 11 healthy age- and weight-matched female minipigs on normal pig chow served as controls (Normal). At sacrifice, coronary flow reserve (CFR) in response to intracoronary infusion of adenosine was measured under anesthesia. Isolated small coronary arteries were used to study the endothelium-dependent response to bradykinin in the presence and absence of eNOS blockade with LNAME, and cyclooxygenase inhibition by indomethacin, and endothelium-independent response to the nitric oxide (NO) donor, SNAP. Results 6 months of sustained hyperglycemia (17.5±1.0 in DM+HC+CKD+OVX vs 8.2±0.9 mmol/l in Normal, P<0.05 by t-test), hypercholesterolemia (15.4±2.0 vs 1.7±0.1 mmol/l, P<0.05), renal dysfunction (NGAL: 205±72 vs 67±5 ng/ml, P<0.05) and low progesterone levels (13.1±5.7 in DM+HC+CKD+OVX vs 92.0±30.0 nmol/l in Normal, P=0.1) were accompanied by systemic inflammation (TNFα: 62±7 vs 47±1 pg/ml, P<0.05). CFR was significantly reduced in DM+HC+CKD+OVX as compared to the healthy animals (panel A). Coronary small arteries from DM+HC+CKD+OVX animals showed impaired endothelium-dependent vasodilation to bradykinin (panel B), which was mediated by a loss of NO (panels C & D). The loss of NO was partly compensated for by activation of the cyclooxygenase pathway, as indomethacin blunted the dilation to bradykinin in the DM+HC+CKD+OVX animals, but had no effect on the dilation to bradykinin in Normals (panels C & D). Endothelium-independent dilation to SNAP was also impaired in DM+HC+CKD+OVX animals, indicating blunted vascular smooth muscle responsiveness to nitric oxide (panel E). Conclusion Postmenopausal minipigs with multiple risk factors, displayed severe coronary microvascular dysfunction as evidenced by a significantly reduced coronary flow reserve and both reduced NO sensitivity/ production, indicating endothelial and smooth muscle cell dysfunction. Loss of NO-mediated vasodilation was partially compensated by activation of cyclooxygenase pathway. Such perturbations may contribute to reduced myocardial perfusion that is often observed in post-menopausal women with multiple cardiovascular risk factors.

Resolving the developmental mechanisms of cardiac microthrombosis of SARS-CoV-2 based on single-cell transcriptome analysis.

The coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) developed into a global health emergency. Systemic microthrombus caused by SARS-CoV-2 infection is a common complication in patients with COVID-19. Cardiac microthrombosis as a complication of SARS-CoV-2 infection is the primary cause of cardiac injury and death in patietns with severe COVID-19. In this study, we performed single-cell sequencing analysis of the right ventricular free wall tissue from healthy donors, patients who died during the hypercoagulable period of characteristic coagulation abnormality (CAC), and patients who died during the fibrinolytic period of CAC. We collected 61,187 cells enriched in 24 immune cell subsets and 13 cardiac-resident cell subsets. We found that in the course of SARS-CoV-2 infected heart microthrombus, MYO1EhighRASGEF1Bhighmonocyte-derived macrophages promoted hyperactivation of the immune system and initiated the extrinsic coagulation pathway by activating chemokines CCL3, CCL5. This series of events is the main cause of cardiac microthrombi following SARS-CoV-2 infection. In a SARS-CoV-2 infected heart microthrombus, excessive immune activation is accompanied by an increase in cellular iron content, which in turn promotes oxidative stress and intensifies intercellular competition. This induces cells to alter their metabolic environment, resulting in increased sugar uptake via the glycosaminoglycan synthesis pathway. In addition, high levels of reactive oxygen species generated by elevated iron levels promote increased endogenous malondialdehyde synthesis in a subpopulation of cardiac endothelial cells. This exacerbates endothelial cell dysfunction and exacerbates the coagulopathy process.

Endothelial KLF11 is a novel protector against diabetic atherosclerosis

BackgroundAtherosclerotic cardiovascular diseases remain the leading cause of mortality in diabetic patients, with endothelial cell (EC) dysfunction serving as the initiating step of atherosclerosis, which is exacerbated in diabetes. Krüppel-like factor 11 (KLF11), known for its missense mutations leading to the development of diabetes in humans, has also been identified as a novel protector of vascular homeostasis. However, its role in diabetic atherosclerosis remains unexplored.MethodsDiabetic atherosclerosis was induced in both EC-specific KLF11 transgenic and knockout mice in the Ldlr−/− background by feeding a diabetogenic diet with cholesterol (DDC). Single-cell RNA sequencing (scRNA-seq) was utilized to profile EC dysfunction in diabetic atherosclerosis. Additionally, gain- and loss-of-function experiments were conducted to investigate the role of KLF11 in hyperglycemia-induced endothelial cell dysfunction.ResultsWe found that endothelial KLF11 deficiency significantly accelerates atherogenesis under diabetic conditions, whereas KLF11 overexpression remarkably inhibits it. scRNA-seq profiling demonstrates that loss of KLF11 increases endothelial-to-mesenchymal transition (EndMT) during atherogenesis under diabetic conditions. Utilizing gain- and loss-of-function approaches, our in vitro study reveals that KLF11 significantly inhibits EC inflammatory activation and TXNIP-induced EC oxidative stress, as well as Notch1/Snail-mediated EndMT under high glucose exposure.ConclusionOur study demonstrates that endothelial KLF11 is an endogenous protective factor against diabetic atherosclerosis. These findings indicate that manipulating KLF11 could be a promising approach for developing novel therapies for diabetes-related cardiovascular complications.

Involvement of aquaporins in Shiga toxin-induced swelling and water transport dysfunction in human renal microvascular endothelial cells

One of the hallmarks of Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is kidney damage. Our previous research demonstrated that Shiga toxin type 2 (Stx2a) decreases cell viability and induces swelling of human glomerular endothelial cells (HGEC). However, Stx2a can disrupt net water transport across HGEC monolayers without affecting cell viability. This work aimed to elucidate the possible mechanisms involved in the water transport disruption caused by Stx2a across HGEC monolayers. We investigated paracellular and transcellular water transfer across HGEC by analyzing the passage of FITC-Dextran and the hydrostatic pressure (Phydr) and measuring the osmotic pressure (Posm), respectively. Stx2a selectively affected the transcellular pathway without impacting the paracellular route. Furthermore, Stx2a cell swelling was prevented by pretreatment with aquaporin inhibitors tetraethylammonium chloride (TEA), Mercury (II) chloride (HgCl2) or TGN-020, suggesting aquaporin involvement in this process. Confocal microscopy revealed that Stx2a increased HGEC total volume, which TEA and TGN-020 counteracted. Additionally, we identified in HGEC not only the expression of aquaporin-1 (AQP1) but also the expression of aquaporin-4 (AQP4). Surprisingly, we observed a decrease in the expression of both AQPs after Stx2a exposure. Our findings suggest that Stx2a may induce water movement into HGEC via AQP1 and AQP4, increasing total cell volume. Subsequently, decreased AQP1 and AQP4 expression could inhibit transcellular water transfer, potentially as a protective mechanism against excessive water entry and cell lysis.

Angiopoietin-2 reverses endothelial cell dysfunction in progeria vasculature.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder in children caused by a point mutation in the lamin A gene, resulting in a toxic form of lamin A called progerin. Accelerated atherosclerosis leading to heart attack and stroke are the major causes of death in these patients. Endothelial cell (EC) dysfunction contributes to the pathogenesis of HGPS related cardiovascular diseases (CVD). Endothelial cell-cell communications are important in the development of the vasculature, and their disruptions contribute to cardiovascular pathology. However, it is unclear how progerin interferes with such communications that lead to vascular dysfunction. An antibody array screening of healthy and HGPS patient EC secretomes identified Angiopoietin-2 (Ang2) as a down-regulated signaling molecule in HGPS ECs. A similar down-regulation of Ang2 mRNA and protein was detected in the aortas from an HGPS mouse model. Addition of Ang2 to HGPS ECs rescues vasculogenesis, normalizes endothelial cell migration and gene expression, and restores nitric oxide bioavailability through eNOS activation. Furthermore, Ang2 addition reverses unfavorable paracrine effects of HGPS ECs on vascular smooth muscle cells. Lastly, by utilizing adenine base editor (ABE)-corrected HGPS ECs and progerin-expressing HUVECs, we demonstrated a negative correlation between progerin and Ang2 expression. Lastly, our results indicated that Ang2 exerts its beneficial effect in ECs through Tie2 receptor binding, activating an Akt-mediated pathway. Together, these results provide molecular insights into EC dysfunction in HGPS and suggest that Ang2 treatment has potential therapeutic effects in HGPS-related CVD.

Baicalein alleviates palmitic acid-induced endothelial cell dysfunction via inhibiting endoplasmic reticulum stress.

Endothelial cells play a critical role in maintaining vascular function and kinetic homeostasis, but excessive accumulation of palmitic acid (PA) may lead to endoplasmic reticulum stress and trigger endothelial cell dysfunction. Baicalin (BCL), a natural plant extract, has received widespread attention for its biological activities in anti-inflammation and anti-oxidative stress. However, the mechanism of BCL on PA-induced endothelial cell dysfunction is unclear. Therefore, the aim of this study was to investigate whether BCL could inhibit PA-induced endoplasmic reticulum stress and thus attenuate endothelial cell dysfunction. Human umbilical vein endothelial cells (HUVECs) were divided into Control, PA, PA + BCL-10 μM, PA + BCL-20 μM, and PA + BCL-50 μM groups. The PA group was treated with PA (200 μM), while the PA + BCL groups were co-treated with different concentrations of BCL (10 μM, 20 μM, 50 μM) for 24 hours. Cell viability was detected by MTT. Cell migration ability was determined by Transwell assay, apoptosis level by flow cytometry, and tube formation ability by tube formation assay. Finally, the levels of apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) and angiogenesis-related proteins (VEGFA and FGF2) were detected by western blot, MMP-9, as well as the protein levels of endoplasmic reticulum stress biomarkers (GRP78, CHOP, PERK, and ATF4). The results at the cellular level showed that cell viability, migration ability and tube formation ability of PA-induced HUVECs were significantly reduced, while apoptosis level was significantly increased. However, administration of different concentrations of BCL significantly enhanced PA-induced cell viability, migration ability and tube formation ability of HUVECs while inhibiting apoptosis. The results of protein levels showed that the protein levels of Bax and cleaved caspase-3 were observably up-regulated in the cells of the PA group, while the protein level of Bcl-2 was significantly down-regulated; compared with the PA group, the protein levels of Bax and cleaved caspase-3 were much lower and the Bcl-2 protein level was much higher in the PA + BCL group. Additionally, the protein levels of VEGFA, FGF2 and MMP-9 were raised and those of GRP78, CHOP, PERK and ATF4 were lowered in the PA + BCL group of cells in a concentration-dependent manner. BCL significantly attenuates PA-induced endothelial cell dysfunction by inhibiting endoplasmic reticulum stress.

SiLOXL2 inhibits endothelial inflammatory response and EndMT induced by Ox-LDL.

Our research aimed to investigate the potential role and mechanism of lysyl oxidase (LOX)-like-2 (LOXL2) in atherosclerosis (AS) by using the human umbilical vein endothelial cells (HUVECs) stimulated by oxidized low-density lipoprotein (ox-LDL). HUVECs were treated with ox-LDL at different concentrations (0, 10, 25, 50, and 100 μg/mL) and incubated for 24 hours. The transfection efficacy of siLOXL2 was investigated by western blot and RT-qPCR. Cell migration, intracellular ROS measurement, oxidative stress, ELISA, and adhesion assays were carried out to examine the ox-LDL-induced HUVECs injury. RT-qPCR and Western blot were used to determine gene and protein expression levels. LOXL2 protein expression increased in ox‑LDL‑induced endothelial cells. Ox-LDL+siLOXL2 significantly inhibited the migration ability of HUVECs and reduced the expression of vascular endothelial growth factor A (VEGFA) and matrix metalloproteinase 9 (MMP-9) gene expressions (all, P <0.05). The ox-LDL+siLOXL2 significantly reduced intracellular ROS production and inhibited the expression of Malondialdehyde (MDA), whereas it markedly enhanced superoxide dismutase (SOD) and catalase (CAT) (all, P <0.05). Supernatant levels of Interleukin-1 beta (IL-1β), Interleukin 6 (IL-6), and Tumor necrosis factor alpha (TNF-α) were significantly attenuated by the ox-LDL+siLOXL2 treatment (all, P <0.05). Ox-LDL+siLOXL2 markedly suppressed the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) (all, P <0.05). ox-LDL+siLOXL2 treatment remarkably reduced the expression of α-smooth muscle actin (α‑SMA) and Vimentin, while increased CD31 and von Willebrand factor (vWF) gene expression (all, P <0.05). LOXL2 silencing is protected against ox-LDL-induced endothelial cell dysfunction, and the mechanism may be related to the inhibition of the EndMT pathway.

Cathepsin L Promotes Pulmonary Hypertension via BMPR2/GSDME-Mediated Pyroptosis.

Pulmonary hypertension (PH) is a fatal progressive disease characterized by pulmonary endothelial injury and occlusive pulmonary vascular remodeling. Lysosomal protease cathepsin L degrades essential molecules to participate in the human pathophysiological process. BMPR2 (bone morphogenetic protein type II receptor) deficiency, an important cause of PH, results from mutational inactivation or excessive lysosomal degradation and induces caspase-3-mediated cell death. Given recent evidence that pyroptosis, as a new form of programmed cell death, is induced by caspase-3-dependent GSDME (gasdermin E) cleavage, we hypothesized that cathepsin L might promote PH through BMPR2/caspase-3/GSDME axis-mediated pyroptosis. Cathepsin L expression was evaluated in the lungs and plasma of patients with pulmonary arterial hypertension. The role of cathepsin L in the progression of PH and vascular remodeling was assessed in vivo. Small interfering RNA, specific inhibitors, and lentiviruses were used to explore the mechanisms of cathepsin L on human pulmonary arterial endothelial cell dysfunction. Cathepsin L expression is elevated in pulmonary artery endothelium from patients with idiopathic pulmonary arterial hypertension and experimental PH models. Genetic ablation of cathepsin L in PH rats relieved right ventricular systolic pressure, pulmonary vascular remodeling, and right ventricular hypertrophy, also restoring endothelial integrity. Mechanistically, cathepsin L promotes caspase-3/GSDME-mediated endothelial cell pyroptosis and represses BMPR2 signaling activity. Cathepsin L degrades BMPR2 via the lysosomal pathway, and restoring BMPR2 signaling prevents the pro-pyroptotic role of cathepsin L in PAECs and experimental PH models. These results show for the first time that cathepsin L promotes the development of PH by degrading BMPR2 to induce caspase-3/GSDME-mediated endothelial pyroptosis.

Mechanisms of Lung Endothelial Cell Injury and Survival in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive, chronic, and incurable inflammatory pulmonary vascular disease characterized by significant sex bias and largely unexplored microbial-associated molecular mechanisms that may influence its development and sex prevalence across various subgroups. PAH can be subclassified as idiopathic, heritable, or associated with conditions such as connective tissue diseases, congenital heart defects, liver disease, infections, and chronic exposure to drugs or toxins. During PAH progression, lung vascular endothelial cells (ECs) undergo dramatic morphofunctional transformations in response to acute and chronic inflammation. These transformations include the appearance and expansion of abnormal vascular cell phenotypes such as those derived from apoptosis-resistant cell growth and endothelial-to-mesenchymal transition (EndoMT). Compelling evidence indicates that these endothelial phenotypes seem to be triggered by chronic lung vascular injury and dysfunction, often characterized by reduced secretion of vasoactive molecules like nitric oxide (NO) and exacerbated response to vasoconstrictors such as Endothelin-1 (ET-1); both long-term known contributors of PAH pathogenesis. This review sheds light on the mechanisms of EC dysfunction, apoptosis, and EndoMT in PAH, aiming to unravel the intricate interactions between ECs, pathogens, and other cell types that drive the onset and progression of this devastating disease. Ultimately, we hope to provide an overview of the complex functions of lung vascular ECs in PAH, inspiring novel therapeutic strategies that target these dysfunctional cells to improve the treatment landscape for PAH, particularly in the face of current and emerging global pathogenic threats.

Disruption of blood-brain barrier and endothelial-to-mesenchymal transition are attenuated by Astragalus polysaccharides mediated through upregulation of ETS1 expression in experimental autoimmune encephalomyelitis

Blood-brain barrier (BBB) breakdown, an early hallmark of multiple sclerosis (MS), remains crucial for MS progression. Our previous works have confirmed that Astragalus polysaccharides (APS) can significantly ameliorate demyelination and disease progression in experimental autoimmune encephalomyelitis (EAE) mice. However, it remains unclear whether APS protects BBB and the potential mechanism. In this study, we found that APS effectively reduced BBB leakage in EAE mice, which was accompanied by a decreased level of endothelial-to-mesenchymal transition (EndoMT) in the central nervous system (CNS). We further induced EndoMT in the mouse brain endothelial cells (bEnd.3) by interleukin-1β (IL-1β) in vitro. The results showed that APS treatment could inhibit IL-1β-induced EndoMT and endothelial cell dysfunction. In addition, the transcription factor ETS1 is a central regulator of EndoMT related to the compromise of BBB. We tested the regulation of APS on ETS1 and identified the expression of ETS1 was upregulated in both EAE mice and bEnd.3 cells by APS. ETS1 knockdown facilitated EndoMT and endothelial cell dysfunction, which completely abolished the regulatory effect of APS. Collectively, APS treatment could protect BBB integrity by inhibiting EndoMT, which might be associated with upregulating ETS1 expression. Our findings indicated that APS has potential value in the prevention of MS.

12489 Senolytics Dasatinib And Quercetin Alleviate Type 1 Diabetes-Related Cardiac Fibrosis In Male Akita Ins-/+Mice

Abstract Disclosure: N. Kadoumi: None. G. Zatarah: None. J. Stokar: None. P. Shivam: None. I. Gurt: None. R. Dresner-Pollak: None. People living with type 1 diabetes (T1D) have a markedly shorter life expectancy compared to non-diabetic age- and sex-matched controls primarily due to cardiovascular disease. Diabetic cardiomyopathy is characterized by structural and functional alterations that occur independently of hypertension or coronary artery leading to congestive heart failure. The pathogenesis of diabetic cardiomyopathy involves endothelial cell dysfunction, microangiopathy, mitochondrial dysfunction, and oxidative stress, resulting in inflammation, extensive remodeling, cardiomyocyte hypertrophy, interstitial fibrosis, and apoptosis. T1D is associated with accelerated aging. Cellular senescence is a hallmark of aging, it is characterized by cell cycle arrest, distinct morphological alterations and the release of molecules referred to as the senescence-associated secretory phenotype (SASP). Senescent cardiomyocytes secrete SASPs that promote activation of fibroblasts, hypertrophy of cardiac muscle cells, and dysfunction of cardiac endothelial cells. An increase in cardiac cellular senescence was demonstrated in mouse models of T1D. Senolytics are compounds that clear senescent cells. Senolytics were shown to improve multiple outcomes in age-associated conditions in mice and humans.We investigated the effects of senolytics Dasatinib (D) and Quercetin (Q) on cardiac fibrosis in the AKITA Ins-/+ mouse model of T1D.We first assessed if there is increased collagen type1 expression in the heart ventricles and perivascular in 3-month-old in AKITA Ins-/+ vs. WT male mice using Immunofluorescence. Increased perivascular and interstitial expression of collagen 1 was found in AKITA Ins-/+ compared to WT age-and sex-matched mice (0.0811±0.008177 µm2 vs. 0.0533± 0.007960 µm2 ,P=0.0476), (105.7±10.29 µm2 vs. 54.85±14.61 µm2, P=0.0476), respectively. Next, 3-month-old AKITA Ins-/+wmale mice were randomly assigned to treatment with Dasatinib (5 mg/kg) and Quercetin (50 mg/kg) or vehicle administered once monthly by oral gavage for a total of four months (n=20, 10 in each group). Heart samples were analyzed for gene expression, collagen 1 protein by western blotting and Immunofluorescence and analyzed using QuPath 0.5.0 Software. Interstitial and perivascular fibrosis was separately assessed in the right and left ventricles. Treatment with D+Q resulted in a 2-fold decrease in COL1A1 gene and Collagen 1 protein expression by RT-PCR and western blot analysis (P= 0.011 and 0.042, respectively) in AKITA Ins-/+ mice. Immunostaining analysis revealed reduced Collagen 1 deposition in both the left and right ventricles (105.7±10.29 µm2 vs. 43.53±16.31 µm2, P=0.0571), as well as in the perivascular area (0.0811±0.008177 µm2 vs. 0.052±0.006163 µm2, P=0.1143). Treatment with senolytics D+Q reduces the expression of Collagen I, a key factor in myocardial fibrosis, in adult male AKITA Ins-/+ mice. Presentation: 6/3/2024