Vascular Endothelial Cell Dysfunction Research Articles

Abstract 11795: CXCL5 Inhibition Recovers Neovascularization in Diabetes Mellitus

Introduction: Diabetes mellitus (DM) is a chronic inflammatory disease accompanied by dysfunction of vascular endothelial cells. Vascular complications are the main causes of death in patients with DM. CXCL5 is an inflammatory chemokine that upregulated in patients with type 2 DM. However, its role in diabetic vasculopathy was not clarified. This study aimed to explore the impacts and mechanistic insights of CXCL5 in neovasculogenesis in DM. Methods: Endothelial progenitor cells (EPCs) and human aortic endothelial cells (HAECs) were used in vitro. Streptozotocin-induced diabetic mice and dbdb mice were used as type 1 and type 2 DM models. CXCL5 knockout mice were also used to generate the diabetic animal model. The hindlimb ischemia surgery, matrigel plug assay, and aortic ring assay were conducted. Flow cytometry were used to evaluate the circulating number of EPCs. Results: CXCL5 concentrations were increased in plasma and EPCs culture medium from type 2 DM patients. CXCL5 inhibition by neutralizing antibody upregulated VEGF/SDF-1 expression and improved migration and tube formation abilities in EPCs from type 2 DM patients and high glucose-treated EPCs from non-diabetic subjects as well as HAECs. Intraperitoneal injection of CXCL5 neutralizing antibody increased circulating EPC number, enhanced capillary density, and recovered the blood flow after hindlimb ischemia surgery in both type 1 and type 2 diabetic animals. Protein levels of VEGF and SDF-1 in ischemic muscle were upregulated in the CXCL5 inhibition group. Additionally, diabetic mice injected with CXCL5 neutralizing antibody showed higher hemoglobin contents and neovascular branches in the matrigel plug assay and aortic ring assay, respectively. Importantly, CXCL5 knockout diabetic mice showed improved ischemia-induced angiogenesis and higher expression of angiogenic proteins in ischemic muscle compared with wild-type diabetic mice. Conclusion: CXCL5 inhibition can improve neovasculogenesis via protecting vascular endothelial function and upregulating angiogenic protein expressions in both type 1 and type 2 DM. Further clinical studies are needed to verify that CXCL5 may be a potential therapeutic target for DM vascular complications.

The Association among Urinary Lead and Cadmium, Serum Adiponectin, and Serum Apoptotic Microparticles in a Young Taiwanese Population.

Previous studies reported that lead (Pb) and cadmium (Cd) exposure are linked to changes in serum adiponectin; an adipokine that promotes glycolysis and inhibits gluconeogenesis to regulate glucose metabolism. However, no study has ever explored the relationship between exposure to these two heavy metals and adiponectin in adolescents and young adults. Additionally, the role of adiponectin in the relationship between Pb and Cd exposure and vascular endothelial cell apoptosis has never been investigated. In this study, 724 Taiwanese participants, aged 12 to 30 years, were enrolled to investigate the association among urinary lead and cadmium, serum adiponectin, and apoptotic microparticles (CD31+/CD42a-, CD31+/CD42a+, and CD14). The results of the current study revealed a statistically significant inverse association between urine Pb and Cd levels and adiponectin levels, as well as a positive association with apoptotic microparticles (CD31+/CD42a-, CD31+/CD42a+, and CD14). Adiponectin was also inversely correlated with CD31+/CD42a- and CD31+/CD42a+. Moreover, when subjects with both Pb and Cd levels above the 50th percentile were compared to those below it, the former group exhibited the lowest average adiponectin value. Additionally, a more pronounced positive association between heavy metals and apoptotic microparticles (CD31+/CD42a- and CD31+/CD42a+) was observed when adiponectin levels were lower. Furthermore, an interaction between adiponectin and heavy metals was identified in the relationship between these metals and CD31+/CD42a-. In conclusion, these findings suggest that Pb and Cd exposure may have an adverse effect on adiponectin, and it may play a role in the link between heavy metal exposure and the dysfunction of vascular endothelial cells. Future studies are needed to establish whether a causal relationship exists.

Isogenic pairs of induced-pluripotent stem-derived endothelial cells identify DYRK1A/PPARG/EGR1 pathway is responsible for Down syndrome-associated pulmonary hypertension.

Down syndrome (DS) is the most prevalent chromosomal disorder associated with a higher incidence of pulmonary arterial hypertension (PAH). The dysfunction of vascular endothelial cells (ECs) is known to cause pulmonary arterial remodeling in PAH, although the physiological characteristics of ECs harboring trisomy 21 (T21) are still unknown. In this study, we analyzed the human vascular ECs by utilizing the isogenic pairs of T21-induced pluripotent stem cells (iPSCs) and corrected disomy 21 (cDi21)-iPSCs. In T21-iPSC-derived ECs, apoptosis and mitochondrial reactive oxygen species (mROS) were significantly increased, and angiogenesis and oxygen consumption rate (OCR) were significantly impaired as compared with cDi21-iPSC-derived ECs. The RNA-sequencing identified that EGR1 on chromosome 5 was significantly upregulated in T21-ECs. Both EGR1 suppression by siRNA and pharmacological inhibitor could recover the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Alternately, the study also revealed that DYRK1A was responsible to increase EGR1 expression via PPARG suppression, and that chemical inhibition of DYRK1A could restore the apoptosis, mROS, angiogenesis, and OCR in T21-ECs. Finally, we demonstrated that EGR1 was significantly upregulated in the pulmonary arterial ECs from lung specimens of a patient with DS and PAH. In conclusion, DYRK1A/PPARG/EGR1 pathway could play a central role for the pulmonary EC functions and thus be associated with the pathogenesis of PAH in DS.

Varied effects of tobacco smoke and e-cigarette vapor suggest that nicotine does not affect endothelium-dependent relaxation and nitric oxide signaling

Chronic smoking causes dysfunction of vascular endothelial cells, evident as a reduction of flow-mediated dilation in smokers, but the role of nicotine is still controversial. Given the increasing use of e-cigarettes and other nicotine products, it appears essential to clarify this issue. We studied extracts from cigarette smoke (CSE) and vapor from e-cigarettes (EVE) and heated tobacco (HTE) for their effects on vascular relaxation, endothelial nitric oxide signaling, and the activity of soluble guanylyl cyclase. The average nicotine concentrations of CSE, EVE, and HTE were 164, 800, and 85 µM, respectively. At a dilution of 1:3, CSE almost entirely inhibited the relaxation of rat aortas and porcine coronary arteries to acetylcholine and bradykinin, respectively, while undiluted EVE, with a 15-fold higher nicotine concentration, had no significant effect. With about 50% inhibition at 1:2 dilution, the effect of HTE was between CSE and EVE. Neither extract affected endothelium-independent relaxation to an NO donor. At the dilutions tested, CSE was not toxic to cultured endothelial cells but, in contrast to EVE, impaired NO signaling and inhibited NO stimulation of soluble guanylyl cyclase. Our results demonstrate that nicotine does not mediate the impaired endothelium-dependent vascular relaxation caused by smoking.

Pathophysiology of Cardiac Cell Injury in Post-COVID-19 Syndrome

Abstract: Recently, the scientific community has realized that COVID-19 effects are not limited to the acute period of infection but continue beyond that to cause more prolonged pathological changes. Post-COVID syndrome is a novel concept that describes the sequelae/persistent pathophysiological changes of post-COVID-19 infection. The current hypothesis suggests the involvement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cardiac arrhythmias, coronary artery aneurism, acute renal injury, central nervous system degenerative diseases, vascular endothelial cell dysfunction, and pulmonary dyspnea, as well as fibrotic lung damage. Therefore, COVID-19 has been identified as a poly-syndromic and poly-systemic inflammatory disease. Post-COVID extrapulmonary complications have been observed in approximately 85% of hospitalized COVID-19 survivors and 35% of COVID-19 outpatients. Furthermore, 25% of hospitalised COVID-19 survivors developed myocardiopathy.

Targeting the cochlin/SFRP1/CaMKII axis in the ocular posterior pole prevents the progression of nonpathologic myopia

Myopia is a major public health issue. However, interventional modalities for nonpathologic myopia are limited due to its complicated pathogenesis and the lack of precise targets. Here, we show that in guinea pig form-deprived myopia (FDM) and lens-induced myopia (LIM) models, the early initiation, phenotypic correlation, and stable maintenance of cochlin protein upregulation at the interface between retinal photoreceptors and retinal pigment epithelium (RPE) is identified by a proteomic analysis of ocular posterior pole tissues. Then, a microarray analysis reveals that cochlin upregulates the expression of the secreted frizzled-related protein 1 (SFRP1) gene in human RPE cells. Moreover, SFRP-1 elevates the intracellular Ca2+ concentration and activates Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling in a simian choroidal vascular endothelial cell line, and elicits vascular endothelial cell dysfunction. Furthermore, genetic knockdown of the cochlin gene and pharmacological blockade of SFRP1 abrogates the reduced choroidal blood perfusion and prevents myopia progression in the FDM model. Collectively, this study identifies a novel signaling axis that may involve cochlin in the retina, SFRP1 in the RPE, and CaMKII in choroidal vascular endothelial cells and contribute to the pathogenesis of nonpathologic myopia, implicating the potential of cochlin and SFRP1 as myopia interventional targets.

Long-term abuse of caffeine sodium benzoate induces endothelial cells injury and leads to coagulation dysfunction.

Our hospital admitted a patient who had difficulty in coagulation even after blood replacement, and the patient had abused caffeine sodium benzoate (CSB) for more than 20 years. Hence, we aimed to explore whether CSB may cause dysfunction in vascular endothelial cells and its possible mechanism. Low, medium, and high concentrations of serum of long-term CSB intake patients were used to treat HUVECs, with LPS as the positive control. MTT and CCK8 were performed to verify CSB's damaging effect on HUVECs. The expression of ET-1, ICAM-1, VCAM-1, and E-selectin were measured by ELISA. TUNEL assay and Matrigel tube formation assay were carried out to detect apoptosis and angiogenesis of HUVECs. Flow cytometry was applied to analyze cell cycles and expression of CD11b, PDGF, and ICAM-1. Expression of PDGF-BB and PCNA were examined by western blot. The activation of MAPK signaling pathway was detected by qRT-PCR and western blot. Intracellular Ca2+ density was detected by fluorescent probes. CCK8 assay showed high concentration of CSB inhibited cell viability. Cell proliferation and angiogenesis were inhibited by CSB. ET-1, ICAM-1, VCAM-1, and E-selectin upregulated in CSB groups. CSB enhanced apoptosis of HUVECs. CD11b, ICAM-1 increased and PDGF reduced in CSB groups. The expression level and phosphorylation level of MEK, ERK, JUN, and p38 in MAPK pathway elevated in CSB groups. The expression of PCNA and PDGF-BB was suppressed by CSB. Intracellular Ca2+ intensity was increased by CSB. Abuse of CSB injured HUVECs and caused coagulation disorders.

Syndecan-4 is the key proteoglycan involved in mediating sepsis-associated lung injury

Vascular endothelial cell dysfunction involving syndecan (SDC) proteoglycans contributes to acute sepsis-associated lung injury (ALI), but the exact SDC isoform involved is unclear. We aimed to clarify which SDCs are involved in ALI. A relevant gene expression dataset (GSE5883) was analysed for differentially expressed genes (DEGs) between lipopolysaccharide (LPS)-treated and control lung endothelial cells and for SDC isoform expression. Bioinformatic analyses to predict DEG function were conducted using R language, Gene Ontology, and the Kyoto Encyclopedia of Genes and Genomes. SDC2 and SDC4 expression profiles were examined under inflammatory conditions in human lung vascular endothelial cell and mouse sepsis-associated ALI models. Transcription factors regulating SDC2/4 were predicted to indirectly assess SDC involvement in septic inflammation. Of the DEGs, 224 and 102 genes were up- and downregulated, respectively. Functional analysis indicated that DEGs were involved in modulating receptor ligand and signalling receptor activator activities, cytokine receptor binding, responses to LPS and molecules of bacterial origin, regulation of cell adhesion, tumour necrosis factor signalling, and other functions. DEGs were also enriched for cytoplasmic ribonucleoprotein granules, transcription regulator complexes, and membrane raft cellular components. SDC4 gene expression was 4.5-fold higher in the LPS group than in the control group, while SDC2 levels were similar in both groups. SDC4 mRNA and protein expression was markedly upregulated in response to inflammatory injury, and SDC4 downregulation severely exacerbated inflammatory responses in both in vivo and in vitro models. Overall, our data demonstrate that SDC4, rather than SDC2, is involved in LPS-induced sepsis-associated ALI.

Lipoprotein(a): A New Intensive Care Unit Admission Predictor in Coronavirus Disease 2019 Patients.

Endothelium-related events in patients with coronavirus disease 2019 are linked to a poor prognosis. Lipoprotein(a) plays a role in vascular endothelial cell dysfunction. This research aims to investigate whether baseline serum lipoprotein(a) levels could be a predictor for intensive care unit admission and related clinical parameters in coronavirus disease 2019 patients. The research covers 126 patients who were hospitalized in intensive care unit or the non-intensive care unit in our hospital. This prospective cohort study was conducted from January 2021 to June 2021. The patients who were positive for severe acute respiratory syndrome coronavirus 2 according to real-time polymerase chain reaction test results were included in the study. Two groups were created according to the status of intensive care unit admission. Lipoprotein(a) was studied from blood samples taken at the time of hospital admission. According to the results of the first clinical evaluation, 46 patients were admitted to the intensive care unit and 80 patients were admitted to non-intensive care unit in the hospital. Patients with intensive care unit admission had significantly higher serum lipoprotein(a) levels than patients without intensive care unit admission (40.9 ng/mL and 17.4 ng/mL, P < .001, respectively). The regres- sion analysis revealed that serum lipoprotein(a) levels were independently related to intensive care unit admission (odds ratio 1.242, 95% CI 1.109-1.391, P < .001). In receiver operating characteristic curve analysis, lipoprotein(a) level ≥31.42 ng/mL had 82.6% sensitivity and 72.5% specificity in predicting intensive care unit admission. The risk of intensive care unit admission was seen to be 12.522-fold higher in cases with lipoprotein(a) level ≥31.42. Lipoprotein(a) could be used as a useful biomarker for the triage of coronavirus disease 2019 patients. Baseline serum lipoprotein(a) levels may serve as a useful prognostic biomarker in patients hospitalized for coronavirus disease 2019.

Interfering with Dusp2 alleviates high glucose-induced vascular endothelial cell dysfunction by promoting p38 MAPK pathway activation

BackgroundHyperglycemia-induced vascular endothelial cell dysfunction is a major factor contributing to diabetic lower extremity ischemia. We intend to investigate the role of Dusp2 in hyperglycemia-induced vascular endothelial cell dysfunction and related mechanisms. MethodsThe human umbilical vein endothelial cells (HUVECs) were treated with high glucose (HG) as the cell model. Streptozotocin injection was performed to induce diabetes and femoral artery ligation was to induce hind limb ischemia in mice. The levels of Dusp2, p-p38 MAPK, E2F4, and p38 MAPK were evaluated by Western blot or quantitative real-time PCR. The laser Doppler perfusion imaging was conducted to measure blood flow recovery. The cell counting kit-8, transwell, and tube formation assay were performed to evaluate cell proliferation, migration, and angiogenesis, respectively. CD31 immunohistochemical staining was carried out to detect the capillary density of gastrocnemius. The dual-luciferase reporter gene assay and Chromatin immunoprecipitation assay were executed to explore the interaction between E2F4 and Dusp2. ResultsDusp2 was highly expressed in HG-induced HUVECs and diabetic lower extremity ischemia model mice. Interference with Dusp2 promoted cell proliferation, migration, and angiogenesis, as well as alleviated mouse diabetic hindlimb ischemia. Dusp2 knockdown up-regulated p-p38 MAPK levels. We verified the binding between E2F4 and Dusp2. Overexpressing E2F4 suppressed Dusp2 levels and promoted cell proliferation, migration, and angiogenesis, co-overexpression of Dusp2 reversed the results. ConclusionsOverexpressing E2F4 promotes endothelial cell proliferation, migration, and angiogenesis by inhibiting Dusp2 expression and activating p38 MAPK to alleviate vascular endothelial cell dysfunction under HG stimulation.

High glucose-induced endothelial STING activation inhibits diabetic wound healing through impairment of angiogenesis

Chronic hyperglycemia-induced impairment of angiogenesis is important in diabetic foot ulcer (DFU). Additionally, the stimulator of interferon gene (STING), which is a key protein in innate immunity, mediates palmitic acid-induced lipotoxicity in metabolic diseases through oxidative stress-induced STING activation. However, the role of STING in DFU is unknown. In this study, we established a DFU mouse model with streptozotocin (STZ) injection and found that the expression of STING was significantly increased in the vascular endothelial cells of wound tissues from diabetic patients and in the STZ-induced diabetic mouse model. We further established high glucose (HG)-induced endothelial dysfunction with rat vascular endothelial cells and found that the expression of STING was also increased by high-glucose treatment. Moreover, the STING inhibitor, C176, promoted diabetic wound healing, whereas the STING activator, DMXAA, inhibited diabetic wound healing. Consistently, STING inhibition reversed the HG-induced reduction of CD31 and vascular endothelial growth factor (VEGF), inhibited apoptosis, and promoted migration of endothelial cells. Notably, DMXAA treatment alone was sufficient to induce endothelial cell dysfunction as a high-glucose treatment. Mechanistically, STING mediated HG-induced vascular endothelial cell dysfunction by activating the interferon regulatory factor 3/nuclear factor kappa B pathway. In conclusion, our study reveals an endothelial STING activation-mediated molecular mechanism in the pathogenesis of DFU and identifies STING as a novel potential therapeutic target for DFU.

Inhibition of the intracellular domain of Notch1 results in vascular endothelial cell dysfunction in sepsis.

Notch signaling is critical for regulating the function of vascular endothelial cells (ECs). However, the effect of the intracellular domain of Notch1 (NICD) on EC injury in sepsis remains unclear. We established a cell model of vascular endothelial dysfunction and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Endothelial barrier function and expression of endothelial-related proteins were determined using CCK-8, permeability, flow cytometry, immunoblot, and immunoprecipitation assays. The effect of NICD inhibition or activation on endothelial barrier function was evaluated in vitro. Melatonin was used for NICD activation in sepsis mice. The survival rate, Evans blue dye of organs, vessel relaxation assay, immunohistochemistry, ELISA, immunoblot were used to explore the specific role of melatonin for sepsis induced vascular dysfunction in vivo. We found that LPS, interleukin 6, and serum collected from septic children could inhibit the expression of NICD and its downstream regulator Hes1, which impaired endothelial barrier function and led to EC apoptosis through the AKT pathway. Mechanistically, LPS decreased the stability of NICD by inhibiting the expression of a deubiquitylating enzyme, ubiquitin-specific proteases 8 (USP8). Melatonin, however, upregulated USP8 expression, thus maintaining the stability of NICD and Notch signaling, which ultimately reduced EC injury in our sepsis model and elevated the survival rate of septic mice. We found a previously uncharacterized role of Notch1 in mediating vascular permeability during sepsis, and we showed that inhibition of NICD resulted in vascular EC dysfunction in sepsis, which was reversed by melatonin. Thus, the Notch1 signaling pathway is a potential target for the treatment of sepsis.

An IGFBP7high endothelial cell subset drives T cell extravasation in psoriasis via endothelial glycocalyx degradation.

Dysfunction of vascular endothelial cells (ECs) facilitates imbalanced immune responses and tissue hyperinflammation. However, the heterogeneous functions of skin ECs and their underlying mechanism in dermatoses remain to be solved. Here, focusing on the pathogenic role of skin ECs in psoriasis, we characterized the molecular and functional heterogeneity of skin ECs from healthy individuals and psoriasis patients at the single-cell level. We found that endothelial glycocalyx destruction, a major feature of EC dysfunction in psoriasis, was a driving force during the process of T cell extravasation. Interestingly, we identified a skin EC subset, IGFBP7high ECs, in psoriasis. This subset actively responded to psoriatic-related cytokine signaling, secreted IGFBP7, damaged the endothelial glycocalyx, exposed the adhesion molecules underneath, and prepared the endothelium for immune cell adhesion and transmigration, thus aggravating skin inflammation. More importantly, we provided evidence in a psoriasis-like mouse model that anti-IGFBP7 treatment showed promising therapeutic effects for restoring the endothelial glycocalyx and alleviating skin inflammation. Taken together, our results depicted the distinct functions of EC clusters in healthy and psoriatic skin, identified IGFBP7high ECs as an active subset modulating vascular function and cutaneous inflammation, and indicated that targeting IGFBP7 is a potential therapeutic strategy in psoriasis.

Phosphoenolpyruvate induces endothelial dysfunction and cell senescence through stimulation of metabolic reprogramming

Endothelial dysfunction is a key early link in the pathogenesis of atherosclerosis, and the accumulation of senescent vascular endothelial cells causes endothelial dysfunction. Phosphoenolpyruvate (PEP), which is a high-energy glycolytic intermediate, protects against ischemia-reperfusion injury in isolated rat lung, heart, and liver tissue by quickly providing ATP. However, it was reported that serum PEP concentrations are 13-fold higher in healthy elderly compare to the young. Unlike that of other cell types, the energy required for the physiological function of endothelial cells is mainly derived from glycolysis. Recently, it is unclear whether circulating accumulation of PEP affects endothelial cell function. In this study, we found for the first time that 50-250μM of PEP significantly promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs) through increased expression of vascular endothelial adhesion factor 1 (VCAM1) and intercellular adhesion factor 1 (ICAM1) in HUVECs. Meanwhile, 50-250μM of PEP decreased the expression of endothelial nitric oxide synthase (eNOS) and cellular level of nitric oxide (NO) in HUVECs. Moreover, PEP increased levels of ROS, enhanced the numbers of SA-β-Gal-positive cells and upregulated the expression of cell cycle inhibitors such as p21, p16 and the phosphorylation level of p53 on Ser15, and the expression of proinflammatory factors including TNF-α, IL-1β, IL-6, IL-8, IL-18 and MCP-1 in HUVECs. Furthermore, PEP increased both oxygen consumption rate (OCR) and glycolysis rate, and was accompanied by reduced NAD+/NADH ratios and enhanced phosphorylation levels of AMPKα (Thr172), p38 MAPK (T180/Y182) and NF-κB p65 (Ser536) in HUVECs. Notably, PEP had no significant effect on hepG2 cells. In conclusion, these results demonstrated that PEP induced dysfunction and senescence in vascular endothelial cells through stimulation of metabolic reprogramming.

Hyperglycemia-Suppressed SMARCA5 Disrupts Transcriptional Homeostasis to Facilitate Endothelial Dysfunction in Diabetes.

Dysfunction of vascular endothelial cells (ECs) plays a central role in the pathogenesis of cardiovascular complications in diabetes. SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) is a key regulator of chromatin structure and DNA repair, but its role in ECs remains surprisingly unexplored. The current study was designed to elucidate the regulated expression and function of SMARCA5 in diabetic ECs. SMARCA5 expression was evaluated in ECs from diabetic mouse and human circulating CD34+ cells using quantitative reverse transcription polymerase chain reaction and Western blot. Effects of SMARCA5 manipulation on ECs function were evaluated using cell migration, in vitro tube formation and in vivo wound healing assays. Interaction among oxidative stress, SMARCA5 and transcriptional reprogramming was elucidated using luciferase reporter assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. Endothelial SMARCA5 expression was significantly decreased in diabetic rodents and humans. Hyperglycemia-suppressed SMARCA5 impaired EC migration and tube formation in vitro, and blunted vasculogenesis in vivo. Contrarily, overexpression of SMARCA5 in situ by a SMARCA5 adenovirus-incorporated hydrogel effectively promoted the rate of wound healing in a dorsal skin punch injury model of diabetic mice. Mechanistically, hyperglycemia-elicited oxidative stress suppressed SMARCA5 transactivation in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Moreover, SMARCA5 maintained transcriptional homeostasis of several pro-angiogenic factors through both direct and indirect chromatin-remodeling mechanisms. In contrast, depletion of SMARCA5 disrupted transcriptional homeostasis to render ECs unresponsive to established angiogenic factors, which ultimately resulted in endothelial dysfunction in diabetes. Suppression of endothelial SMARCA5 contributes to, at least in part, multiple aspects of endothelial dysfunction, which may thereby exacerbate cardiovascular complications in diabetes.

BMP4 aggravates mitochondrial dysfunction of HRMECs

Mitochondria are important places for the oxidative phosphorylation of glucose and the maintenance of cell oxidation and antioxidant stability. However, mitochondrial dysfunction causes cell dysfunction. Meanwhile, retinal vascular endothelial cell dysfunction may cause vascular inflammation, hemorrhage, angiogenesis, and other manifestations. Our previous studies have shown that Bone morphogenetic protein 4 (BMP4) is an important target for the treatment of retinal neovascularization, but the mechanism remains unclear. Therefore, our study aims to observe the effects of BMP4 on vascular endothelial cells and hopes to provide a new target for diabetic retinopathy. 4-Hydroxynonenal (4HNE), a kind of lipid peroxide, was used to induce the oxidative stress model. Human retinal microvascular endothelial cells (HRMECs) were randomly divided into control, 4HNE, negative control, and siBMP4 groups. Si-BMP4 significantly reduced leukocyte adhesion and 4HNE-induced high ROS level and restored the mitochondrial membrane potential and OCR. This indicates that BMP4 plays an important role in inducing leukocyte adhesion, oxidative stress, and mitochondrial dysfunction. The relationship between BMP4 and retinal vascular endothelial cell dysfunction is preliminarily confirmed by this study. Mitochondrial dysfunction and oxidative stress may be involved in BMP4-mediated retinal vascular endothelial cell dysfunction.

Exosomal circTUBGCP4 promotes vascular endothelial cell tipping and colorectal cancer metastasis by activating Akt signaling pathway

BackgroundExosome is crucial mediator and play an important role in tumor angiogenesis. Tip cell formation is a prerequisite for persistent tumor angiogenesis which causes tumor metastasis. However, the functions and underlying mechanisms of tumor cell-derived exosomes in angiogenesis and tip cell formation remain less understood.MethodsExosomes derived from serum of colorectal cancer (CRC) patients with metastasis/non-metastasis and CRC cells were isolated by ultracentrifugation. CircRNAs in these exosomes were analyzed by circRNA microarray. Then, exosomal circTUBGCP4 was identified and verified by quantitative real-time PCR (qRT–PCR) and in situ hybridization (ISH). Loss- and gain-of-function assays were performed to explore the effect of exosomal circTUBGCP4 on vascular endothelial cell tipping and colorectal cancer metastasis in vitro and in vivo. Mechanically, bioinformatics analysis, biotin-labeled circTUBGCP4/ miR-146b-3p RNA pulldown, RNA immunoprecipitation (RIP), and luciferase reporter assay were used to confirm the interaction among circTUBGCP4, miR-146b-3p, and PDK2.ResultsHere, we showed that exosomes derived from CRC cells enhanced vascular endothelial cell migration and tube formation via inducing filopodia formation and endothelial cell tipping. We further screened the upregulated circTUBGCP4 in serum of CRC patients with metastasis compared to non-metastasis. Silencing circTUBGCP4 expression in CRC cell-derived exosomes (CRC-CDEs) inhibited endothelial cell migration, tube formation, tip cell formation, and CRC metastasis. Overexpression of circTUBGCP4 had opposite results in vitro and in vivo. Mechanically, circTUBGCP4 upregulated PDK2 to activate Akt signaling pathway by sponging miR-146b-3p. Moreover, we found that miR-146b-3p could be a key regulator for vascular endothelial cell dysfunction. Exosomal circTUBGCP4 promoted tip cell formation and activated the Akt signaling pathway by inhibiting miR-146b-3p.ConclusionsOur results suggest that colorectal cancer cells generate exosomal circTUBGCP4, which causes vascular endothelial cell tipping to promote angiogenesis and tumor metastasis by activating Akt signaling pathway.

LINC00926 is involved in hypoxia-induced vascular endothelial cell dysfunction via miR-3194-5p regulating JAK1/STAT3 signaling pathway.

Vascular endothelial cell (VEC) dysfunction is associated with the development of coronary heart disease (CHD). Long intergenic non-protein coding RNA 926 (LINC00926), a kind of long noncoding RNA (lncRNA), has been found to be abnormally expressed in CHD patients. However, the biological role of LINC00926 has not been reported. In our research, we intended to explore the regulatory mechanism of LINC00926 in hypoxia-exposed HUVEC cells (HUVECs). In our in vitro study, HUVECs were exposed under hypoxic conditions (5% O2) for 24 h. RT-qPCR and Western blotting assay were used to detect the mRNA and protein levels. CCK-8 assay, flow cytometry, transwell assay and in vitro angiogenesis assay were performed to measure cell proliferation, apoptosis, migration and tube formation, respectively. Bioinformatics analysis was applied to predict the target of LINC00926 and miR-3194-5p, which was verified by dual-luciferase reporter assays. The results showed that LINC00926 was highly expressed in CHD patients and hypoxia-exposed HUVECs. LINC00926 overexpression suppressed cell proliferation, migration and tube formation and increased cell apoptosis. MiR-3194-5p was a target of LINC00926 and can target binding to JAK1 3'UTR. LINC00926 could up-regulate JAK1 and p-STAT3 levels via miR-3194-5p. In addition, overexpressed LINC00926 suppressed cell proliferation, migration and tube formation and increased cell apoptosis via miR-3194-5p/JAK1/STAT3 axis. In summary, LINC00926 aggravated endothelial cell dysfunction via miR-3194-5p regulating JAK1/STAT3 signaling pathway in hypoxia-exposed HUVECs.

NEDD4 attenuates oxidized low‑density lipoprotein‑induced inflammation and dysfunction in vascular endothelial cells via regulating APEX1 expression.

Atherosclerosis chiefly results from inflammation as well as vascular endothelial cell dysfunction. Methylation levels of neuronally expressed developmentally downregulated 4 (NEDD4) were found to be fortified in atherosclerosis patients and NEDD4 deficiency enhanced vascular calcification. However, the exact function of NEDD4 in inflammation and vascular endothelial dysfunction remains to be elucidated. In the present study, CCK-8 assay was used to estimate cell viability. Reverse transcription-quantitative PCR was adopted to examine the expression of NEDD4, inflammation-associated enzymes and apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1). Western blotting was used to test NEDD4, endothelial nitric oxide synthase, inducible nitric oxide synthase and APEX1 protein levels. Cytotoxicity was detected by a lactate dehydrogenase (LDH) kit. Reactive oxygen species level was tested by a corresponding kit. Vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 contents were examined with ELISA. Cell adhesion assays evaluated the adhesion of endothelial cells. Co-immunoprecipitation assay was used to test the relationship between NEDD4 and APEX1. The data revealed that NEDD4 expression rapidly declined in oxidized low density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Following NEDD4 overexpression, the active damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were attenuated. After co-transfection of APEX1 interference plasmids and NEDD4 overexpression plasmids, cell damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were improved again. Taken together, NEDD4 attenuated ox-LDL-induced inflammation and endothelial dysfunction by regulating APEX1 expression.

LncRNA NEAT1 Promotes Vascular Endothelial Cell Dysfunction via miR-218-5p/GAB2 and Serves as a Diagnostic Biomarker for Deep Vein Thrombosis.

Deep vein thrombosis (DVT) is a common peripheral disease. This study aimed to elucidate the diagnostic biomarker of lncRNA nuclear-enriched abundant transcript 1 (NEAT1) in the DVT, and explore possible mechanisms in Human umbilical vein endothelial cells (HUVECs). 101 patients with lower extremity DVT and 82 healthy controls were enrolled. RT-qPCR was designed to resolve the mRNA levels of NEAT1, miR-218-5p, and GAB2. ROC was applied for the diagnosis of DVT. Systemic inflammation (IL-1β, IL-6, and TNF-α) and adhesion factor (SELP, VCAM-1, and ICAM-1) were examined by the ELISA. And cell proliferation, migration, and apoptosis were conducted by the CCK-8, Transwell, flow cytometry assay. The targeting relationship was validated by Dual luciferase reporter and RIP analysis. NEAT1 and GAB2 were upregulated in patients with DVT, while miR-218-5p was decreased (P < .01). Serum NEAT1 can identify DVT patients from healthy individuals. NEAT1 was positively correalted with fibrinolysis factors, coagulation factors, and vasoconstrictors. NEAT1 inhibited the proliferation, migration, and promoted apoptosis as well as inflammation and adhesion factors secretion of HUVECs (P < .05), but all were impaired by overexpression of miR-218-5p (P < .05). NEAT1 promoted GAB2 expression in DVT by acting as a sponge for miR-218-5p. Elevated NEAT1 is a possible DVT diagnostic biomarker, and is implicated in vascular endothelial cell dysfunction via miR-218-5p/GAB2 axis.