Apoptosis Of Human Umbilical Vein Endothelial Cells Research Articles

MicroRNA-301a-3p is a potential biomarker in venous ulcers vein and gets involved in endothelial cell dysfunction

ABSTRACT Venous ulcer is a common contributor to chronic venous insufficiency (CVI) of lower limbs, which seriously affects the life quality of patients. In this study, we researched the expression characteristics of microRNA-301a-3p (miR-301a-3p) in patients with CVI and investigated the impact of miR-301a-3p on the dysfunction of human umbilical vein endothelial cells (HUVECs). The plasma level of miR-301a-3p in normal controls, patients with varicose great saphenous vein, and patients with the venous ulcer of lower limbs were measured. We adopted Interleukin-1β (IL-1β), H2O2, and oxygen and glucose deprivation (OGD) to induce endothelial cell injury in vitro. In this way, we evaluated the influence of miR-301a-3p on HUVEC viability, apoptosis, inflammatory response, and oxidative stress. Our data showed that miR-301a-3p was substantially overexpressed in patients with lower limb venous ulcers. The viability of HUVECs decreased, and miR-301a-3p was up-regulated after IL-1β, H2O2, and OGD treatment. miR-301a-3p inhibition greatly ameliorated the dysfunction and cell damage of HUVECs, promoted IGF1/PI3K/Akt/PPARγ, and down-regulated NF-κB/MMPs. The phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) or the peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor (GW9661) reversed the anti-inflammatory, antioxidant, and anti-apoptotic effects mediated by miR-301a-3p down-regulation. The nuclear factor-κB (NF-κB) inhibitor lessened cell injury mediated by miR-301a-3p overexpression. In terms of the mechanism, miR-301a-3p targeted the 3ʹUTR of Insulin-like growth factor-1 (IGF1) and repressed the profile of IGF1. Thus, miR-301a-3p mediates venous endothelial cell damage by targeting IGF1 and regulating the IGF1/PI3K/Akt/PPARγ/NF-κB/MMPs pathway.

MiR-4463 Regulates Hypoxia-Induced Autophagy and Apoptosis by Targeting ULK1 in Endothelial Cells

Our previous study revealed aberrant miR-4463 expression in the vascular tissues of patients with arteriosclerosis obliterans of the lower extremities (ASO), but the role of miR-4463 was largely ambiguous. In the current study, we aimed to explore the function of miR-4463 in hypoxia-induced endothelial cells and determine its molecular mechanisms. CCK-8 assay and flow cytometry were performed to evaluate cell viability and apoptosis. Adenovirus carrying mRFP-GFP-LC3 was employed to monitor cellular autophagy, and mitochondrial membrane potential was determined by JC-1 staining. Moreover, dual-luciferase reporter gene assay, qPCR, western blot and siRNA analysis were carried out to explore the potential molecular mechanisms. Hypoxia significantly elevated the miR-4463 expression in primary human umbilical vein endothelial cells (HUVEC). Overexpression of miR-4463 inhibited hypoxia-induced autophagy by suppressing the formation of autophagosomes and autolysosomes, resulting in reduced cell viability and increased apoptosis, and these effects were reversed by miR-4463 inhibitor. Furthermore, activation of autophagy induced by miR-4463 inhibitor attenuated HUVECs apoptosis in hypoxic conditions. Mechanically, the results of the dual-luciferase reporter gene assay discovered that miR-4463 directly targeted Unc-51 like kinase 1 (ULK1). The silence of ULK1 blocked miR-4463 inhibitor-activated autophagy and further facilitated apoptosis under hypoxic conditions. Our findings indicate that miR-4463 is an essential regulator of hypoxia-induced autophagy and apoptosis in endothelial cells via directly targeting ULK1. Inhibition of miR-4463 might be a potential strategy to protect endothelial cells and maintain vascular function in patients with lower limb ischemia and its complications.

Identification of pathways and key genes in male late-stage carotid atherosclerosis using bioinformatics analysis.

Late-stage carotid atherosclerosis has a high incidence rate and may lead to various cerebrovascular diseases. The gene expression profile GSE100927 was selected to identify differentially expressed genes (DEGs) in carotid atherosclerosis. Subsequently, protein-protein interaction, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were conducted. Furthermore, experimental verification was performed using human umbilical vein endothelial cells (HUVECs), human aortic vascular smooth muscle cells (HAVSMCs) and Tohoku Hospital Pediatrics-1 (THP-1)-induced macrophages. The groups were as follows: Control group, solvent control group and palmitic acid group. The levels of reactive oxygen species (ROS) in the three cell types were detected by flow cytometry or fluorescence microscopy. Furthermore, apoptosis of HUVECs and HAVSMCs was assessed by flow cytometry and the nuclear Hoechst 33258 staining of THP-1-induced macrophages was performed. Male late-stage carotid atherosclerosis samples, including 10 control samples and 21 atherosclerosis samples, were selected. Pathway enrichment analysis demonstrated that ‘Toll-like receptor signaling pathway’ was the top pathway associated with the DEGs. MMP7, MMP9, IL1β, C-C motif chemokine ligand 4 (CCL4), secreted phosphoprotein 1 (SPP1), CCL3 and interferon regulatory factor 5 (IRF5) were selected for experimental verification. Palmitic acid increased the ROS levels and the apoptosis rates of HUVECs and HAVSMCs. However, it did not increase the levels of ROS and did not shrink the nuclei of THP-1-induced macrophages. Furthermore, palmitic acid increased the mRNA levels of IL1β, CCL4, SPP1, CCL3, IRF5, MMP7 and MMP9 in HUVECs and THP-1-induced macrophages, and increased the mRNA levels of CCL4 and MMP9 in HAVSMCs. In conclusion, IL1β, CCL3, CCL4, SPP1, IRF5, MMP7 and MMP9 are important markers of late-stage carotid atherosclerosis.

Dapagliflozin improves endothelial cell dysfunction by regulating mitochondrial production via the SIRT1/PGC-1α pathway in obese mice

Obesity is closely related to the occurrence of cardiovascular diseases, and an important reason for this is the induction of endothelial cell dysfunction. Mitochondria play an important role in maintaining the function of endothelial cells. In the present study, we examined the effects of the sodium-dependent glucose transporters 2 inhibitor dapagliflozin (DAPA) on the vascular endothelium in obese mice in vivo and on the structure and function of human umbilical vein endothelial cells (HUVECs) in vitro. The results revealed that DAPA rescued vascular endothelial damage in obese mice. Moreover, DAPA reversed the effects of palmitic acid (PA) on the reduction in angiogenesis and the increase in apoptosis in HUVECs. Furthermore, DAPA rescued the reduced mitochondrial membrane potential, mitochondrial viability, energy metabolism, mitochondrial biogenesis and the mitochondrial structural injury caused by PA. DAPA also activated the SIRT1/PGC-1α signaling pathway, while the SIRT1 inhibitor EX-527 abrogated the effects of DAPA on the mitochondria of HUVECs. In summary, our study suggests that DAPA improves endothelial cell mitochondrial function in obese mice by activating the SIRT1/PGC-1α pathway.

Circ_0124644 enhances ox-LDL-induced cell damages in human umbilical vein endothelial cells through upregulating FOXO4 by sponging miR-370-3p

Circular RNA circ_0124644 has crucial regulation in the progression of coronary artery diseases, including atherosclerosis (AS). The aim of this study was to explore the regulatory mechanism of circ_0124644 in oxidized low-density lipoprotein (ox-LDL)-induced endothelial injury in human umbilical vein endothelial cells (HUVECs). Cell viability and proliferation were assessed using cell counting kit-8 (CCK-8) assay and EdU assay. The apoptosis detection was performed by flow cytometry. Angiogenesis was evaluated through tube formation assay. The protein analysis was conducted via western blot. Inflammatory cytokines were examined by enzyme-linked immunosorbent assay (ELISA). The expression determination of circ_0124644, microRNA-370-3p (miR-370-3p) and forkhead box protein O4 (FOXO4) was performed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to analyze the interaction between targets. Treatment of ox-LDL resulted in the inhibition of cell viability, proliferation and angiogenesis but the promotion of apoptosis and inflammation in HUVECs. These ox-LDL-induced cell damages were alleviated after the downregulation of circ_0124644. Circ_0124644 interacted with miR-370-3p, and the regulatory role of circ_0124644 was associated with the sponge function of miR-370-3p. Additionally, miR-370-3p targeted FOXO4 and circ_0124644 increased the expression of FOXO4 through acting as a sponge of miR-370-3p. Overexpression of miR-370-3p protected from ox-LDL-induced injury via the downregulation of FOXO4. All results revealed that circ_0124644 accelerated endothelial injury in ox-LDL-treated HUVECs by mediating miR-370-3p-related FOXO4 expression.

Long noncoding RNA TDRG1 aggravates doxorubicin-induced cardiomyopathy by binding with miR-873-5p to upregulate PRKAR2.

Doxorubicin-induced cardiomyopathy (DCM) is a life-threatening event. The long noncoding RNAs (lncRNAs) have been reported with close associations with DCM, which may provide novel insight into pathophysiological mechanisms of DCM. DCM rat model and cell models were established using doxorubicin. Echocardiography analyses were performed to assess cardiac function. We found that testis developmental-related gene 1 (TDRG1) expression was upregulated in DCM rats and in doxorubicin-treated human umbilical vein endothelial cells (HUVECs). TDRG1 knockdown enhanced cell viability, promoted tube formation, and inhibited apoptosis of doxorubicin-treated HUVECs. Additionally, knockdown of TDRG1 alleviated cardiac injury in DCM rats. Mechanistically, miR-873-5p was identified to bind with TDRG1. In addition, protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2) was confirmed to bind with miR-873-5p as a target mRNA. MiR-873-5p negatively regulated PRKAR2 mRNA and protein levels. At last, rescue assays indicated that the overexpression of PRKAR2 restored the effect of TDRG1 knockdown on doxorubicin-treated HUVEC angiogenesis and apoptosis. To conclude, TDRG1 aggravates DCM progression by binding with miR-873-5p to upregulate PRKAR2. This work suggested the potential of TDRG1 as a target for DCM treatment.

Investigation of nickel sulfate-induced cytotoxicity and underlying toxicological mechanisms in human umbilical vein endothelial cells through oxidative stress, inflammation, apoptosis, and MAPK signaling pathways.

Growing evidence indicates that nickle and its compounds have adverse effects on the cardiovascular system. In this study, the cytotoxic insults caused by nickel sulfate (NiSO4 ) in human umbilical vein endothelial cells (HUVECs) were explored by examining cell viability, oxidative stress, inflammation, apoptosis, and MAPK signaling pathway activity. Cultured HUVECs were treated with varying concentrations of NiSO4 (0, 62.5, 250, and 1000 μM) for 24 h. Subsequently, markers of oxidative stress, inflammation, apoptosis, and MAPK signaling pathways were analyzed using biochemical assays, real-time quantitative polymerase chain reaction, and western blot. Rates of apoptosis were evaluated using flow cytometry. The results showed that NiSO4 exerted dose- and time-dependent inhibitory effects on cell growth. It induced oxidative stress and lipid peroxidation by increasing the generation of reactive oxygen species, the oxidized glutathione to reduced glutathione ratio (GSSG/GSH ratio), and malondialdehyde levels. Further, it inhibited superoxide dismutase activity in HUVECs. Flow cytometry analysis results revealed that NiSO4 (62.5-1000 μM) could induce apoptosis in HUVECs. The protein and gene expressions of cleaved Caspase 3 and Bax were elevated, and those of Bcl-2 and Bcl-XL were reduced after NiSO4 treatment. Additionally, NiSO4 triggered inflammation in HUVECs, increasing the protein and mRNA levels of IL-6 and TNF-α and reducing those of TGF-β. Furthermore, western blot findings revealed that NiSO4 could activate MAPK signaling pathways, upregulating p38, JNK, and ERK1/2 in HUVECs by increasing the levels of p-P38，p-JNK, and p-ERK1/2 in a dose-dependent manner. MAPK pathway inhibitors (10μM SB203580 and 10μM SP600125) could attenuate the NiSO4 -induced increase in apoptosis and inflammation in HUVECs. They could also attenuate the dysregulation of inflammatory factors and related proteins caused by high-dose NiSO4 exposure. Interestingly, while the MEK inhibitor U0126 (10μM) enhanced NiSO4 -induced apoptosis in HUVECs, it reduced cell inflammation. Taken together, these experimental results suggest that NiSO4 can inhibit cell growth, induce oxidative stress, and trigger subsequent inflammatory responses and apoptosis in HUVECs. These effects may be mediated by the P38 and JNK MAPK stress response pathways.

The Combined Use of Platelet-Rich Plasma Clot Releasate and Allogeneic Human Umbilical Cord Mesenchymal Stem Cells Rescue Glucocorticoid-Induced Osteonecrosis of the Femoral Head.

Glucocorticoid-induced osteonecrosis of the femoral head (ONFH) is a refractory disease. The treatment options for ONFH, especially nonsurgical ones, merit further investigation. To evaluate the combinatorial therapeutic effects of platelet-rich plasma clot releasate (PRCR) and umbilical cord mesenchymal stem cells (UC-MSCs) on glucocorticoid-induced ONFH, a dexamethasone (DEX)-treated cell model and a high-dose methylprednisolone (MPS)-treated rat model were established. Cell counting kit-8 (CCK-8) assay was performed in vitro to determine the optimum dosage of PRCR for UC-MSC viability. The effects of PRCR, UC-MSCs, and PRCR + UC-MSCs on cell viability, apoptosis, migration, and differentiation capacities of DEX-treated bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cell (HUVECs) were explored via Transwell assays. Western blotting was conducted to evaluate the expression levels of RUNX2, VEGF, caspase-3, and Bcl-2 in the coculture systems. Ultrasound-guided intra-articular PRCR, UC-MSCs, and PRCR + UC-MSC injections were performed on the ONFH model rats. Microcomputed tomography, histological and immunohistochemical analyses, tartrate-resistant acid phosphatase (TRAP) staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess the therapeutic effects of PRCR and UC-MSCs on bone loss and necrosis induced by high-dose MPS. Results of this study revealed that the in vitro application of PRCR, UC-MSCs, and PRCR + UC-MSCs reversed the impaired proliferation and migration capacities and resisted apoptosis of BMSCs and HUVECs induced by DEX. Moreover, the PRCR and UC-MSC application significantly improved the alkaline phosphatase (ALP) and alizarin red (ALR) staining of BMSCs and tube formation capacity of HUVECs and promoted the protein expression of RUNX2 in BMSCs and VEGF in HUVECs. Similarly, in the ONFH rat model, the intra-articular injection of UC-MSCs and PRCR improved the subchondral bone mass parameters; promoted the expression of ALP, RUNX2, and VEGF; suppressed osteoclast overactivity; and resisted cell apoptosis. The combination of PRCR and UC-MSCs shows promising therapeutic effects in treating glucocorticoid-induced ONFH. The current study provides important information on intra-articular therapy, paving the way for the clinical management of ONFH in the future.

Quercetin regulates vascular endothelium function in chronic renal failure via modulation of Eph/Cav-1 signaling.

Arteriovenous fistula (AVF) is frequently believed to be the best vascular access for chronic renal failure (CRF) patients. Vascular endothelial cell dysfunction has been implicated in AVF maturation. Quercetin (Quer) is a natural polyphenolic compound widely used in traditional Chinese medicine. We aimed to uncover the impacts of Quer on vascular endothelial cells in a CRF rat model and human umbilical vein endothelial cells (HUVECs) stimulated by lipopolysaccharide (LPS) and serum from rat with CRF. Blood urea nitrogen and serum creatinine levels were tested in CRF rat model after administration of Quer. H&E staining was used to estimate endothelial damage. Nitric oxide (NO), endothelial NO synthase (eNOS), EPH receptor B4 (EphB4), EphrinB2, and p-caveolin-1 (p-Cav-1) levels in the serum were examined by enzyme-linked immunosorbent assay. Western blot was employed to analyze the expressions of eNOS, phosphorylated (p)-eNOS, EphB4, and Cav-1 in arterial tissues and HUVECs. Cell counting kit-8 was applied for assessing cell proliferation. TUNEL(terminal-deoxynucleotidyl transferase-mediated nick end labeling) assay was employed to estimate cell apoptosis. Results showed that Quer ameliorated renal function impairment and endothelial injury in vivo. Meanwhile, Quer boosted the proliferation and suppressed the apoptosis of HUVECs stimulated by LPS and serum from rat with CRF. Additionally, Quer elevated NO and eNOS levels, upregulated p-eNOS expression but downregulated EphB4, EphrinB2, and p-Cav-1 expressions. Moreover, EphB4 inhibitor had the similar effect as Quer treatment in HUVECs stimulated by LPS and serum from rat with CRF. Collectively, Quer might effectively regulate vascular function to prevent AVF failure in CRF via modulation of Eph/Cav-1 signaling.

Pigment Epithelium-Derived Factor-Loaded PEGylated Nanoparticles as a New Antiangiogenic Therapy for Neovascularization.

Background Pathological neovascularization, which involves a disruption in the balance between angiogenic and antiangiogenic factors under pathological conditions, is the basis of many intraocular diseases. Pigment epithelium-derived factor (PEDF) is a potent natural, endogenous inhibitor of neovascularization because of its antiangiogenic and neuroprotective benefits. However, its application is restricted by its instability and short half-life. The present study is aimed at investigating the cytotoxicity and antiangiogenic effects of PEDF-loaded PEGylated nanoparticles (NP-PEG-PEDF) on high glucose-stimulated human umbilical vein endothelial cells (HUVECs). Methods In this study, NP-PEG-PEDF were fabricated using the multiple emulsion method for the first time. HUVECs were cultured in a high concentration of glucose (30 mmol/L D-glucose), simulating diabetic conditions. The antiangiogenic effects of vascular endothelial growth factor (VEGF), pure PEDF, and NP-PEG-PEDF on proliferation, migration, and tube formation were evaluated. VEGF secretion in high glucose-stimulated HUVECs was further tested in vitro. Results NP-PEG-PEDF exhibited low cytotoxicity in HUVECs. Our results indicated that in vitro, NP-PEG-PEDF attenuated diabetes-induced HUVEC proliferation, migration, and tube formation and suppressed VEGF secretion. The apoptosis of diabetes-induced HUVECs occurred in a dose-dependent manner, which showed a statistically significant difference compared with the PEDF treatment group. Conclusion Our study is the first to demonstrate that NP-PEG-PEDF exert antiangiogenic effects on high glucose-stimulated HUVECs and have the potential to alleviate microvascular dysfunction. These data suggest that the NP-PEG-PEDF delivery system may offer an innovative therapeutic strategy for preventing neovascularization of the fundus.

Far-infrared radiation alleviates cisplatin-induced vascular damage and impaired circulation via activation of HIF-1α.

Severe vascular damage and complications are often observed in cancer patients during treatment with chemotherapeutic drugs such as cisplatin. Thus, development of potential options to ameliorate the vascular side effects is urgently needed. In this study, the effects and the underlying mechanisms of far‐infrared radiation (FIR) on cisplatin‐induced vascular injury and endothelial cytotoxicity/dysfunction in mice and human umbilical vein endothelial cells (HUVECs) were investigated. An important finding is that the severe vascular stenosis and poor blood flow seen in cisplatin‐treated mice were greatly mitigated by FIR irradiation (30 minutes/day) for 1‐3 days. Moreover, FIR markedly increased the levels of phosphorylation of PI3K and Akt, and VEGF secretion, as well as the expression and the activity of hypoxia‐inducible factor 1α (HIF‐1α) in cisplatin‐treated HUVECs in a promyelocytic leukemia zinc finger protein (PLZF)‐dependent manner. However, FIR‐stimulated endothelial angiogenesis and VEGF release were significantly diminished by transfection with HIF‐1α siRNA. We also confirmed that HIF‐1α, PI3K, and PLZF contribute to the inhibitory effect of FIR on cisplatin‐induced apoptosis in HUVECs. Notably, FIR did not affect the anticancer activity and the HIF‐1α/VEGF cascade in cisplatin‐treated cancer cells under normoxic or hypoxic condition, indicating that the actions of FIR may specifically target endothelial cells. It is the first study to demonstrate that FIR effectively attenuates cisplatin‐induced vascular damage and impaired angiogenesis through activation of HIF‐1α–dependent processes via regulation of PLZF and PI3K/Akt. Taken together, cotreatment with the noninvasive and easily performed FIR has a therapeutic potential to prevent the pathogenesis of vascular complications in cancer patients during cisplatin treatment.

FGA Controls VEGFA Secretion to Promote Angiogenesis by Activating the VEGFR2-FAK Signalling Pathway.

BackgroundOur previous work revealed the high expression of fibrinogen alpha chain (FGA) in patients with endometriosis (EM) and that it could promote the migration and invasion of endometrial stromal cells. Angiogenesis is the key condition for the development of EM. This study was aimed to elucidate the role of FGA in endometrial stromal cells involved in angiogenesis in EM.MethodsImmunohistochemistry was used to detect the microvessel density (MVD) and VEGF expression in the eutopic endometrium samples from EM and non-EM. The conditioned medium (CM) of human primary eutopic endometrial stromal cells (EuESC) and immortalized endometrial stromal cell line hEM15A with FGA knockdown were collected and used to treat human umbilical vein endothelial cells (HUVECs). Then, tube formation assay, EdU assay, wound assay, transwell assay and flow cytometry assays were performed to assess the function of HUEVCs in vitro. The angiogenic capability of HUVECs was further measured using a matrigel plug assay with BALB/c nude mice in vivo. Immunofluorescence was used to detect the expression of F-actin and VE-cadherin. RT-PCR and western blotting were used to detect the expression of angiogenesis-related factors in endometrial stromal cells and downstream signalling pathways in HUVECs.ResultsMVD and VEGF expression in the eutopic endometrium of EM patients were significantly higher than those in the normal endometrium of non-EM patients, and the increased MVD in EM indicates an increased risk of recurrence. Functionally, we found that CM of endometrial stromal cells with FGA knockdown could inhibit HUEVCs migration and tube formation in vitro and in vivo, while having no significant effect on HUVECs proliferation, apoptosis and cell cycle. Mechanically, the expression of VEGFA, PDGF, FGF-B, VEGF, MMP-2 and MMP-9 was reduced in hEM15A cells with FGA knockdown. CM of hEM15A cells with FGA knockdown reduced the number of microfilaments and pseudopodia, as well as the expression of VE-cadherin, and inhibited the activity of VEGFR2 and the FAK signalling pathway in HUVECs.ConclusionOur study demonstrated FGA could enhance the interaction between endometrial stromal cells and HUVECs via the potential VEGA-VEGFR-FAK signalling axis and promote EM angiogenesis, revealing a promising therapeutic approach for EM.

Dapagliflozin attenuates high glucose-induced endothelial cell apoptosis and inflammation through AMPK/SIRT1 activation.

Hyperglycaemia is a major cause of pathophysiological processes such as oxidative stress, inflammation, and apoptosis in diabetes. Dapagliflozin (DAPA), a novel hypoglycaemic drug, has been shown to have anti-apoptotic, anti-inflammatory, and antioxidant effects in multiple experimental studies. In this study, we investigated the protective effects of DAPA in the hyperglycaemic condition to identify associated molecular mechanisms. human umbilical vein endothelial cells (HUVEC) endothelial cells were treated with 40 mM glucose for 72 h to establish an in vitro high glucose (HG) condition model, and then additional groups co-treated with or without DAPA before glucose treatment. Then, cell viability, reactive oxygen species (ROS), pro-inflammatory cytokines (IL-6 and TNF-α), apoptosis, and SIRT1 expression were measured. The results showed that DAPA pretreatment resulted in increased cell viability. Additionally, DAPA pretreatment decreased endothelial ROS, IL-6, and TNF-α levels in endothelial cells subjected to HG conditions. Moreover, DAPA pretreatment significantly prevented HG-induced apoptosis and caspase-3 activity in HUVECs. Furthermore, DAPA increased the expression of SIRT1, PGC-1α, and increased the phosphorylation levels of AMPK (p-AMPK) in a set of HG conditions in HUVECs. However, the endothelial protective effects of DAPA were abolished when cells were subjected to the SIRT1 inhibitor (EX-527) and AMPK inhibitor (Compound C). These findings suggest that DAPA can abrogate HG-induced endothelial cell dysfunction by AMPK/SIRT1 pathway up-regulation. Therefore, suggesting that the activation of AMPK/SIRT1 axis by DAPA may be a novel target for the treatment of HG-induced endothelial cell injury.

Protocatechualdehyde Rescues Oxygen-Glucose Deprivation/Reoxygenation-Induced Endothelial Cells Injury by Inducing Autophagy and Inhibiting Apoptosis via Regulation of SIRT1

Background: Oxidative stress-induced endothelial cell death, such as apoptosis and autophagy, plays a critical role in ischemia-reperfusion injury. Protocatechualdehyde (PCA) is a major bioactive component of the traditional Chinese medicine Salvia miltiorrhiza Bunge (Lamiaceae), and it has been proved to be effective in the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases. However, its role in oxidative stress-induced endothelial cell death and its underlying mechanisms remains unclear. This study aims to investigate the effects and mechanisms of PCA on endothelial cell apoptosis and autophagy induced by oxygen-glucose deprivation/reoxygenation (OGD/R) injury. Methods: After OGD/R induction, human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of PCA. Cell viability, apoptosis, and autophagy were detected by Cell Counting Kit-8 assay, flow cytometry, and monodansylcadaverine assay, respectively. Western blot was applied to explore the effects of PCA on the expression levels of relevant protein factors. Results: The results show that PCA significantly promoted cell survival rate and cell proliferation and enhanced the antioxidant activity in OGD/R-induced HUVECs. PCA inhibited HUVECs apoptosis, as evidenced by decreased expression of cleaved-caspase-3, Bcl2-associated X (BAX), and increased expression of Bcl-2. PCA induced autophagy by reducing the expression of P62 while increasing the expression of Beclin-1 and LC3 II/I. Meanwhile, PCA enhanced the expression of Sirtuin 1 (SIRT1) and suppressed the expression of P53. When SIRT1 was inhibited by selisistat or SIRT1 small-interfering RNA, the anti-apoptotic and pro-autophagy abilities of PCA were attenuated. Conclusion: These results demonstrated that PCA rescued HUVECs from OGD/R-induced injury by promoting autophagy and inhibiting apoptosis through SIRT1 and could be developed as a potential therapeutic agent against ischemic diseases.

KLF2 mediates the suppressive effect of BDNF on diabetic intimal calcification by inhibiting HK1 induced endothelial-to-mesenchymal transition

Diabetic vascular calcification in the arterial intima is closely associated with endothelial-to-mesenchymal transition (EndMT). Glucose metabolism reprogramming is involved in EndMT. Although brain-derived neurotrophic factor (BDNF) and Krüppel-like family of transcription factor 2 (KLF2) play protective roles in the physiological activity of the vascular endothelium, the underlying mechanisms are unclear. Human umbilical vein endothelial cells (HUVECs) were incubated with diabetic osteogenic medium (DOM) to induce EndMT and accelerate osteogenic differentiation. Glycolysis in HUVECs was assessed by monitoring glucose uptake, lactate production, extracellular acidification rate and expression of key glycolytic enzymes. DOM induced EndMT and accelerated osteo-induction in HUVECs, which was alleviated by BDNF/tropomyosin receptor kinase B (TrkB) pathway. Mechanistically, DOM caused hyperactivation of glycolysis in HUVECs and inhibition of the BDNF/TrkB pathway. BDNF preserved KLF2 and downregulated hexokinase 1 (HK1) in HUVECs after DOM treatment. Furthermore, KLF2 interacted with HK1. Increased KLF2 alleviated HK1-mediated glucose metabolism abnormality. HK1 knockdown or a targeted glycolysis inhibitor suppressed EndMT, apoptosis, inflammation and vascular calcification of HUVECs after DOM exposure. This study suggests that KLF2 mediates the suppressive effect of BDNF on diabetic intimal calcification by inhibiting HK1-induced glucose metabolism reprogramming and the EndMT process.

Hsa_circ_0058122 knockdown prevents steroid-induced osteonecrosis of the femoral head by inhibiting human umbilical vein endothelial cells apoptosis via the miR-7974/IGFBP5 axis.

BackgroundSteroid‐induced osteonecrosis of femoral head (SONFH) is a serious complication of glucocorticoid overused. Recent evidence has demonstrated that circRNAs exert key pathophysiological roles in a variety of disease processes. However, the role of circRNA in SONFH remains largely unknown. The current study sought to evaluate how hsa_circ_0058122 affects SONFH in dexamethasone (DEX) treated human umbilical vein endothelial cells (HUVECs) model.MethodsRT‐PCR was used to demonstrate the hsa_circ_0058122 expression level in Dex‐treated HUVECs cells. The effects of hsa_circ_0058122 on HUVECs apoptosis were evaluated via overexpression plasmid and siRNA. Using dual‐luciferase and fluorescence in situ hybridization assays, we demonstrated that hsa_circ_0058122 binds to miR‐7974 thereby facilitating HUVECs apoptosis. Bioinformatics analysis and western blot were performed to confirm target genes of hsa‐miR‐7974.ResultsIn our previous work, we revealed the top 20 elevated circRNAs in SONFH patients were hsa_circ_0010027, hsa_circ_0058115, hsa_circ_0010026, hsa_circ_0058839, hsa_circ_0056886, hsa_circ_0056885, hsa_circ_0058146, hsa_circ_0058105, hsa_circ_0058112, hsa_circ_0058143, hsa_circ_0058102, hsa_circ_0058090, hsa_circ_0075353, hsa_circ_0058126, hsa_circ_0058130, hsa_circ_0058140, hsa_circ_0058122, hsa_circ_0058123, hsa_circ_0058103, and hsa_circ_0058121. Among these, hsa_circ_0058122 was finally selected for further investigation. We found hsa_circ_0058122 expression was markedly elevated in Dex‐treated HUVECs cells, and the Dex‐mediated HUVEC apoptosis was impaired in hsa_circ_0058122‐silenced cells and increased in hsa_circ_0058122‐overexpressing cells. hsa_circ_0058122 competitively binds to hsa‐miR‐7974, which in turn interacts with insulin‐like growth factor binding protein 5 (IGFBP5).Conclusionshsa_circ_0058122/miR‐7974/IGFBP5 was proposed to be a key regulatory pathway for SONFH. DEX treatment upregulated hsa_circ_0058122 expression in HUVECs, which sponged miR‐7974, thereby increasing IGFBP5 expression, the hsa_circ_0058122/miR‐7974/IGFBP5 axis contributed to the Dex‐mediated apoptosis. These findings may identify novel targets for SONFH molecular therapy.

SESN1 attenuates the Ox‑LDL‑induced inflammation, apoptosis and endothelial‑mesenchymal transition of human umbilical vein endothelial cells by regulating AMPK/SIRT1/LOX1 signaling.

Endothelial cells are an important component of the heart and vasculature and form a crucial link between the cardiovascular system and the immune system. Sestrin 1 (SESN1) has an important role in atherosclerosis by inhibiting NOD-like receptor family pyrin domain containing 3 inflammasome activation. However, whether SESN1 is involved in human umbilical vein endothelial cell (HUVEC) injury caused by atherosclerosis has remained to be elucidated. The present study aimed to investigate the functions of SESN1 in the inflammatory response, apoptosis and endothelial-mesenchymal transition (EndMT) of HUVECs following stimulation with oxidized low-density lipoprotein (Ox-LDL). SESN1 expression at the mRNA and protein levels was detected using reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. Following SESN1 overexpression in Ox-LDL-stimulated HUVECs, cell viability was determined using a Cell Counting Kit-8 assay. Terminal deoxynucleotidyl transferase-mediated nick-end labeling staining was employed to detect cell apoptosis and western blot analysis was used to determine the levels of apoptosis-related proteins. RT-qPCR, ELISA and western blot were utilized to determine the levels of inflammatory factors. Immunofluorescence staining, RT-qPCR and western blot analysis were employed to assess the EndMT of Ox-LDL-stimulated HUVECs. The results revealed that SESN1 exhibited a low expression in HUVECs following Ox-LDL stimulation. SESN1 overexpression suppressed inflammation, apoptosis and EndMT in Ox-LDL-induced HUVECs. In addition, SESN1 stimulated adenosine monophosphate-activated protein kinase catalytic subunit α1/sirtuin 1 signaling to suppress Ox-LDL receptor-1 expression. An AMPK and SIRT1 inhibitor reversed the effects of SESN1 overexpression on the inflammatory response, apoptosis and EndMT of HUVECs exposed to Ox-LDL. Taken together, the present study demonstrated that SENS1 exerts a suppressive effect on Ox-LDL-induced inflammation, apoptosis and EndMT of HUVECs, suggesting that SENS1 may be used as a novel biomarker for endothelial injury-related disorders.

Exosomes derived from stem cells of human deciduous exfoliated teeth inhibit angiogenesis in vivo and in vitro via the transfer of miR-100-5p and miR-1246

BackgroundAnti-angiogenic therapy has been shown to be a promising strategy for anti-tumor treatment. Increasing evidence indicates that tumor angiogenesis is affected by exosomes that are secreted by mesenchymal stem cells (MSCs), but whether exosomes derived from MSCs suppress or promote angiogenesis remain paradoxical. The purpose of this study focused on understanding the potential role of exosomes derived from stem cells of human deciduous exfoliated teeth (SHED-Exos) in regulating angiogenesis and the underlying molecular mechanism.MethodsExosomes were isolated from supernatants of SHED cells using an exosome purification kit and were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Cell Counting Kit-8, flow cytometric assays, western blots, wound healing and transwell migration assays were performed to characterize the roles of SHED-Exos on cell proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs). The anti-angiogenic activity of SHED-Exos was assessed via a tube formation assay of endothelial cells and angiogenesis-related factors were analyzed by western blotting. In vivo, we used the chick chorioallantoic membrane (CAM) assay and an oral squamous cell carcinoma (OSCC) xenograft transplantation model with nude mice that received multi-point injections at three-day intervals to evaluate the effects on angiogenesis. Furthermore, the sequencing of microRNAs (miRNAs) in SHED-Exos was performed to investigate the underlying anti-angiogenic mechanism.ResultsThe results showed that SHED-Exos inhibit cell proliferation and migration and induce apoptosis in HUVECs. SHED-Exos suppress the tube-like structure formation of HUVECs in vitro. SHED-Exos downregulate several angiogenesis-related factors, including VEGFA, MMP-9 and ANGPT1. In vivo, the chick CAM assay verified that treatment with SHED-Exos inhibits micro-vascular formation, and importantly, significantly reduces the micro-vascular formation of tumors generated from xenografted OSCC cells, which was associated with the inhibition of tumor growth in vivo. Mechanistically, our data suggested that SHED-Exos are enriched with miR-100-5p and miR-1246 and are transferred to endothelial cells, which results in decreased tube formation via the down-regulation of VEGFA expression.ConclusionsThese results demonstrate that SHED-Exos inhibit angiogenesis in vitro and in vivo, which suggests that SHED-Exos could potentially serve as a novel and effective therapeutic approach for anti-angiogenic treatment.

SAR131675 Receptor Tyrosine Kinase Inhibitor Induces Apoptosis through Bcl- 2/Bax/Cyto c Mitochondrial Pathway in Human Umbilical Vein Endothelial Cells.

Tyrosine Kinase Inhibitors (TKIs) can be used to inhibit cancer cell proliferation by targeting the vascular endothelial growth factor receptor (VEGFR) family. SAR131675 is a highly selective receptor tyrosine kinase inhibitor to VEGFR3 that reveals the inhibitory effect on proliferation in human lymphatic endothelial cells. However, the molecular mechanisms underlying this process are generally unclear. This study was performed to investigate the possible involvement of the Bcl-2/Bax/Cyto c apoptosis pathway in Human Umbilical Vein Endothelial Cells (HUVECs). In addition, the role of Reactive Oxygen Species (ROS) and mitochondrial membrane potential was evaluated. The effect of SAR131675 on HUVEC cell viability was evaluated by MTT assay. The activity of SAR131675 in inducing apoptosis was carried out through the detection of Annexin V-FITC/PI signal by flow cytometry. To determine the mechanisms underlying SAR131675 induced apoptosis, the mitochondrial membrane potential, ROS generation, the activity of caspase-3, and expression of apoptosis-related proteins such as Bcl-2, Bax, and cytochrome c were evaluated in HUVECs. SAR131675 significantly inhibited cell viability and induced apoptosis in HUVECs in a dose-dependent manner. Moreover, SAR131675 induced mitochondrial dysfunction, ROS generation, Bcl-2 down-regulation, Bax upregulation, cytochrome c release, and caspase-3 activation, which displays features of mitochondria-dependent apoptosis signaling pathway. Our present data demonstrated that SAR131675-induced cytotoxicity in HUVECs associated with the mitochondria apoptotic pathway. These results suggest that further studies are required to fully elucidate the role of TKIs in these cellular processes.

Kawasaki disease: lncRNA Slco4a1 regulates the progression of human umbilical vein endothelial cells by targeting the miR-335-5p/POU5F1 axis.

BackgroundKawasaki disease (KD) is an autoimmune disease with systemic vasculitis as the main pathological change, and is most common in children under 5. The role of long non-coding RNAs (lncRNAs) in human diseases has been highlighted. LncRNA Slco4a1 was reported to promote cell growth and act as an oncogenic regulator in cancer. However, the role of lncRNA Slco4a1 in KD remains unclear. This study aimed to investigate the role and mechanism of lncRNA Slco4a1 in KD.MethodsEnzyme linked immunosorbent assay (ELISA), qRT-PCR, Western blot, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining were conducted to explore the function of lncRNA Slco4a1. The interaction between POU5F1 and miR-335-5p was analyzed by the RIP assay and dual luciferase assay.ResultsLncRNA Slco4a1 was significantly upregulated in the serum of KD patients compared with healthy controls. LncRNA Slco4a1 was upregulated in human umbilical vein endothelial cells (HUVECs) stimulated with KD serum. LncRNA Slco4a1 overexpression could promote the expression of inflammatory factors and apoptosis in HUVECs. The number of inflammatory cells and the infiltration area of the coronary artery in KD rats were decreased after lncRNA Slco4a1 silencing. Furthermore, lncRNA Slco4a1 is a sponge of miR-335-5p and negatively regulated the expression of miR-335-5p. POU5F1 was the downstream target of miR-335-5p, and miR-335-5p overexpression could upregulate the expression of POU5F1. Additionally, miR-335-5p overexpression could inhibit the expression of inflammatory factors and apoptosis in HUVECs. We further investigated the effect of lncRNA Slco4a1 on the mitogen-activated protein kinase (MAPK) signaling pathway, and the results showed that lncRNA Slco4a1 could promote the activation of the MAPK signaling pathway.ConclusionsTogether, these results indicated that lncRNA Slco4a1 could regulate the progression of HUVECs in KD by targeting the miR-335-5p/POU5F1 axis, providing new insights for KD treatment.