Apoptosis In Umbilical Vein Endothelial Cells Research Articles

NLRC3 deficiency promotes hypoxia-induced pulmonary hypertension development via IKK/NF-κB p65/HIF-1α pathway

Hypoxia-induced pulmonary hypertension is a subgroup of type 3 pulmonary hypertension (PH) with the recommended treatment limited to oxygen therapy and lacks potential therapeutic targets. To investigate the role of NLRC3 in hypoxia-induced PH and its potential mechanism, we first collected lung tissues of high-altitude pulmonary hypertension (HAPH) patients. Immunohistochemistry and immunofluorescence showed that NLRC3 was downregulated and was mainly co-localized with the smooth muscle cells of the pulmonary vessels in HAPH patients. Besides, we found that NLRC3 was also expressed in endothelial cells in HAPH patients for the first time. Then, wild type (WT) and NLRC3 knockout (NLRC3−/−) mice were used to construct hypoxia models and primary pulmonary arterial smooth muscle cells (PASMCs) of rats and endothelial cells were cultured for verification. Right heart catheterization and echocardiography suggested that NLRC3 knockout promoted right ventricular systolic pressure (RVSP) up-regulation, right ventricular hypertrophy and fibrosis in hypoxia-induced mice. This study first demonstrated that NLRC3 deficiency promoted hypoxia-stimulated PASMCs proliferation, Human umbilical vein endothelial cells (HUVECs) apoptosis, migration and inflammation through IKK/NF-κB p65/HIF-1α pathway in vitro and in vivo, further promoted vascular remodeling and PH progression, which provided a new target for the treatment of hypoxia-induced PH.

Diesel exhaust particles induce human umbilical vein endothelial cells apoptosis by accumulation of autophagosomes and caspase-8 activation

Diesel exhaust particles (DEP) are risk factors for endothelial cells (ECs) dysfunction. However, the mechanism by which DEP induce ECs apoptosis remains unclear. Here, we investigated how DEP induce death of human umbilical vein ECs (HUVECs), with a focus on the autophagy-mediated apoptotic pathway. DEP induced dose-dependent HUVECs death and exposure to the IC50 concentration of DEP (70 µg/ml) led to apoptosis. DEP phosphorylated Beclin-1 (Ser93) and increased protein levels of p62 and LC3BII and the number of LC3B puncta, indicating autophagy initiation. DEP increased expression of pro- and mature forms of cathepsin D, which increases lysosomal activity. However, DEP suppressed expression of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (STX17, VAMP8, SNAP29, YKT6, and STX7) to inhibit autolysosome formation, resulting in accumulation of autophagosomes. LC3B, p62, and caspase-8 form a tertiary complex in accumulated autophagosomes, which is known to serve as a platform for caspase-8 activation. Indeed, DEP activates caspase-8 and pretreatment with a caspase-8 inhibitor suppressed DEP-induced apoptosis. Furthermore, depletion of p62 decreased caspase-8 and caspase-3 activation and inhibited the DEP-induced apoptosis. Taken together, these findings demonstrated that DEP induced HUVECs apoptosis by inhibiting autophagosome maturation and identified caspase-8 as a novel mediator of DEP-induced ECs apoptosis.

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.

Inhibition of the miR-193b-3p protects against oxidized low-density lipoprotein-induced HUVECs injury by upregulating ALDH2.

Atherosclerosis (AS) is the most dangerous factor for human death, which is a lipid-driven chronic inflammatory disorder of the arteries. Growing evidence has showed that microRNAs play an important role in AS. However, the role of mir-193b-3p in atherosclerosis has been poorly studied to date. Therefore, we focused on the potential role of miR-193b-3p in atherosclerosis. The expressions of miR-193b-3p in the serum of AS patients were detected. We also established an oxidized low density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptosis model in vitro. The mRNA and protein levels of target molecules were detected by RT-qPCR and Western blotting. Apoptosis of HUVECs was determined by Annexin V/PI staining on a flow cytometry. The potential molecular targets of miR-193b-3p were investigated by applying such technologies as dual-luciferase reporter and RIP assay. Our study showed that miR-193b-3p expression level was significantly lower in AS patients than controls. ROC curve analysis showed that the areas under the curve (AUC) of plasma miR-193b-3p was 0.859. We also found that miR-193b-3p was decreased in ox-LDL-induced HUVECs and knockdown of miR-193b-3p suppressed ox-LDL-induced HUVECs injury. By using bioinformatics analysis, aldehyde dehydrogenase (ALDH2) was predicted as a downstream target of miR-193b-3p. The ALDH2 gene is also involved in the development of atherosclerosis. Meanwhile, inhibition of miR-193b-3p and ALDH2 protects ox-LDL-induced HUVECs against endoplasmic-reticulum (ER) stress. In conclusion, inhibition of miR-193b-3p was able to suppress ox-LDL-induced injury in AS through targeting ALDH2 and reducing ER stress.

Proteome-scale profiling reveals MAFF and MAFG as two novel key transcription factors involved in palmitic acid-induced umbilical vein endothelial cell apoptosis

BackgroundVascular endothelial cell apoptosis is the leading risk factor of atherosclerosis (AS). The purpose of our study was to use a new generation high-throughput transcription factor (TF) detection method to identify novel key TFs in vascular endothelial cell apoptosis induced by palmitic acid (PA).MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with 0, 300, or 500 µM PA. Candidate TFs in the three groups were identified by differential expression, pathway enrichment, Western Blot (WB), and RT-qPCR analyses. Apoptosis was assessed by fluorescence-activated cell sorting (FACS) using FITC-annexin V and propidium iodide staining.ResultsWe established a HUVEC apoptosis model to simulate the process of atherosclerosis onset and identified 51 significant TFs. of the 51 TFs, v-maf musculoaponeurotic fibrosarcoma oncogene family protein G (MAFG) and v-maf musculoaponeurotic fibrosarcoma oncogene family protein F (MAFF), were matched to known AS signalling pathways and were validated by WB and RT-qPCR analyses in our study. Overexpression of MAFG or MAFF in HUVECs significantly inhibited PA-induced early apoptosis.ConclusionsWe identified MAFF and MAFG as novel key TFs in vascular endothelial cell apoptosis.

MicroRNA-532-5p-programmed cell death protein 4 (PDCD4) axis regulates angiotensin II-induced human umbilical vein endothelial cell apoptosis and proliferation

BackgroundThis study was carried out to investigate the effect of microRNA miR-532-5p on the proliferation of hypertension endothelial cells. MethodsAngiotensin II (Ang II)-treated human umbilical vein endothelial cells (HUVECs) and primary human aortic endothelial cells (HAECs) were used as cell models to imitate the pathological changes in endothelial cells under hypertensive conditions. The expression levels of miR-532-5p and programmed cell death protein 4 (PDCD4) were detected by Quantitative Real-time PCR (qRT-PCR). The effects of miR-532-5p and PDCD4 on the proliferation of HUVECs and HAECs treated with Ang II were detected by Methyl Thiazolyl Tetrazolium (MTT) assay. The effects of miR-532-5p and PDCD4 on the apoptosis and cell cycle of HUVECs and HAECs treated with Ang II were detected by flow cytometry. Western blot was used to detect the expression levels of PDCD4, apoptosis-related proteins and cycle-related proteins in HUVECs and HAECs treated with Ang II. Bioinformatics analysis and Luciferase gene reporter assay were used to assess the relationship between miR-532-5p and PDCD4. ResultsThe expression levels of miR-532-5p were reduced, while the expression levels of PDCD4 were raised in Ang II-treated HUVECs and HAECs. MiR-532-5p mimic and si-PDCD4 restrained the apoptosis, promoted the proliferation of Ang II-treated HUVECs and HAECs and caused S-phase arrest of cells. PDCD4 was identified as a potential target for miR-532-5p. Knockdown of PDCD4 significantly affected apoptosis and proliferation of Ang II-treated HUVECs. MiR-532-5p regulates apoptosis and proliferation of Ang II-induced HUVECs and HAECs. In addition, overexpression of PDCD4 attenuated the effect of miR-532-5p on the proliferation of Ang II-treated HUVECs and HAECs. ConclusionMiR-532-5p inhibited the expression of PDCD4, thereby inhibiting apoptosis and promoting proliferation of Ang II-treated HUVECs and HAECs.

MiR-100 alleviates the inflammatory damage and apoptosis of H2O2-induced human umbilical vein endothelial cells via inactivation of Notch signaling by targeting MMP9.

Thromboangiitis obliterans is a nonatherosclerotic segmental inflammatory disease, and miR-100 plays an anti-inflammatory role in chronic inflammation. Therefore, we hypothesized that miR-100 might alleviate the inflammatory damage and apoptosis of H2O2-induced ECV304 cells and aimed to investigate the relationship between miR-100 and thromboangiitis obliterans and the related molecular mechanism. Cell counting kit-8 was used to detect cell viability, and the expression of inflammatory factors and oxidative stress was measured by ELISA. TUNEL assay was used to detect the apoptosis of human umbilical vein endothelial cells after induction by H2O2. Furthermore, the interaction between miR-100 and matrix metalloproteinase-9 was verified by dual-luciferase assay. Quantitative reverse transcription polymerase chain reaction and western blot were used to detect the expression of the adhesion factors, apoptosis-related proteins and Notch pathway-related protein. The results revealed that miR-100 was decreased in H2O2-induced human umbilical vein endothelial cells. Overexpression of miR-100 attenuated inflammatory response and cell apoptosis in H2O2-induced human umbilical vein endothelial cells. The overexpression of miR-100 inhibited matrix metalloproteinase-9 expression in H2O2-induced human umbilical vein endothelial cells. miR-100 inhibited H2O2-induced human umbilical vein endothelial cell inflammation, oxidative stress, and cell apoptosis via inactivation of Notch signaling by targeting matrix metalloproteinase. Our study demonstrated that miR-100 reduced the inflammatory damage and apoptosis of H2O2-induced human umbilical vein endothelial cells via inactivation of Notch signaling by targeting matrix metalloproteinase. These findings suggested that miR-100 might be a novel therapeutic target for the prevention of thromboangiitis obliterans.

Downregulation of interleukin-6 and C-reactive protein underlies a novel inhibitory role of microRNA-136-5p in acute lower extremity deep vein thrombosis.

Deep vein thrombosis (DVT) comprises a critical and common health condition with high incidence, mortality, and long-term adverse sequelae. Several differentially expressed microRNAs (miRNAs) have emerged as promising prognostic markers in DVT. The present study intended to explore the functional relevance of miR-136-5p in acute lower extremity DVT (LEDVT). Rat models of acute LEDVT were established and miR-136-5p expression was altered by agomir or antagomir to assess its effects. In addition, in vitro gain- and loss-experiments, prior to exposure to CoCl2, were performed to investigate effects of miR-136-5p on human umbilical vein endothelial cell (HUVEC) apoptosis and levels of interleukin-6 (IL-6) and C-reactive protein (CRP). miR-136-5p was downregulated, whereas IL-6 and CRP were elevated in acute LEDVT patients. Notably, miR-136-5p was confirmed to target both IL-6 and CRP. Overexpression of miR-136-5p led to reduced length, weight, and ratio of weight to length of the venous thrombus. Furthermore, overexpressed miR-136-5p downregulated the expression of IL-6 and CRP, consequently inhibiting HUVEC apoptosis. Conjointly, our data indicate that the overexpression of miR-136-5p has the potential to bind to the 3’-UTR in the mRNAs for IL-6 and CRP and mitigate acute LEDVT, which provides a basis for new therapeutic targets in acute LEDVT treatment.

Targeting Endothelial Cell Metabolism by Inhibition of Pyruvate Dehydrogenase Kinase and Glutaminase-1.

Targeting endothelial cell (EC) metabolism should impair angiogenesis, regardless of how many angiogenic signals are present. The dependency of proliferating ECs on glucose and glutamine for energy and biomass production opens new opportunities for anti-angiogenic therapy in cancer. The aim of the present study was to investigate the role of pyruvate dehydrogenase kinase (PDK) inhibition with dichloroacetate (DCA), alone or in combination with the glutaminase-1 (GLS-1) inhibitor, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), on Human umbilical vein endothelial cells (HUVECs) metabolism, proliferation, apoptosis, migration, and vessel formation. We demonstrated that both drugs normalize HUVECs metabolism by decreasing glycolysis for DCA and by reducing glutamate production for BPTES. DCA and BPTES reduced HUVECs proliferation and migration but have no impact on tube formation. While DCA increased HUVECs respiration, BPTES decreased it. Using both drugs in combination further reduced HUVECs proliferation while normalizing respiration and apoptosis induction. Overall, we demonstrated that DCA, a metabolic drug under study to target cancer cells metabolism, also affects tumor angiogenesis. Combining DCA and BPTES may reduce adverse effect of each drug alone and favor tumor angiogenesis normalization.

An inhibitor of endothelial ETS transcription factors promotes physiologic and therapeutic vessel regression

During the progression of ocular diseases such as retinopathy of prematurity and diabetic retinopathy, overgrowth of retinal blood vessels results in the formation of pathological neovascular tufts that impair vision. Current therapeutic options for treating these diseases include antiangiogenic strategies that can lead to the undesirable inhibition of normal vascular development. Therefore, strategies that eliminate pathological neovascular tufts while sparing normal blood vessels are needed. In this study we exploited the hyaloid vascular network in murine eyes, which naturally undergoes regression after birth, to gain mechanistic insights that could be therapeutically adapted for driving neovessel regression in ocular diseases. We found that endothelial cells of regressing hyaloid vessels underwent down-regulation of two structurally related E-26 transformation-specific (ETS) transcription factors, ETS-related gene (ERG) and Friend leukemia integration 1 (FLI1), prior to apoptosis. Moreover, the small molecule YK-4-279, which inhibits the transcriptional and biological activity of ETS factors, enhanced hyaloid regression in vivo and drove Human Umbilical Vein Endothelial Cells (HUVEC) tube regression and apoptosis in vitro. Importantly, exposure of HUVECs to sheer stress inhibited YK-4-279-induced apoptosis, indicating that low-flow vessels may be uniquely susceptible to YK-4-279-mediated regression. We tested this hypothesis by administering YK-4-279 to mice in an oxygen-induced retinopathy model that generates disorganized and poorly perfused neovascular tufts that mimic human ocular diseases. YK-4-279 treatment significantly reduced neovascular tufts while sparing healthy retinal vessels, thereby demonstrating the therapeutic potential of this inhibitor.

Quercetin inhibits TNF-α induced HUVECs apoptosis and inflammation via downregulating NF-kB and AP-1 signaling pathway in vitro.

Background:Quercetin, a major flavonol, wildly exists in plantage, which has been reported to have an anti-apoptosis and anti-inflammation effects on vascular endothelial cells, but its underlying molecular mechanisms remain unclear.Objective:The aim of this study was to investigate the mechanisms of how quercetin inhibits tumor necrosis factor alpha (TNF-α) induced human umbilical vein endothelial cells (HUVECs) apoptosis and inflammation.Methods and Results:HUVECs were preconditioned with quercetin for 18 hours, and subsequently treated with TNF-α for 6 hours to induce apoptosis. The expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, β-actin mRNA was then detected by RT-PCR. Flow cytometry was used to estimate the apoptosis rates, and the expression of activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) was measured by Western blot. TNF-α induced elevated apoptosis rates and upregulation of VCAM-1, ICAM-1, and E-selectin were meaningfully reduced in HUVECs by pretreatment with quercetin. In addition, quercetin also inhibited the activation of AP-1and NF-κB.Conclusion:Results indicate that quercetin could suppress TNF-α induced apoptosis and inflammation by blocking NF-κB and AP-1 signaling pathway in HUVECs, which might be one of the underlying mechanisms in treatment of coronary heart disease.

The soluble VEGF receptor sFlt-1 contributes to endothelial dysfunction in IgA nephropathy.

Endothelial injury is a common manifestation in IgA nephropathy (IgAN). After the previous identification of the upregulated soluble fms-like tyrosine kinase-1 (sFlt-1) correlated with endothelial injury in IgAN, in the present study, we further explored the role of sFlt-1 in endothelial injury in IgAN. We enrolled 72 patients with IgAN and detected the sFlt-1 levels. The polymeric IgA1 (pIgA1) complexes were isolated from the pooled plasma samples of another 10 patients with IgAN. Apoptosis proteins were detected in cultured human umbilical vein endothelial cells (HUVECs) with the stimulation of recombinant sFlt-1 or the caspase-9 inhibitor Z-LEHD-FMK. We identified there were positive correlations between sFlt-1 and IgA-IgG complex as well as vWF levels in patients with IgAN. The sFlt-1 levels in HUVECs were significantly upregulated by pIgA1 complex derived from IgAN patients in a concentration-dependent manner. The proliferation ability of HUVECs was damaged when stimulated with sFlt-1 protein in a time- and dose- dependent manner. And the apoptosis rate was up-regulated significantly as the stimulation concentrations of sFlt-1 increased. We found sFlt-1 challenge could significantly increase the expression of vWF. In addition, sFlt-1 increased the levels of caspase-9, caspase-3, Bax and mitochondrial membrane potential; facilitated the release of cytochrome C from mitochondria to cytoplasma. In contrast, Z-LEHD-FMK attenuated high sFlt-1-induced HUVECs apoptosis. In conclusion, our study demonstrated that sFlt-1 expression was up-regulated by the challenge of pIgA1 complex derived from patients with IgAN. Furthermore, increased sFlt-1 facilitated human umbilical vein endothelial cells apoptosis via the mitochondrial-dependent pathway.

Corrigendum] Protective effect of sonic hedgehog against oxidized low‑density lipoprotein-induced endothelial apoptosis: Involvement of NF-κB and Bcl-2 signaling.

Following the publication of this article, the authors have realized that the affiliation for the first author, Huashan Huang, was presented incorrectly as a multiple affiliation: Because the student was under the supervision of Professor Pengli Zhu, the only affiliation that should have been presented in this paper for this author was for the first affiliation, i.e., Shengli Clinical Medical college of Fujian Medical University. Therefore, the author affiliations for this paper should have appeared as follows: HUASHAN HUANG1, HUIZHEN YU1-3, LIANG LIN4, JUNMING cHEN1,2 and PENGLI ZHU1,2 1Shengli Clinical Medical College of Fujian Medical University; 2Key Laboratory of Geriatrics, Fujian Provincial Hospital, Fuzhou, Fujian 350001; Departments of 3Cardiology and 4Gynecology and Obstetrics, Fujian Provincial Hospital South Branch, Fuzhou, Fujian 350028, P.R. China. [the original article was published in International Journal of Molecular Medicine 45: 1864-1874, 2020; DOI: 10.3892/ijmm.2020.4542].

Protective effect of sonic hedgehog against oxidized low‑density lipoprotein‑induced endothelial apoptosis: Involvement of NF‑κB and Bcl‑2 signaling.

Sonic hedgehog (Shh) is pivotally important in embryonic and adult blood vessel development and homeostasis. However, whether Shh is involved in atherosclerosis and plays a role in endothelial apoptosis induced by oxidized low-density lipoprotein (ox-LDL) has not been reported. The present study used recombinant Shh-N protein (rShh-N) and a plasmid encoding the human Shh gene (phShh) to investigate the role of Shh in ox-LDL-mediated human umbilical vein endothelial cell (HUVEC) apoptosis. The present study found that ox-LDL was able to induce apoptosis in HUVECs and that Shh protein expression was downregulated. Furthermore, pretreatment with rShh-N or transfection with phShh increased anti-apoptosis protein Bcl-2 expression and decreased cell apoptosis. These protective effects of rShh-N could be abolished by cyclopamine, which is a hedgehog signaling inhibitor. Furthermore, a co-immunoprecipitation assay was performed to demonstrate that Shh interacted with NF-κB p65 in HUVECs. Additionally, ox-LDL upregulated the phosphorylation of NF-κB p65 and inhibitor of NF-κB-α (IκBα), and these effects decreased notably following rShh-N and phShh treatment. Together, the present findings suggested that Shh serves an important protective role in alleviating ox-LDL-mediated endothelial apoptosis by inhibiting the NF-κB signaling pathway phosphorylation and Bcl-2 mediated mitochondrial signaling.

ERK1/2-PPARγ pathway is involved in Chlamydia pneumonia-induced human umbilical vein endothelial cell apoptosis through increased LOX-1 expression

Chlamydia pneumonia (C.pn) is a common respiratory pathogen that is involved in human cardiovascular diseases and promotes the development of atherosclerosis in hyperlipidemic animal models. C.pn reportedly up-regulated lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells. Recently, the anti-atherosclerotic activity of peroxisome proliferator-activated receptor γ (PPARγ) has been documented. In the present study, we investigated the effect of C.pn on LOX-1 expression in human umbilical vein endothelial cells (HUVECs) and identified the involvement of the PPARγ signaling pathway therein. The results showed that C.pn increased the expression of LOX-1 in HUVECs in a dose- and time-dependent manner. C.pn-induced up-regulation of LOX-1 was mediated by ERK1/2, whereas p38 MAPK and JNK had no effect on this process. C.pn induced apoptosis, inhibited cell proliferation, and decreased the expression PPARγ in HUVECs. Additionally, LOX-1 activity and cell injury caused by C.pn through activation of ERK1/2 was completely inhibited by rosiglitazone, a PPARγ agonist. In conclusion, we inferred that activation of PPARγ in HUVECs suppressed C.pn-induced LOX-1 expression and cell damage by inhibiting ERK1/2 signaling.

Upregulation of microRNA-136-5p Alleviates Acute Lower Extremity Deep Vein Thrombosis Through Negatively Regulating Interleukin-6 and C-Reactive

Background: Deep vein thrombosis (DVT) is known as a critical common health issue with high incidence, mortality, and long-term sequelae. Many differentially expressed microRNAs (microRNAs) have been proven to be promising prognostic markers for DVT patients. Herein, this study intended to explore the functional relevance of miR-136-5p on acute lower extremity DVT (LEDVT). Methods: Following the establishment of acute LEDVT rat model, the expression of miR-136-5p was altered by agomir or antagomir to assess its effects on regulating venous thrombus. Moreover, the gain- and loss-experiments, prior to the exposure to CoCl2, were performed in vitro to clarify the effects of miR-136-5p on human umbilical vein endothelial cell (HUVEC) apoptosis and levels of interleukin-6 (IL-6) and C-reactive protein (CRP). Results: Accordingly, miR-136-5p was found to be downregulated, while IL-6 and CRP were elevated in acute LEDVT patients. Conclusion: Notably, miR-136-5p was confirmed to target both IL-6 and CRP. Overexpression miR-136-5p led to reduced length, weight, and the ratio of weight to length of venous thrombus. Additionally, overexpressed miR-136-5p regulated both IL-6 and CRP to inhibit HUVECs apoptosis. Taken conjointly, the overexpression of miR-136-5p has the potential to bind to IL-6 and CRP to mitigate acute LEDVT, which provided new therapeutic targets for acute LEDVT treatment. Funding Statement: This study was supported by the General Projects of National Natural Science Foundation of China — the Function and Regulation Mechanism of ETBR in Aortic Endothelium and Smooth Muscle Cells in Pregnancy Induced Hypertension (No. 81871187), the Regional Projects of the National Natural Science Foundation of China — Correlation Analysis Between Endothelin Receptor and Pregnancy Induced Hypertension and Abnormal Blood Pressure Regulation (No. 81460239), and the Natural Science Foundation Project of Ningxia Province — Correlation Analysis Between Anastomotic Regional Blood Flow Velocity and Intimal Hyperplasia after Revascularization (No. NZ17195). Declaration of Interests: The authors have declared that no competing interests exist. Ethics Approval Statement: This study protocol was approved by the Ethics Committee and Experimental Animal Ethics Committee of Qingdao Municipal Hospital. In addition, written informed consent was obtained from all participants or their relatives that were involved in the study. The animal experiment strictly adhered to the principles of the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health to minimize the pain, suffering and discomfort to experimental animals.

Notoginsenoside R1 upregulates miR-221-3p expression to alleviate ox-LDL-induced apoptosis, inflammation, and oxidative stress by inhibiting the TLR4/NF-κB pathway in HUVECs.

Atherosclerosis (AS) is a common vascular disease, which can cause apoptosis of vascular endothelial cells. Notoginsenoside R1 (NGR1) is considered an anti-AS drug. MicroRNAs (miRNAs) are believed to play a vital role in cell apoptosis and angiogenesis. This study aimed to explore the mechanism of NGR1 for treating AS through miRNAs. Flow cytometry was used to detect the apoptosis rate. The levels of inflammatory cytokines interleukin (IL)-6 and IL-1β were detected using ELISA. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured using corresponding assay kits. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to detect miR-221-3p expression. Dual-luciferase reporter and RNA immunoprecipitation assays were carried out to examine the relationship between miR-221-3p and toll-like receptors 4 (TLR4). Also, western blot analysis was performed to determine the levels of TLR4 and nuclear factor kappa B (NF-κB) signaling pathway-related proteins. Oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs) apoptosis, inflammation, and oxidative stress. NGR1 alleviated the negative effect of ox-LDL through promoting the expression of miR-221-3p in HUVECs. TLR4 was a target of miR-221-3p, and its overexpression could reverse the inhibition effects of miR-221-3p on apoptosis, inflammation, and oxidative stress. NGR1 improved miR-221-3p expression to inhibit the activation of the TLR4/NF-κB pathway in ox-LDL-treated HUVECs. NGR1 decreased ox-LDL-induced HUVECs apoptosis, inflammation, and oxidative stress through increasing miR-221-3p expression, thereby inhibiting the activation of the TLR4/NF-κB pathway. This study of the mechanism of NGR1 provided a more theoretical basis for the treatment of AS.

Beneficial Effects of Ticagrelor on Oxidized Low-Density Lipoprotein (ox-LDL)-Induced Apoptosis in Human Umbilical Vein Endothelial Cells.

BackgroundTicagrelor is the first oral anti-platelet agent which has direct anti-platelet aggregation effect by combining with ADP P2Y12 receptors in platelets. It has been approved to reduce the incidence of thrombus cardiovascular events in acute coronary syndrome patients. However, the effects of ticagrelor on endothelial apoptosis have not been investigated.Material/MethodsOxidized low-density lipoprotein (ox-LDL) was used to establish a human umbilical vein endothelial cell (HUVEC) apoptosis model. To investigate the effects of ticagrelor on endothelial apoptosis, the HUVECs were treated with different dose of ticagrelor. Apoptosis rates of HUVECs was evaluated by flow cytometry, and the expression levels of Akt, p-Akt, Bcl-2, Bax, caspase-3, endothelial nitric oxide synthase (eNOS), and nitric oxide (NO) concentration were assessed.ResultsAfter treatment with 50 ug/mL ox-LDL or 100 ug/mL ox-LDL, we found that the late apoptosis and necrosis rate and the expression levels of Bax and caspase-3 were significantly increased in HUVECs, whereas the expression levels of Akt, p-Akt, Bcl-2, eNOS, and NO were significantly decreased. Ticagrelor restored the apoptosis rate of ox-LDL-induced HUVECs in a dose-dependent manner. In addition, compared with ox-LDL group, ticagrelor treatment significantly increased the expression levels of Akt, p-Akt, Bcl-2, eNOS, and NO concentration, and significantly decreased the expression levels of Bax and caspase-3.ConclusionsWe found that ox-LDL induced significant apoptosis and necrosis in our model, which was dose-dependently improved by ticagrelor. These changes might be explained by alterations in apoptosis and antioxidant pathways.

Inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells on apoptosis of oxygen-glucose deprived reoxygenation model of venous endothelial cells

Objective To explore the inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells(HUCMSC) on apoptosis of human umbilical vein endothelial cells(HUVEC) after model group(oxygen-glucose deprivation reoxygenation), and to clarify its possible mechanism. Methods Human umbilical cord mesenchymal stem cells were cultured. The collected cell supernatant was stored in a centrifugal tube. The exosomes secreted by human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and identified. Human umbilical vein endothelial cells were randomly divided into control group, model group and different concentrations of HUCMSC-EXO(20 μg/ml, 40 μg/ml, 60 μg/ml) treatment groups(adding HUCMSC-EXO into the model group) . The morphological changes of HUVEC cells in each group were observed by inverted phase contrast microscope, and the proliferation inhibition rate of HUVEC in each group was measured by CCK-8 reagent. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3, Bax, Bcl-2 and hypoxia-associated protein hypoxia inducible factor 1α(HIF-1α). Inhibitor(HIF-1α inhibitor) + model group and HUCMSC-EXO + inhibitor + model group were added on the basis of the above experiments. Western blot analysis was performed to observe the effects of HUCMSC-EXO, inhibitor and both of them on HIF-1α and Bax expressions in HUVEC. Results HUCMSC-EXO was successfully extracted and identified. Compared with the control group, the volume of HUVEC in the model group and the HUCMSC-EXO group with different concentrations decreased, became round, connected and evacuated, and the growth state was poor under the inverted phase contrast microscope.CCK-8 detection showed that the cell viability in the HUCMSC-EXO group was significantly higher than that in the model group, the difference was statistically significant (t=9.23, P 0.05)and Bax protein ((0.363±0.069), (0.370±0.064); t=0.18, P>0.05). But both of them were down-regulated compared with the model group (HIF-1α protein (0.919±0.064), Bax protein (0.902±0.071)), the differences were significant( t=13.56, t=13.03, both P<0.05). Conclusion HUCMSC-EXO has a protective effect on OGD/R model of HUVEC, and its mechanism may be related to the down-regulation of HIF-1α expression. Key words: Human umbilical mesenchymal stem cells; Exosomes; Oxygen-glucose deprivation reoxygenation; Human umbilical venous endothelial cells; Apoptosis

Effect of miR-27b-5p on apoptosis of human vascular endothelial cells induced by simulated microgravity

Weightlessness-induced cardiovascular dysfunction can lead to physiological and pathological consequences. It has been shown that spaceflight or simulated microgravity can alter expression profiles of some microRNAs (miRNAs). Here, we attempt to identify the role of miRNAs in human umbilical vein endothelial cells (HUVECs) apoptosis under simulated microgravity. RNA-sequencing and quantitative real-time PCR (qRT-PCR) assays were used to identify differentially expressed miRNAs in HUVECs under simulated microgravity. Then we obtained the target genes of these miRNAs through target analysis software. Moreover, GO and KEGG enrichment analysis were performed. The effects of these miRNAs on HUVECs apoptosis were evaluated by flow cytometry, Western blot and Hoechst staining. Furthermore, we obtained the target gene of miR-27b-5p by luciferase assay, qRT-PCR and Western blot. Finally, we investigated the relationship between this target gene and miR-27b-5p in HUVECs apoptosis under normal gravity or simulated microgravity. We found 29 differentially expressed miRNAs in HUVECs under simulated microgravity. Of them, the expressions of 3 miRNAs were validated by qRT-PCR. We demonstrated that miR-27b-5p affected HUVECs apoptosis by inhibiting zinc fingers and homeoboxes 1 (ZHX1). Our results reported here demonstrate for the first time that simulated microgravity can alter the expression of some miRNAs in HUVECs and miR-27b-5p may protect HUVECs from apoptosis under simulated microgravity by targeting ZHX1.