High Glucose-induced Endothelial Cell Apoptosis Research Articles

Paeoniflorin alleviates high glucose-induced endothelial cell apoptosis in diabetes mellitus by inhibiting HRAS-activated RAS pathway.

Paeoniflorin (Pae) can improve diabetes mellitus (DM), especially endothelial dysfunction induced by high glucose (HG). Molecularly, the mechanism pertinent to Pae and DM lacks further in-depth research. Hence, this study determined the molecular mechanism of Pae in treating DM through network pharmacology. The target of Pae was analyzed by TCMSP database, and DM-related genes were dissected by Genecards database and Omim database. PPI network was constructed for cross targets through Cytoscape 3.9.1 and STRING platform. GO and KEGG analyses were carried out on the cross targets. Protein molecular docking verification was completed by AutoDockTools and Pymol programs. Human umbilical vein endothelial cells (HUVECs) were separately treated with HG, Pae (5, 10, 20 μM) and/or HRAS overexpression plasmids (oe-HRAS). The cell viability, apoptosis and the protein expressions of HRAS and Ras-GTP were evaluated. There were 50 cross targets between Pae and DM, and VEGFA, EGFR, HRAS, SRC and HSP90AA1 were the key genes identified by PPI network analysis. GO and KEGG analyses revealed signal paths such as Rap1 and Ras. Molecular docking results confirmed that Pae had a good binding ability with key genes. In HG-treated HUVECs, Pae dose-dependently facilitated cell viability, attenuated cell apoptosis, and dwindled the expressions of HRAS and Ras-GTP, but these effects of Pae were reversed by oe-HRAS. In conclusion, Pae regulates the viability and apoptosis of HG-treated HUVECs by inhibiting the expression of HRAS.

Integrin β5 subunit regulates hyperglycemia-induced vascular endothelial cell apoptosis through FoxO1-mediated macroautophagy.

Hyperglycemia frequently induces apoptosis in endothelial cells and ultimately contributes to microvascular dysfunction in patients with diabetes mellitus (DM). Previous research reported that the expression of integrins as well as their ligands was elevated in the diseased vessels of DM patients. However, the association between integrins and hyperglycemia-induced cell death is still unclear. This research was designed to investigate the role played by integrin subunit β5 (ITGB5) in hyperglycemia-induced endothelial cell apoptosis. We used leptin receptor knockout (Lepr-KO) ( db / db ) mice as spontaneous diabetes animal model. Selective deletion of ITGB5 in endothelial cell was achieved by injecting vascular targeted adeno-associated virus via tail vein. Besides, we also applied small interfering RNA in vitro to study the mechanism of ITGB5 in regulating high glucose-induced cell apoptosis. ITGB5 and its ligand, fibronectin, were both upregulated after exposure to high glucose in vivo and in vitro . ITGB5 knockdown alleviated hyperglycemia-induced vascular endothelial cell apoptosis and microvascular rarefaction in vivo.In vitro analysis revealed that knockdown of either ITGB5 or fibronectin ameliorated high glucose-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). In addition, knockdown of ITGB5 inhibited fibronectin-induced HUVEC apoptosis, which indicated that the fibronectin-ITGB5 interaction participated in high glucose-induced endothelial cell apoptosis. By using RNA-sequencing technology and bioinformatic analysis, we identified Forkhead Box Protein O1 (FoxO1) as an important downstream target regulated by ITGB5. Moreover, we demonstrated that the excessive macroautophagy induced by high glucose can contribute to HUVEC apoptosis, which was regulated by the ITGB5-FoxO1 axis. The study revealed that high glucose-induced endothelial cell apoptosis was positively regulated by ITGB5, which suggested that ITGB5 could potentially be used to predict and treat DM-related vascular complications.

Albiflorin attenuates high glucose-induced endothelial apoptosis via suppressing PARP1/NF-κB signaling pathway.

Paeonia lactiflora Pall has long been recognized as an anti-inflammatory traditional Chinese herbal medicine. We aimed to study the pharmacological action of albiflorin, an active ingredient extracted from the roots of Paeonia lactiflora Pall, on diabetic vascular complications. Human umbilical vein endothelial cells (HUVECs) were stimulated with high glucose and treated with 5, 10, and 20μM albiflorin. CCK-8 assay, EdU staining, Annexin V-FITC staining, transwell assay, scratch test, RT-PCR, ELISA, Western blot, and immunofluorescence were carried out. SwissTargetPrediction database was used for screening targets of albiflorin and molecular docking was done using Autodock Vina software. Albiflorin treatment dose-dependently alleviated high glucose-induced viability loss of HUVECs. In addition, albiflorin promoted the proliferation and migration, while inhibited apoptosis and the release of TNF-α, IL-6, and IL-1β in HUVECs. PARP1 was predicted and confirmed to be a target for albiflorin in vitro. Albiflorin targeted PARP1 to inhibit the activation of NF-κB. Transfection of HUVECs with PARP1 overexpression plasmids effectively reversed the effects of albiflorin on high glucose-treated HUVECs. Albiflorin suppressed high glucose-induced endothelial cell apoptosis and inflammation, suggesting its potential in treating diabetic vascular complications. The action of albiflorin possibly caused by its regulation on inhibiting PARP1/NF-κB signaling.

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.

Vitexin protects against high glucose-induced endothelial cell apoptosis and oxidative stress via Wnt/β-catenin and Nrf2 signalling pathway

Vitexin, a polyphenolic flavonoid, has been reported to be traditionally applied in the treatment of diabetes, cancer and cardiovascular diseases. Objective The aim of this study was to investigate the anti-apoptosis and anti-oxidation effect and the potential mechanism of vitexin on high glucose-induced HUVECs. Materials and methods A high dose of glucose was added to HUVECs to establish an in vitro model. The cell viability was detected by CCK8 and flow cytometry assays. 2,7-dichlorodihydrofluorescein diacetate, colorimetry, and enzyme-linked immunosorbent assay were performed to detect oxidative stress. Besides, top flash and western blotting were employed to evaluate the effect of vitexin on Wnt/β-catenin. Furthermore, a Wnt/β-catenin inhibitor (KYA1797K) was used to confirm whether Wnt/β-catenin is involved in the protection of vitexin. At the same time, RT-PCR and western blot were performed to determine the effect of vitexin on Nrf2, while immunofluorescence assays were employed for the assessment of Nrf2 localisation. Then, in order to validate that Nrf2 plays an important role in the anti-oxidant effect of vitexin, methods were utilised to silence Nrf2 gene. Results Herein, vitexin inhibited the proliferation and apoptosis of HG-mediated HUVECs. Mechanically, vitexin disrupted Wnt/β-catenin signalling pathway, thus resulting in the decrease of apoptosis in HG-induced HUVECs. A Wnt/β-catenin inhibitor (KYA1797K), was used for reverse verification. In the meantime, vitexin administration decreased reactive oxygen species (ROS) production and malondialdehyde (MDA) content and increased superoxide dismutase (SOD) activity in HG-induced HUVECs. Further investigations have revealed vitexin activated Nrf2 in HUVEC under high glucose, which was involved in its anti-oxidant effects. Conclusion Our investigation demonstrated that vitexin protected HUVECs from high glucose-induced injury via up-regulation of Wnt/β-catenin and Nrf2 signalling pathway. These results suggested that vitexin might serve as a potential drug for atherosclerosis and cardiovascular complications of diabetes.

RPS4Y1 Promotes High Glucose-Induced Endothelial Cell Apoptosis and Inflammation by Activation of the p38 MAPK Signaling.

AimEndothelial dysfunction is a key pathological basis for diabetes mellitus complications, including diabetic nephropathy, diabetic retinopathy, and diabetic cardiomyopathy. This study aimed to reveal the functional role of ribosomal protein S4 Y-linked 1 (RPS4Y1) in endothelial dysfunction.MethodsHuman umbilical vein endothelial cells (HUVECs) were subjected to high glucose. The expression of RPS4Y1 in cells was overexpressed or silenced by plasmid or siRNA transfection. MTT assay, flow cytometry, JC-1 probe, scratch test, tube formation, and ELISA were conducted to assess the effects of RPS4Y1 on cell. Western blot was performed to assay the downstream signaling of RPS4Y1. The inhibitors of p38, ERK, and Jnk were used to treat cells to validate the involvement of them in RPS4Y1-mediated endothelial dysfunction.ResultsRPS4Y1 was upregulated in HUVECs in response to high glucose in both dose- and time-dependent manners. Overexpression of RPS4Y1 induced viability loss, apoptosis, and inflammation, but inhibited cell migration and tube formation. Silence of RPS4Y1 impacted these aspects in a contrary trend. The phosphorylation of p38 rather than ERK and Jnk was activated by RPS4Y1. In addition, the dysfunction of HUVECs mediated by RPS4Y1 was attenuated by SB203580 (a specific inhibitor of p38 signaling).ConclusionThe highly expressed RPS4Y1 in endothelial cells may contribute to high glucose-induced dysfunction through regulating p38 MAPK signaling. RPS4Y1 might be a potential therapeutic target for treating diabetes mellitus complications.

LncRNA MALAT1 affects high glucose-induced endothelial cell proliferation, apoptosis, migration and angiogenesis by regulating the PI3K/Akt signaling pathway.

To investigate the effects of long non-coding ribonucleic acid (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on the high glucose-induced proliferation, apoptosis, migration and angiogenesis of endothelial cells and its potential mechanism. Human umbilical vein endothelial cells (HUVECs) were divided into 3 groups, including control group (medium with 5.5 mmol/L glucose), high glucose group (HG group, medium with 33.5 mmol/L glucose) and lncRNA MALAT1 knockdown group [HG + MALAT1 small interfering RNA (siRNA) group, medium with 33.5 mmol/L glucose]. Cell Counting Kit-8 (CCK-8) assay was performed to observe the proliferation of HUVECs in each group at different time points. Meanwhile, the wound-healing assay was applied to detect the migratory ability of HUVECs in each group at 0 h and 24 h. The apoptosis rate of each group of cells was measured by means of flow cytometry, and the expression of Bcl-2-associated X protein (Bax) was detected via immunofluorescence at the same time. In addition, the amount of neovascularization in each group of cells was observed through the tube formation assay. Finally, Western blotting was utilized to determine the expression level of proteins in phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in each group of cells. Compared with that in the control group, the expression level of lncRNA MALAT1 in the HG group was elevated markedly (p<0.05). The proliferative capacity of HUVECs in the HG group was increased notably after knocking down lncRNA MALAT1 with siRNA (p<0.05). According to wound-healing assay, the knockdown of lncRNA MALAT1 could prominently reverse the declined HUVECs migratory ability induced by high glucose (p<0.05). Flow cytometry results manifested that the apoptosis level of HUVECs in the HG group was increased markedly, but inhibition on lncRNA MALAT1 could lower the apoptosis level evidently (p<0.05). The results of immunofluorescence showed that the expression of Bax in the HG + MALAT1 siRNA group was remarkably lower than that in the HG group (p<0.05). It was revealed in Western blotting that the knockdown of lncRNA MALAT1 could reverse the inhibition of high glucose on the PI3K/Akt signaling pathway in HUVECs (p<0.05). Inhibiting lncRNA MALAT1 can promote endothelial cell proliferation, migration and angiogenesis and repress endothelial cell apoptosis simultaneously, whose mechanism may be related to the activation of the PI3K/Akt signaling pathway.

Resveratrol attenuates high glucose-induced endothelial cell apoptosis via mediation of store-operated calcium entry.

The aim of this study was to evaluate the influence of resveratrol on HG-induced calcium entry in islet microvascular (MS-1) endothelial cells. MS-1 cells were pretreated with resveratrol or 2-APB (an inhibitor of store-operated calcium entry) and then incubated with high glucose. Cell viability was determined using the cell counting kit-8 method. Reactive oxygen species, endothelial apoptosis, and NO production were detected by DHE probe, TUNEL detection, and nitrate reductase assay kit. Protein levels of SOCE were detected by western blotting. Pretreatment with resveratrol significantly attenuated HG-induced endothelial apoptosis and improved cell viability. However, pretreatment with resveratrol and 2-APB abolished this effect, suggesting that the attenuation of HG-induced apoptosis by resveratrol may be associated with SOCE. Subsequent analyses indicated that HG induced the SOCE-related proteins, including TRPC1, Orai1, and Stim1. These results suggest that resveratrol pretreatment is associated with relieved HG-induced endothelial apoptosis at least partly via inhibition of SOCE-related proteins.

Salusin-β Is Involved in Diabetes Mellitus-Induced Endothelial Dysfunction via Degradation of Peroxisome Proliferator-Activated Receptor Gamma.

The pathophysiological mechanisms for vascular lesions in diabetes mellitus (DM) are complex, among which endothelial dysfunction plays a vital role. Therapeutic target against endothelial injury may provide critical venues for treatment of diabetic vascular diseases. We recently identified that salusin-β contributed to high glucose-induced endothelial cell apoptosis. However, the roles of salusin-β in DM-induced endothelial dysfunction remain largely elusive. Male C57BL/6J mice were used to induce type 2 diabetes mellitus (T2DM) model. Human umbilical vein endothelial cells (HUVECs) were cultured in high glucose/high fat (HG/HF) medium. We demonstrated increased expression of salusin-β in diabetic aortic tissues and high-glucose/high-fat- (HG/HF-) incubated HUVECs. Disruption of salusin-β by shRNA abrogated the reactive oxygen species (ROS) production, inflammation, and nitrotyrosine content of HUVECs cultured in HG/HF medium. The HG/HF-mediated decrease in peroxisome proliferator-activated receptor γ (PPARγ) expression was restored by salusin-β shRNA, and PPARγ inhibitor T0070907 abolished the protective actions of salusin-β shRNA on endothelial injury in HG/HF-treated HUVECs. Salusin-β silencing obviously improved endothelium-dependent vasorelaxation, oxidative stress, inflammatory response, and nitrative stress in diabetic aorta. Taken together, our results highlighted the essential role of salusin-β in pathological endothelial dysfunction, and salusin-β may be a promising target in treatment of vascular complications of DM.

Upregulation of Periostin Prevents High Glucose-Induced Mitochondrial Apoptosis in Human Umbilical Vein Endothelial Cells via Activation of Nrf2/HO-1 Signaling

Background/Aims: High glucose-induced oxidative damage to endothelial cells plays a central role in the pathogenesis of diabetic vascular complications. This study was undertaken to explore the role of periostin in high glucose-induced endothelial cell apoptosis and associated molecular mechanisms. Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to high glucose (33.3 mmol/L) and examined for the expression of periostin. The effects of periostin upregulation on high glucose-induced apoptosis, mitochondrial dysfunction, and reactive oxygen species (ROS) production were determined. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) by periostin was checked. HO-1 knockdown experiments were done to confirm its role in the action of periostin in high glucose-exposed HUVECs. Results: High glucose significantly upregulated the expression of periostin in HUVECs. Enforced expression of periostin attenuated high glucose-induced apoptosis in HUVECs, as determined by TUNEL staining and caspase-3 activity assay. Periostin overexpression prevented loss of Δψm, release of mitochondrial cytochrome c, and dysregulation of Bcl-2 and Bax in high glucose-exposed HUVECs. Periostin upregulation suppressed high glucose-induced ROS generation and activated the Nrf2/HO-1 signaling. HO-1 silencing restored high glucose-induced ROS generation and apoptotic response in periostin-overexpressing HUVECs. Conclusion: Periostin mitigates high glucose-induced mitochondrial apoptosis in endothelial cells, via activation of Nrf2/HO-1 signaling and reduction of ROS formation. Further studies are warranted to explore the therapeutic potential of periostin in diabetic vascular complications.

Coenzyme Q10 Attenuates High Glucose-Induced Endothelial Progenitor Cell Dysfunction through AMP-Activated Protein Kinase Pathways.

Coenzyme Q10 (CoQ10), an antiapoptosis enzyme, is stored in the mitochondria of cells. We investigated whether CoQ10 can attenuate high glucose-induced endothelial progenitor cell (EPC) apoptosis and clarified its mechanism. EPCs were incubated with normal glucose (5 mM) or high glucose (25 mM) enviroment for 3 days, followed by treatment with CoQ10 (10 μM) for 24 hr. Cell proliferation, nitric oxide (NO) production, and JC-1 assay were examined. The specific signal pathways of AMP-activated protein kinase (AMPK), eNOS/Akt, and heme oxygenase-1 (HO-1) were also assessed. High glucose reduced EPC functional activities, including proliferation and migration. Additionally, Akt/eNOS activity and NO production were downregulated in high glucose-stimulated EPCs. Administration of CoQ10 ameliorated high glucose-induced EPC apoptosis, including downregulation of caspase 3, upregulation of Bcl-2, and increase in mitochondrial membrane potential. Furthermore, treatment with CoQ10 reduced reactive oxygen species, enhanced eNOS/Akt activity, and increased HO-1 expression in high glucose-treated EPCs. These effects were negated by administration of AMPK inhibitor. Transplantation of CoQ10-treated EPCs under high glucose conditions into ischemic hindlimbs improved blood flow recovery. CoQ10 reduced high glucose-induced EPC apoptosis and dysfunction through upregulation of eNOS, HO-1 through the AMPK pathway. Our findings provide a potential treatment strategy targeting dysfunctional EPC in diabetic patients.

Attenuation of Glomerular Endothelial Cells from High Glucose-Induced Injury by Blockade of MAD2B

Background/Aims: To assess the role of mitotic arrest-deficient 2-like protein 2 (MAD2B) in high glucose-induced injury in mouse glomerular endothelial cells (GEnCs). Methods: GEnCs were cultured in vitro, and MAD2B protein levels were measured by Western blot in cells stimulated with high glucose (30 mM) for various periods of time. MAD2B and scrambled shRNA were introduced into GEnCs by liposomal transfection. Cell proliferation, apoptosis, nitric oxide (NO) production, and monolayer permeability were then measured in cells grown in the following conditions: control, high glucose treatment, MAD2B shRNA transfection with high glucose treatment, and scrambled shRNA transfection with high glucose treatment. Results: High glucose increased the protein levels of MAD2B in GEnCs. Compared with control cells, apoptosis was increased by high glucose treatment, which was attenuated by transfection with MAD2B shRNA transfection. Cells treated with high glucose produced less NO than control cells, whereas MAD2B shRNA transfection increased NO production. Cell monolayer permeability was enhanced in high glucose treated cells, but MAD2B shRNA transfection reduced permeability. Conclusion: High glucose levels induced the expression of MAD2B in GEnCs, whereas suppressing its expression reduced high glucose-induced endothelial cell apoptosis and high permeability, and promoted cell proliferation and NO production.

Adaptive Induction of Growth Differentiation Factor 15 Attenuates Endothelial Cell Apoptosis in Response to High Glucose Stimulus

Growth differentiation factor 15 (GDF15), a direct target gene of p53, is a multifunctional member of the TGF-β/BMP superfamily. GDF15 can be induced and is implicated as a key secretory cytokine in response to multiple cellular stimuli. Accumulating evidence indicates that GDF15 is associated with the development and prognosis of diabetes mellitus, while whether GDF15 can be induced by high glucose is unknown. In the present study, we revealed that high glucose could induce GDF15 expression and secretion in cultured human umbilical vein endothelial cells in a ROS- and p53-dependent manner. Inhibition of high glucose-induced GDF15 expression by siRNA demonstrated that adaptively induced GDF15 played a protective role against high glucose-induced human umbilical vein endothelial cell apoptosis via maintaining the active state of PI3K/Akt/eNOS pathway and attenuating NF-κB/JNK pathway activation. The protective effects of GDF15 were probably achieved by inhibiting ROS overproduction in high glucose-treated human umbilical vein endothelial cells in a negative feedback manner. Our results suggest that high glucose can promote GDF15 expression and secretion in human umbilical vein endothelial cells, which in turn attenuates high glucose-induced endothelial cell apoptosis.

Overexpression of angiopoietin-2 impairs myocardial angiogenesis and exacerbates cardiac fibrosis in the diabetic db/db mouse model

The angiopoietins/Tie-2 system is essential for the maintenance of vascular integrity and angiogenesis. The functional role of angiopoietin-2 (Ang-2) in the regulation of angiogenesis is dependent on other growth factors such as VEGF and a given physiopathological conditions. This study investigates the potential role of Ang-2 in myocardial angiogenesis and fibrosis formation in the diabetic db/db mouse. Diabetic db/db mice received intramyocardial administration of either adenovirus Ang-2 (Ad-CMV-Ang-2) or Ad-β-gal. The levels of Tie-2, VEGF, caspase-3, Wnt7b, fibroblast-specific protein-1 (FSP-1), and adhesion molecules (ICAM-1 and VCAM-1) expression were measured. Apoptosis, capillary density, and cardiac fibrosis were also analyzed in the db/db mouse hearts. Overexpression of Ang-2 suppressed Tie-2 and VEGF expression in db/db mouse hearts together with significant upregulation of Wnt7b expression. Overexpression of Ang-2 also sensitizes ICAM-1 and VCAM-1 expression in db/db mouse hearts. Immunohistochemical analysis revealed that overexpression of Ang-2 resulted in a gradual apoptosis as well as interstitial fibrosis formation, these leading to a significant loss of capillary density. Data from these studies were confirmed in cultured mouse heart microvascular endothelial cells (MHMEC) exposed to excessive Ang-2. Exposure of MHMEC to Ang-2 resulted in increased caspase-3 activity and endothelial apoptosis. Knockdown of Ang-2 attenuated high glucose-induced endothelial cell apoptosis. Further, counterbalance of Ang-2 by overexpression of Ang-1 reversed loss of capillary density and fibrosis formation in db/db mouse hearts. Our data demonstrate that Ang-2 increases endothelial apoptosis, sensitizes myocardial microvascular inflammation, and promotes cardiac fibrosis and thus contributes to loss of capillary density in diabetic diseases.

High glucose-induced apoptosis in human coronary artery endothelial cells involves up-regulation of death receptors

BackgroundHigh glucose can induce apoptosis in vascular endothelial cells, which may contribute to the development of vascular complications in diabetes. We evaluated the role of the death receptor pathway of apoptotic signaling in high glucose-induced apoptosis in human coronary artery endothelial cells (HCAECs).MethodsHCAECs were treated with media containing 5.6, 11.1, and 16.7 mM of glucose for 24 h in the presence or absence of tumor necrosis factor (TNF)-α. For detection of apoptosis, DNA fragmentation assay was used. HCAEC expression of death receptors were analyzed by the PCR and flow cytometry methods. Also, using immunohistochemical techniques, coronary expression of death receptors was assessed in streptozotocin-nicotinamide-induced type 2 diabetic mice.ResultsExposure of HCAECs to high glucose resulted in a significant increase in TNF-R1 and Fas expression, compared with normal glucose. High glucose increased TNF-α production by HCAECs and exogenous TNF-α up-regulated TNF-R1 and Fas expression in HCAECs. High glucose-induced up-regulation of TNF-R1 and Fas expression was undetectable in the presence of TNF-α. Treatment with TNF-R1 neutralizing peptides significantly inhibited high glucose-induced endothelial cell apoptosis. Type 2 diabetic mice displayed appreciable expression of TNF-R1 and Fas in coronary vessels.ConclusionsIn association with increased TNF-α levels, the death receptors, TNF-R1 and Fas, are up-regulated in HCAECs under high glucose conditions, which could in turn play a role in high glucose-induced endothelial cell apoptosis.

Rosiglitazone inhibits high glucose-induced apoptosis in human umbilical vein endothelial cells through the PI3K/Akt/eNOS pathway

Previous studies have shown that the phosphatidylinositol 3-kinase / Akt / endothelial nitric oxide synthase / NO (PI3K/Akt/eNOS/NO) pathway is involved in high glucose-induced endothelial cell apoptosis and rosiglitazone has a protective effect on endothelium. In the present study, we investigated the antiapoptotic effect of rosiglitazone on human umbilical vein endothelial cells (HUVECs) exposed to high glucose and explored its possible mechanism. Treatment of high glucose (33 mmol/L) for 48 h significantly induced the apoptosis of HUVECs, concomitantly with increased caspase-3 activity. High glucose treatment also decreased Akt and eNOS phosphorylation levels with subsequent NO production. All these alterations induced by high glucose were attenuated by rosiglitazone (1 micromol/L). Interestingly, the antiapoptotic effect of rosiglitazone was inhibited by PI3K inhibitor (LY294002, wortmannin) or eNOS inhibitor NG-<span class="smallcap">l</span>-nitro-arginine methyl ester (<span class="smallcap">l</span>-NAME). The reverse effects of rosiglitazone on phosphorylation of Akt and eNOS with subsequent NO production were also inhibited by LY294002, wortmannin or <span class="smallcap">l</span>-NAME, respectively. These findings suggest that rosiglitazone inhibits high glucose-induced apoptosis in HUVECs through the PI3K/Akt/eNOS pathway.

Effect of the antioxidant α-lipoic acid on apoptosis in human umbilical vein endothelial cells induced by high glucose

High glucose plays an important role in the pathogenesis of atherosclerosis. In this study, we assessed the effects of high glucose on human umbilical vein endothelial cell (HUVEC) apoptosis. Additionally, we investigated whether alpha-lipoic acid, an antioxidant, prevents high glucose-induced apoptosis of HUVECs. HUVECs were treated with high glucose in the presence or absence of alpha-lipoic acid. Treatment of HUVECs with high glucose changed cell morphology and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by high glucose in a dose-and time-dependent fashion. High glucose markedly elevated Bax, and decreased NF-kappaB and Bcl-2 expression. Most importantly, pretreatment with alpha-lipoic acid protected against high glucose-induced apoptosis in the endothelial cells. alpha-Lipoic acid significantly promoted the expression of NF-kappaB while decreasing the expression of Bax and the activities of caspase-3 and-9 without significantly affecting the Bcl-2 level. Our data suggest that high glucose induces apoptosis in endothelial cells. alpha-Lipoic acid effectively attenuates high glucose-induced endothelial cell apoptosis. These findings provide new perspectives on the role of alpha-lipoic acid in cardiovascular disease.

Erythropoietin attenuated high glucose-induced apoptosis in cultured human aortic endothelial cells

High glucose-induced apoptosis in vascular endothelial cells may contribute to the acceleration of atherosclerosis associated with diabetes. Here, we show that erythropoietin attenuates high glucose-induced apoptosis in cultured human aortic endothelial cells (HAECs). Exposure of HAECs to high glucose level for 72 h significantly increased the number of apoptotic cells compared with normal glucose level, as evaluated by TUNEL assay. Simultaneous addition of erythropoietin (100 U/ml) significantly attenuated high glucose-induced apoptosis. In parallel, exposure to high glucose level induced caspase-3 activation and erythropoietin also prevented it. Erythropoietin stimulated Akt phosphorylation in a dose-dependent manner (1–100 U/ml). PI3 kinase inhibitor, wortmannin or LY294002 eliminated erythropoietin’s inhibitory effect on caspase-3 activity. In conclusion, erythropoietin may attenuate high glucose-induced endothelial cell apoptosis via PI-3 kinase pathway. Replacing therapy with erythropoietin is often used for correction of renal anemia, but may have potential in preventing atherosclerosis in diabetic patients with end-stage renal failure.

Up‐regulation of the association between heat shock protein 90 and endothelial nitric oxide synthase prevents high glucose‐induced apoptosis in human endothelial cells

Hyperglycemia is the hallmark of diabetes mellitus. Poor glycemic control is correlated with increased cardiovascular morbidity and mortality. High glucose can trigger endothelial cell apoptosis by de-activation of endothelial nitric oxide synthase (eNOS). eNOS was recently demonstrated to be extensively regulated by Akt and heat shock protein 90 (HSP90). Yet, little is known about the molecular mechanisms that regulate eNOS activity during high glucose exposure. The present study was designed to determine the involvement of protein interactions between eNOS and HSP90 in high glucose-induced endothelial cell apoptosis. The protein interaction of eNOS/HSP90 and eNOS/Akt were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to either control-level (5.5 mM) or high-level (33 mM) glucose for different durations (2, 4, 6, and 24 h). The results showed that the protein interactions between eNOS and HSP90 and between eNOS and Akt and the phosphorylation of eNOS were up-regulated by high glucose exposure for 2-4 h. With longer exposures, these effects decreased gradually. During early hours of exposure, the protein interactions of eNOS/HSP90 and eNOS/Akt and the phosphorylation of eNOS were all inhibited by geldanamycin, an HSP90 inhibitor. High glucose-induced endothelial cell apoptosis was also enhanced by geldanamycin and was reversed by NO donors. LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor, inhibited the association of eNOS/Akt and the phosphorylation of eNOS but had no effect on the interaction between eNOS and HSP90 during early hours of exposure. From our results we propose that, in HUVECs, during early phase of high glucose exposure, apoptosis can be prevented by enhancement of eNOS activity through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt. With longer exposure, dysregulation of eNOS activity would result in apoptosis. The present study provides a molecular basis for the effects of eNOS in the prevention of endothelial cells apoptosis during early phase of high glucose exposure. These observations may contribute to the understanding of the pathogenesis of vascular complications in diabetes mellitus.

Effect of Scutellariae radix extract on the high glucose-induced apoptosis in cultured vascular endothelial cells.

Endothelial cell apoptosis has been postulated as the initial trigger of the progression of microvascular disease in patients with diabetes mellitus. To investigate the role of Scutellariae radix extract, we examined its effect on the endothelial cell proliferation using the [3H]-thymidine incorporation method. Scutellariae radix extract significantly stimulated endothelial cell proliferation in a dose-dependent manner. We focused on the protective action of Scutellariae radix extract on the endothelial cell apoptosis induced by high glucose concentrations. Determination of endothelial cell apoptosis was performed using DNA gel electrophoresis, terminal deoxynuclotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and an ELISA kit. Exposure of vascular endothelial cell to high glucose (16.7 mM) for 72 h resulted in a significant increase in apoptosis, compared with the normal glucose concentrations (5.5 mM). Scutellariae radix extract inhibited high glucose-induced endothelial cell apoptosis. This result suggests that Scutellariae radix extract may contribute to antiapoptotic action against vascular endothelial cells, resulting in a beneficial effect in preventing diabetes-associated microvascular complications.