The expression and proangiogenic effect of nucleolin during the recovery of heat-denatured HUVECs

Background

The present study aims to examine the expression patterns and roles of nucleolin during the recovery of heat-denatured human umbilical vein endothelial cells (HUVECs).

Methods

Deep partial thickness burn model in Sprague–Dawley rats and the heat denatured cell model (52 °C, 35 s) were used. The expression of nucleolin was measured using Western blot analysis and real-time PCR. Angiogenesis was assessed using in vitro parameters including endothelial cell proliferation, transwell migration assay, and scratched wound healing. Gene transfection and RNA interference approaches were employed to investigate the roles of nucleolin.

Results

Nucleolin mRNA and protein expression showed a time-dependent increase during the recovery of heat-denatured dermis and HUVECs. Heat-denaturation time-dependently promoted cell growth, adhesion, migration, scratched wound healing and formation of tube-like structures in HUVECs. These effects of heat denaturation on endothelial wound healing and formation of tube-like structures were prevented by knockdown of nucleolin, whereas over-expression of nucleolin increased cell growth, migration, and formation of tube-like structures in cultured HUVEC endothelial cells. In addition, we found that the expression of vascular endothelial growth factor (VEGF) increased during the recovery of heat-denatured dermis and HUVECs, and nucleolin up-regulated VEGF in HUVECs.

Conclusions

The present study reveals that the expression of nucleolin is up-regulated, and plays a pro-angiogenic role during the recovery of heat-denatured dermis and its mechanism is probably dependent on production of VEGF.

General significance

We find a novel and important pro-angiogenic role of nucleolin during the recovery of heat-denatured dermis.     

肝细胞生长因子抑制糖基化终产物诱导人脐静脉内皮细胞的凋亡作用

目的探讨肝细胞生长因子(HGF)抑制糖基化终产物(AGEs)诱导人脐静脉内皮细胞凋亡的作用及其相关分子机制。方法体外培养ECV-304人脐静脉内皮细胞,采用噻唑蓝(MTT)法测定HGF对AGEs作用后ECV-304细胞生长抑制率的影响;通过Hoechst33258荧光染色观察细胞形态学改变、流式细胞术测定AnnexinV-FITC/PI双染标记的细胞凋亡率,检测HGF对AGEs诱导ECV-304细胞凋亡的影响;Western印迹法检测Bax、Bcl-2蛋白的表达。结果HGF能明显降低AGEs对ECV-304细胞生长的抑制作用;AGE诱导培养的ECV-304细胞出现明显的凋亡形态学改变,在一定浓度范围内,ECV-304细胞凋亡率与AGEs的浓度和作用时间呈依赖关系,加入HGF处理后可显著降低不同时间的内皮细胞凋亡率;HGF作用ECV-304细胞后Bcl-2蛋白表达明显升高,而Bax蛋白表达无明显变化。结论AGEs能诱导内皮细胞凋亡,而HGF能部分抑制AGEs诱导的内皮细胞凋亡,其作用机制可能与上调Bcl-2蛋白的表达水平有关。     

Hsa-miRNA-29a protects against high glucose-induced damage in human umbilical vein endothelial cells

Sustained exposure to high glucose (HG) results in dysfunction of vascular endothelial cells. Hence, diabetic patients often suffer from secondary vascular damages, such as vascular sclerosis and thrombogenesis, which may eventually cause cardiovascular problems. Thus, elucidating how HG results in vascular endothelial cell damage and finding an approach for prevention are important to prevent and treat vascular damages in diabetic patients. In the current study, we first showed that 72-hour exposure to HG-decreased hsa-miRNA-29a and increased the expression of Bcl-2 associated X protein (Bax), which subsequently inhibited Bcl-2 and promoted the expression of apoptotic protease activating factor-1 and activation of caspase-3, thus directly triggering the mitochondrial apoptotic pathway in human umbilical vein endothelial cells (HUVECs). Study of the underlying mechanism showed that hsa-miRNA-29a/Bax plays an essential role in the decreased proliferation and increased apoptosis of HUVECs induced by HG, and overexpression of hsa-miRNA-29a effectively inhibits HG-induced apoptosis and restores the proliferation and tube formation of HUVECs exposed to HG by inhibiting its target gene Bax. In short, our study demonstrates that hsa-miRNA-29a is a promising target for the prevention and treatment of vascular injury in diabetic patients.     

Hypoxia-induced apoptosis in endothelial cells and embryonic stem cells

Background: To evaluate the influence of hypoxia and molecular events in endothelial and embryonic stem cells.Materials and Methods: Human umbilical vein endothelial cells (HUVECs) and mouse embryoid body (EB) cells were subjected to hypoxic conditions for different time courses. DNA fragmentation assay, quantification of apoptotic cells by TUNEL assay measured by flowcytometry, and Western blot analysis for the molecular events of apoptosis were performed.Results: DNA fragmentation could be identified under hypoxic conditions in HUVECs and mouse EBs. The DNA fragmentation increased when the hypoxic interval was extended.In situ internucleosomal DNA fragmentation-TUNEL assay also found that the percentages of apoptotic cells increased gradually in HUVECs and mouse EBs when the hypoxic interval was extended. Furthermore, the levels of expression of p53 and Bax both increased in hypoxic conditions.Conclusions: Hypoxia increases both HUVEC and mouse EB apoptosis, which is associated with increase in p53/Bax expression.     

Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoptosis

Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-kappaB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-kappaB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease.     

Plasminogen K5 activates mitochondrial apoptosis pathway in endothelial cells by regulating Bak and Bcl-x<Subscript>L</Subscript> subcellular distribution

Plasminogen Kringle 5(K5) is a proteolytic fragment of plasminogen, which displays potent anti-angiogenic activities. K5 has been shown to induce apoptosis in proliferating endothelial cells; however the exact mechanism has not been well explored. The present study was designed to elucidate the possible molecular mechanism of K5-induced endothelial cell apoptosis. Our results showed that K5 inhibited basic fibroblast growth factors activated in human umbilical vein endothelial cells (HUVECs), indicating proliferation in a dose-dependent manner and induced endothelial cell death via apoptosis. K5 exposure activated caspase 7, 8 and 9. These results suggested that both the intrinsic mitochondrial apoptosis pathway and extrinsic pathway might be involved in K5-induced apoptosis. K5 reduced mitochondrial membrane potential (MMP) of HUVECs, demonstrating mitochondrial depolarization in HUVECs. K5 increased the ratio of Bak to Bcl-x_L on mitochondria, decreased the ratio in cytosol, and had no effect on the total amounts of these proteins. K5 also did not effect on Bax/Bcl-2 distribution. K5 increased the ratio of Bak to Bcl-x_L on mitochondrial that resulted in mitochondrial depolarization, cytochrome c release and consequently the cleavage of caspase 9. These results suggested that K5 induces endothelial cell apoptosis at least in part via activating mitochondrial apoptosis pathway. The regulation of K5 on Bak and Bcl-x_L distribution may play an important role in endothelial cell apoptosis. These results provide further insight into the anti-angiogenesis roles of K5 in angiogenesis-related ocular diseases and solid tumors.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Beraprost sodium attenuates cigarette smoke extract-induced apoptosis in vascular endothelial cells

Apoptosis is now widely recognized as an important part of chronic obstructive pulmonary disease (COPD) pathogenesis. Our previous study demonstrated that a prostacyclin (PGI₂) analogue (beraprost sodium, BPS) prevented cigarette smoke extract (CSE) induced apoptosis of the pulmonary endothelium in rats. So we determined to clarify the apoptosis of vascular endothelial cells in COPD patient and the role of prostacyclin in the protection against apoptosis in vascular endothelial cells induced by CSE. Surgical specimens were obtained from 12 patients with COPD and 10 controls, and the level of apoptosis, prostacyclin synthase (PGI₂S) expression and 6-keto-PGF1α (a stable metabolite of PGI₂) were detected. The apoptotic index (AI), caspase-3 activity, expression of caspase-3 and 6-keto-PGF1α were examined in human umbilical vein endothelial cells (HUVECs) under exposure to varied concentrations of CSE for 24?h as well as under exposure to 2.5?% CSE for varied durations. Then, HUVECs under 2.5?% CSE were exposed to varied concentrations of BPS for 24?h and observed the alteration and the level of cAMP. Increased AI, decreased expression of PGI₂S and 6-keto-PGF1α, were found in the lungs of patients with COPD compared with controls. Moreover, CSE induced apoptosis in means of both dose-dependent and time-dependent manners, and reduced the level of 6-keto-PGF1α in HUVECs. And with the treatment of BPS, an enhanced level of cAMP and decreased apoptosis were detected. The deficiency of PGI₂ critically contributes to the COPD-associated endothelial dysfunction and apoptosis. And BPS protects against the apoptosis in the vascular endothelial cells induced by CSE.     

lncRNA PVT1沉默对高糖诱导的血管内皮细胞增殖、凋亡及氧化应激的影响*

目的: 探讨抑制lncRNA PVT1对高糖诱导的血管内皮细胞的增殖,凋亡和氧化应激的影响。方法: 体外培养人脐静脉内皮细胞(HUVECs),分为四组:对照组(5.5 mmol/L葡萄糖),高糖组(30 mmol/L葡萄糖),高糖+siNC组(30 mmol/L葡萄糖+siNC,细胞转染阴性对照组),高糖+siPVT1组(30 mmol/L葡萄糖+siPVT1,抑制lncRNA PVT1组)。采用荧光定量PCR的方法检测转染后PVT1的表达水平。MTT检测siPVT1(短片段干扰RNA PVT1)对高糖诱导的HUVECs细胞增殖能力的影响。流式细胞术检测siPVT1对高糖诱导的HUVECs细胞ROS和凋亡水平。Western blot检测HUVECs细胞中凋亡相关蛋白如Bax,Bcl-2和cleaved-caspase-3的表达水平。结果: 与对照组比较,转染siPVT1后,PVT1的表达水平显著降低(P＜0.05)。MTT结果显示,与对照组比较,培养24 h和48 h后高糖组中HUVECs细胞增殖活力均显著降低,与高糖+siNC组(阴性对照组)比较,培养24 h和48 h后,高糖+siPVT1组中的HUVECs细胞增殖活力显著增加(P＜0.05)。流式细胞术检测结果表明,与对照组比较,高糖组HUVECs细胞中ROS和凋亡率均显著增加;和高糖+siNC组比较,高糖+siPVT1组中HUVECs细胞中ROS和凋亡率均有减少(P＜0.05)。Western blot结果表明,与对照组比较,高糖组中cleaved-caspase-3和Bax表达水平均显著上调,Bcl-2的表达水平显著下调(P＜0.05,P＜0.01)。与高糖+siNC组比较,高糖+siPVT1组cleaved-caspase-3和Bax表达水平显著下调,Bcl-2的表达显著上调(P＜0.05,P＜0.01)。结论: 抑制lncRNA PVT1可以显著增加高糖诱导的HUVECs细胞增殖活力,减轻氧化应激,抑制细胞凋亡。     

PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax–caspase9–caspase3 pathway

This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24?h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24?h, cells were treated with ox-LDL for 24?h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.     

20(S)-protopanaxadiol induces apoptosis in human umbilical vein endothelial cells by activating the PERK-eIF2alpha-ATF4 signaling pathway

20(S)-protopanaxadiol (PPD)-type ginsenosides are generally believed to be the most pharmacologically active components of Panax ginseng. These compounds induce apoptotic cell death in various cancer cells, which suggests that they have anti-cancer activity. Anti-angiogenesis is a promising therapeutic approach for controlling angiogenesis-related diseases such as malignant tumors, age-related macular degeneration, and atherosclerosis. Studies showed that 20(S)-PPD at low concentrations induces endothelial cell growth, but in our present study, we found 20(S)-PPD at high concentrations inhibited cell growth and mediated apoptosis in human umbilical vein endothelial cells (HUVECs). The mechanism by which high concentrations of 20(S)-PPD mediate endothelial cell apoptosis remains elusive. The present current study investigated how 20(S)-PPD induces apoptosis in HUVECs for the first time. We found that caspase-9 and its downstream caspase, caspase-3, were cleaved into their active forms after 20(S)-PPD treatment. Treatment with 20(S)-PPD decreased the level of Bcl-2 expression but did not change the level of Bax expression. 20(S)-PPD induced endoplasmic reticulum stress in HUVECs and stimulated UPR signaling, initiated by protein kinase R-like endoplasmic reticulum kinase (PERK) activation. Total protein expression and ATF4 nuclear import were increased, and CEBP-homologous protein (CHOP) expression increased after treatment with 20(S)-PPD. Furthermore, siRNA-mediated knockdown of PERK or ATF4 inhibited the induction of CHOP expression and 20(s)-PPD-induced apoptosis. Collectively, our findings show that 20(S)-PPD inhibits HUVEC growth by inducing apoptosis and that ATF4 expression activated by the PERK-eIF2α signaling pathway is essential for this process. These findings suggest that high concentrations of 20(S)-PPD could be used to treat angiogenesis-related diseases.     

Salidroside protects retinal endothelial cells against hydrogen peroxide-induced injury via modulating oxidative status and apoptosis

Oxidative stress can cause injury in retinal endothelial cells. Salidroside is a strong antioxidative and cytoprotective supplement in Chinese traditional medicine. In this study, we investigated the effects of salidroside on H₂O₂-induced primary retinal endothelial cells injury. Salidroside decreased H₂O₂-induced cell death, and efficiently suppressed cellular ROS production, malondialdehyde generation, and cell apoptosis induced by H₂O₂ treatment. Salidroside induced the intracellular mRNA expression, protein expression, and enzymatic activities of catalase and Mn-SOD and increased the ratio of Bcl2/Bax. Our results demonstrated that salidroside protected retinal endothelial cells against oxidative injury through increasing the Bcl2/Bax signaling pathway and activation of endogenous antioxidant enzymes. This finding presents salidroside as an attractive agent with potential to attenuate retinopathic diseases.     

Degradation of heparan sulfate proteoglycans enhances oxidized-LDL-mediated autophagy and apoptosis in human endothelial cells

BackgroundCell surface heparan sulfate proteoglycans (HSPG) play an important role in atherogenesis. We hypothesized that degradation of HSPG may increase the binding of atherogenic oxidized low density lipoprotein (ox-LDL) to endothelial cells, and result in extensive HSPG degradation as well as autophagy and apoptosis.MethodsPrimary human umbilical vein endothelial cells (HUVECs) were used to study the expression of lectin-like ox-LDL receptor-1 (LOX-1), HSPG, autophagy and apoptosis in response to ox-LDL and heparinase III (Hep III).ResultsAs expected, ox-LDL treatment resulted in LOX-1 expression, ox-LDL uptake and reactive oxygen species (ROS) generation. Ox-LDL treatment also resulted in a modest degradation of HSPG and increase in autophagy (expression of LC3, beclin-1 and Atg5) and apoptosis (enhanced expression of caspases and Bax, and reduced expression of Bcl-2 and Bcl-xL). The effects of ox-LDL were blocked by pretreatment of cells with LOX-1 antibody or apocynin, an NADPH oxidase inhibitor. Hep III alone caused HSPG degradation and slightly, but significantly, increased ROS generation, and induced autophagy and caspase expression. However, autophagy and apoptosis induced by Hep III were not affected by apocynin or LOX-1 antibody. Importantly, Hep III pretreatment of cells significantly enhanced ox-LDL-induced HSPG degradation, LOX-1 expression, ox-LDL uptake and ROS generation as well as autophagy and apoptosis.ConclusionThese data demonstrate that Hep III enhances the pro-atherosclerotic characteristics in HUVECs induced by ox-LDL.     

2, 3, 7, 8‐Tetrachlorodibenzo‐p‐dioxin promotes endothelial cell apoptosis through activation of EP3/p38MAPK/Bcl‐2 pathway

Endothelial injury or dysfunction is an early event in the pathogenesis of atherosclerosis. Epidemiological and animal studies have shown that 2, 3, 7, 8‐tetrachlorodibenzo‐p‐dioxin (TCDD) exposure increases morbidity and mortality from chronic cardiovascular diseases, including atherosclerosis. However, whether or how TCDD exposure causes endothelial injury or dysfunction remains largely unknown. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to different doses of TCDD, and cell apoptosis was examined. We found that TCDD treatment increased caspase 3 activity and apoptosis in HUVECs in a dose‐dependent manner,at doses from 10 to 40 nM. TCDD increased cyclooxygenase enzymes (COX)‐2 expression and its downstream prostaglandin (PG) production (mainly PGE₂ and 6‐keto‐PGF_1α) in HUVECs. Interestingly, inhibition of COX‐2, but not COX‐1, markedly attenuated TCDD‐triggered apoptosis in HUVECs. Pharmacological inhibition or gene silencing of the PGE₂ receptor subtype 3 (EP3) suppressed the augmented apoptosis in TCDD‐treated HUVECs. Activation of the EP3 receptor enhanced p38 MAPK phosphorylation and decreased Bcl‐2 expression following TCDD treatment. Both p38 MAPK suppression and Bcl‐2 overexpression attenuated the apoptosis in TCDD‐treated HUVECs. TCDD increased EP3‐dependent Rho activity and subsequently promoted p38MAPK/Bcl‐2 pathway‐mediated apoptosis in HUVECs. In addition, TCDD promoted apoptosis in vascular endothelium and delayed re‐endothelialization after femoral artery injury in wild‐type (WT) mice, but not in EP3^?/? mice. In summary, TCDD promotes endothelial apoptosis through the COX‐2/PGE₂/EP3/p38MAPK/Bcl‐2 pathway. Given the cardiovascular hazard of a COX‐2 inhibitor, our findings indicate that the EP3 receptor and its downstream pathways may be potential targets for prevention of TCDD‐associated cardiovascular diseases.     

Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices

Vascular endothelial cells are highly sensitive to oxidative stress, and this is one of the mechanisms by which widespread endothelial dysfunction is induced in most cardiovascular diseases and disorders. However, how these cells can survive in oxidative stress environments remains unclear. Salidroside, a traditional Chinese medicine, has been shown to confer vascular protective effects. We aimed to understand the role of autophagy and its regulatory mechanisms by treating human umbilical vein endothelial cells (HUVECs) with salidroside under oxidative stress. HUVECs were treated with salidroside and exposed to hydrogen peroxide (H₂O₂). The results indicated that salidroside exerted cytoprotective effects in an H₂O₂-induced HUVEC injury model and suppressed H₂O₂-induced apoptosis of HUVECs. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased oxidative stress-induced HUVEC apoptosis, while the autophagy activator rapamycin induced anti-apoptosis effects in HUVECs. Salidroside increased autophagy and decreased apoptosis of HUVECs in a dose-dependent manner under oxidative stress. Moreover, 3-MA attenuated salidroside-induced HUVEC autophagy and promoted apoptosis, whereas rapamycin had no additional effects compared with salidroside alone. Salidroside upregulated AMPK phosphorylation but downregulated mTOR phosphorylation under oxidative stress; however, administration of compound C, an AMPK inhibitor, abrogated AMPK phosphorylation and increased mTOR phosphorylation and apoptosis compared with salidroside alone. These results suggest that autophagy is a protective mechanism in HUVECs under oxidative stress and that salidroside might promote autophagy through activation of the AMPK pathway and downregulation of mTOR pathway.     

Involvement of Kv1.5 Protein in Oxidative Vascular Endothelial Cell Injury

Endothelial injury related to oxidative stress is a key event in cardiovascular diseases, such as hypertension and atherosclerosis. The activation of the redox-sensitive Kv1.5 potassium channel mediates mitochondrial reactive oxygen species (ROS)-induced apoptosis in vascular smooth muscle cells and some cancer cells. Kv1.5 channel is therefore taken as a new potential therapeutic target for pulmonary hypertension and cancers. Although Kv1.5 is abundantly expressed in vascular endothelium, there is little knowledge of its role in endothelial injury related to oxidative stress. We found that DPO-1, a specific inhibitor of Kv1.5, attenuated H₂O₂-evoked endothelial cell apoptosis in an in vivo rat carotid arterial model. In human umbilical vein endothelial cells (HUVECs) and human pulmonary arterial endothelial cells (HPAECs), angiotensin II and oxLDL time- or concentration-dependently enhanced Kv1.5 protein expression in parallel with the production of intracellular ROS and endothelial cell injury. Moreover, siRNA-mediated knockdown of Kv1.5 attenuated, whereas adenovirus-mediated Kv1.5 cDNA overexpression enhanced oxLDL–induced cellular damage, NADPH oxidase and mitochondria-derived ROS production and restored the decrease in protein expression of mitochondria uncoupling protein 2 (UCP2). Collectively, these data suggest that Kv1.5 may play an important role in oxidative vascular endothelial injury.     

Ganoderma lucidum polysaccharides peptide inhibits the growth of vascular endothelial cell and the induction of VEGF in human lung cancer cell

Ganoderma lucidum Polysaccharide Peptide (Gl-PP) has shown some effects as anti-tumors in mice and potential anti-angiogenesis. In this study, we elucidated the possible mechanism of Gl-PP action on anti-angiogenesis of tumor. Our research indicated that the proliferation of HUVECs was inhibited by Gl-PP in a dose-dependent fashion, but not because of cytotoxicity. Flow cytometric studies revealed that Gl-PP treatment of HUVECs could induce cell apoptosis directly. Moreover, addition of Gl-PP also led to a reduction of Bcl-2 anti-apoptotic protein expression and an increase of Bax pro-apoptotic protein expression of HUVECs. Therefore, inducing cell apoptosis by Gl-PP might be the mechanism of inhibiting HUVEC proliferation. Human lung carcinoma cells PG when exposed to high dose of Gl-PP in hypoxia for 18 h resulted in a decrease in the secreted VEGF. Taken together, these findings support the hypothesis that the key attribute of the anti-angiogenic potential of Gl-PP is that it may directly inhibit vascular endothelial cell proliferation or indirectly decrease growth factor expression of tumor cells.     

Phosphocreatine protects against LPS-induced human umbilical vein endothelial cell apoptosis by regulating mitochondrial oxidative phosphorylation

Phosphocreatine (PCr) is an exogenous energy substance, which provides phosphate groups for adenosine triphosphate (ATP) cycle and promotes energy metabolism in cells. However, it is still unclear whether PCr has influenced on mitochondrial energy metabolism as well as oxidative phosphorylation (OXPHO) in previous studies. Therefore, the aim of the present study was to investigate the regulation of PCr on lipopolsaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs) and mitochondrial OXPHO pathway. PCr protected HUVECs against LPS-induced apoptosis by suppressing the mitochondrial permeability transition, cytosolic release of cytochrome c (Cyt C), Ca²⁺, reactive oxygen species and subsequent activation of caspases, and increasing Bcl₂ expression, while suppressing Bax expression. More importantly, PCr significantly improved mitochondrial swelling and membrane potential, enhanced the activities of ATP synthase and mitochondrial creatine kinase (CK_mt) in creatine shuttle, influenced on respiratory chain enzymes, respiratory control ratio, phosphorus/oxygen ratio and ATP production of OXPHO. Above PCr-mediated mitochondrial events were effectively more favorable to reduced form of flavin adenine dinucleotide (FADH₂) pathway than reduced form of nicotinamide-adenine dinucleotid pathway in the mitochondrial respiratory chain. Our results revealed that PCr protects against LPS-induced HUVECs apoptosis, which probably related to stabilization of intracellular energy metabolism, especially for FADH₂ pathway in mitochondrial respiratory chain, ATP synthase and CK_mt. Our findings suggest that PCr may play a certain role in the treatment of atherosclerosis via protecting endothelial cell function.     

Delphinidin attenuates stress injury induced by oxidized low-density lipoprotein in human umbilical vein endothelial cells

Moderate consumption of natural dietary polyphenolic compounds can reduce the risk of cardiovascular diseases. Here we investigated the protective effects of delphinidin against oxidized low-density lipoprotein (oxLDL)-induced damage in human umbilical vein endothelial cells (HUVECs). The MTT assay showed that 2 h pre-incubation with delphinidin markedly restored the oxLDL-induced viability loss in HUVECs in a concentration-dependent manner. This effect was accompanied by a significant decrease in intracellular reactive oxygen species. Moreover, delphinidin imposed preventive effects on suppressing the production of lipid peroxidation, restoring the activities of endogenous antioxidants, and increasing the level of nitric oxide. Pre-incubation of delphinidin with HUVECs led to the reduction of apoptosis. Finally, delphinidin can efficiently prevent the down-regulation of Bcl-2 protein and up-regulation of Bax protein. Together, our findings suggest that delphinidin can effectively protect HUVECs against oxidative stress induced by oxLDL, which may be important for preventing both plaque development and stability in atherosclerosis.