Angiotensin II induces focal adhesion kinase/paxillin phosphorylation and cell migration in human umbilical vein endothelial cells

In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.     

Paeoniflorin attenuates oxidized low-density lipoprotein-induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is recognized as a driving force in the development of atherosclerosis (AS). Paeoniflorin (Pae), a typical traditional herbal medicine, possesses anti-inflammatory, antioxidative, antihyperglycaemic, and antiapoptotic properties. Our study aimed to investigate the effects of Pae on ox-LDL-induced injury of the human umbilical vein endothelial cells (HUVECs) and to explore its molecular mechanism. We found that ox-LDL stimulation inhibited cell viability, activated autophagy, and induced apoptosis and adhesion molecule expression in HUVECs. Pae rescued ox-LDL-induced viability reduction and enhanced the ox-LDL-induced autophagy activation in HUVECs. Pae inhibited ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement in HUVECs. In addition, inhibition of SIRT1 by EX-527 abolished the promoting effect of Pae on autophagy and restored the inhibitory effect of Pae on apoptosis and adhesion molecule expression in the presence of ox-LDL. In conclusion, Pae attenuated ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement via upregulation of SIRT1 in HUVECs, shedding light on the mechanism underlying the protective effect of Pae on ox-LDL-induced injury of HUVECs.     

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.     

Metabolic responses induced by thrombin in human umbilical vein endothelial cells

Metabolic responses induced by thrombin in human umbilical vein endothelial cells (HUVECs) were investigated by using the cytosensor technique. Thrombin increased the extracellular acidification rate of endothelial cells, measured as an index of metabolic activity with a cytosensor microphysiometer, in a concentration-dependent fashion with an EC(50) of 1.27+/-0.59 IU/ml, which was abolished by the MAP kinase inhibitor PD98059. When intracellular Ca(2+) was chelated or PKC was inactivated, PD98059 failed to abolish the thrombin-induced acidification rate response in HUVECs. In addition, the tyrosine kinase inhibitor genistein, PKC inhibitor calphostin C, and Na(+)/H(+)exchanger antagonist MIA also partly inhibited thrombin-induced acidification rate responses. It is suggested that thrombin stimulated rapid metabolic responses via MAP kinase in HUVECs, which are calcium- and PKC-dependent.     

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.     

cAMP inhibits cytokine-induced biosynthesis of tetrahydrobiopterin in human umbilical vein endothelial cells

We studied the effects of cAMP on cytokine (interferon-gamma plus tumor necrosis factor-alpha)-induced stimulation of tetrahydrobiopterin (BH4) synthesis in human umbilical vein endothelial cells (HUVEC). The cytokine mixture caused a marked increase in the biosynthesis and release of BH4 by HUVEC. Dibutyryl-cAMP produced a dose-dependent inhibition of this cytokine-induced stimulation of synthesis and release of BH4 by these cells. 8-Bromo-cAMP also caused a significant inhibition, although the effects were less marked than those of dibutyryl-cAMP. Both forskolin and the stable analog of prostacyclin, iloprost, caused cAMP accumulation and a concomitant diminution of the cytokine-induced BH4 synthesis in HUVEC. Dibutyryl-cAMP and iloprost also significantly inhibited the cytokine-induced stimulation of GTP cyclohydrolase I (GCHI) activity and mRNA production. We concluded that the suppression by the cAMP messenger system of cytokine-induced stimulation of synthesis and release of BH4 by HUVEC can be attributed to the inhibition of the activity of GCHI, the rate-limiting enzyme in BH4 biosynthetic pathway, in HUVEC. The data also suggest that the cAMP-mediated reduction in the GCHI mRNA level may at least partially explain the decline in GCHI activity. It is reasoned that under inflammatory conditions, cAMP-elevating agents such as prostacyclin exert regulatory effects on circulation by inhibiting cytokine-induced synthesis and release of BH4 by HUVEC.     

Antiapoptotic effect of ouabain on human umbilical vein endothelial cells

The present study investigates the effect of ouabain on caspase-3 activation in human umbilical vein endothelial cells (HUVEC). Ouabain (EC(50) 20 nM) reduced caspase-3 activity in HUVEC treated for 24h in a medium deprived of fibroblast growth factor (FGF). Incubation for 5h in the absence of both FGF and serum produced an increase in caspase-3 activity that was completely abolished by 100 nM ouabain. Pretreatment with the phosphatidylinositol 3 kinase (PI-3K) inhibitor, wortmannin, prevented the protective effect of ouabain against serum deprivation. Furthermore, Western blotting analysis revealed an increase in phosphorylation of extracellular signal-regulated kinases (ERK-1 and ERK-2) induced by 100nM ouabain in serum-deprived cells. In accord, pretreatment of HUVEC with PD98059, inhibitor of the ERK pathway, abrogated the effect of ouabain. Our results show that ouabain has an antiapoptotic effect on HUVEC through the activation of PI-3K and ERK dependent pathways.     

脂联素抑制t-BHP诱导的内皮细胞凋亡作用及其可能机制探讨

目的：探讨重组脂联素对第三丁基过氧化氢（t-BHP）诱导的人脐静脉内皮细胞（HUVECs）凋亡的影响及其相关的分子机制。方法：以HUVECs作为研究对象,给予t-BHP处理,模拟体外HUVECs氧化损伤细胞凋亡模型。在此基础上,用携带重组脂联素基因的腺病毒转染HUVECs,观察重组脂联素对t-BHP诱导的HUVECs凋亡的影响。用MTT法检测细胞增殖活力。Hochest/PI荧光染色检测细胞凋亡率。Western blot检测细胞凋亡相关蛋白p-JNK、JNK和Caspase 3表达水平的变化。结果：100 μmol/L的t-BHP作用8 h可诱导HUVECs发生凋亡。与对照组相比,t-BHP组p-JNK、active caspase 3表达增多（P<0.01）。HUVECs高表达重组脂联素基因后,可明显抑制t-BHP诱导的HUVECs凋亡（P<0.01）,下调t-BHP诱导的p-JNK、active caspase 3表达。结论：持续t-BHP氧化损伤可诱导HUVECs发生凋亡。重组脂联素可有效抑制t-BHP诱导的HUVECs凋亡,其机制与p-JNK、active caspase 3的表达下调有关。     

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.     

Caspase pathway of elaidic acid (9t-C18:1)-induced apoptosis in human umbilical vein endothelial cells

Although TFAs (trans fatty acids) do have effects on many endothelial functions, systemic inflammation and immune disorders, only limited experimental evidence is available that TFAs participate in the pathogenesis of endothelial cell apoptosis. HUVEC (human umbilical vein endothelial cells) were grown in medium with elaidic acid (9t-C18:1) at 50, 100, 200 and 400 μmol/l for 24 h. Apoptosis was measured by flow cytometry, and caspase 3, 8 and 9 activities by colorimetric assay and their mRNA expression by qRT-PCR (quantitative real-time PCR). Results showed that 9t-C18:1 induced apoptosis of HUVEC in a dose-dependent manner. The activities and mRNA expression of caspases 8, 9 and 3 were significantly increased compared with that of the control. Z-IETD-FMK and Z-LEHD-FMK inhibited the activation of caspase 3 and apoptosis induced by 9t-C18:1. Also Z-IETD-FMK inhibited the activation of caspase 9. mRNA expressions of Bid and Smac (second mitochondria-derived activator of caspase)/DIABLO [direct IAP (inhibitor of apoptosis)-binding protein with low pI] were also significantly elevated. We conclude that 9t-C18:1 induces apoptosis of HUVEC through activating caspases 8, 9 and 3. The death receptor pathway and the mitochondrial pathway both participated in the apoptosis course induced by 9t-C18:1.     

Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation

Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me₂SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols.     

AMPK inhibits fatty acid-induced increases in NF-kappaB transactivation in cultured human umbilical vein endothelial cells

The fuel sensing enzyme AMP-activated protein kinase (AMPK) enhances processes that generate ATP when stresses such as exercise or glucose deprivation make cells energy deficient. We report here a novel role of AMPK, to prevent the activation of NF-kappaB in endothelial cells exposed to the fatty acid palmitate or the cytokine TNF-alpha. Incubation of cultured human umbilical vein endothelial cells (HUVEC) with elevated levels of palmitate (0.4mM) increased NF-kappaB reporter gene expression by 2- to 4-fold within 8h and caused a 7-fold increase in VCAM-1 mRNA expression at 24h. In contrast, no increase in reporter gene expression was detected for AP-1, glucocorticoid-, cyclic AMP-, or serum response elements. Similar increases in NF-kappaB activation and VCAM-1 expression were not observed in cells incubated with an elevated concentration of glucose (25mM). The increases in NF-kappaB activation and VCAM-1 expression caused by palmitate were markedly inhibited by co-incubation with the AMPK activator AICAR and, where studied, by expression of a constitutively active AMPK. Likewise, AMPK activation inhibited the increase in NF-kappaB reporter gene expression observed in HUVEC incubated with TNF-alpha. The results suggest that AMPK inhibits the activation of NF-kappaB caused by both palmitate and TNF-alpha. The mechanism responsible for this action, as well as its relevance to the reported anti-atherogenic actions of exercise, metformin, thiazolidinediones, and adiponectin, all of which have been shown to activate AMPK, remains to be determined.     

Scutellarin promotes in vitro angiogenesis in human umbilical vein endothelial cells

Angiogenesis is critical to a wide range of physiological and pathological processes. Scutellarin, a major flavonoid of a Chinese herbal medicine Erigeron breviscapus (Vant.) Hand. Mazz. has been shown to offer beneficial effects on cardiovascular and cerebrovascular functions. However, scutellarin’s effects on angiogenesis and underlying mechanisms are not fully elucidated. Here, we studied angiogenic effects of scutellarin on human umbilical vein endothelial cells (HUVECs) in vitro. Scutellarin was found by MTT assay to induce proliferation of HUVECs. In scutellarin-treated HUVECs, a dramatic increase in migration was measured by wound healing assay; Transwell chamber assay found significantly more invading cells in scutellarin-treated groups. Scutellarin also promoted capillary-like tube formation in HUVECs on Matrigel, and significantly upregulated platelet endothelial cell adhesion molecule-1 at both mRNA and protein levels. Scutellarin’s angiogenic mechanism was investigated in vitro by measuring expression of angiogenic factors associated with cell migration and invasion. Scutellarin strongly induced MMP-2 activation and mRNA expression in cultured HUVECs in a concentration-dependent manner. Taken together, these results suggest that scutellarin promotes angiogenesis and may form a basis for angiogenic therapy.     

CD226单克隆抗体诱导人脐静脉内皮细胞胞质钙离子浓度的变化

为观察CD226单克隆抗体(mAb)对培养人脐静脉内皮细胞(HUVECs)胞质钙离子变化的影响,我们用Fluo-3作为钙指示剂,用激光共聚焦显微镜观测不同状态下CD226 mAb作用后HUVECs胞质钙离子[Ca2 ]i的变化。结果发现：(1)用Hanks液平衡,CD226 mAb作用后HUVECs[Ca2 ]i水平缓慢升高后回到原位;加入二抗(羊抗鼠IgG)交联后[Ca2 ]i水平有较大幅度的升高,随后回到原位,与此同时,细胞外液中[Ca2 ]。水平有一定程度的下降;(2)用D-Hanks液平衡,CD226 mAb作用后HUVECs[Ca2 ]i水平无显著变化,加入二抗发生交联作用后,[Ca2 ]：水平有较大幅度的下降;(3)用EGTA预处理后,CD226 mAb及其二抗交联对HUVECs[Ca2 ]i变化无显著影响。以上结果提示,CD226mAb及其二抗交联可诱导不同状态的HUVECs胞质钙离子水平发生不同程度的变化,从而参与一系列的生理和病理过程。     

Reactive oxygen species activate focal adhesion kinase, paxillin and P130CAS tyrosine phosphorylation in endothelial cells

Reactive oxygen species (ROS), particularly hydroxyl radical (HO), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 μM) and herbimycin A (0.9 μM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120–130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125^FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 μM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.     

单核细胞趋化蛋白-1诱导人脐静脉内皮细胞凋亡的分子机制(英文)

本研究旨在探讨单核细胞趋化蛋白-1(monocyte chemotacitic protein-1,MCP-1)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,hUVECs)凋亡的分子机制。胶原酶消化收集hUVECs,体外培养细胞,用胰蛋白酶-EDTA混合液消化传代,用血管性假血友病因子(von Willebrand factor,vWF)和VEGF受体2(KDR)免疫染色证实培养细胞为内皮细胞;用不同浓度MCP-1(0.1、1.0、10、100ng/mL)分别作用hUVECs24h、48h;用流式细胞术及蛋白免疫印迹法检测凋亡相关蛋白Fas、Bcl-2、Bax的表达。如我们前期结果所示,MCP-1能诱导hUVECs的凋亡,其效应随浓度和时间的增加而增强;与对照组比较,MCP-1下调抑凋亡蛋白Bcl-2的表达,上调促凋亡蛋白Fas、Bax的表达。以上结果表明,MCP-1能诱导hUVECs凋亡,其作用机制可能与上调Bax、Fas蛋白及下调Bcl-2蛋白表达有关。     

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。     

Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, but the mechanism by which this occurs is unknown. Src suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after cell adhesion and that SSeCKS translocated from the membrane to the cytosol during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we show that RPMVEC cells in which SSeCKS expression was inhibited reduce adhesion and spread on LN through blocking the formation of actin stress fibers and focal adhesions. These results demonstrated SSeCKS modulate endothelial cells adhesion and spreading by reorganization of the actin cytoskeleton.     

Erbin plays a critical role in human umbilical vein endothelial cell migration and tubular structure formation via the Smad1/5 pathway

Angiogenesis is an important process in atherosclerosis. ErbB2 was proved to have an important role in vascular development, but it is still unclear whether Erbin expresses in vessels as well as its location and function in the vessels. In the current study, we investigated the location and function of Erbin in human umbilical veins. The human umbilical veins were prepared, and immunofluorescent analysis was performed to determine the expression of Erbin. Human umbilical vein endothelial cells (HUVECs) were cultured and the lentivirus (LV) containing Erbin RNAi was also prepared. After transfection with the lentivirus, CCK-8 assay and Annexin V-PI assay were used for cell proliferation and apoptosis, respectively. Cell migration was studied using the scratch wound healing assay and the transwell assay. The capillary-like tube formation assay was performed to illustrate the effect of Erbin on HUVEC tube formation. Expression of signaling pathway molecules was assessed with Western blot. The immunofluorescent analysis suggested that Erbin expressed in human umbilical veins and the majority of the Erbin is strongly colocalized in endothelial cells. Although knockdown of Erbin did not affect HUVEC proliferation and apoptosis, it significantly suppressed HUVEC migration and tubular structure formation. Erbin knockdown showed no effect on the ERK1/2 and Smad2/3 signaling pathways but significantly promoted Smad1/5 phosphorylation and nuclear translocation. Ablation of the Smad1/5 pathway decreased the effects of Erbin on endothelial cells. Erbin is mainly localized in endothelial cells in human umbilical veins and plays a critical role in endothelial cell migration and tubular formation via the Smad1/5 pathway.