Procyanidin B2 improves endothelial progenitor cell function and promotes wound healing in diabetic mice via activating Nrf2

One of the major reasons for the delayed wound healing in diabetes is the dysfunction of endothelial progenitor cells (EPCs) induced by hyperglycaemia. Improvement of EPC function may be a potential strategy for accelerating wound healing in diabetes. Procyanidin B2 (PCB2) is one of the major components of procyanidins, which exhibits a variety of potent pharmacological activities. However, the effects of PCB2 on EPC function and diabetic wound repair remain elusive. We evaluated the protective effects of PCB2 in EPCs with high glucose (HG) treatment and in a diabetic wound healing model. EPCs derived from human umbilical cord blood were treated with HG. The results showed that PCB2 significantly preserved the angiogenic function, survival and migration abilities of EPCs with HG treatment, and attenuated HG-induced oxidative stress of EPCs by scavenging excessive reactive oxygen species (ROS). A mechanistic study found the protective role of PCB2 is dependent on activating nuclear factor erythroid 2-related factor 2 (Nrf2). PCB2 increased the expression of Nrf2 and its downstream antioxidant genes to attenuate the oxidative stress induced by HG in EPCs, which were abolished by knockdown of Nrf2 expression. An in vivo study showed that intraperitoneal administration of PCB2 promoted wound healing and angiogenesis in diabetic mice, which was accompanied by a significant reduction in ROS level and an increase in circulating EPC number. Taken together, our results indicate that PCB2 treatment accelerates wound healing and increases angiogenesis in diabetic mice, which may be mediated by improving the mobilization and function of EPCs.     

Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells

Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.     

Vascular endothelial growth factor and substrate mechanics regulate in vitro tubulogenesis of endothelial progenitor cells

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co‐regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1‐MMP (MT1‐MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs.     

香椿子正丁醇提取物通过激活Nrf2改善高糖引起的肾小球内皮细胞氧化应激

目的探讨香椿子正丁醇提取物(n-butyl alcohol extract of toona sinensis,NBAE)对高糖引起的肾小球内皮细胞氧化应激的影响及机制。方法分别以高糖(high glucose,HG)、HG+NBAE刺激人肾小球内皮细胞,采用DCFDA法检测细胞内活性氧(reactive oxygenspecies,ROS)生成情况,Nitrate/Nitrite Colorimetric法检测细胞内NO的含量,Western blot检测Nrf2、NQO1及HO-1的蛋白表达,免疫荧光方法检测Nrf2、P47phox在细胞内的定位及表达。结果高糖刺激肾小球内皮细胞后出现氧化应激损伤,ROS升高,NO减少,p47phox表达量增高,Nrf2及下游蛋白NQO1、HO-1表达减少;NBAE可明显提高Nrf2的表达,并增加下游蛋白NQO1和HO-1的表达,从而抑制高糖导致的ROS升高,抑制p47phox的表达,并稳定NO的含量。结论NBAE通过激活Nrf2及其下游靶蛋白来改善高糖对肾小球内皮细胞造成的氧化应激损伤。     

Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A(2) (cPLA(2)). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor alpha (PPARalpha) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARalpha null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA(2) and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases.     

Culture of endothelial cells by transfection with plasmid harboring vascular endothelial growth factor

Vascular endothelial cells (ECs) are usually difficult to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement (ECGS). The expression of VEGF by HUVEC tansfected with VEGF gene was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS., However, when the culture medium was supplied with 2.5 ng/mL of basic fibroblast growth factor (bFGF), a synergistic effect of VEGF and bFGF was observed. In this case, the final cell density was recovered up to about 78% of maxium value.     

VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the Rho family of small GTPases. As a member of this family, Rac1 promotes VEGF-induced endothelial cell migration by stimulating the formation of lamellipodia and membrane ruffles. To form these membrane protrusions, Rac1 is activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP. The goal of this study was to identify the GEF responsible for activating Rac1 in response to VEGF stimulation. We have found that VEGF stimulates biphasic activation of Rac1 and for these studies we focused on the peak of activation that occurs at 30 min. Inhibition of VEGFR-2 signaling blocks VEGF-induced Rac1 activation. Using a Rac1 nucleotide-free mutant (G15ARac1), which has a high affinity for binding activated GEFs, we show that the Rac GEF Vav2 associates with G15ARac1 after VEGF stimulation. Additionally, we show that depleting endothelial cells of endogenous Vav2 with siRNA prevents VEGF-induced Rac1 activation. Moreover, Vav2 is tyrosine phosphorylated upon VEGF treatment, which temporally correlates with Rac1 activation and requires VEGFR-2 signaling and Src kinase activity. Finally, we show that depressing Vav2 expression by siRNA impairs VEGF-induced endothelial cell migration. Taken together, our results provide evidence that Vav2 acts downstream of VEGF to activate Rac1.     

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.     

OSAHS患者腭咽组织中COX-2、VEGF的表达及其与新生血管生成的关系

目的：探讨阻塞性睡眠呼吸暂停低通气综合症（OSAHS）患者腭咽组织中环氧化酶-2（COX-2）、血管内皮生长因子（VEGF）的表达及其与新生血管生成的关系及意义。方法：经多导睡眠监测仪（PSG）确诊的40例OSAHS患者（其中轻度组7例,中度12例,重度21例）及6例无鼾症患者的软腭组织,采用HE染色光镜观察腭咽部组织的病理组织学改变,免疫组化技术检测COX-2,VEGF及微血管密度（MVD）的表达情况。结果：COX-2、VEGF主要表达于OSAHS患者软腭组织的黏膜鳞状上皮和导管腺上皮,中、重度OSAHS组与对照组比较,COX-2,VEGF,MVD表达均有显著差异（P〈0.01）,OSAHS组明显高于对照组,轻度组与对照组差别无统计学意义（P〉0.05）;COX-2、VEGF均与MVD的表达呈正相关（P〈0.01）,COX-2与VEGF的表达呈正相关（P〈0.01）,COX-2,VEGF,MVD均与睡眠呼吸暂停低通气指数（AHI）呈正相关（P〈0.01）,与夜间最低氧饱和度呈负相关（P〈0.01）。结论：OSAHS患者腭咽部存在新生血管增生,与缺氧程度有关,COX-2及VEGF在其发生发展过程中可能起到重要作用。     

Cross‐talk between the VEGF‐A and HGF signalling pathways in endothelial cells

Background information. Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre‐existing vascular bed. VEGF‐A (vascular endothelial growth factor‐A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF‐A‐driven angiogenesis, although the signalling mechanisms underlying this co‐operation are not completely understood. Results. We analysed the effects of the combination of VEGF‐A and HGF on the activation of VEGFR‐2 (VEGF receptor‐2) and c‐met receptors, and on the stimulation of downstream signalling pathways in endothelial cells. We found that VEGFR‐2 and c‐met do not physically associate and do not transphosphorylate each other, suggesting that co‐operation involves signalling events more distal from receptor activation. We demonstrate that the VEGF isoform VEGF‐A₁₆₅ and HGF stimulate a similar set of MAPKs (mitogen‐activated protein kinases), although the kinetics and strengths of the activation differ depending on the growth factor and pathway. An enhanced activation of the signalling was observed when endothelial cells were stimulated by the combination of VEGF‐A₁₆₅ and HGF. Moreover, the combination of VEGF‐A and HGF results in a statistically significant synergistic activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) and p38 kinases. We demonstrated that VEGF‐A₁₆₅ and HGF activate FAK (focal adhesion kinase) with different kinetics and stimulate the recruitment of phosphorylated FAK to different subsets of focal adhesions. VEGF‐A₁₆₅ and HGF regulate distinct morphogenic aspects of the cytoskeletal remodelling that are associated with the preferential activation of Rho or Rac respectively, and induce structurally distinct vascular‐like patterns in vitro in a Rho‐ or Rac‐dependent manner. Conclusions. Under angiogenic conditions, combining VEGF‐A with HGF can promote neovascularization by enhancing intracellular signalling and allowing more finely regulated control of the signalling molecules involved in the regulation of the cytoskeleton and cellular migration and morphogenesis.     

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.     

Control of vascular endothelial growth factor angiogenic activity by the extracellular matrix

In adult vessels the proliferation rate of differentiated endothelial cells is very low. In response to several environmental stimuli the expression of so-called ‘angiogenic factors’ is upregulated and the messenger RNAs are actively translated in secreted factors which induce the proliferation of endothelial cells; the digestion of their basement membrane then allows their migration and differentiation. Considerable progress has been made during the past years in elucidating the molecular actors of angiogenesis. Vascular endothelial growth factor turned out to represent the major inducer of angiogenesis. Optional splicing of its pre-messenger RNA generates various isoforms which differ not only by their storage in the extracellular matrix but also by their signaling pathways.     

NDRG2 induced by oxidized LDL in macrophages antagonizes growth factor productions via selectively inhibiting ERK activation

During atherogenesis, macrophage foam cells produce prodigious growth factors, cytokines, and chemokines, which play the central roles in inflammatory process in atherosclerotic plaque formation. In the present study, we identified a new protein marker, N-Myc downstream-regulated protein 2 (NDRG2), which is significantly up-regulated in oxidized low density lipoprotein (oxLDL) treated macrophages and in human atherosclerotic plaques. Over-expression and siRNA knockdown studies showed that NDRG2 is a negative regulator of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) productions in macrophages. Furthermore, we investigated the effects of NDRG2 on MAPK signal activation. Our results showed ERK1/2 activation, but not P38 or JNK1/2 activation, is responsible for regulation of NDRG2 on VEGF and PDGF productions. Consistent with the PDGF levels, the vascular smooth muscle cell (VSMC) proliferation was also regulated by the conditional medium of the oxLDL treated macrophages with NDRG2 knockdown or over-expression. Neutralizing anti-PDGF antibody can significantly inhibit the enhanced VSMC proliferation by macrophage medium with NDRG2 knockdown. Our present results demonstrate that NDRG2 participates in oxLDL-induced macrophage activation and modulates ERK1/2-dependent PDGF and VEGF production, which has potential application in atherogenesis.