An essential role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris

Clinical and experimental observations indicate a role for VEGF secreted by the retinal pigment epithelium (RPE) in the maintenance of the choriocapillaris (CC). VEGF in mice is produced as three isoforms, VEGF120, VEGF164, and VEGF188, that differ in their ability to bind heparan sulfate proteoglycan. RPE normally produces the more soluble isoforms, VEGF120 and VEGF164, but virtually no VEGF188, reflecting the fact that molecules secreted by the RPE must diffuse across Bruch''s membrane (BrM) to reach the choriocapillaris. To determine the role of RPE-derived soluble VEGF on the choriocapillaris survival, we used mice that produce only VEGF188. VEGF188/188 mice exhibited normal choriocapillaris development. However, beginning at 7 months of age, we observed a progressive degeneration characterized by choriocapillaris atrophy, RPE and BrM abnormalities, culminating in areas of RPE loss and dramatic choroidal remodeling. Increased photoreceptor apoptosis in aged VEGF188/188 mice led to a decline in visual acuity as detected by electroretinogram (ERG). These changes are reminiscent of geographic atrophy (GA) and point to a role for RPE-derived VEGF in the maintenance of the choriocapillaris.     

Important role of erythropoietin receptor to promote VEGF expression and angiogenesis in peripheral ischemia in mice

We have recently demonstrated that endogenous erythropoietin (Epo)/Epo receptor (EpoR) system plays an important protective role in hypoxia-induced pulmonary hypertension. However, it remains to be examined whether vascular EpoR system contributes to angiogenesis in response to ischemia. We examined angiogenesis in EpoR(-/-)-rescued mice that lack EpoR in most organs including cardiovascular system except erythroid-lineage cells. Two weeks after femoral artery ligation, blood flow recovery, activation of VEGF/VEGF receptor system, and mobilization of endothelial progenitor cells were all impaired in EpoR(-/-)-rescued mice as compared with wild-type (WT) mice. Bone marrow (BM) transplantation with WT-BM cells in EpoR(-/-)-rescued mice partially but significantly improved blood flow recovery after hindlimb ischemia. The extent of VEGF upregulation and the number of BM-derived cells in ischemic tissue were significantly less in EpoR(-/-)-rescued mice compared with WT mice even after BM reconstitution with WT-BM cells. Similarly, the recovery of blood flow was significantly impaired in recipient EpoR(-/-)-rescued mice that had been transplanted with WT-BM or EpoR(-/-)-rescued-BM as compared with recipient WT mice. Furthermore, the Matrigel implantation assay and aortic ring assay showed that microvessel growth in vitro was significantly reduced in EpoR(-/-)-rescued mice as compared with WT mice. These results indicate that vascular EpoR system also plays an important role in angiogenesis in response to hindlimb ischemia through upregulation of VEGF/VEGF receptor system, both directly by enhancing neovascularization and indirectly by recruiting endothelial progenitor cells and BM-derived proangiogenic cells.     

PEDF-derived peptide inhibits corneal angiogenesis by suppressing VEGF expression

Pigment epithelium-derived factor (PEDF) a glycoprotein that belongs to the superfamily of serine protease inhibitors, has been recently shown to be the most potent inhibitor of angiogenesis in the mammalian eye. However, which active domain of PEDF protein could be involved in its anti-angiogenic properties remains unknown. Therefore, in this study, we examined which PEDF-derived synthetic peptides could inhibit corneal neovascularization induced by chemical cauterization in vivo. Rats treated with topical application of PEDF protein had 31% less corneal neovascularization at day 7 after the injury than phosphate-buffered saline (PBS)-treated rats. P5-2 and P5-3 peptides (residues 388-393 and 394-400 of PEDF protein, respectively) significantly suppressed the corneal neovascularization after chemical cauterization at day 7, and its anti-angiogenic potential was almost equal to that of full-length PEDF protein. Further, full-length PEDF protein and P5-3 peptide significantly decreased 8-hydroxy-2'-deoxyguanosine and vascular endothelial growth factor (VEGF) levels in the corneal. Our present study suggests that PEDF-derived synthetic peptide, P5-3 could inhibit the corneal neovascularization induced by chemical cauterization in rats by suppressing VEGF expression via its anti-oxidative properties.     

AMP-activated protein kinase signaling stimulates VEGF expression and angiogenesis in skeletal muscle

AMP-activated protein kinase (AMPK) is regulated by various cellular stresses. Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, is also upregulated by several stress-inducible factors such as hypoxia and stimulation by cytokines and growth factors. Here, we investigated whether AMPK signaling in muscle has a role in regulating VEGF-mediated angiogenic processes. AICAR stimulated VEGF mRNA and protein levels in C2C12 myotube cultures. Transduction with dominant-negative AMPK abolished AICAR-induced VEGF expression at both steady state mRNA and protein levels. AICAR increased VEGF mRNA stability without affecting VEGF promoter activity. AICAR also stimulated p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Activation of p38 MAPK was suppressed by transduction with dominant-negative AMPK, suggesting that AMPK is upstream of p38 MAPK. The p38 MAPK inhibitor SB203580 blocked AICAR-induced increase in VEGF mRNA and protein levels, indicating that AICAR-mediated VEGF induction is dependent on p38 MAPK signaling. AICAR treatment increased VEGF expression and accelerated angiogenic repair of ischemic hindlimbs in mice in an AMPK-dependent manner. These data indicate that AMPK-p38 MAPK signaling cascade can increase VEGF production in muscle and promote angiogenesis in response to ischemic injury.     

血管内皮生成因子165诱导血管形成中镁离子作用的研究

目的探讨血管内皮生成因子165（VEGF165）对人脐带静脉内皮细胞（HUVEC）内游离镁离子浓度（[Mg^2＋];）的调节机制及镁离子（Mg^2＋）与血管形成的相关性。方法采用荧光指示剂mag-fura-2,运用PTi阳离子测定系统动态测HUVEC内的[Mg^2＋];。新鲜脐带内灌注胶原酶消化,获得内皮细胞,用含20％胎牛血清的M199液进行培养,当细胞外Mg^2＋浓度分别为0、1和2mmol/L时,观察VEGF。65促进HUVEC血管形成的能力。结果VEGF。65诱导的[Mg^2＋]i增加与细胞外Mg^2＋浓度无关。在细胞外无Mg^2＋时,VEGF165能剂量依赖性地增加[Mg^2＋]i。VEGF165诱导的[Mg^2＋];增加与细胞外Na^＋浓度和细胞内Ca^2＋浓度无关。经VEGF的受体亚型2（KDR）阻断剂SU1498预处理,能明显阻断VEGF165诱导的[Mg^2＋]i增加。当细胞外Mg^2＋为0mmol/L时,HUVEC血管形成作用受到明显抑制,VEGF165也不刺激血管形成。当细胞外Mg^2＋为1或2mmol/L时,HUVEC能形成血管,两组间差异无统计学意义,VEGF165则可促进HUVEC形成血管,两组间差异无统计学意义。当细胞外Mg^2＋为1或2mmol/L时,KDR阻断剂SU1498明显抑制VEGF165促进HUVEC血管形成的作用。结论VEGF165通过KDR信号传递途径使细胞内的Mg^2＋库释放Mg^2＋,从而增加HUVEC的[Mg^2＋]i,并对促进血管形成起重要作用。     

癌胚抗原相关细胞黏附分子1和血管内皮生长因子在HBV相关性肝癌血管生成中的作用

目的 研究癌胚抗原相关细胞黏附分子1 (CEACAMl)和血管内皮生长因子(VEGF)在HBV相关性肝癌组织中的表达与HBV相关性肝癌血管生成的关系.方法 采用免疫组织化学SP法检测81例HBV相关性肝癌组织中CEACAM1、VEGF的表达,光镜下计数CD105标记的微血管密度(MVD).结果 81例HBV相关性肝癌组...     

VEGF function for upregulation of endogenous PlGF expression during FGF-2-mediated therapeutic angiogenesis

Vascular endothelial growth factor (VEGF) is a major positive angiogenic factor. Using a murine hindlimb ischemia model, we previously showed that fibroblast growth factor-2 (FGF-2) enhances the expression of endogenous VEGF which highly contribute to the therapeutic effect of FGF-2 gene transfer. Recently, placental growth factor (PlGF) has been shown to play an important role in angiogenesis. However, the molecular mechanism of endogenous PlGF during FGF-2-mediated angiogenesis has not been elucidated. Severe hindlimb ischemia stimulated PlGF expression that was more strongly enhanced by FGF-2 gene transfer, and a blockade of PlGF activity diminished the recovery of blood flow by FGF-2-mediated angiogenesis. The PlGF expression in cultured endothelial cells was significantly enhanced by VEGF stimulation, but not by FGF-2. The upregulation of endogenous PlGF expression was significantly decreased by the inhibition of endogenous VEGF activity in vivo. Subsequent signal inhibition experiments revealed that the PKC, MEK, and possibly NF-κB-related pathways were essential in stimulating PlGF expression with VEGF, while p70S6K is the regulator for VEGF expression. These results indicate that the FGF-2-mediated enhancement of PlGF expression was dependent on VEGF function, and the FGF-2/VEGF axis participates in FGF-2-mediated angiogenesis indirectly via PlGF as well as directly.     

血管瘤中血管内皮生长因子及其受体KDR的表达和血管生成的研究

目的 探讨血管内皮生长因子(VEGF)及其受体KDR在血管瘤中的表达以及其与微血管密度(MVD)的关系.方法 采用免疫组化SP法检测35例婴幼儿血管瘤VEGF及KDR的表达和MVD.结果 35例血管瘤中VEGF、KDR的蛋白表达阳性率分别是66.5%,73.4%.其中增生期和消退期VEGF阳性率分别为92.1%、31.4%(P<0.01),KDR阳性率分别为89.3%、42.5%(P<0.01);MVD分别为75.7±13.71、28.9±8.5(P<0.01).KDR的表达与VEGF成正相关(P<0.01),KDR和VEGF与MVD都成正相关(P<0.01).结论 血管瘤增生期血管内皮细胞存在着血管内皮细胞生长因子及其受体KDR的相互作用,促进了新生微血管生成.     

roundabout4 is essential for angiogenesis in vivo

Stereotypical patterns of vascular and neuronal networks suggest that specific genetic programs tightly control path determination and, consequently, angiogenesis and axon-guidance mechanisms. Our study focuses on one member of the roundabout family of receptors, which traditionally mediate repulsion from the midline. Here, we characterize a fourth member of this family, roundabout4 (robo4), which is the predominant roundabout (robo) that is expressed in embryonic zebrafish vasculature. Gene knockdown and overexpression approaches show that robo4 is essential for coordinated symmetric and directed sprouting of intersomitic vessels and provide mechanistic insights into this process. Also, human robo4 gene functionally compensates for loss of robo4 gene function, suggesting evolutionary conservation. This article reports an endothelial-specific function for a robo gene in vertebrates in vivo.     

An essential role for orexins in emergence from general anesthesia

The neural mechanisms through which the state of anesthesia arises and dissipates remain unknown. One common belief is that emergence from anesthesia is the inverse process of induction, brought about by elimination of anesthetic drugs from their CNS site(s) of action. Anesthetic-induced unconsciousness may result from specific interactions of anesthetics with the neural circuits regulating sleep and wakefulness. Orexinergic agonists and antagonists have the potential to alter the stability of the anesthetized state. In this report, we refine the role of the endogenous orexin system in impacting emergence from, but not entry into the anesthetized state, and in doing so, we distinguish mechanisms of induction from those of emergence. We demonstrate that isoflurane and sevoflurane, two commonly used general anesthetics, inhibit c-Fos expression in orexinergic but not adjacent melanin-concentrating hormone (MCH) neurons; suggesting that wake-active orexinergic neurons are inhibited by these anesthetics. Genetic ablation of orexinergic neurons, which causes acquired murine narcolepsy, delays emergence from anesthesia, without changing anesthetic induction. Pharmacologic studies with a selective orexin-1 receptor antagonist confirm a specific orexin effect on anesthetic emergence without an associated change in induction. We conclude that there are important differences in the neural substrates mediating induction and emergence. These findings support the concept that emergence depends, in part, on recruitment and stabilization of wake-active regions of brain.     

An essential role for mitochondrial aldehyde dehydrogenase in nitroglycerin bioactivation

The identity of the cellular mechanisms through which nitroglycerin (glyceryl trinitrate, GTN) elicits nitric oxide (NO)-based signaling to dilate blood vessels remains one of the longest standing foci of investigation and sources of controversy in cardiovascular biology. Recent evidence suggests an unexpected role for mitochondria. We show here that bioconversion by mitochondria of clinically relevant concentrations of GTN results in activation of guanylate cyclase, production of cGMP, vasodilation in vitro, and lowered blood pressure in vivo, which are eliminated by genetic deletion of the mitochondrial aldehyde dehydrogenase (mtALDH). In contrast, generation of vasoactivity from alternative nitro(so)-vasodilators is unaffected. In mtALDH(-/-) mice and their isolated vascular tissue, GTN bioactivity can still be generated, but only at substantially higher concentrations of GTN and by a mechanism that does not exhibit tolerance. Thus, mtALDH is necessary and sufficient for vasoactivity derived from therapeutic levels of GTN, and, more generally, mitochondria can serve as a source of NO-based cellular signals that may originate independently of NO synthase activity.     

Prognostic significance of VEGF immunohistochemical expression and tumor angiogenesis in head and neck squamous cell carcinoma

PURPOSE: Tumor angiogenesis is crucial for both the growth of the primary tumor and the development of metastases. Among the factors causing tumor angiogenesis, vascular endothelial growth factor (VEGF) is considered as a leading candidate. We aimed to assess the prognostic significance of VEGF and tumor angiogenesis in head and neck squamous cell carcinoma (HNSCC). METHODS: We performed a retrospective study of 69 patients with HNSCC, in order to investigate whether VEGF immunohistochemical expression and tumor angiogenesis correlate with clinicopathological parameters and outcome. Tumor angiogenesis was estimated by determining microvessel density (MVD), and VEGF expression was assessed quantitatively. RESULTS: Vascular endothelial growth factor and MVD correlated statistically significant with the clinical stage, but not with the presence of lymph node metastasis at the time of diagnosis. Tumors located in the oral cavity and larynx more often expressed high VEGF immunostaining compared with tumors located in the lower lip. High VEGF expression was associated with higher clinical stage and worse overall survival in this cohort of patients. CONCLUSIONS: Vascular endothelial growth factor expression may have prognostic significance for patients with HNSCC.     

Relationship between the expression of iNOS,VEGF,tumor angiogenesis and gastric cancer

AIM：To in vestigate the relationship between the expression of inducible nitric oxide synthase(iNOS),vascular endothelial growth factor(VEGF),the microvascular density(MVD)and the pathological features and clinical staging of gastric cancer.METHODS:Immunohistochemical staining was used for detecting the expression of iNOS and VEGFin46resected specimens of gastric carcinoma;the monoclonal antibody against CD34 was used for displaying vascular endothelial cells,and MVD was detected by counting of CD34-positive vascular endothelial cells.RESULTS:Of 46resected specimens of gastric carcinoma,the rates of expressions of iNOS and VEGF were 58.70%and76.09%,respectively,and MVDaveraged55.59&#177;19.39,Judged by the standard TNM criteria,the rate of expression of iNOS in stageⅣ（84.46%)was higher than those in stageⅠ,Ⅱ,Ⅲ（Fish exact probabilities test,P=0.019,0.023and 0.033,respectively);the rates of expression of VEGFin stage Ⅲ,Ⅳ（76.0%,92.31%,respectively)were higher than those in stageⅠ,Ⅱ（Fis exact probabilities test,P=0.031,0.017,0.022and0.019).MVDs in stageⅢ,Ⅳ（64.72&#177;14.96,67.09&#177;18.29,respectively)were higher than those in stageⅠ,Ⅱ(t\2.378,4.015,2.503and2.450,P&lt;0.05,P&lt;0.001,P&lt;0.001,P&lt;0.05,respectively),In37gastric carcinoma specimens with lymph node metastasis,MVD(68.69&#177;18.07)and the rates of expression of iNOS and VEGF(70.27%,83.78%,respectively)were higher than those in the specimens with absence of metastasis(t=2.205,X^2=6.3587,X^2=6.2584,P&lt;0.01,P&lt;0.05,P&lt;0.05,respectively),MVD and the expressions of iNOS and EGF were not correlated to the location,size or grade of tumor,nor with the depth of invasion of tumor;MVDs in the positive iNOS and VEGF specimens(59.88&#177;18.02,58.39&#177;17.73,repectively)were higher than those in the negative iNOS and VEGF specimens(X^2=6.3587and 6.1574,P&lt;0.05,P&lt;0.05,respectively);thus the expressions of iNOS and VEGF was correlated to MVD,but the expression of iNOS was not correlated to that of VEGF,In addition.of the 46 surviving patients,the 5-year survival rate of patients with positive iNOS or VEGF tumors was significantly less than that of patients with negative iNOS-or VEGF tumors(X^2=4.3842and 5.4073,P&lt;0.05,P&lt;0.05.respectively).CONCLUSION:The expressions of iNOS and VEGF are colosely related to tumor angiogenesis,and are involved in the advancement and the lymph node metastasis;thusMVD and the expressions of iNOS and EGF may serve indexes for evaluating staging of gastric carcinoma and forecasting its risk of metastasis,which will help establish a comprehensive therapeutical measure of post-operative patients and provide a new approach to tumor therapy.