Moleculo-Pathological studies on intracellular cross-talk signal in angiogenic process.

血管生成因子胞内串扰的机制及其在生理性和病理性血管生成中的病理生理作用尚不清楚。为了阐明这种时间依赖性和空间依赖性的血管生成串扰，并在已有证据的基础上建立新的有效的治疗阳性和阴性血管生成疾病的方法，我们研究了血管生成/血管生成的分子机制，我们得到了以下结果：(1)分子和细胞交叉过程中血管生成因子的时间依赖性和空间层系：fgf2诱导的血管生成在VEGF和HGF表达的早期和后期由MEK介导，p70^< 6k bb0， Ras/PDGF介导。在小鼠肿瘤移植模型中，p70^<s6k>和Ras/PDGF通路是基质间充质细胞和肿瘤细胞表达VEGF-A等血管生成因子的重要促进因子。此外，将FGF-2基因转移到缺血肢体后，可作为其他血管生成因子（VEGF、VEGF- c、VEGF- d、HGF和PDGF）的分级增强剂，促进血管生成。(2)动脉粥样硬化及新壁下血管生成：vegf - c阳性细胞的数量与动脉粥样硬化程度及动脉粥样硬化内膜新生血管的数量相关。通过募集单核/巨噬细胞，在腔内插管后实验性再狭窄的发展中，VEGF的表达和活性的增加是必不可少的。色素上皮衍生因子（PEDF）在人冠状动脉粥样硬化内膜有两种分布模式。分布模式的差异与内膜血管生成模式密切相关。以巨噬细胞为主表达的il -10阳性细胞数量与浸润于同一区域的淋巴细胞数量呈显著负相关。这些发现提示VEGF、VEGF- c、PEDF和IL-10在动脉粥样硬化的进展中起重要作用。(3)新型基因转移载体的生物学特性及其治疗意义：通过在成年大鼠视网膜下注射靶基因约1年的时间内评估转基因表达活性、视网膜功能和组织学，我们发现SIV载体可有效、安全地用于视网膜基因转移。我们还证明了使用SIV-PEDF的神经保护基因治疗可以保护患有视网膜色素炎的小鼠的视网膜变性和功能丧失。这些数据表明siv介导的稳定基因表达可能是一种高潜力的基因治疗工具。使用SeV hFGF-2治疗血管生成可能适用于缺血器官，如肢体和冠状动脉功能不全。在猪心肌梗死模型中，我们通过一种新型导管将SeV-FGF-2基因从心室管腔转移到心肌，证明SeV-FGF-2可以抑制心肌缺血、破裂和心力衰竭。这些发现表明血管生成因子的细胞和空间闭合/反馈回路在血管生成中起着重要作用。和谐平衡的分裂导致了几种血管生成疾病。我们的新型基因转移载体有望成为强有力的治疗工具。少

The mechanism of intracellular crosstalk of angiogenic factors and its patho-physiological role in physiological and pathological angiogenesis remain unclear. To elucidate this time-dependent and spatial angiogenic crosstalk and to establish the novel and effective therapy on the basis of our evidences obtained for positive and negative angiogenic diseases, we studied the molecular mechanisms of angiogenesis/vasculogenesis, and we have got the following results.(1)Time-dependent and spatial hierarchy of angiogenic factors durhg molecular and cellular crosstalks :FGF 2-induced angiogenesis is mediated by MEK in early phase and p70^<s6k>, and Ras/PDGF in late phase of VEGF and HGF expressions. In murine tumor transplantation models, p70^<s6k> and Ras/PDGF pathways were important enhancers of VEGF-A and other angiogenic factors expressed by not only stromal mesenchymal cells but also tumor cells. In addition, FGF-2 gene transferred to ischemic limbs could induce a well harmonizing and fun … More ctional angiogeness partly as a hierarchical enhancer of other angiogenic factors such as VEGF,VEGF-C,VEGF-D,HGF and PDGF.(2)Atherosderosis and infra-neohtimal angiogenesis :The number of VEGF-C-positive cells correlate with the degree of atherosclerosis and the number of newly formed vessels in atherosclerotic intima. Increased expression and activity of VEGF are essential in the development of experimental restenosis after intraluminal catheterization by recruiting monocytes/macrophages. There were two disribution patterns of pigment epithelium derived factor (PEDF) in atherosclerotic intima of human coronary arteries. The difference of distribution pattern is closey related to intimal angiogenesis pattern. Moreover, significant inverse correlation between the number of IL-10-positive cells expressed mainly by macrophages and the number of lymphocytes infiltrated in the same areas. These findings suggest that VEGF,VEGF-C,PEDF and IL-10 play important roles in progression of atherosclerosis.(3)Biological characteristics of our novel gene transfer vectors and their therapeutic implications :Simian immunodeficiency virus(SIV) vector is efficiently and safely applicable to retinal gene transfer by assessing the transgene expression activity, retinal function and histologies over about 1-year period following subretinal injection of target genes into adult rat retinas. We also demonstrated that neuroprotective gene therapy using SIV-PEDF could be protective from retinal degeneration and functional loss in mice with retintis pigmentosa. These data indicate that SIV-mediated stable gene expression might be a high potential tool for gene therapy. Therapeutic angiogeness using SeV hFGF-2 may be applicable for ischemic organs such as limbs and coronary insuficiency. In porcine model of myocardial infarction, we demonstrated that SeV-FGF-2 could suppress the cardiac ischemia, rupture and heart failure, by using a novel catheter for gene transfer to myocardium from ventricular lumen.These findings suggest that well harmonized cellular and spatial closstalks/feedback loops of angiogenic factors play important roles in angiogenesis. Rhexis of the harmonized balance leads to several angiogenic diseases. Our novel gene transfer vectors could be expected to be powerful therapeutic tools. Less