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

血管生成因子的细胞内串扰机制及其在生理和病理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，并在我们获得的阳性和阴性血管生成疾病的证据的基础上建立新的有效的治疗方法，我们研究了血管生成/血管生成的分子机制，并得到了以下结果。（1）分子和细胞交联过程中血管生成因子的时间依赖性和空间层次性：FGF 2诱导的血管生成早期由MEK介导，晚期由p70 β<s6k>和Ras/PDGF介导。在小鼠肿瘤移植模型中，p70 β<s6k>和Ras/PDGF途径是VEGF-A和其他血管生成因子的重要增强子，这些因子不仅由间充质细胞表达，而且也由肿瘤细胞表达。此外，FGF-2基因转移到缺血肢体可以诱导良好的协调和乐趣 ...更多信息 部分作为其他血管生成因子如VEGF、VEGF-C、VEGF-D、HGF和PDGF的分级增强剂。（2）动脉粥样硬化与内膜下血管生成：VEGF-C阳性细胞数与动脉粥样硬化程度及内膜新生血管数相关。血管内皮生长因子的表达和活性的增加是必要的实验性再狭窄后，管腔内导管通过招募单核细胞/巨噬细胞的发展。色素上皮衍生因子（PEDF）在人冠状动脉粥样硬化内膜中有两种分布模式。分布模式的差异与内膜血管生成模式密切相关。此外，IL-10阳性细胞主要由巨噬细胞表达的数量与相同区域中浸润的淋巴细胞数量之间存在显著的负相关。提示VEGF、VEGF-C、PEDF和IL-10在动脉粥样硬化的发生发展中起重要作用。（3）新型基因转移载体的生物学特性及其治疗意义：通过对成年大鼠视网膜下注射目的基因后约1年的转基因表达活性、视网膜功能和组织学的评估，证实了猴免疫缺陷病毒（Simian immunodeficiency virus，SIV）载体可有效和安全地用于视网膜基因转移。我们还证明，使用SIV-PEDF的神经保护基因治疗可以防止患有色素性视网膜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个高潜力的基因治疗工具。使用SeV hFGF-2的治疗性血管生成可适用于缺血性器官，如肢体和冠状动脉功能不全。在猪心肌梗死模型中，我们通过一种新型的心室腔基因转移导管，证明了SeV-FGF-2对心肌缺血、破裂和心力衰竭的抑制作用，这些发现表明血管生成因子的细胞和空间协调的闭环/反馈环在血管生成中起重要作用。协调平衡的破裂导致几种血管生成疾病。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