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，并在我们对阳性和阴性血管生成疾病的研究的基础上建立新的有效的治疗方法，我们对血管生成/血管生成的分子机制进行了研究，得到了以下结果：(1)在分子和细胞的串扰过程中，血管生成因子的时间和空间层次：FGF2诱导的血管生成早期由MEK介导，p70^-lt；S6K&GT；Ras/PDGF在晚期表达。在小鼠肿瘤移植模型中，p70、lt、S6K、GT和RAS/PDGF通路是血管内皮生长因子和其他血管生成因子的重要增强子，不仅在间质细胞中表达，而且在肿瘤细胞中也表达。此外，将成纤维细胞生长因子-2基因转移到缺血肢体可诱导良好的协调和有趣的…动脉粥样硬化和血管新生：血管内皮生长因子C阳性细胞的数量与动脉粥样硬化的程度和动脉粥样硬化内膜新生血管的数量有关。血管内皮生长因子的表达和活性增加在实验性血管内插管术后再狭窄的发生发展中起重要作用。色素上皮源性因子(PEDF)在人冠状动脉粥样硬化内膜中有两种分布模式。分布模式的差异与血管内膜血管生成模式密切相关。此外，以巨噬细胞为主的IL-10阳性细胞数与同一区域内浸润的淋巴细胞数呈显著负相关。这些发现表明，血管内皮生长因子、血管内皮生长因子-C、PEDF和IL-10在动脉粥样硬化的进展中起重要作用。(3)我们新型基因转移载体的生物学特性及其治疗意义：猴免疫缺陷病毒(SIV)载体通过评估转基因表达活性、视网膜功能和组织学在成年大鼠视网膜下注射靶基因后大约一年的时间内，有效和安全地应用于视网膜基因转移。我们还证明，使用SIV-PEDF的神经保护性基因治疗可以保护视网膜色素变性小鼠的视网膜免受功能丧失的影响。这些数据表明，SIV介导的稳定的基因表达可能是一种很有潜力的基因治疗工具。使用SeV hFGF-2的治疗性血管成形术可能适用于肢体和冠状动脉功能不全等缺血器官。在猪心肌梗死模型上，我们通过使用一种新型的从室腔向心肌转移基因的导管，证明了SeV-FGF-2可以抑制心肌缺血、破裂和心力衰竭。这些发现表明，良好协调的细胞和空间闭合/血管生成因子反馈环在血管生成中起着重要作用。这种协调平衡的失调会导致几种血管新生疾病。我们的新型基因转移载体有望成为强有力的治疗工具。较少

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