Homocysteine and Angiotensin II in Renovascular Remodeling

描述(由申请人提供)：虽然尚不清楚高同型半胱氨酸血症(HHcy)是否会导致高血压，但最近的临床数据表明，高同型半胱氨酸血症(HHcy)与HHcy之间存在联系。血管紧张素II(Ang II)和同型半胱氨酸(Hcy)均可减少血流量，但Hcy在Ang II介导的血流量和血管密度下降中的作用尚不清楚。有趣的是，对这一建议的初步研究表明，Ang II提高了小鼠的血浆Hcy水平。用叶酸(FA)治疗血管紧张素Ⅱ(Ang II)动物可降低血浆同型半胱氨酸水平，缓解高血压。在我们的初步研究的基础上，在这个方案中，我们假设HHcy在一定程度上通过减少血管密度，增加Ang II介导的肾血管重构中的氧化、促炎、促纤维化和抗血管生成因子来减少血流量。FA通过同型半胱氨酸清除机制降低血浆Hcy水平，从而减轻肾脏重塑。这一假说将通过以下具体目标进行检验：(1)确定Hcy是否部分通过诱导氧化应激(NOX2、gp47Phox、NOX4和mtROS)，通过诱导MCP-1、MIP-2、ICAM-1和VCAM-1而加剧肾脏炎症，以及FA是否改善这些变化；(2)确定Hcy是否部分通过诱导IV型胶原、MMP-2、-9、-13；TIMP-1、-2、-3、-4；以及FA是否减轻这种肾血管纤维化而引发Ang II高血压和肾血管纤维化；以及(3)确定Hcy是否通过血管生成因子(VEGF)和抗血管生成因子(Angiostatin和Endostatin)失衡，以及FA是否增加血管密度和血流量，从而促进Ang II高血压和减少肾血管血流量。本研究将采用WT(C57BL/6J)小鼠、遗传性HHcy小鼠模型(CBS)和血管紧张素受体I缺陷小鼠(AT1R-/-)。在WT和CBS小鼠中，通过Alzet微型泵注入Ang II(1000 ng/kg/min，连续4周)，造成高血压。对照动物将只接受车辆。在另一组动物中，FA(0.015g/L，饮用水中)将在Ang II输注2周后引入，并将持续到实验结束(共2周)。将使用适当的FA控制。对AT1R-/-小鼠给予或不给予Hcy(1.8g/L，连续4周)，以确定HHcy的作用是否依赖AT1R。动态血压将用DSI无线电遥测仪(TA11PA-C10型)测量。血浆同型半胱氨酸采用高效液相色谱法测定。血管密度将通过活体软组织硫酸钡对比X线血管造影术测量，肾皮质血流量将通过MoorFLPI全填充激光灌注成像仪测量。肾功能将通过测量肾小球滤过率(GFR)来确定。肾组织切片将用于检测ROS、胶原、系膜增宽和足细胞损伤。采用双抗体夹心法测定血浆MCP-1、MIP-2、VCAM-1和ICAM-1水平。免疫印迹和免疫染色检测组织胶原、MMPs、TIMPs、NOX2、p47Phox、NOX4、ICAM-1、VCAM-1的蛋白表达。MMPs活性用凝胶内酶谱法测定，TIMPs活性用反向酶谱法测定，mRNA丰度用Q-PCR法测定。除了确认初步研究外，这项研究的范围还将扩大，以了解FA治疗血管紧张素Ⅱ诱导的高血压对调节促炎、促纤维化和抗血管生成因子的意义。本研究结果将加深我们对Hcy在Ang II高血压和肾血管重塑中的作用的了解。此外，研究结果将提供HHcy作为肾血管纤维化的潜在危险因素的缺失信息，从而加剧高血压，并将导致开发或修改当前肾血管疾病的治疗策略。

DESCRIPTION (provided by applicant): Although it is not clear whether hyperhomocysteinemia (HHcy), an elevated plasma homocysteine (Hcy) level, causes hypertension, recent clinical data suggest an association between systolic hypertension and HHcy. Both angiotensin II (Ang II) and Hcy decrease blood flow; however, the role of Hcy in Ang II mediated decrease in blood flow and vascular density is unclear. Interestingly, preliminary studies of this proposal suggest that Ang II increases plasma Hcy level in mice. Treatment of Ang II animals with folic acid (FA) decreases plasma Hcy and mitigates hypertension. Based on our preliminary studies, in this proposal, we hypothesize that HHcy decreases blood flow in part by decreasing vascular density, increasing oxidative, pro-inflammatory, pro-fibrotic and anti-angiogenic factors in Ang II mediated renovascular remodeling. FA treatment through Hcy clearance mechanism reduces plasma Hcy level that mitigates renal remodeling. The hypothesis will be tested by following their specific aims: (1) To determine whether the Hcy contributes to Ang II hypertension, in part, by inducing oxidative stress (Nox2, gp47phox, Nox4 and mtROS), exacerbating renal inflammation by inducing MCP-1, MIP-2, ICAM-1, VCAM-1, and if FA ameliorates these changes; (2) To determine whether the Hcy instigates Ang II hypertension and renovascular fibrosis, in part, by inducing collagen IV, MMP-2, -9, -13; TIMP-1,-2, -3, -4; and if FA mitigates this renovascular fibrosis; and (3) To determine whether the Hcy promotes Ang II hypertension and decreases renovascular blood flow, in part, by unbalancing of angiogenic (VEGF) and anti-angiogenic factors (angiostatin and endostatin), and if FA increases vascular density and blood flow. WT (C57BL/6J) mouse, genetic mouse model of HHcy (CBS) and mouse deficient with angiotensin receptor I (AT1R-/-) will be used in this study. In WT and CBS mice, hypertension will be created by infusing Ang II (1000 ng/kg/min for 4 weeks) through alzet mini pump. Control animals will receive only vehicle. In a separate group of animals FA (0.015g/L, in drinking water) will be introduced after 2 weeks of Ang II infusion and will be continued until the end of experiments (total 2 weeks). Appropriate FA controls will be used. AT1R-/- mice will be treated with or without Hcy (1.8 g/L for 4 weeks) to determine whether the effect of HHcy is AT1R dependent. Ambulatory blood pressure will be measured by DSI radiotelemetry (model TA11PA- C10). Plasma Hcy will be measured by HPLC. Vascular density will be measured by in vivo soft-tissue Barium sulfate-contrast X-ray angiography and renal cortical blood flow by moorFLPI full-filled laser perfusion imager. Renal function will be determined by measuring glomerular filtration rate (GFR). Histological kidney sections will be used to detect ROS, collagen, mesangial widening and podocyte injury. Plasma MCP-1, MIP-2, VCAM- 1 and ICAM-1 will be measured by ELISA. Tissue collagen, MMPs, TIMPs, Nox2, p47phox, Nox4, ICAM-1, VCAM-1 protein expressions will be measured by Western blot and immunostaining. MMPs activities will be measured by in gel zymography, TIMPs activity by reverse zymography, and mRNA abundance by Q-PCR. In addition to confirming the preliminary studies, the scope of the research will be extended to understand the implications of FA treatment in Ang II-induced hypertension to modulate pro-inflammatory, pro-fibrotic and anti- angiogenic factors. The results of this study will increase our understanding of role of Hcy in Ang II hypertension and renovascular remodeling. Additionally, the research outcome will provide the missing information of HHcy as a potential risk factor of renovascular fibrosis, which exacerbates hypertension, and will lead to develop or modify current therapeutic strategies of renovascular disease.