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）（血浆同型半胱氨酸（Hcy）水平升高）是否会导致高血压，但最近的临床数据表明收缩期高血压与HHcy之间存在相关性。血管紧张素II（Ang II）和Hcy均降低血流量;然而，Hcy在Ang II介导的血流量和血管密度降低中的作用尚不清楚。有趣的是，这一提议的初步研究表明，Ang II增加小鼠血浆Hcy水平。用叶酸（FA）治疗Ang II动物降低血浆Hcy并减轻高血压。基于我们的初步研究，在这个提议中，我们假设HHcy通过降低血管密度、增加Ang II介导的肾血管重塑中的氧化、促炎、促纤维化和抗血管生成因子来部分地减少血流量。FA治疗通过Hcy清除机制降低血浆Hcy水平，从而减轻肾脏重塑。本研究的主要目的是：（1）确定Hcy是否通过氧化应激参与Ang Ⅱ高血压的形成（Nox 2、gp 47 phox、Nox 4和mtROS），通过诱导MCP-1、MIP-2、ICAM-1、VCAM-1而加重肾脏炎症，以及FA是否改善这些变化;（2）确定Hcy是否部分通过诱导胶原IV、MMP-2、-9、-13; TIMP-1、-2、-3、-4而引起Ang II高血压和肾血管纤维化;以及FA是否减轻这种肾血管纤维化;以及（3）确定Hcy是否部分地通过血管生成因子（VEGF）和抗血管生成因子（血管抑素和内皮抑素）的不平衡而促进Ang II高血压并减少肾血管血流量，以及FA是否增加血管密度和血流量。本研究将使用WT（C57 BL/6 J）小鼠、HHcy遗传小鼠模型（CBS）和血管紧张素受体I（AT 1 R-/-）缺陷小鼠。在WT和CBS小鼠中，通过alzet微型泵输注Ang II（1000 ng/kg/min，持续4周）来产生高血压。对照动物将仅接受溶剂。在另一组动物中，在Ang II输注2周后引入FA（0.015 g/L，在饮用水中），并持续至实验结束（共2周）。将使用适当的FA质控品。AT 1 R-/-小鼠将用或不用Hcy（1.8g/L，持续4周）处理以确定HHcy的作用是否是AT 1 R依赖性的。将通过DSI无线电遥测（型号TA 11 PA-C10）测量动态血压。将通过高效液相色谱法测量血浆同型半胱氨酸。将通过体内软组织硫酸钡造影X射线血管造影术测量血管密度，并通过moorFLPI全填充激光灌注成像仪测量肾皮质血流。将通过测量肾小球滤过率（GFR）确定肾功能。组织学肾脏切片将用于检测ROS、胶原蛋白、系膜增宽和足细胞损伤。将通过ELISA测量血浆MCP-1、MIP-2、VCAM- 1和ICAM-1。通过Western blot和免疫组织化学染色检测组织胶原、MMPs、TIMPs、Nox 2、p47 phox、Nox 4、ICAM-1、VCAM-1蛋白表达。MMP活性将通过凝胶内酶谱法测量，TIMP活性通过反向酶谱法测量，mRNA丰度通过Q-PCR测量。除了确认初步研究外，研究范围还将扩展到了解FA治疗在Ang II诱导的高血压中调节促炎、促纤维化和抗血管生成因子的意义。本研究结果将有助于我们进一步了解Hcy在Ang Ⅱ高血压和肾血管重构中的作用。此外，研究结果将提供缺失的信息，高同型半胱氨酸作为肾血管纤维化的潜在危险因素，这加剧了高血压，并将导致发展或修改目前的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.