Pathomechanisms and modulation of impaired angiogenesis in chronic kidney disease (CKD)

慢性肾脏疾病(CKD)患者心血管并发症和心源性死亡的发生率不成比例。其发病机制尚不清楚，现有的预防心血管并发症的策略在CKD患者中往往失败。基于我们以前的工作，我们认为CKD形成新的毛细血管的能力受损是心血管疾病负担的原因之一。毛细血管生成是通过恢复受影响器官的灌流来应对大血管狭窄/闭塞或心脏肥大等过程中不可或缺的适应性过程。我们建议的总体目标是更好地了解慢性肾脏病血管生成受损的病理生理学，并开发治疗方法来改善慢性肾脏病的毛细血管形成。利用已建立的大鼠肾大部切除术(CKD)模型，并使用读数系统评估心肌毛细血管密度、缺血诱导的骨骼肌毛细血管生成以及心肌梗死面积，我们提出了以下具体目标：首先，我们将使用脯氨酰羟化酶(ICA)抑制剂来稳定低氧诱导因子(HIF)，以促进毛细血管形成。在初步实验中，我们已经证明：(A)CKD大鼠缺血后HIF靶点的激活减少，(B)缺血前应用的非药理学方法(0.1%CO气体)和使用ICA稳定HIF的药理学方法极大地改善了CKD大鼠缺血后肢的毛细血管形成。我们现在将开发这种方法作为治疗方案中的一种药理学工具，用于诱导缺血后的应用。此外，我们将评估ICA治疗是否改善了CKD大鼠的心肌毛细血管供应并限制了心肌梗死范围。第二，我们将测试两个候选分子靶点(CCL7和金属硫蛋白-1a)在CKD血管生成障碍中的功能作用。这两个候选分子靶标来自于Affymetrix微阵列基因表达筛选，在CKD大鼠与对照组大鼠的缺血和非缺血肢体中，在诱导缺血24小时后获得。这两个候选基因都有已知的血管生成特性；他们的时间和空间表达模式将被研究。此外，还将进行体外实验，以研究Pro-羟基酶抑制剂CCL7和金属硫蛋白-1a在毛细血管形成中的作用。将使用增殖、凋亡和不同的细胞迁移分析来阐明我们的分子靶点的潜在促血管生成作用是否是由于对内皮细胞的直接作用(而不是通过例如渗透的单个核细胞的间接作用)。随后，我们将阐明介导观察到的促血管生成效应的信号转导途径。从我们提出的实验中，我们期待着对CKD血管生成障碍的发病机制有新的见解，并展望新的治疗方法。

Patients with chronic kidney disease (CKD) suffer disproportionately from cardiovascular complications and cardiac death. The pathogenesis is not well understood, and existing strategies to prevent cardiovascular complications have often failed in CKD patients. Based on our previous work, we propose that an impaired capacity to form new capillary vessels in CKD contributes to the burden of cardiovascular disease. Capillary angiogenesis is an indispensable adaptive process to cope with processes such as macrovascular stenosis/occlusion or heart hypertrophy by restoring the perfusion of the affected organs. The overall aim of our proposal is to better understand the pathophysiology of impaired angiogenesis in CKD, and to develop therapeutic approaches to improve capillary formation in CKD. Using a well-established rat model of CKD (subtotal nephrectomy) and previously employed readout systems to assess myocardial capillary density, ischemia-induced capillary angiogenesis in skeletal muscle as well as myocardial infarct size, we propose the following specific aims:First, we will use an inhibitor of prolyl hydroxylase (ICA) to stabilize hypoxia-inducible factors (HIF) in order to improve capillary formation. In preliminary experiments, we have already demonstrated that (a) activation of HIF targets by ischemia is diminished in CKD, and that (b) a non-pharmacological approach (0.1% CO gas) and a pharmacological approach using ICA both applied before ischemia to stabilize HIF greatly improve capillary formation in the ischemic hindlimb of CKD rats. We will now develop this approach as a pharmacological tool in a therapeutic protocol for application after the induction of ischemia. Further, we will assess whether ICA treatment improves myocardial capillary supply and limits myocardial infarct size in CKD rats.Second, we will test the functional role of 2 candidate molecular targets (CCL7 and Metallothionein-1a) for impaired angiogenesis in CKD which were derived from an Affymetrix microarray gene expression screen in ischemic versus non-ischemic limbs from CKD versus control rats, obtained 24 hours after induction of ischemia. Both candidates have known angiogenic properties; their temporal and spatial expression pattern will be investigated. Furthermore, in vitro experiments will be performed to investigate the function of the prolylhydroxylase inhibitor, CCL7 and metallothionein-1a for capillary formation. Proliferation-, apoptosis- and different cell migration assays will be used to elucidate if the potential pro-angiogenic effects of our molecular targets are due to direct actions on endothelial cells (as opposed to indirect effects via e.g. infiltrating mononuclear cells). Subsequently, we will elucidate the signal transduction pathwaysmediating the observed pro-angiogenic effects.From our proposed experiments, we expect novel insight in the pathogenesis of impaired angiogenesis in CKD, and a perspective toward new therapeutic approaches.