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

慢性肾脏疾病（CKD）患者遭受不成比例的心血管并发症和心源性死亡。发病机制尚不清楚，现有的预防心血管并发症的策略在CKD患者中经常失败。基于我们之前的工作，我们提出CKD中形成新毛细血管的能力受损有助于心血管疾病的负担。毛细血管新生通过恢复受累器官的灌注，是应对大血管狭窄/闭塞或心脏肥厚等过程不可缺少的适应性过程。我们建议的总体目标是更好地了解CKD中血管生成受损的病理生理学，并开发改善CKD中毛细血管形成的治疗方法。利用一个完善的大鼠CKD（肾次全切除术）模型和先前使用的读数系统来评估心肌毛细血管密度、骨骼肌缺血诱导的毛细血管生成以及心肌梗死大小，我们提出以下具体目标：首先，我们将使用脯氨酰羟化酶（ICA）抑制剂来稳定缺氧诱导因子（HIF），以改善毛细血管形成。在初步实验中，我们已经证明(a) CKD中缺血对HIF靶点的激活减弱，(b)在缺血前使用非药物方法（0.1% CO气体）和使用ICA的药物方法来稳定HIF，可以极大地改善CKD大鼠缺血后肢的毛细血管形成。我们现在将把这种方法作为一种药理学工具，应用于诱导缺血后的治疗方案中。此外，我们将评估ICA治疗是否能改善CKD大鼠心肌毛细血管供应并限制心肌梗死面积。其次，我们将测试2个候选分子靶点（CCL7和金属硫蛋白-1a）在CKD血管生成受损中的功能作用，这些靶点来自于Affymetrix微阵列基因表达筛选，在诱导缺血24小时后，在CKD和对照大鼠的缺血性和非缺血性肢体中获得。这两种候选物都有已知的血管生成特性；我们将研究它们的时空表达模式。此外，体外实验将研究脯氨酸羟化酶抑制剂CCL7和金属硫蛋白1a对毛细血管形成的作用。增殖、凋亡和不同的细胞迁移试验将用于阐明我们的分子靶点的潜在促血管生成作用是否由于对内皮细胞的直接作用（而不是通过浸润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作用的信号转导途径。从我们提出的实验中，我们期望对CKD血管生成受损的发病机制有新的认识，并对新的治疗方法有新的看法。

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.