Precision Medicine for Therapeutic Angiogenesis in Peripheral Arterial Disease: Targeting of the IL21R Pathway

外周动脉疾病（PAD）由动脉粥样硬化引起，有两种不同的临床表现： 间歇性跛行（IC，休息后行走疼痛缓解）和严重肢体缺血（CLI，组织损失）。 即使在调整PAD的主要人口统计学和临床变量后， 临床变异性与解剖学和血流动力学结果相当。我们鉴定了白细胞介素21 受体（IL 21 R）作为一种基因，改变血管生成，灌注恢复和组织损失的程度 后肢缺血（HLI）后，从近交系，和同类，小鼠品系。内皮细胞（EC） 常氧和生长因子丰富的环境中IL 21 R具有血管抑制作用，但在PAD相关条件下，缺氧 加上血清饥饿（HSS），IL 21 R促进血管生成（更大的EC生长和管形成，而更小的EC生长和管形成）。 细胞凋亡）与STAT 3活化。先前报道的体内研究证实，IL 21 R上调在 缺血肌肉中的低氧EC导致更好的灌注恢复和更少的组织损失，相反， IL 21 R途径的丧失导致灌注恢复较差和更多的组织损失。在将治疗从小鼠转移到 在人类中，许多PAD的临床试验都失败了，并且无法完全理解 人PAD确实是一个主要原因。我们将证明，来自CLI患者的缺血性肌肉 在缺血性肌肉中IL 21 R表达方面，非裔美国人CLI可能具有较低的 这种潜在的有益受体的表达比高加索人。我们将展示a）体内和体外， IL-21 R系统可促进缺氧依赖性血管生成，而不改变VEGF蛋白、其受体或 其Akt/eNOS/一氧化氮（NO）信号传导途径; B）体外IL 21 R不增加血管通透性， 不能用NO抑制剂阻断;和c）IL 21 R途径在HLI模型中显示出体内功效 VEGF失败的地方。我们已经鉴定了一种微小RNA（miR），其似乎是IL 21 R介导的细胞凋亡所必需的。 低氧依赖性血管生成，并且可以用作不能靶向IL 21 R的治疗剂。一 单个长非编码（lnc）RNA（IL 21 R-AS 1）存在，在PAD患者中差异表达，并获得， IL 21 R-AS 1的缺失对HSS下HUVEC中的血管生成具有方向相反的作用。在配对的中央 假设，我们认为，在人类中，这种意想不到的血管生成途径的治疗靶向将需要， 基于PAD状态和个体内而不同，并且IL 21 R介导的血管生成通过eNOS发生。 当受体不可用时，miR-30 b可以作为独立途径的治疗剂。目标 目的1）：评估人类治疗靶向，确定是否：针对所有患者的单一策略， PAD临床亚组的策略，或完全个性化的策略允许IL 21 R的最佳靶向 介导的缺氧依赖性血管生成。目的2）：缺氧时IL 21 R和VEGF的体内、体外对比研究 依赖血管生成。目的3）确定miR-30 b是否是IL 21 R介导的缺氧所必需的 在PAD病症中，IL 21 R可促进血管生成，并可作为IL 21 R的治疗替代物。

Peripheral arterial disease (PAD) results from atherosclerosis and has two distinct clinical manifestations: intermittent claudication (IC, pain with walking relieved with rest), and critical limb ischemia (CLI, tissue loss). Even after adjusting for major demographic and clinical variables in PAD there is marked patient to patient clinical variability with comparable anatomic and hemodynamic findings. We identified the interleukin 21 receptor (IL21R) as a gene that modified the extent of angiogenesis, perfusion recovery and tissue loss following hind-limb ischemia (HLI) from inbred, and congenic, mouse strains. In endothelial cells (EC) in normoxic and growth factor rich settings IL21R had angiostatic effects but in PAD relevant conditions, hypoxia plus serum starvation (HSS), IL21R promoted angiogenesis (greater EC growth and tube formation, and less apoptosis) with STAT3 activation. Previously reported in-vivo, studies confirmed that IL21R up-regulation in hypoxic EC in ischemic muscle resulted in better perfusion recovery and less tissue loss and conversely the loss of the IL21R pathway poorer perfusion recovery and more tissue loss. In moving therapies from mouse to humans, numerous clinical trials in PAD have failed and the inability to fully appreciate the complexity of human PAD can indeed be a major reason. We will show that ischemic muscle from humans with CLI differs from IC in regards to IL21R expression in ischemic muscle and African Americans with CLI may have lower expression of this potentially beneficial receptor than Caucasians. We will show a) in-vivo and in-vitro that the IL21R system can promote hypoxia dependent angiogenesis without changes in VEGF protein, its receptor, or its Akt/eNOS/nitric oxide (NO) signaling pathway; b) in-vitro IL21R does not increase vascular permeability and cannot be blocked with NO inhibitors; and c) the IL21R pathway demonstrates efficacy in-vivo in HLI models where VEGF has failed. We have identified a micro-RNA (miR) that appears necessary for IL21R mediated hypoxia-dependent angiogenesis and can serve as a therapeutic where the IL21R cannot be targeted. A single long noncoding (lnc) RNA (IL21R-AS1) exists, is differentially expressed in PAD patients, and gain, and loss, of IL21R-AS1 had directionally opposite effects on angiogenesis in HUVEC under HSS. In paired central hypotheses, we posit that in humans therapeutic targeting of this unexpected angiogenic pathway will need to differ based on the PAD state and within individuals and IL21R mediated angiogenesis occurs via an eNOS independent pathway and miR-30b can serve as a therapeutic when the receptor is not available. The aims are: Aim 1): To assess human therapeutic targeting establish whether: a single strategy for all patients, distinct strategies by PAD clinical subgroups, or fully personalized strategies allows optimal targeting of IL21R mediated hypoxia-dependent angiogenesis. Aim 2): In-vitro and in-vivo contrast IL21R to VEGF in hypoxia dependent angiogenesis. Aim 3) Determine whether miR-30b is necessary for IL21R mediated hypoxic angiogenesis and can serve as a therapeutic alternative to IL21R in PAD conditions.