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

外周动脉疾病(PAD)是动脉粥样硬化的结果，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，组织丢失)。 即使在对PAD中的主要人口学和临床变量进行调整后，仍存在明显的患者对患者 临床变异性与可比的解剖和血流动力学结果。我们鉴定出白介素21 受体(IL21R)作为一种基因改变血管生成、灌注恢复和组织丢失的程度 近交系和同源系小鼠的后肢缺血(HLI)。在内皮细胞(EC)中 在常氧和富含生长因子的环境中，IL21R具有血管抑制作用，但在PAD相关条件下，低氧 加上血清饥饿(HSS)，IL21R促进了血管生成(更多的EC生长和管子形成，而更少 细胞凋亡)，并激活STAT3。先前报道在体内，研究证实IL21R在 缺氧肌中低氧EC能更好地恢复血流灌注，减少组织损失，反之， IL21R途径缺失，血流恢复较差，组织丢失较多。在将疗法从鼠标转移到 人类，PAD的无数临床试验都失败了，无法充分认识到PAD的复杂性 人类的PAD确实可能是一个主要原因。我们将展示与患有CLI的人不同的缺血肌肉 来自IC的IL21R在缺血肌和患有CLI的非裔美国人中的表达可能较低 这种潜在有益的受体的表达比高加索人要好。我们将展示a)体内和体外 IL21R系统可在不改变血管内皮生长因子及其受体的情况下促进缺氧依赖的血管生成 其Akt/eNOS/NO信号通路；b)体外IL21R不增加血管通透性和 不能被NO抑制剂阻断；以及c)IL21R通路在HLI模型中显示出体内的有效性 而在这一点上，血管内皮生长因子却失灵了。我们已经确定了一种似乎对IL21R介导的微RNA(MiR)是必要的 低氧依赖的血管生成，在IL21R不能靶向的情况下可以作为治疗药物。一个 单个长非编码(LNC)RNA(IL21R-AS1)存在，在PAD患者中差异表达，Gain，和 IL21R-AS1缺失对HSS诱导的人脐静脉内皮细胞血管生成的影响与之相反。在配对的中环 假设，我们假设在人类中，这种意想不到的血管生成途径的治疗靶点将需要 根据PAD状态和个体内的不同而不同，IL21R介导的血管生成通过eNOS发生 当受体不可用时，独立途径和miR-30b可以起到治疗作用。目标 目标1)：评估人类治疗靶向是否：针对所有患者的单一策略，不同 PAD临床亚组的策略或完全个性化的策略允许IL21R的最佳靶向 介导的低氧依赖的血管生成。目的：体外和体内比较IL-21R和血管内皮细胞生长因子在低氧条件下的作用 依赖血管生成。目的3)确定miR-30b在IL21R介导的低氧中是否必需 血管生成，在PAD条件下可作为IL21R的替代治疗。

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.