Mechanisms of Angiogenesis in ROP

在早产儿视网膜病变（ROP）中，生理性血管分布受损和生理性视网膜病变延迟， I期的血管发育（PRVD）先于玻璃体内血管增生性新生血管（IVNV）， 第二阶段。在之前的研究期间，我们发现抑制VEGF信号传导的玻璃体内抗体重新排列， 血管生成，减少IVNV，并允许一些PRVD，也损害幼鼠的生长，减少生理 在啮齿类动物氧诱导的视网膜病变（OIR）模型中，靶向 过表达VEGF或VEGF信号通路中的活化蛋白与细胞特异性shRNA抑制 IVNV不会降低幼仔生长或生理性血管分布，但不会完全延长PRVD。人 我们发现患有严重ROP的婴儿由于PRVD不完全和受损而发生复发性IVNV， 玻璃体内抗VEGF抗体后的生理性血管形成。总之，这些发现表明，生理 重新定向血管生成以治疗病理性IVNV不足以延长PRVD，但完全PRVD - 保留生理性血管-对预防复发性IVNV很重要。我们的下一个目标是 延长PRVD和保护生理性血管供应以预防严重ROP的机制， 保护机制内源性或通过外源性治疗。使用产妇模型 子宫胎盘功能不全（UPI）和幼鼠OIR，我们确定上调的系统性促红细胞生成素（EPO）是一种 潜在的保护因素，延长PRVD。我们还发现了ERBB 2驱动的蛋白质， 运动（MEMO 1），定位于IVNV，但不正常发育的血管，并干扰EPO诱导的 抑制视网膜内皮细胞（EC）凋亡和淬灭活性氧（ROS）的机制。 MEMO 1是VEGF诱导的血管生成所必需的，通过转录因子STAT 3介导， EC。使用慢病毒基因治疗方法特异性地敲低体内视网膜EC中的STAT 3，我们发现， EC-STAT 3敲低抑制大鼠OIR中的IVNV。这些发现支持我们下一个资助期的目标， 检验PRVD和生理性血管分布可以通过EPO信号传导调节的假设， 受体（EPOR）在血管生成。具体目的1是测试是否诱导来自UPI的内源性EPO信号传导 pup-OIR通过减少ROS和细胞凋亡对生理性血管分布和再生很重要。具体 目的2是检测外源性EPO是否足以促进PRVD并改善视网膜结构和功能 在EC EPOR下降的情况下，不增加IVNV。具体目标3是测试MEMO 1是否 干扰EPO介导的机制，促进PRVD，如果MEMO 1维持VEGF诱导的 血管生成信号导致IVNV。方法包括：新型慢病毒基因治疗大鼠;氧诱导 大鼠视网膜病模型和新型复合鼠dam UPI/pup OIR模型;人源化敲入小鼠 其中EPO信号传导是低活性的; Micron IV成像;视网膜下注射;光学相干断层扫描; 视网膜电描记术;平片和切片的免疫组织化学;蛋白质印迹，实时PCR。

In retinopathy of prematurity (ROP), compromised physiologic vascularity and delay in physiologic retinal vascular development (PRVD) in Phase I precede vasoproliferative intravitreal neovascularization (IVNV) in Phase II. During prior grant periods, we found that intravitreal antibodies that inhibit VEGF-signaling reorder angiogenesis, which reduces IVNV and allows some PRVD, also impair pup growth, reduce physiologic vascularity and disorganize retinal structure in rodent oxygen-induced retinopathy (OIR) models. Targeting over-expressed VEGF or activated proteins in the VEGF-signaling pathway with cell-specific shRNA inhibits IVNV without reducing pup growth or physiologic vascularity but does not completely extend PRVD. In human infants with severe ROP we found that recurrent IVNV occurs due to incomplete PRVD and compromised physiologic vascularity after intravitreal anti-VEGF antibody. Together, these findings suggest that physiologic reorientation of angiogenesis to treat pathologic IVNV is insufficient to extend PRVD, but complete PRVD - with preserved physiologic vascularity - is important to prevent recurrent IVNV. Our next goal is to identify mechanisms to extend PRVD and preserve physiologic vascularity to prevent severe ROP by exploiting protective mechanisms endogenously or through exogenous treatment. Using models of maternal uteroplacental insufficiency (UPI) and pup OIR, we identified upregulated systemic erythropoietin (EPO) as a potentially-protective factor, which extended PRVD. We also found the protein, mediator of ERBB2-driven motility (MEMO1), localized to IVNV but not to normal developing vessels and interfered with EPO-induced mechanisms to inhibit retinal endothelial cell (EC) apoptosis and quench reactive oxygen species (ROS). MEMO1 was necessary for VEGF-induced angiogenesis mediated through the transcription factor, STAT3, in ECs. Using a lentiviral gene therapy method to specifically knockdown STAT3 in retinal ECs in vivo, we found EC-STAT3 knockdown inhibited IVNV in rat OIR. These findings support our goal for the next grant period to test the hypothesis that PRVD and physiologic vascularity can be modulated by EPO signaling through EPO- receptor (EPOR) in angiogenesis. Specific Aim 1 is to test if induced endogenous EPO signaling from UPI and pup-OIR is important for physiologic vascularity and regrowth by reducing ROS and apoptosis. Specific Aim 2 is to test if exogenous EPO is sufficient to promote PRVD and improve retinal structure and function without increasing IVNV in the setting of knocked down EC EPOR. Specific Aim 3 is to test if MEMO1 interferes with EPO-mediated mechanisms to promote PRVD, and if MEMO1 sustains VEGF-induced angiogenic signaling leading to IVNV. Methods include: novel lentiviral gene therapy in rat; oxygen-induced retinopathy models in rat and in novel composite murine dam UPI/pup OIR models; humanized knockin mice in which EPO signaling is hypoactive; Micron IV imaging; subretinal injections; optical coherence tomography; electroretinography; immunohistochemistry of flat mounts and sections; western blot, real-time PCR.