Epigenetic and Transcriptional Regulation of Angiogenic Regulator CD36 and Transformation of Capillaries into Small Arteries

项目摘要 小动脉分化和从非常微小的血管形成小动脉（毛细血管动脉化） 是缺血性器官从有害损伤中恢复的基本过程。尽管其明显 临床和生物学的重要性，很少有人知道这些事件的分子机制和它们的作用。 调控我们最近发现，CD 36的转录抑制参与了促血管生成的作用。 和毛细血管内皮细胞（EC）或微血管EC（MVEC）的促动脉发生重编程， 与毛细血管动脉化有关具体地说，我们已经证明LPA/PKD-1信号转导介导的CD 36 转录抑制在促进MVEC中的动脉形成基因表达中起关键作用， 体内微血管重塑。此外，EC特异性pkd-1缺失显示了受损的恢复， 缺血性损伤这些意想不到的发现导致了一种假设，即通过LPA/PKD-1抑制CD 36基因表达。 信号传导轴重编程MVEC以分化成小动脉EC并促进毛细血管动脉化。到 为了验证这一假设，我们已经建立了二维和三维MVEC培养系统，并在体内 基质胶测定。更重要的是，我们建立了独特的cd 36基因的后肢缺血模型， 用EC特异性翻译核糖体亲和纯化（TRAP）和EC特异性pkd-1 缺陷型小鼠或EC特异性pkd-1缺陷型TRAP小鼠。这些新的TRAP转基因系具有靶向 基因缺陷，并携带EC特异性EGFP标签，使我们能够直接纯化核糖体结合的mRNA 用于下一代RNA测序，促进EC特异性转录组的探针， 非编码RNA的核糖体结合。这种小鼠模型的可用性首次为我们提供了 设计实验以获得成人小动脉生长生物学的新的和基本的见解的能力 在局部缺血的情况下。使用这些工具，我们的目标是：1）证明在MVEC中，LPA/PKD-1-CD 36 信号轴在调节MVEC重编程和小动脉分化中起重要作用; 2）验证假设 通过LPA/PKD-1-FoxO 1信号轴的CD 36转录抑制是一个关键的组成部分， 基因重编程开关，以促进毛细血管动脉化（从头动脉生成）。连同 使用一系列分子生物学和成像方法的测定，单细胞RNA测序以及 斑马鱼模型，我们希望实现这些目标，并提供一个全面的评估动脉 分化和毛细血管动脉化。这将阐明探索不足和理解不足的方面， 血管生物学这项建议的重点是在成人从头动脉生成，因为它非常实用 意义更深入地了解成人组织中的毛细血管动脉化将提供重要的 深入了解寻找新的和有效的治疗靶点，对缺血性心脏和血管疾病。

Project Summary Arteriolar differentiation and formation of small arteries from very tiny blood vessels (capillary arterialization) are fundamental processes underlying ischemic organ recovery from a noxious insult. Despite its obvious clinical and biological importance, little is known about the molecular mechanisms of these events and their regulation. We have recently discovered that transcriptional repression of CD36 is involved in a proangiogenic and proarteriogenic reprogramming of capillary endothelial cells (EC) or microvascular EC (MVECs), and is implicated in capillary arterialization. Specifically, we have shown that LPA/PKD-1 signaling-mediated CD36 transcriptional repression plays a key role in promoting arteriogenic gene expression in MVECs, and microvascular remodeling in vivo. Moreover, EC-specific deletion of pkd-1 showed impaired recovery from ischemic insult. These unexpected findings led to the hypothesis that CD36 gene repression via LPA/PKD-1 signaling axis reprograms MVECs to differentiate into arteriolar ECs and promotes capillary arterialization. To test this hypothesis, we have established two- and three-dimensional MVEC culture systems, and in vivo Matrigel assays. More importantly, we have established hindlimb ischemia models in unique cd36 gene deficient mice with the EC-specific translating ribosome affinity purification (TRAP), and EC-specific pkd-1 deficient mice or EC-specific pkd-1 deficient TRAP mice. These novel TRAP transgenic lines have targeted gene deficiency and carry an EC-specific EGFP-tag that allows us to directly purify ribosome-bound mRNA from ECs in vivo for next generation RNA-sequencing, facilitating probes of EC-specific transcriptome and ribosome binding of noncoding RNAs. The availability of this mouse model provides us for the first time with the ability to devise experiments to gain new and fundamental insights into biology of adult arteriolar growth under ischemic conditions. Using these tools,, we aim to 1) prove that in MVECs the LPA/PKD-1-CD36 signaling axis is essential to regulate MVEC reprogramming and arteriolar differentiation; 2) test the hypothesis that CD36 transcriptional repression via the LPA/PKD-1-FoxO1 signaling axis is a critical component of a genetic reprogramming switch to promote capillary arterialization (de novo arteriogenesis). Together with assays using a series of molecular biology and imaging methods single cell RNA-sequencing as well as zebrafish models, we expect to accomplish these aims and provide a comprehensive evaluation of arteriolar differentiation and capillary arterialization. This will illuminate poorly explored and poorly understood aspects of vascular biology. This proposal focuses on de novo arteriogenesis in adult because of its remarkably practical significance. Getting a greater understanding of capillary arterialization in adult tissues will provide important insights into finding novel and effective therapeutic targets against ischemic heart and vascular diseases.