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

项目摘要 小动脉的分化和从非常微小的血管形成小动脉(毛细血管动脉化) 是从伤害性侮辱中恢复缺血器官的基本过程。尽管很明显 临床和生物学重要性，对这些事件的分子机制和它们的作用机制知之甚少 监管。我们最近发现CD36的转录抑制参与了一种促血管生成 和毛细血管内皮细胞(EC)或微血管内皮细胞(MVECs)的促动脉生成重编程，并且是 牵涉到毛细血管动脉化。具体地说，我们已经证明了LPA/PKD-1信号介导的CD36 转录抑制在促进血管内皮细胞动脉形成基因表达中起关键作用，并且 体内微血管重塑。此外，EC特异性的PKD-1缺失显示出从 缺血性侮辱。这些意想不到的发现导致了CD36基因通过LPA/PKD-1进行抑制的假设 Signal Axis对MVECs重新编程，使其分化为小动脉内皮细胞，促进毛细血管动脉化。至 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 马特里格尔化验。更重要的是，我们已经建立了独特的CD36基因的后肢缺血模型 EC特异性翻译核糖体亲和纯化(TRAP)和EC特异性PKD-1缺陷小鼠 缺陷小鼠或EC特异性PKD-1缺陷陷阱小鼠。这些新型的陷阱转基因品系已经瞄准了 基因缺陷，并携带EC特异性的EGFP标签，使我们能够直接纯化核糖体结合的mRNA 来自活体内皮细胞的新一代RNA测序，促进了EC特异性转录组和 非编码RNA的核糖体结合。这款鼠标模型的问世首次为我们提供了 设计实验以获得对成人小动脉生长生物学的新的和基本的见解的能力 在缺血条件下。使用这些工具，我们的目标是1)证明在MVEC中LPA/PKD-1-CD36 信号轴是调节微血管内皮细胞重编程和微动脉分化的关键；2)检验假设 通过LPA/PKD-1-FoxO1信号轴的CD36转录抑制是 基因重编程开关促进毛细血管动脉化(新生动脉生成)。与.一起 使用一系列分子生物学和成像方法的分析单细胞RNA测序以及 斑马鱼模型，我们期望实现这些目标并提供对微动脉的全面评估 分化和毛细血管动脉化。这将阐明未被探索和理解的方面 血管生物学。这项建议侧重于成人的新生动脉生成，因为它非常实用 意义。对成人组织中的毛细血管动脉化有更深入的了解将提供重要的 对寻找新的和有效的治疗缺血性心脏和血管疾病靶点的见解。

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.