Role of endothelial glycolysis in pericyte recruitment and function

基本原理：血管新生、新血管生长和内皮细胞特化的协调对血管的形成和器官的正常功能至关重要。老鼠视网膜的血管网络是独一无二的，有一个浅层和两个较深的神经视网膜层，从出生时就开始发育。神经视网膜的内稳态依赖于高度选择性的血视网膜屏障（BRB），该屏障由内皮细胞和周围周细胞（PCs）之间的相互作用调节。尽管它很重要，但在发芽血管生成过程中调节BRB形成的机制仍不清楚。内皮尖细胞是新生毛细血管末端的特化细胞，引导毛细血管生长。视网膜浅表尖端细胞（s -尖端细胞）不与pc相互作用，BRB低。我们最近发现侵入小鼠神经视网膜的潜水尖细胞（d -尖细胞）不同于s -尖细胞。D-tip细胞具有独特的转录特征，包括高TGFß信号和低糖酵解速率。与s -尖端细胞相比，d -尖端细胞与pc相互作用强，具有BRB特性。TGFß受体I （Alk5iEKO）的内皮缺失抑制d -尖端细胞的特异性、BRB和深血管丛的形成。此外，内皮细胞Alk5的缺失促进尖端细胞糖酵解和异常周细胞分化。调节尖端细胞- pc相互作用的机制尚不清楚。S-和Alk5iEKO尖端细胞比d -尖端细胞表达更多的乳酸脱氢酶A (LDHA)，提示尖端细胞产生的乳酸可以调节PC的募集和功能。因此，本研究旨在研究尖端细胞代谢是否能够控制PC的募集和BRB的获取。目的和假设：为了解决这一假设，我们提出了三个具体目标：1)研究周细胞是否对d尖细胞分化和神经视网膜血管化至关重要；2)确定尖端细胞糖酵解是否调节周细胞募集和识别；3)评估乳酸介导的周细胞身份和行为。相关性：周细胞的募集和功能对视网膜和大脑血管生成至关重要，但机制尚不清楚。本研究将使我们提出一种新的调控视网膜血管生成的综合机制，并为该领域今后的研究奠定基础。值得注意的是，在视网膜和大脑发育过程中，发芽血管生成和BRB形成耦合的概念在很大程度上仍未被探索。因此，识别内皮细胞中调节PCs募集和功能的线索，对视网膜生物学以外的领域，如类器官血管化，具有重要意义，对加拿大血管研究具有战略意义。

Rationale: The coordination of sprouting angiogenesis, the growth of new blood vessels, and specialization of endothelial cells (ECs) is essential for vascular patterning and the proper function of organs. The mouse retina's vascular network is unique, with one superficial and two deeper neuroretina layers, which start developing at birth. The neuroretina's homeostasis strongly depends on the highly selective blood-retina barrier (BRB), which is regulated by the interaction between the ECs and the surrounding pericytes (PCs). Despite its importance, the mechanisms regulating BRB formation during sprouting angiogenesis remain poorly characterized. Endothelial tip cells are specialized cells at the extremity of newly formed capillaries that guide capillary outgrowth. The retinal superficial tip cells (S-tip cells) do not interact with PCs and have a low BRB. We recently revealed that diving tip cells invading the mouse neuroretina (D-tip cells) are distinct from S-tip cells. D-tip cells have a unique transcriptional signature, including high TGFß signaling and low glycolysis rate. Compared with S-tip cells, D-tip cells show strong interaction with PCs and acquire BRB properties. Endothelial deletion of TGFß receptor I (Alk5iEKO) inhibits D-tip cell specification, BRB, and deep vascular plexus formation. Moreover, loss of endothelial Alk5 promotes tip cell glycolysis and aberrant pericyte differentiation. Mechanisms regulating tip cell-PC interaction remain unclear. S- and Alk5iEKO tip cells express more lactate dehydrogenase A (LDHA) than D-tip cells, suggesting that lactate produced by tip cells could regulate PC recruitment and function. Therefore, this proposal aims to investigate whether tip cell metabolism could control PC recruitment and BRB acquisition. Objective and hypothesis: To address this hypothesis, we propose three specific aims: 1) Investigate whether pericytes are essential for D-tip cell specification and neuroretina vascularization; 2) Determine whether tip cell glycolysis regulates pericyte recruitment and identity; and 3) Assess lactate-mediated pericyte identity and behavior. Relevance: Pericytes recruitment and function are essential for retina and brain angiogenesis, but mechanisms remain unclear. This study will allow us to propose a new comprehensive mechanism that regulates retina angiogenesis and will constitute the basis for future research in the field. Notably, the concept of sprouting angiogenesis and BRB formation coupling remains largely unexplored during retina and brain development. Therefore, identifying endothelial cues regulating PCs recruitment and function will be of great significance to fields beyond retina biology, such as organoid vascularization, and strategic interest to vascular research in Canada.