Role of endothelial glycolysis in pericyte recruitment and function

原理：新生血管生成、新血管的生长和内皮细胞(ECs)的特化之间的协调对于血管构型和器官的正常功能是必不可少的。小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，从出生时开始发育。神经视网膜的动态平衡强烈依赖于高选择性血视网膜屏障(BRB)，该屏障受内皮细胞与周围周细胞(PC)相互作用的调节。尽管它很重要，但在萌发血管形成过程中调节BRB形成的机制仍然很少被描述。内皮尖细胞是新形成的毛细血管末端的特化细胞，引导毛细血管生长。视网膜浅层细胞(S尖细胞)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-TIP细胞)与S-TIP细胞不同。D-TIP细胞具有独特的转录特征，包括高的转化生长因子？信号和低的糖酵解率。与S-TIP细胞相比，D-TIP细胞与PC具有很强的相互作用，并具有BRB特性。内皮细胞缺失的转化生长因子受体I(Alk5iEKO)抑制D-末端细胞规范、BRB和深血管神经丛的形成。此外，内皮细胞Alk5的缺失促进了TIP细胞的糖酵解和周细胞的异常分化。调控TIP细胞-PC相互作用的机制尚不清楚。S和Alk5iEKO TIP细胞比D-TIP细胞表达更多的乳酸脱氢酶A(LDHA)，提示TIP细胞产生的乳酸可能调节PC的募集和功能。因此，本研究旨在探讨TIP细胞代谢是否能控制PC的募集和BRB的获得。目的和假设：为了解决这一假设，我们提出了三个具体的目标：1)研究周细胞是否对D-TIP细胞的特化和神经视网膜血管形成是必要的；2)确定TIP细胞糖酵解是否调节周细胞的招募和鉴定；以及3)评估乳酸介导的周细胞的特性和行为。相关性：周细胞的募集和功能对视网膜和脑血管生成是必不可少的，但机制尚不清楚。这项研究将使我们能够提出一种新的全面的机制来调节视网膜血管生成，并将构成该领域未来研究的基础。值得注意的是，在视网膜和大脑发育过程中，萌芽血管生成和BRB形成耦合的概念在很大程度上仍未被探索。因此，识别调节PC募集和功能的内皮信号将对视网膜生物学以外的领域具有重要意义，如器官血管形成，以及加拿大血管研究的战略兴趣。

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.