Vascular development within the central nervous system (CNS) progresses through extremely active sprouting angiogenesis. Nascent vascular sprouts at the angiogenic front are sensitive to bleeding, but the mechanisms that maintain the developing neurovascular integrity are not fully understood, and the blood-brain barrier is not functionally mature until after birth. The O-glycoprotein podoplanin (PDPN) and its platelet receptor C-type lectin-like receptor 2 (CLEC-2) are required for vascular integrity in the developing CNS, but the defects that result in spontaneous CNS hemorrhages in Pdpn-/- and Clec-2-/- mice and the mechanism of PDPN-CLEC-2-mediated platelet signaling are unknown. Preliminary observations show that loss of PDPN- CLEC-2 results in defective vascular sprout ingression with dilated nascent sprouts that are a source of hemorrhaging. Observations also indicate that CLEC-2-activated platelet secretory products are able to reverse VEGF-induced VE-cadherin internalization and that Pdpn-/- CNS have significantly upregulated VEGF signaling pathway gene expression. CLEC-2-activated platelets secrete the molecules sphingosine-1- phosphate (S1P) and angiopoietin-1 (Ang1), but it is not known if their respective signaling mechanisms function in cooperation for maintaining CNS vascular integrity during development. These preliminary results support the hypothesis that neural PDPN-CLEC-2-mediated platelet activation and signaling is crucial for maintaining structural stability of the developing vasculature in the CNS. This will be tested by determining 1) the nature of the vascular defect leading to hemorrhaging in the absence of PDPN-CLEC-2 interaction using fluorescent vascular reporter mice and two-photon confocal microscopy, as well as determining defects in the endothelial morphology of nascent sprouts using transmission electron microscopy. In addition, the hypothesis will be tested by determining 2) the mechanism of PDPN-CLEC-2-mediated platelet signaling that regulates vascular integrity during development. Preliminary observations support the hypothesis that S1P and Ang1 released from platelets after PDPN-CLEC-2-mediated activation cooperate to counteract VEGF-signaling- induced VE-cadherin phosphorylation and internalization from the adherins junction. This will be tested by determining if threshold concentration S1P agonist and Ang1 signaling cooperates in an in vitro endothelial permeability assay. In addition, using an ex vivo embryonic brain slice model, the downstream effectors of S1P and Ang1 signaling, Rac1 and RhoA, will be inhibited in order to study the combined effects on angiogenic response and junction stability. If the proposed studies support these hypotheses, it will define a novel mechanism of tissue-specific platelet signaling in regulation of vascular sprout integrity in the developing CNS.
Immature vascular sprouts are vulnerable to bleeding, and must be tightly protected in the developing brain. This proposal will investigate the role of platelets, activated by a protein expressed in the developing brain, in maintaining immature sprout integrity and how loss of that neural protein leads to brain bleeding. Understanding the role of platelets in vascular sprout development in the brain will provide new directions for the treatment of important human developmental bleeding disorders in the brain, such as germinal matrix- intraventricular hemorrhage.
