Determine the role of ALK5 signaling in murine brain pericytes at embryonic day E13.5

Pericytes (PCs) are microvascular mural cells which constituent the embryonic blood brain barrier (BBB) along with endothelial cells (ECs). During brain development, germinal matrix (GM) - a highly vascularized region rich in neuronal-glial precursors, is selectively vulnerable to hemorrhage in premature infants. The transforming growth factor β (TGFβ) pathway plays a crucial role in barrier development by regulating cellular cross talk between PCs and ECs. Indeed, murine embryos lacking TGFβ receptor activin receptor-like kinase 5 (Alk5) in brain PCs (mutants) develop gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH) and culminate in perinatal lethality. Mutant GM vessels display reduced PC and collagen coverage, abnormal vessel dilation and EC hyperproliferation. However, the mechanistic link between PC-specific deletion of ALK5 and aberrant EC behavior remains elusive. Herein, using bulk RNA sequencing from human brain PCs lacking ALK5 (siALK5) as well as murine vascular cells [PCs and ECs] isolated from embryonic brain at embryonic days E11.5 and E13.5 we establish that angiopoietin 2 (ANGPT2), a secreted angiogenic growth factor, is robustly repressed by the TGFβ pathway in PCs. Conversely, mutants lacking PC-ALK5 secrete higher levels of ANGPT2 resulting in overactivation of tyrosine protein kinase receptor (TIE2) and culminating in EC hyperproliferation, vessel dilation, BBB breakdown and GMH. PC-specific Angpt2 deletion or pharmacological inhibition in mutants improves GM vessel morphology, reduces EC proliferation and attenuates GMH pathogenesis. Taken together, we demonstrate that loss of TGFβ-mediated ANGPT2 repression in PCs is detrimental for BBB integrity and identify ANGPT2 as an important pathological target for GMH-IVH.