Endothelial O-Glycoproteins Cooperate with Platelet CLEC-2 Signaling to Maintain Cerebrovascular Integrity during Mouse Development
Authors: Fu Jianxin , Song Jianhua , Xia Lijun 
Published: October 2019

Abstract
Protein glycosylation plays vital roles in many biological processes. We previously discovered that embryos with global deficiencies of mucin-type O-glycans (i.e. C1galt1-/- mice) develop fatal brain hemorrhage during embryogenesis. However, mice lacking endothelial cell O-glycans exhibit blood-lymphatic mixing with no brain bleeding phenotype, whereas mice lacking either O-glycoprotein podoplanin or its receptor CLEC-2 develop similar spontaneous bleeding in developing brain. To further investigate whether endothelial O-glycans contribute to cerebrovascular barrier development and/or maintenance, we generated two independent transgenic mouse lines, in which the expression of C1galt1 is under the control of endothelial-specific Tie2 promoter/enhancer (EC-C1galt1 Tg mice). When breeding the EC-C1galt1 Tg mice into C1galt1-/- background, we found that the embryonic lethality of C1galt1-/- mice was completely rescued, supporting a critical role of endothelial O-glycans in governing the cerebral vascular integrity during embryogenesis. Platelets have been identified as one of the key players for vascular integrity under physiological and pathological conditions although the underlying mechanisms remain elusive. Based on the existing knowledge, we hypothesize that endothelial O-glycoproteins cooperate with neural podoplanin, an O-glycoprotein highly expressed on neural progenitor cells in early developing brain, through activating its platelet receptor CLEC-2, to maintain cerebral vascular integrity during active sprouting angiogenesis. To test the hypothesis, we generated mice with compound deficiencies of endothelial O-glycans and podoplanin. Interestingly, double knockout mice faithfully phenocopied the global C1galt1-/- mice, including a full penetration of fatal hemorrhage. These results strongly support a cooperative role between endothelial O-glycans and neural podoplanin. Mechanistically, we demonstrated that loss of podoplanin or CLEC-2 in vivo leads to an impaired expression of VE-cadherin, a major endothelial intercellular junctional protein. Moreover, in vitro cell-based analysis showed that podoplanin-CLEC-2 interaction and subsequent platelet release counteracts the effects of VEGF on VE-cadherin expression and endothelial permeability, indicating that podoplanin-dependent activation of platelet CLEC-2 signaling is critical for the stability of newly formed fragile vessels in the early developing brain by promoting endothelial junctions via platelet release. Taken together, our results provide a new model for the maintenance of cerebrovascular integrity during early brain development, in which the interactions between endothelial cells, neural cells and platelets are essential for the brain vascular homeostasis. These findings not only provide new insights into the regulation of cerebrovascular development, but could also allow us to test new therapies for hemorrhagic disorders with pathway-targeted drugs.

Disclosures
No relevant conflicts of interest to declare.