Tmem30a Deficiency in the retinal endothelial cells impairs cell proliferation and angiogenesis

Abstract

Purpose : Phosphatidylserine (PS) is PS asymmetry in the eukaryotic cell membrane is maintained by a group of proteins belonging to the P4-ATPase family, namely, PS flippases. The folding and transporting of P4-ATPases to their cellular destination requires a beta-subunit member of the TMEM30 protein family. Loss of Tmem30a has been shown to cause multiple disease conditions. However, its roles in vascular development have not been elucidated.

Methods : We analyzed the role of Tmem30a in endothelial cells using primary human retinal endothelial cells (HREC). Tmem30a expression was analyzed by realtime-PCR. shRNA mediated knockdown of Tmem30a was performed. Tube formation was assessed using In vitro Matrigel tube formation assays. Endothelial cell specific knock out mouse model was generated by crossing a conditional allele of Tmem30a to an inducible PDGFbeta-Cre ER line. Retinal vascular development was assessed in wholemount retinas staining with isolectin, which labels the blood vessels. Blood vessel barrier integrity was evaluated by Ter119 staining of red blood cells. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EDU)lableing. Transcritome analysis was performed by RNA-seq using Tmem30a knockdown HREC and knockout tissues.

Results : Our data indicated that knockdown of TMEM30A in primary human retinal endothelial cells led to reduced tube formation. In mice, endothelial cell (EC)-specific deletion of Tmem30a led to retarded retinal vascular development with a hyperpruned vascular network as well as blunted-end, aneurysm-like tip endothelial cells (ECs) with fewer filopodia at the vascular front and reduced number of tip cells. Deletion of Tmem30a also impaired vessel barrier integrity. Mechanistically, deletion of TMEM30A caused reduced EC proliferation by inhibiting VEGF-induced signaling.

Conclusions : Our data show that TMEM30A plays critical roles in retinal vascular angiogenesis, which is a fundamental process in vascular development. Our findings reveal essential roles of TMEM30A in angiogenesis, and providing a potential therapeutic target.

This abstract was presented at the 2019 ARVO Imaging in the Eye Conference, held in Vancouver, Canada, April 26-27, 2019.

 

In vitro studies of the function of TMEM30A in tube formation.

 

Tmem30a is essential for sprouting angiogenesis and vessel barrier integrity in mouse retinas.Reduced tip ECs and filopodia in retinal vessels of Tmem30a^i△EC and Tmem30a-iKO mice was shown. The number of tip cells and filopodia was counted in each genotypes and plotted in G.