Abstract P2058: LncRNA PCAT19 Limits Angiogenesis And Safeguards Quiescent Endothelial DNA By Preventing Uncontrolled Phosphorylation Of Replication Protein A2

Abstract

Background: Long noncoding RNAs (lncRNA) provide an additional layer of molecular control in pathways crucial for vascular development and cardiovascular disease. We set out to identify novel endothelial lncRNAs that could be exploited to treat vascular disease. Methods and Results: We identified the endothelial cell-specific long noncoding RNA (lncRNA) PCAT19, which contributes to the endothelial proliferation-quiescence switch and acts as a safeguard for the endothelial genome during quiescence. PCAT19 was upregulated with endothelial cell confluence and quiescence. Knockdown of PCAT19 promoted endothelial proliferation and angiogenic sprouting. Conversely, PCAT19 overexpression reduced endothelial proliferative capacity and angiogenic sprouting. Using a cardiac organoid model system, we observed a markedly denser vascular network after the removal of PCAT19. RNA-sequencing after PCAT19 knockdown revealed multiple differentially regulated cell cycle genes. An antisense-oligonucleotide pulldown of PCAT19 followed by mass spectrometry identified multiple DNA damage response and cell cycle-related proteins as PCAT19 interaction partners; among them was the full DNA replication protein A complex. PCAT19 knockdown was found to sensitise DNA to damage, as measured by heightened p53 and γH2AX levels as well as a positive TUNEL signal and longer comet tail olive moments. Mechanistically, PCAT19 limited the phosphorylation of RPA2 on the serine 33 (S33) residue by inhibiting RPA2 interaction with the ATR kinase. Conclusions: In healthy vessels, endothelial cells maintain a stable, differentiated and growth-arrested phenotype. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. PCAT19 was identified as a highly enriched endothelial lncRNA that acts as a dynamic guardian of the endothelial genome, promotes cellular longevity and facilitates rapid switching to proliferation. Therapeutic targeting of PCAT19 could potentially alter the course of vascular disease development and progression by modulating endothelial growth and angiogenic sprouting.