Endothelial RAP1A attenuates sinusoidal capillarisation and liver fibrosis by inhibiting RAF1-mediated Notch activation.

Abstract

Capillarisation of liver sinusoidal endothelial cells (LSECs) constitutes an early pathological event that promotes hepatic stellate cell activation and initiates liver fibrogenesis. Previous studies suggest that Ras-associated protein 1A (RAP1A) might be involved in liver fibrosis. However, the role of RAP1A in LSEC capillarisation remains unclear. This study aimed to investigate the role of the endothelial GTPase RAP1A in sinusoidal capillarisation and liver fibrosis. Liver fibrosis models were induced by HFHCD, CDAHFD, CCl₄ or DDC treatment. EC-specific <i>Rap1a</i> knockout (<i>Rap1a</i> ^ΔEC) mice were generated by breeding <i>Rap1a</i> ^fl/fl mice with <i>Cdh5</i>-Cre mice. Therapeutic intervention was performed using the RAP1 activator 8-pCPT-2'-O-Me-cAMP. LSEC capillarisation, along with the extent of liver fibrosis and inflammation, was evaluated. RAP1A expression in LSECs was downregulated in all murine fibrosis models, as well as in primary LSECs undergoing spontaneous capillarisation during in vitro culturing. Young <i>Rap1a</i> ^ΔEC mice exhibited spontaneous capillarisation, which progressed to liver fibrosis with age. Endothelial <i>Rap1a</i> deficiency exacerbated sinusoidal capillarisation and liver fibrosis in CCl₄-/DDC-treated mice. Mechanistically, RAP1A loss promoted RAF1 degradation via the ubiquitin-proteasome pathway, enhancing nuclear translocation of Notch intracellular domain (NICD) and activating Notch signalling. Pharmacological activation of RAP1A attenuated LSEC capillarisation and mitigated liver fibrosis in CCl₄-/DDC-induced models. Endothelial <i>Rap1a</i> deficiency aggravates capillarisation and liver fibrosis by activating Notch signalling through RAF1 degradation. These findings indicate that <i>Rap1a</i> is essential for maintaining LSEC homeostasis and represents a potential intervention target for liver fibrosis.