Abstract 514: Netrin 4 Deficiency Leads to Endothelial Cell Senescence

Senescence phenotype of endothelial cells (ECs) has pathophysiological consequences, such as decreased regeneration capacity, pro-atherogenic tendency and dysregulated vessel tune. We find that netrin 4 (NTN4), recognized in neural and vascular development, is highly expressed by mature ECs. Remarkably, little is known about its role in vascular biology after development. NTN4 is deposited in the extracellular matrix. Using human decellularized kidney extracelluar matrix scaffolds, we found that pre-treatment of the scaffolds with NTN4 increased numbers of EC adhesion to the matrix, showing a pro-survival effect of NTN4. Subsequently we explored the regulation of NTN4 expression in ECs. We found a 1.8-fold (±0.3; p<0.05) upregulation in NTN4 expression in ECs cultured under laminar flow conditions compared to static culture conditions. In contrast, ECs stimulated with TNFα resulted in decreased NTN4 expression (0.17 ± 0.06 fold; p<0.05), indicating a role for NTN4 in quiescent healthy endothelium. Silencing of NTN4 in ECs, to investigate the necessity of NTN4 in ECs, markedly resulted in more senescent associated β-galactosidase activity (20-50%; p<0.05) that could be rescued by NTN4 protein coating. Consistent with increased senescence, NTN4 reduction is accompanied with increased expression of senescence-associated transcription factors, CDKN1A and CDKN2A, as well as decreased ability to proliferate. Importantly, ECs with reduced levels of NTN4 have also increased expression of ICAM-1 and VCAM-1, are more prone to adhesion of human monocyte and have impaired barrier function, measured in an electric cell-substrate impedance sensing system as well as ‘organ-on-a-chip’ microfluidic system. In conclusion, our results identified the anti-senescence function of NTN4 and thereby provides novel insights in the role of NTN4 in EC function. In situations like acute inflammation and unfavourable hemodynamic conditions, NTN4 expression decreases, so that there is a possible window for us to improve EC function by normalizing NTN4 expression.