Background- New vessel formation is the sequential process of attachment-detachment-reattachment of endothelial cells (ECs) and endothelial progenitor cells (EPCs), and dynamic interaction between cell and extracellular matrix (ECM) is critical point. Intergrin-linked kinase (ILK) plays pivotal role in ECM-mediated signaling. However, the function of this gene in the angiogenic process of ECs and EPCs is largely unknown. So, we investigated the role of ILK in the EC and EPC during neovascularization.
Methods and Results- In human umbilical cord vein endothelial cells (HUVECs) and EPCs, endogeneous ILK expression was reduced in anchorage or nutrient deprivation. Overall expression pattern of ILK was similar between mature ECs and progenitor cells. But, in serial immunoblot exam, basal expression level of ILK in EPC was much higher than EC, and maintained basal high level of ILK after various stress and then decreased slowly. And EPC was more resistant at detached or nutrient deprived stress. So, we made an adenoviral construct of wild type ILK to enhance efficiency of transduction and overexpress ILK. After detachement and nutrient deprivation, ILK-overexpessing HUVECs and EPCs increased survival, stimulated downstream survival signal molecules (phospho-Akt and phospho-GSK3[Beta]) and decreased apoptosis. ILK-overexpression also increased cell proliferation and enhanced capillary formation when reattachment occurred. To determine if such genetic modification may facilitate therapeutic neovascularization, ILK-overexpressing EPCs were administered to nude mice with hindlimb ischemia via local or systemic delivery. ILK-EPCs, even at the 20 times less dose than GPF-EPCs, improved limb salvage rate and blood flow recovery. Furthermore, efficacy of systemic delivery was comparable with local injection when ILK overexpressed.
Conclusions- ILK gene transfer protects ECs and EPCs from anchorage- or nutrient-deprived stress, and enhances neovascularization, suggesting that ILK is an optimal target gene for genetically modified cell-based therapy.