Abstract 508: The Role of Dual Specificity Phosphatase 5 in Post Ischemic Angiogenesis

Abstract

Background: Dual specificity phosphatase 5 (DUSP5) is a member of the protein phosphatase subfamily that inactivate their target kinases by dephosphorylating both the phosphoserine/threonine and phosphotyrosine residues. DUSP5 regulates the mitogen activated protein (MAP) kinase ERK1/2, and although it has been shown to play a key role in embryonic vascular development, its role in post ischemic angiogenesis is not known. This study sort to investigate the role of DUSP5 is endothelial cell function and angiogenesis in ischemia. Methods: Hindlimb ischemia (HLI) was induced in mice and at day 3 post HLI, expression of DUSP5 was assessed in the ischemic hind limbs. We assessed DUSP5 expression in 3 major cell types found the ischemic hindlimb, endothelial cells or ECs (human umbilical vein endothelial cells, HUVECs), skeletal muscle myoblast (C2C12) and vascular smooth muscles cells (VSMC). Cells were exposed to simulated ischemia (2% oxygen with serum starvation) for 24 hours analyzed for DUSP5 mRNA and protein expression. The effect of loss of DUSP5 on EC function in ischemia was assessed by CRISPR/Cas9 mediated knock down of DUSP5 followed by analysis of EC proliferation, apoptosis and tube formation in simulated ischemia. Results: DUSP5 protein levels were significantly upregulated in mouse post ischemic hind limbs (ischemic (I) vs non-ischemic (NI); 1.63 ± 0.39 vs. 0.22 ± 0.02, p<0.05, n=3). Post ischemic exposure, ECs and C2C12 showed significant upregulation of DUSP5 protein. (DUSP5/Tubulin, I vs NI, HUVEC: 0.43 ± 0.09 vs. 0.09 ± 0.03, p<0.02; C2C12: 1.77 ± 0.10 vs. 0.42 ± 0.01, p< 0.01, n=5). VSMCs showed no significant change in DUSP5 expression. Knock down of DUSP5 in HUVECs resulted in significant decrease in cell proliferation in ischemia (O.D 450 control vs knock-down : 0.46 ± 0.01 vs. 0.34 ± 0.01, p<0.01, n=7) but no significant change in apoptosis (O.D 450 control vs knock-down: 0.13 ± 0.01 vs. 0.13 ± 0.00, p>0.05, n=7). It also resulted in significant impairment in, in vitro angiogenesis (tube/sq cm: 28.6 ± 1.2 vs. 16.43 ± 1.6, p<0.01, n=7). Conclusion: DUSP5 is highly upregulated in ischemic endothelial cells and plays an important role in EC proliferation and post ischemic angiogenesis.