Abstract 5564: Sequencial Activation of VEGF/Flk-1/MKK2/NFkappaB Signaling in Ischemic Preconditioning Induced Neovascularization: A Study with Flk-1+/− and MKK2−/− Knockout Mice

Abstract

Ischemic preconditioning mediated activation of VEGF-FLK-1-MKK2/NFkappaB pathway identifies these components as a critical regulator of myocardial angiogenesis in an infarcted myocardium. In the present study we used Flk-1+/− and MKK2−/− knockout mice in an attempt to address and prove this important clinical issue by identifying potential downstream candidates of VEGF signaling through Flk-1 receptor that trigger angiogenic signal during ischemic preconditioning (IP). Both wild type (WT) and Flk1+/− mice were subjected to either permanent LAD ligation (MI) or Ischemic preconditioning (IP) followed by MI. Flk-1+/− mouse hearts displayed significant increase in infarct size in Flk-1+/−PCMI (46%) group as compared to WTPCMI (33%) groups. Immunohistochemical analysis demonstrated decreased capillary and arteriolar density in both Flk-1+/−MI and Flk-1+/−PCMI groups as compared to WTMI and WTPCMI. Echocardiography demonstrated significant decrease in fractional shortening ( 21% vs 28%) and ejection fraction (42% vs 54%) following 4 weeks of myocardial infarction in Flk-1+/−PCMI group as compared with WTPCMI. As expected we found Flk-1+/− resulted in disruption of the cardioprotective effect by IP compared to WT. IP induced phosphorylation of p38MAPK, MKK2 was found to be inhibited (1.8 and 2 folds) significantly in both the Flk-1+/−MI and Flk-1+/−PCMI groups by Western blot analysis. Similarly Gelshift analysis showed down regulation of transcription factor NFkappaB (2 fold) in the Flk-1+/− mice as compared to WT. We further extended our experiments to validate our data in MKK2−/− mice. Hearts were subjected to LAD ligation. After 4-days of MI BSL-lectin infusion demonstrated significant disruption of neovascularization (capillary density) in the MKK2−/−PCMI (1520 counts/mm2) group compared to the WTPCMI ( 1850 counts/mm2). Gel-shift analysis in MKK2−/−PCMI group documented reduced NFkappaB activity (1.5 fold) compared to WTPCMI (2.4 fold). Taken together, this study validated for the first time that NFkappaB is a novel downstream mediator of VEGF/FLK-1/p38MAPK-MKK2 signaling and it induces IP mediated cardioprotection and angiogenesis via MKK2 signaling.