Integrin α2β1 Is Required for Synergistic Effect of Cells and Matrix Therapy in Improving the Perfusion, Viability and Function of Infarcted Hearts

Abstract

s S377 BACKGROUND: Milrinone (Mil) is a potent cardiac inotrope that improves cardiac function and has essential anti-inflammatory effects. Mil has a half life of about 2 hrs hence it is given normally via central line as continuous infusion. We hypothesize that intravenous injection of Mil prepared in microparticles prolongs its pharmakokinetics by sustained slow-release mechanism that enhances myocardial function in a rat model of ischemic cardiomyopathy. METHODS: Polylactic-co-glycolic acid (PLGA) microparticles (MPs) loaded with Mil (Mil-PLGA microparticles) were prepared by double emulsion-solvent evaporation technique. The 2,3 dichloro-5,6-dicyanobenzoquinone (DDQ) test and spectrophotometry were used to estimate the percentage of milrinone encapsulation and for quantification, respectively. Myocardial infarctionwas induced in all rats by ligation of the left anterior descending (LAD) artery. One week after ligation, rats were randomized into one of three groups: Control group (n1⁄4 8) received intravenous injection of empty PLGA microparticles (250 mL); Group II (n 1⁄4 8) received bolus intravenous Mil (50mg/kg/min); and Group III (n 1⁄4 8) received bolus intravenous Mil-PLGA microparticles with the same dose as Group II. All intravenous injection was given via tail vein slowly over 10 mins. Transthoracic echocardiography and non-invasive heart rate and blood pressure measurements were performed at different time intervals for 24hrs after the injection. The rats were scarified 24 hrs after the injection. The plasma level of Mil at 24 hrs following the injection was measured using HPLC. Enzymelinked immunosorbent assay was used to measure the serum cytokines levels at 24hrs after administration. RESULTS: In-vitro Mil-release kinetic studies showed a sustained release of Mil from the particles over a 24-hour period. Rats treated with Mil-PLGA microparticles had significant increases in left-ventricular ejection fraction at 90mins, 3hrs, 6hrs, 12, 24 hrs post-treatment compared with other groups (P 0.05). CONCLUSION: Encapsulation of Mil in microparticles provides a novel drug delivery method that can potentially prolong its positive inotropic and anti-inflammatory effects. This may benefit patients with end stage heart failure who need prolong inotrpic support. 728 INTEGRIN a2b1 IS REQUIRED FOR SYNERGISTIC EFFECT OF CELLS AND MATRIX THERAPY IN IMPROVING THE PERFUSION, VIABILITY AND FUNCTION OF INFARCTED HEARTS A Ahmadi, B McNeill, B Vulesevic, M Kordos, L Mesana, S Thorn, JM Renaud, E Manthorp, D Kuraitis, H Toeg, TG Mesana, RS Beanlands, JN DaSilva, RA deKemp, M Ruel, EJ Suuronen Ottawa, Ontario BACKGROUND: Injectable delivery matrices hold promise for enhancing engraftment and the overall efficacy of cardiac cell therapies; however, the mechanisms responsible remain largely unknown. Here, we studied a combined therapy of collagen matrix and circulating angiogenic cells (CACs), which was applied to MI mice. We evaluated the role of integrins (Itg) and integrin-linked kinase (ILK) in CACmatrix interaction. METHODS AND RESULTS: Seven days after left anterior descending coronary artery ligation, female C57BL6/J mice received one of the following treatments delivered to the infarcted myocardium: CACs, collagen matrix, CACs+collagen matrix, or PBS. CACs were green fluorescent protein (GFP)+ bone marrow cells from male C57BL/6-Tg(eGFP) mice. Echocardiography demonstrated that the left ventricular ejection fraction (EF) of infarcted mouse hearts was 36 2% and improved to 56 3% 3wk after treatment of CACs+matrix; whereas no improvement occurred with other treatments ( 40 2%; p<0.001). PET analysis revealed elevated glucose metabolism (18F-FDG) and perfusion (13N-NH3) only with CACs+matrix by 35% and 29%, respectively (p 0.04). Histology showed that the anterior to posterior LV wall thickness ratio was greater for CACs+matrix (0.7 0.1) than for all other groups ( 0.3 0.01; p<0.001). More arterioles were detected in hearts injected with CACs+matrix (10.9 1.1/FOV) compared to other treatments ( 6.2 0.5; p<0.001). Moreover, Y chromosome q-PCR indicated increased intramyocardial retention of transplanted cells in CACs+matrix group (by 8.6 1.4-fold; p1⁄40.001) relative to CACs only. ILK expression was higher in hearts treated with CACs+matrix (1.4 0.1-fold) or matrix (1.6 0.1-fold) compared to CACs-only or PBS treatment (p 0.02). In vitro, collagen matrix culture increased ILK protein levels by w2-fold in CACs compared to fibronectin (p1⁄40.003). An integrin (Itg) expression screen revealed an increase in the collagenbinding receptor Itga2 in matrix-cultured CACs. Blocking Itga2b1 resulted in ILK down-regulation (30% less), and reduced adhesion (82% less), proliferation (77% less) and secretion of SDF-1 (50% less) in matrix-cultured CACs, compared to non-blocked CACs (all p<0.05). Blocking Itga2b1 in CACs also abolished the in vivo therapeutic effects of CACs+matrix treatment in infarcted hearts: there was no improvement in EF (baseline1⁄441 3%; follow-up EF1⁄442 3%), vascular density was reduced and CAC engraftment was negligible, despite normal cell viability upon treatment delivery. CONCLUSION: The collagen matrix does not simply provide passive delivery of CACs, but rather, it interacts with the cells through integrin a2b1 to activate intracellular signaling leading to enhanced proliferation, adhesion, and cytokine production. This was essential for the synergistic therapeutic benefit of CACs+matrix treatment post-MI.