Interaction of alpha-6 integrin with laminin-1 is essential for cardiac regeneration mediated by cardiac primitive cells.

According to the recent findings, CD117(+) cells reside in the subepicardium and epicardially-derived cells may represent a population of cardiac primitive cells. We have previously shown the presence of laminin-1, typical of developing heart, in the subepicardium of human adult heart with chronic ischemic cardiomyopathy. Hence, the hypothesis arises that laminin-1 may influence cardiac primitive cells biology. CD117(+) cells from adult human heart were cultured on laminin-1, laminin-2 or albumin. Specific human alpha6 integrin small interfering RNA (siRNA) was used to examine the contribution of alpha6 integrin and laminin interactions to the cell proliferation, apoptosis and migration in vitro. Both laminin-1 and laminin-2 increased the proliferation of cells, as evaluated with BrdU incorporation. In the presence of laminin-2 the proliferation rate reached 7.9±0.4% (n=4), whereas during the culture of cardiac primitive cells on laminin-1 the incorporation of BrdU was over 4-fold higher (n=4, p<0.001). Transfection with alpha6 integrin siRNA did not influence significantly proliferation rate. Apoptosis, detected in TUNEL assay, in the presence of laminin-1 was 2-fold lower (2,6±0.3%, n=5) than in the culture of cells on laminin-2 (4.3±0.5%, n=5, p<0.05) and 6-fold lower than in the presence of albumin (12.2±1.8%, n=3, p<0.001). These effects of both laminin isoforms were inhibited with alpha6 integrin siRNA, when the apoptosis rate increased 7-fold (n=3, p<0.05) and 5-fold (n=3, p<0.05) in the presence of laminin-1 and -2, respectively. The presence of laminin-1 or laminin-2 increased both transverse and invasive displacement of cardiac primitive cells in vitro. In the scratch wound assay the speed of migration was 36,8±2,4μm/h (n=3) in the presence of albumin and it reached 51,5±5,4μm/h (n=4) on laminin-2 and 94,7±7,1μm/h (n=4) on laminin-1 coated dishes. The number of migrating CD117(+) cells increased 2,5-fold (n=3, p<0.05) on laminin-2 and 4-fold (n=3, p<0.05) on laminin-1 with respect to control. In all cases, the migration on laminin-1 outweighed significantly that on laminin-2. When CD117(+) cells from adult human hearts were transfected with alpha6 integrin siRNA, the invasive displacement in the laminins presence diminished significantly. In conclusion, the signaling mediated by alpha6 integrin in the presence of laminin is essential for cardiac primitive cells survival and migration towards myocardium. Among laminin isoforms, mostly laminin-1 is responsible for the protection of CD117(+) cells pool. The abundance of laminin-1 in the subepicardium may be directly related to the stimulation of cardiac regeneration.