Effect And Mechanism Of Lipopolysaccharides Pretreatment On Cardiac Stem Cells Migration

BackgroundAdult mammalian heart tissue is traditionally thought to be terminally differentiated cells without self-renewal potential. This classic concept leads to a consensus that the damaged heart tissue cannot be self-repaired, scar tissue replaces damaged myocardial tissue resulting in the impairment of cardiac function. It has been reported that cardiac stem cells (CSCs) can repair the damage to the muscle, which occurs during many pathological conditionings such as heart attack, due to its ability of regeneration and migration. The fate of CSCs under various heart disorders is crucial for the cardiac function preservation or deterioration. Lipopolysaccharide (LPS), the major molecular component of the outer membrane of gram-negative bacteria, is a main causative agent of septic shock which could result in cardiac dysfunction. However, LPS-preconditioning has recently been reported to induce the reduction of infarct size and promote the recovery of post-ischemic cardiac function in the ischemia-reperfusion heart. The mechanism that underlies the paradoxical role of inflammation in cardiac injury and repair is unclear. We hypothesized that it might be due to activation or inactivation of CSCs proliferation/migration. The Wnt/β-catenin signaling pathway plays a crucial role during embryonic development by controlling cell proliferation, stem cell maintenance and cell fate decisions, and migration. Whether activation of P-catenin is also involved in the LPS-pretreatment induced changes in proliferation and migration of CSCs is unclear.ObjectivesThe aims of this study were to investigate the effect of LPS on the proliferation and migration of CSCs, and to explore whether β-catenin is involved in its underlying mechanism, and finally to observe whether LPS pretreatment-induced CSCs proliferation or migration play an important role in the cardiac repair in the rat model of ischemia-reperfusion injury.MethodsCSCs were isolated from the hearts of neonatal Sprague-Dawley rats. CSCs were exposed to different dose of LPS (0,0.01,0.1,1 μg/ml) with or without pretreatment of 17-AAG or FH535. CCK-8 assay kit was used to evaluate the cell proliferation of CSCs. Migration was detected using 24-well transwell system in vitro cultured CSCs. Male adult SD rat hearts were suffered 30 min of ischemia and 120 min of reperfusion. At the end of ischemia, cultured CSCs pretreated with or without LPS (0.01 μg/ml) were injected into four sites along the border zone of the ischemia area (area at risk). Migration of CSCs was detected using carboxyfluorescein diacetate (CFDA)-labelled method in myocardial infarction rat model. The expression of toll like receptor 4 (TLR4), connexin 43 (CX43), β-catenin, active β-catenin, heat shock protein 90 (HSP90) were analyzed using western blotting.Results(1) Compared with control group, treatment of CSCs with LPS (0.01 or 0.1 μg/mL) for 1,3, or 7 d had no effect on cell proliferation. A transwell-based migration assay was performed after 24 h treatment of LPS. Compared with control group, lower and medium doses of LPS significantly increased the number of migrated CSCs, which reached a peak at 0.01 μg/mL.(2) The effect of LPS (0.01 μg/mL) on CSCs migration in the isolated rat hearts were assessed by using CFDA-labeled method. At the end of reperfusion, the number of LPS-pretreated CSCs migrated into the risk area was higher than the LPS-untreated CSCs. Injection of LPS-pretreated CSCs also caused a significant decrease in infarct size than that of LPS-untreated CSCs group.(3) Compared with control group, lower dose of LPS did not influence the expression of CX43, TLR4, and total amount of β-catenin protein in CSCs. However, it enhanced the levels of active β-catenin and nuclear β-catenin protein. Meanwhile, β-catenin inhibitor FH535 inhibited the enhancement of lower dose of LPS-induced migration.(4) HSP90 protein expression markedly increased in CSCs treated with LPS (0.01μg/mL). Compared with LPS group, after preincubated with HSP90 inhibitor 17-AAG, the LPS-induced enhancement of active β-catenin protein and nuclear P-catenin protein was abolished. And 17-AAG also prevented the lower dose of LPS-induced cell migration.ConclusionIn summary, the present study showed that low dose of LPS induced increase in migration of CSCs, reduced the infarct size in ischemic-reperfusion heart. The mechanism might be due to the activation and translocation of β-catenin via HSP90-dependent manner. These results suggested the LPS-pretreated CSCs as potential treatment for the repair of damaged heart tissue.