The Effects Of Heat Shock Protein 90 On The Ability Of Survival And Movement Of Rat Mesenchymal Stem Cells

ParⅠHeat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathwaysBackground:Transplantation of bone marrow-derived mesenchymal stem cells (MSCs) has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability after transplantation limited the reparative capacity of these cells in vivo (der Bogt KE et al.2009). Myocardial necrosis induces complement activation and free radical generation, triggering a cytokine cascade, and then donor cell apoptosis (Frangogiannis et al.2002)。Neovascularization can provide the implanted cells with adequate microenvironment to enhance survival and function, whereas exchange vessels loss and scar formation attenuate the ability to nourish the implanted cells. The donor cell growth appears tenuous and their cardiac reparative benefits are transient (Dai et al. 2005). Preconditioning MSCs with some physical or cytochemical stimuli may improve the therapeutic efficacy of cell therapy, including hypoxia (Hu et al.2008), growth factors (Hahn et al.2008), and some cytokines (Gui et al.2007; Pasha et al.2008), more available for translational application than gene transfection.Heat shock protein 90 (Hsp90) is deemed as the most active molecular chaperone which plays a critical role in the development and progression of cancer (Tsutsumi et al. 2009). It also acts as a checkpoint, leading to survival or death under stress stimulus.Recently, Hsp90 was found to exert a cardioprotective effect via the PI3K/Akt pathway (1)(Wang et al.2009).Here, we established an in vitro apoptosis model induced by hypoxia and serum deprivation to investigate the role of exogenous Hsp90 in rat bone marrow MSCs on the apoptosis and signaling molecules involved.Objective:To explore the cytopretection of HSp90 against hypoxia and serum deprivation induced apoptosis and the possible mechanisms in rat mesenchymal stem cells.Methods:Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was assessed by Hoechest 33258 nuclear staining and flow cytometric analysis with annexin V/PI staining. The gene expression of TLR4 and ErbB2 was detected by real-time PCR. The protein level of cleaved-caspase-3, bcl-2, bcl-xL, bax, total-Erk, phospho-Erk, total-Akt, phospho-Akt, and hsp90 were detected by Western-blot. The production of nitric oxide was measured by spectrophotometric assay.Results:Hsp90 improves MSCs viability and protects MSCs against apoptosis induced by serum deprivation and hypoxia. The protective role of Hsp90 not only elevates bcl-2/bax and bcl-xL/bax expression and attenuates cleaved caspase-3 expression via down-regulating membrane TLR-4 and ErbB2 receptors and then activating their downstream PI3K/Akt and ERK1/2 pathways, but also enhances the paracrine effect of MSCs.Hsp90 significantly protected MSCs against apoptosis induced by hypoxia and serum deprivation.Conclusions:These findings demonstrated a novel and effective treatment strategy against MSC apoptosis in cell transplantation.PartⅡHeat shock protein 90 enhances rat mesenchymal stem cells migration via PI3K/Akt and ERK1/2 pathwaysBackground:Bone marrow mesenchymal stem cells (MSCs) are pluripotent stem cells that localize in the stromal compartment of the bone marrow, where they support hematopoiesis and differentiate into mesenchymal lineages such as bone, cartilage, muscle tissues. Accumulating evidence suggests that MSCs are attractive candidates for cardiovascular therapy because of their capacity to facilitate myocardial repair and neovascularization in models of cardiac injury. MSCs are able to migrate into the injured myocardium from the circulation and contribute to cardiac repair post myocardial infarction.Some clinical trial suggest that the benefits of stem cells therapy derive primarily from secretion of paracrine factors, appropriate homing of these cells is essential to enable such factors to function, because their effects are, limited to a short range, requiring relatively high local concentrations. Some chemokine such as granulocyte colony stimulating factor (G-CSF), and the closely related granulocyte/macrophage colony-stimulating factor (GM-CSF), SDF-l,erythropoietin, and stem cell factor (SCF) (the ligand for c-kit) and some growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-1, can regulate the migration of MSCs. The SDF-1/CXCR4 axis is central for stem cell mobilization from the bone marrow and homing to ischemic tissues.Some clinical trial suggest that the benefits of stem cells therapy derive primarily from secretion of paracrine factors, appropriate homing of these cells is essential to enable such factors to function, because their effects are, limited to a short range, requiring relatively high local concentrations. Some chemokine such as granulocyte colony stimulating factor (G-CSF), and the closely related granulocyte/macrophage colony-stimulating factor (GM-CSF), SDF-l,erythropoietin, and stem cell factor (SCF) (the ligand for c-kit) and some growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-1, can regulate the migration of MSCs. and Adhesive activation such as VCAM-1 and ICAM-1.The SDF-1/CXCR4 axis is central for stem cell mobilization from the bone marrow and homing to ischemic tissues.Migration of cells requires degradation of basement membrane, which is dependent on the production of matrix-degrading enzymes, especially those capable of degrading type IV collagen, such as MMPs, especially MMP-2 and MMP-9.Heat shock protein90 (Hsp90) is a highly conserved, constitutively expressed protein in eucaryotic cell, which account for 2 5% of total cellular proteins even in the absence of stress. There are two isoforms:Hsp90 a and Hsp90β, which are essential for the viability of eukaryotic cells.The function of Hsp90 is for maintaining the stability and function of numerous proteins referred to as client proteins.Previous studyshowed that using mAb 4C5, a function-blocking monoclonal antibody against Hsp90 in human breast cancer cells, which altered actin dynamics to inhibit cell invasion.Whether Hsp90 play an important role in MSCs migration is still unclear.Objective:In the present study, we hypothesized that Hsp90 would be involved in the migration of MSCs. Our data may provide new mechanistic insights into the hsp90-mediated migration of MSCs.and, may provide new perspectives in the development of therapeutic strategies targeting MSCs migration and angiogenesis.Methods:Rat bone marrow-derived mesenchymal stem cells were harvested from femora and tibia by density gradient centrifugation.The espremiental was divided five groups:control group, Hsp90 a group, siRNAHsp90 a group,17-AAG group, wortmannin group, U0126 group. The ability of MSCs cell migration is determined using the wound healing assay and transwell assay. The activity of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) were estimated by gelatin zymography. The mRNA levels of MMP-2,MMP-9,CXCR4 and VCAM-1 were detected by real-time PCR. The protein expression of MMP-2,MMP-9 and ERK1/2, phospho-ERK1/2, Akt, phospho-Akt was determined by Western-blot.Results:Treatment with RhHsp90a significantly enhances MSCs migration. However, sirhsp90 a significantly decreased MSCs migration compared with treatment of hsp90 a. Treatment with RhHsp90 a enhance the MSCs secrete MMP-2 and MMP-9; especially increased the activity of MMP-9.and increased the expression of CXCR4 and VCAM-1. Our data showed that hsp90αmodulate the rat MSCs migration via MMPs and CXCR4. PI3K/Akt and ERK signaling pathways mediates these effects.Conclusions:These findings indicated that RhHsp90a promoted MSCs migration, elevated the expression of MMP-2 and MMP-9, and via PI3K/Akt and ERK signaling pathways mediates these effects. Hsp90 is a candidate drug for enhancing MSCs migration.