Endoplasmic Reticulum Stress Apoptotic Pathways And Paracrine Effects Of MSCs Stimulated By Hypoxia And Serum Deprivation

Cardiomyocytes have been traditionally regarded as terminally differentiated cells that compensate for cardiac dysfunction through hypertrophy. Myocardial infarction (MI) induces the irreversible loss of cardiomyocytes, scar formation, and may ultimately result in congestive heart failure. Pharmacological therapies for the treatment of heart failure have traditionally targeted pump function and quality of life for endstage heart failure patients, and although several medications are available to limit the progression of the disease, the current therapies or interventional procedures do not lead to replacement of tissue and, thus, do not stop or reverse progression of adverse left ventricular (LV) remodeling in all patients. Cell therapy for heart failure has the potential to restore cardiac function by inducing neovascularization, regenerating and protecting cardiomyocytes, and may contribute to a causal therapy for heart failure after MI.Mesenchymal stem cells (MSCs) reside in the bone marrow nonhematopoietic tissues. It is well documented MSCs can be ease of isolation and expanded highly in culture and display genetic stability, reproducible attributes from isolate to isolate, reproducible characteristics in widely dispersed laboratories and compatibility with tissue engineering principles. Furthermore, MSCs are able to differentiate into endothelial cells, vascular smooth muscle cells, and perhaps even cardiac-like myocytes when transplanted into the ischemic heart. However, the therapeutic contribution of MSCs to myocardial repair can be caused by multiple factors including:direct differentiation into cardiac tissue including cardiomyocytes, smooth muscle cell, and vascular endothelial cells; secreting a variety of cytokines and growth factors that have paracrine activities; spontaneous cell fusion; and stimulating endogenous repair. A major dilemma in stem cell therapy for ischemic heart diseases is the low survival of transplanted cells in the ischemic and peri-infarcted region. Most implanted cells may die within 4 days after transplantation into the ischemic heart. Thus, improving grafted cell survival after transplantation is critical for enhancing the efficacy and efficiency of stem cell therapy and it is essential to have a clear understanding of the events and factors that may predispose MSCs to undergo apoptosis in ischemic tissue.We previously demonstrated that in response to hypoxia and serum deprivation (hypoxia/SD), both of which are components of ischemia in vivo, MSCs underwent the mitochondria-dependent apoptosis, while LPA inhibited hypoxia/SD-induced mitochondrial dysfunction by activating ERK1/2 and PI3K/Akt pathways. However, whether there are other proapoptotic pathways existing that mediate hypoxia/SD-induced MSC apoptosis remains largely unknown. In addition, we also found that the conditioned medium for hypoxia/SD-stimulated MSCs (MSCs-CM) could induce in vitro cardiomyocyte hypertrophy. However, the effects of MSCs-CM on the biological propoties of cardiac fibroblasts are not clear. Herein, we focused on the following questions:(1) Identify the roles of ER stress-associated apoptotic pathways and the effects of LPA on these pathways in hypoxia/SD-induced MSC apoptosis, and clarify the signaling mechanisms that mediated such actions of LPA. (2) Identify the effects of MSCs-CM on the biological properties of cardiac fibroblasts as well as the functional factors mediating such effects.This research can be divided into two sections:1. LPA rescues ER stress-associated apoptosis in hypoxia/SD-stimulated MSCsIn this section, we found that hypoxia/SD-induced MSC apoptosis was associated with ER stress, as shown by the induction of CHOP expression and procaspase-12 cleavage, while the effects were abrogated by LPA treatment, suggesting ER stress is also a target of LPA. Furthermore, hypoxia/SD induced p38 activation, and inhibition of p38 activation resulted in decreases of apoptotic cells, procaspase-12 cleavage and mitochondrial cytochrome c release but an enhancement of CHOP expression. Interestingly, p38 activation, a common process mediating various biological effects of LPA, was inhibited by LPA in this study, and the regulation of p38 pathway by LPA was dependent on LPA1/3/Gi/ERK1/2 pathway-mediated MKP-1 induction but independent of PI3K/Akt pathway. Collectively, our findings indicate that ER stress is a target of LPA to antagonize hypoxia/SD-induced MSC apoptosis, and the modulation of mitochondria and ER stress-associated apoptosis by LPA is at least partly dependent on LPA1/3/Gi/ERK/MKP-1 pathway-mediated p38 inhibition. This study may provide new anti-apoptotic targets for elevating the viability of MSCs for therapeutic potential of cardiac repair.2. The effects of MSCs-CM on cardiac fibroblast proliferation and collagen synthesisIn this section, we found that hypoxia/SD induced a NF-κBp65-dependent transcriptional upregulation of IL-1βand TNF-α. Furthermore, hypoxia/SD induced increased translation of pro-IL-1βand cleavages of pro-caspase-1 and pro-IL-1β, while the translational level of TNF-αremained unchanged. Unexpectedly, the releases of IL-1βand TNF-αwere undetectable unless in the presence of ATP or LPS. This suggests that the autocrine or paracrine effects of TNF-αand IL-1βby transplanted MSCs maybe negligible. We also found that hypoxia/SD also induced a transcriptional upregulation and a small amount secretion of IL-10, which were significantly enhanced by lipopolysaccharide (LPS) and the proteasome inhibitor MG132. Moreover, both the conditioned medium from MSCs and IL-10 could efficiently inhibit cardiac fibroblast proliferation and collagen expression, suggesting hypoxia/SD-induced IL-10 secretion from MSCs may play a paracrine role in cardiac remodeling. This study may help to understand the biochemical, cellular and molecular basis of anti-inflammatory and paracrine effects of MSCs.