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Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis.

ABSTRACT: Myocardial ischaemia (MI) results in extensive cardiomyocyte death and reactive oxygen species (ROS)-induced damage in an organ with little or no regenerative capacity. Although the use of adult bone marrow mesenchymal stem cells (BMMSCs) has been proposed as a treatment option, the high cell numbers required for clinical use are difficult to achieve with this source of MSCs, and animal studies have produced inconsistent data. We recently demonstrated in small and large animal models of acute MI that the application of human term placenta-derived multipotent cells (PDMCs), a foetal-stage MSC, resulted in reversal of cardiac injury with therapeutic efficacy. However, the mechanisms involved are unclear, making it difficult to strategize for therapeutic improvements. We found that PDMCs significantly reduced cardiomyocyte apoptosis and ROS production through the paracrine factors GRO-α, HGF and IL-8. Moreover, culturing PDMCs on plates coated with laminin, an extracellular matrix (ECM) protein, resulted in significantly enhanced secretion of all three paracrine factors, which further reduced cardiomyocyte apoptosis. The enhancement of PDMC paracrine function by laminin was mediated through αvβ3 integrin, with involvement of the signalling pathways of JNK, for GRO-α and IL-8 secretion, and PI3K/AKT, for HGF secretion. Our results demonstrated the utility of PDMC therapy to reduce cardiomyocyte apoptosis through modulation of ECM proteins in in vitro culture systems as a strategy to enhance the therapeutic functions of stem cells.

SUBMITTER: Peng KY

PROVIDER: S-EPMC5543513 | biostudies-other | 2017 Aug

REPOSITORIES: biostudies-other

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Project description:Integrins provide the primary link between mesenchymal stem cells (MSCs) and their surrounding extracellular matrix (ECM), with different integrin pairs having specificity for different ECM molecules or peptide sequences contained within them. It is widely acknowledged that the type of ECM present can influence MSC differentiation; however, it is yet to be determined how specific integrin-ECM interactions may alter this or how they change during differentiation. We determined that human bone marrow-derived mesenchymal stem cells (hMSCs) express a broad range of integrins in their undifferentiated state and show a dramatic, but transient, increase in the level of ?5 integrin on day 7 of osteogenesis and an increase in ?6 integrin expression throughout adipogenesis. We used a nonfouling polystyrene-block-poly(ethylene oxide)-copolymer (PS-PEO) surface to present short peptides with defined integrin-binding capabilities (RGD, IKVAV, YIGSR, and RETTAWA) to hMSCs and investigate the effects of such specific integrin-ECM contacts on differentiation. hMSCs cultured on these peptides displayed different morphologies and had varying abilities to differentiate along the osteogenic and adipogenic lineages. The peptide sequences most conducive to differentiation (IKVAV for osteogenesis and RETTAWA and IKVAV for adipogenesis) were not necessarily those that were bound by those integrin subunits seen to increase during differentiation. Additionally, we also determined that presentation of RGD, which is bound by multiple integrins, was required to support long-term viability of hMSCs. Overall we confirm that integrin-ECM contacts change throughout hMSC differentiation and show that surfaces presenting defined peptide sequences can be used to target specific integrins and ultimately influence hMSC differentiation. This platform also provides information for the development of biomaterials capable of directing hMSC differentiation for use in tissue engineering therapies.

Project description:BACKGROUND:Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS:hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis. RESULTS:Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS:Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.

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Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis.

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