Tissue inhibitor of matrix metalloproteinase-3 or vascular endothelial growth factor transfection of aged human mesenchymal stem cells enhances cell therapy after myocardial infarction

Abstract

Mesenchymal stem cell (MSC) transplantation has been proposed as a potential therapeutic approach for ischemic heart disease, but the regenerative capacity of these cells decreases with age. In this study, we genetically engineered old human MSCs (O-hMSCs) with tissue inhibitor of matrix metalloproteinase-3 (TIMP3) and vascular endothelial growth factor (VEGF) and evaluated the effects on the efficacy of cell-based gene therapy in a rat myocardial infarction (MI) model. Cultured O-hMSCs were transfected with TIMP3 (O-TIMP3) or VEGF (O-VEGF) and compared with young hMSCs (Y-hMSCs) and non-transfected O-hMSCs for growth, clonogenic capacity, and differentiation potential. In vivo, rats were subjected to left coronary artery ligation with subsequent injection of Y-hMSCs, O-hMSCs, O-TIMP3, O-VEGF, or medium. Echocardiography was performed prior to and at 1, 2, and 4 weeks after MI. Myocardial levels of matrix metalloproteinase-2 (MMP2), MMP9, TIMP3, and VEGF were assessed at 1 week. Hemodynamics, morphology, and histology were measured at 4 weeks. In vitro, genetically modified O-hMSCs showed no changes in growth, colony formation, or multi-differentiation capacity. In vivo, transplantation with O-TIMP3, O-VEGF, or Y-hMSCs increased capillary density, preserved cardiac function, and reduced infarct size compared to O-hMSCs and medium control. O-TIMP3 and O-VEGF transplantation enhanced TIMP3 and VEGF expression, respectively, in the treated animals. O-hMSCs genetically modified with TIMP3 or VEGF can increase angiogenesis, prevent adverse matrix remodeling, and restore cardiac function to a degree similar to Y-hMSCs. This gene-modified cell therapy strategy may be a promising clinical treatment to rejuvenate stem cells in elderly patients.

FIG. 1.

Proliferation of young human mesenchymal stem cells (Y-hMSCs) and old human mesenchymal stem cells (O-hMSCs). (a) Photomicrographs of cultured hMSCs on day 3, 7, and 11 of the initial passage. Magnification, 100×. (b) The Cell Counting Kit-8 (CCK-8) colorimetric assay was performed on cells (passage 3) to assess proliferation. Growth curves showed a significant difference between Y-hMSCs and O-hMSCs (**p<0.01) whether or not they were genetically modified (O-hMSCs transfected with vascular endothelial growth factor [O-VEGF] and tissue inhibitor of matrix metalloproteinase-3 [O-TIMP3]). (c) Cultured cells (passage 3, 100 cells/well) stained with Crystal Violet 2 weeks after plating (arrows indicate representative colonies). (d) Quantification of colonies revealed significantly more colony-forming units (CFUs) in the Y-hMSC cultures. (**) p<0.01 vs. all other groups.

FIG. 2.

Transfection of old human mesenchymal stem cells (O-hMSCs) with plasmid vectors. (a) Photomicrographs of O-hMSCs transfected with the tissue inhibitor of matrix metalloproteinase-3 (TIMP3) or vascular endothelial growth factor (VEGF) green fluorescent protein (GFP)-expressing vector. Magnification, 200×. (b) Flow cytometry assessed the transfection efficiency for O-hMSCs transfected with TIMP3 (14.5%±1.1%) and VEGF (13.3%±1.5%). (c and d) RT-PCR showed that TIMP3 and VEGF mRNA levels in O-hMSCs were highest 3 days after transfection. A significant overexpression of TIMP3 and VEGF in transfected O-hMSCs lasted for 7 days (**) p<0.01 vs. prior to transfection. (e and f ) Western blots showed that TIMP3 and VEGF protein levels in O-hMSCs were highest 3 days after transfection and lasted for 7 days. (**) p<0.01 vs. prior to transfection. (β-Actin served as internal control; day 0 was prior to transfection).

FIG. 3.

Genetically modified old human mesenchymal stem cells (O-hMSCs) enhanced cardiac function. (a and b) Analysis of ejection fraction (a) and fractional shortening (b) showed that, compared to the medium control and O-hMSC groups, Y-hMSC, and O-hMSCs transfected with vascular endothelial growth factor (O-VEGF) and tissue inhibitor of matrix metalloproteinase-3 (O-TIMP3) groups showed significant functional improvement. (**) p<0.01. Day 0 was the day of myocardial infarction (MI) and cell transplantation. (c and d) Quantitative analysis of hemodynamic parameters dP/dt max (c) and dP/dt min (d) showed significant improvement in the O-hMSC, Y-hMSC, O-VEGF, and O-TIMP3 groups. (*) p<0.05 vs. medium control; (#) p<0.05 vs. O-hMSCs.

FIG. 4.

Vascular endothelial growth factor (VEGF), tissue inhibitor of matrix metalloproteinase-3 (TIMP3), matrix metalloproteinase (MMP) mRNA expression and MMP activity. (a) Expression of VEGF, TIMP3, MMP2, and MMP9 mRNA in the rat myocardium was evaluated by RT-PCR 1 week after MI and cell transplantation. β-Actin served as internal control. (b) VEGF mRNA levels were significantly higher in the young human mesenchymal stem cells (Y-hMSCs) and old (O)-VEGF groups. (c) TIMP3 mRNA levels were significantly higher in the Y-hMSC and O-TIMP3 groups. (d and e) MMP2 (d) and MMP9 (e) mRNA levels were significantly lower in the Y-hMSC and O-TIMP3 groups. (f ) TIMP3 activity was evaluated using reverse zymography and was significantly higher in the Y-hMSC and O-TIMP3 groups. (g) MMP2 and MMP9 activities were evaluated using zymography, and both were significantly lower in the Y-hMSC and O-TIMP3 groups. (*) p<0.05 vs. all other groups. C, Medium control; O, O-hMSCs; Y, Y-hMSCs; O-V, O-VEGF; O-T, O-TIMP3).

FIG. 5.

Cell survival and capillary density increased with transplantation of genetically modified old human mesenchymal stem cells (O-hMSCs). (a) Photomicrographs of myocardial sections 4 weeks after myocardial infarction (MI) and cell transplantation. Scale bar, 200 μm. The sections were stained with a human mitochondrial antibody (brown, indicated by arrows). (b) A greater number of transplanted cells was present in the infarcted myocardium of the young (Y)-hMSCs and O-hMSCs transfected with vascular endothelial growth factor (O-VEGF) and tissue inhibitor of matrix metalloproteinase-3 (O-TIMP3) groups. (**) p<0.01 vs. O-hMSCs. (c) Peri-infarct zone stained with CD31 antibody (brown, indicated by arrows) 4 weeks after MI and cell transplantation. Scale bar, 200 μm. (d) Capillary density was increased in the Y-hMSC, O-VEGF, and O-TIMP3 groups. (*) p<0.05, (**) p<0.01 vs. medium control and O-hMSCs.

FIG. 6.

Collagen content and infarct size decreased with transplantation of genetically modified old human mesenchymal stem cells (O-hMSCs). (a) Masson's Trichrome staining of the infarct zone 4 weeks after cell transplantation (blue collagen; red myocardium). Scale bar, 200 μm. (b) Quantitative analysis of collagen content in the infarcted area showed less collagen in the young (Y)-hMSC and O-hMSCs transfected with vascular endothelial growth factor (O-VEGF) and tissue inhibitor of matrix metalloproteinase-3 (O-TIMP3) groups. vs. the medium control and O-hMSC groups. (*0 p<0.05; (**) p<0.01. (c) Mid-ventricular heart slices 4 weeks after cell transplantation. Thinning of the left ventricular free wall and dilation of the left ventricle were noted in the medium control and O-hMSC groups. (d) Scar size was significantly reduced in the Y-hMSC, O-VEGF, and O-TIMP3 groups compared with medium control and O-hMSC groups (**) p<0.01.

FIG. 7.

Post-myocardial infarction (MI) tumor necrosis factor-α (TNF-α) level increased less with transplantation of genetically modified old human mesenchymal stem cells (O-hMSCs). TNF-α level was similar among groups before MI (day 0) but increased in all groups after MI. After MI and cell transplantation, TNF-α was significantly lower in the young (Y)-hMSC and O-hMSCs transfected with vascular endothelial growth factor (O-VEGF) and tissue inhibitor of matrix metalloproteinase-3 (O-TIMP3) groups. O-VEGF, and O-TIMP3 groups vs. the medium control and O-hMSC groups (*p<0.05).