mesenchymal stem cell and endothelial progenitor cells on the status of CXCR-4 signaling and expression of related genes

Abstract

Abstract: Background: Today, many efforts have been focused on the reduction of cell mortality and an increase of angiogenesis during transplantation to target tissues. Tissue engineering modalities such as encapsulation are touted as an emerging approach for high throughput transplantation. The emergence of numerous tissue engineering and regenerative medicine techniques cell encapsulation paves a way to heal and restore the function of various injured tissues mainly cardiovascular system.Here, we aimed to investigate the role of alginate-gelatin encapsulation on the dynamic of rat cardiomyoblasts in vitro. we investigated the angiogenic potential of endothelial progenitor cells juxtaposed with mesenchymal stem cells inside alginategelatin microspheres with stromal derived factor-1α for 7 days.

Methods: Rat cardiomyoblasts cell line H9C2 were enclosed by using alginate-gelatin microspheres and incubated for 7 days. MTT method was used to examine cell viability. The level of genes associated with cardiomyoblasts maturation MYL7, NPPA, NKX2-5, and GATA4 real-time PCR. ELISA was used to measure the protein levels of Bcl-2 and Bax factor post-encapsulation. The level of SOD, GPx, and TAC was detected by biochemical analyses. Western blotting was performed to measure the content of AMP-activated protein kinase.we designated six encapsulated groups including EPCs, EPCs/SDF-1α, MSCs, MSCs/SDF-1α, EPCs+MSCs and EPCs+MSCs/SDF-1α. Cells were encapsulated with a mixture of 1% alginate and 2% gelatin hydrogel. Cell survival was examined by MTT assay. Endothelial differentiation was determined by flowcytometry and ELISA. Matrigel assay was used to detect tubulogenesis activity. Thefunctional behavior of cells was detected by Ac-Dil-LDL uptake. Cell migration was analyzed by Transwell insert and gelatin zymography analyses. By using real-time PCR, we measured the transcription of Akt and PK1.

Results: We found that encapsulation was able to increase the viability of rat cardiomyocytes after 7 days. The decreased level of Bcl-2 (p < 0.001) coincided with non-significant differences in the level of Bax (p > 0.05).The transcription level of all genes MYL7, NPPA, NKX2-5, and GATA4 were found to down-regulate compared to the control non-treated cells (p < 0.05). No significant differences were found regarding the level of SOD, GPx,and TAC compared to the control (p>0.05). According to western blotting, revealed a reduced level of AMPK following 7-day incubation of rat cardiomyoblasts (p < 0.05).Furthermore We found an increase in cell viability in MSCs/SDF-1α microspheres compared to EPCs group (p<0.05). EPC/MSCs co-culture increased the percent of CD133 cells while we found high CD31 levels in MSCs group (p<0.05) while the protein level of VE-cadherin dominant in encapsulated EPCs. Juxtaposition of EPC/MSCs increased the formation of tube area compared to SDF-1a free condition (p<0.001). SDF-1α had the potential to increase in AC-LDL uptake in MSCs and EPCs/MSCs groups. In addition, SDF-1α increased migration and MMP-9 activities. SDF-1α could induce the expression of PK1 and Akt in encapsulated cells, especially in a co-culture system.

Conclusion: Data confirmed that the encapsulation of rat cardiomyoblasts with alginate-gelatin microspheres maintained the cells multipotentiality Alginate-gelatin had the potential to alter the angiogenic potential of progenitor cells in the presence of SDF-1α.The use of alginate / gelatin microcapsules is not only an appropriate cellular pathway for treatment, but also has an effect on cellular viability and cellular potency such as angiogenesis, which increases the cell's function in the therapeutic process.