[Rat bone marrow mesenchymal stem cells-differentiated endothelial like cells facilitate angiogenesis and the Rho kinase contribution].

ABSTRACT

The aim of this study was to investigate the influence of endothelial like cells differentiated from rat bone marrow mesenchymal stem cells (rBMSC-ECs) on angiogenesis and the effect of Rho kinase (ROCK) inhibitor using an in vitro model of cells co-cultured with rat aorta ring. Cell proliferation capability was detected by MTT method. The rBMSC-ECs were co-cultured with rat aorta ring in rat tail collagen and endothelial medium. A ROCK specific inhibitor, HA-1077 at different concentrations (0, 10, 30 and 60 mmol/L, respectively) was added into the medium of ring-cell co-culture. The protein expression of ROCK I and ROCK II were detected by Western blot. On the third day of cultivation, the branch number of neogenetic microvessels increased by 34.5% in ring-cell co-culture group compared with that in simple aorta ring group (P < 0.01). Compared with that in ring-cell co-culture group, it was significantly decreased by 57.70%, 64.13% and 48.23% respectively in three concentrations of HA-1077 groups (all P < 0.01). However, on the sixth day, rBMSC-ECs proliferated and migrated to the nearby aorta ring, and the growth of microvessels became slow. On the ninth day, some of neogenetic microvessels were retracted, some became thicken, coarsen and lengthen, and some of rBMSC-ECs were sprouting and forming capillary like picture. The protein expression of ROCK I/II was slightly higher in ring-cell co-culture group than that in simple aorta ring group. But, in three concentrations of HA-1077 groups, it was slightly lower than that in ring-cell co-culture group. By using rhodamine-phalloidin staining and laser scanning confocal fluorescence microscope, it showed that there were a lot of the F-actin cytoskeletons in neogenesis microvessels of aorta ring, and there were a lot of thick and long stress fibers in the cells. F-actin-rich surface protrusions at the leading edge of the cell were also shown. Another ROCK inhibitor, Y-27632 (10 µmol/L) induced the actin cytoskeleton reorganization F-actins appeared to be peripheral fibers at outer area of cell; stress fiber and filopodia disappeared. These results suggest that rBMSC-ECs themselves can be differentiated into new microvessels and facilitate angiogenesis when they are co-cultured with rat aorta ring. The mechanisms involve ROCK activation and F-actin cytoskeleton recombination.