The Mechanism Of Neointimal Smooth Muscle Like Cell Recruitment Induced By Endothelial Cell

BACKGROUND: Graft vascular disease is a common pathological changes invarious organ graft chronic loss of function, is also one of the most important andindependent factors that affect the long-term survival. The main pathological featureis diffused arterial intimal hyperplasia within the graft artery, consequently resultingin stenosis of the transplant artery in a few months to years after transplantation,causing ischemia, hypoxia and parenchyma tissue fibrosis of transplant organs,eventually leading to the transplant organ dysfunction. Recent studies have shown thatmost of the neointimal cells are smooth muscle cells, however, these traditionalvascular medial smooth muscle cells are significantly different from those inneointimal. The main important difference is that neointimal smooth muscle cells canhighly expressed CCR1chemokine receptor but medial smooth muscle cells do not.According to different animal models, the precursor cells of neointimal smoothmuscle like cells probably are derived from bone marrow mesenchymal stem cells,medial smooth muscle cells, vascular adventitial cells and perivascular matrix. Theseprecursor cells migrate to neointimal and settle down through various pathways,overproliferating, finally resulting in neointimal hyperplasia, transplant arterystenosis. Recent studies have showm that IFN-gamma can induce endothelial cells tosecret many kinds of chemokines, and RANTES was probably involved ininflammatory exudation, and other researchers showed RANTES can affect thedevelopment of graft vascular disease. Nevertheless the mechanism of neointimalsmooth muscle like cells recruitment in graft vascular disease is still unknown. Thusour study can help elucidate the mechanism of smooth muscle cell recruitment andprovide an effective target for graft vascular disease therapeutic strategies in thefuture. OBJECTIVE: To find an effective approach to isolate and cultivate primaryendothelial cells, smooth muscle cells and smooth muscle like cells from mouse heart,aorta and artery transplant model. Use IFN-gamma to stimulate endothelial cells, thendetect whether the chemokines secreted by them can induce medial smooth musclecells or intimal smooth muscle-like cells migration. Elucidate endothelial cells effecton the mechanism of neointimal smooth muscle like cells recruitment in graft vasculardisease.METHODS: Primary culture of endothelial cells from mouse heart bycollagenase digestion method and magnetic beads sorting, purification of endothelialcells by continuous proliferation method; primary culture of smooth cells from mouseaorta by collagenase digestion method and magnetic beads sorting, purification ofendothelial cells by continuous proliferation method; primary culture of neointimalsmooth muscle like cells from transplant aorta of mouse transplant model, purificationof smooth muscle like cells by continuous proliferation method and identification ofsmooth like cells by FACS; use microfluidic device to co-culture endothelial cells andsmooth muscle cells or smooth muscle like cells; identify the efficacy of themicrofluidic device by comparison with scratch assay and Boyden chamber assay; useIFN-γ to treat endothelial cells, detect the migration induced by the chemokinessecreted by IFN-γ treated endothelial cells in microfluidic device.RESULTS: Collagenase digestion method and magnetic beads sorting are veryeffective for primary cell isolation; continuous proliferation method is good and easyto purify the primary cells; microfluidic device is the best for cell migration detectioncompared with scratch assay and Boyden chamber assay; siRNA can effectivelyreduce RANTES expression by IFN-γ treated endothelial cells; RANTES can inducesmooth muscle like cells migration but not smooth muscle cells.CONCLUSION: Microfluidic device is much better than scratch assay andBoyden chamber assay in detect cell migration towards a chemokine concentration gradients. RANTES secreted by IFN-γ treated endothelial cells can induce smoothmuscle cell migration but not smooth muscle cell in the microfluidic device.