Responses Of Adventitial CD34~+ Vascular Wall-resident Stem/Progenitor Cells And Smooth Muscle Cells To Carotid Injury

Part 1:The main source of cell expansion in traditional smooth muscle cell cultureObjectives:To investigate whether the media-isolated smooth muscle cells (SMC) possess the ability of proliferation and whether the cell expansion in traditional SMC culture is due to the proliferation and differentiation of small population of vascular wall-resident stem/progenitor cells (VRS/Pcs).Methods:The sections of SD rats'aortas were immunofluorescently stained with anti-CD34, anti-SM MHC or anti-CD31, followed by related secondary antibodies to examine the distribution of SMCs and CD34+VRS/Pcs. The SM MHC+SMCs were isolated from the media using traditional enzyme-isolation method, and the purity of the cells was evaluated by both fluorescent-immunocytochemistry and flow cytometry, with the proliferating cells detected by BrdU incorporation assay.Results:Flow cytometry analysis showed that traditional enzyme-isolated SMCs from the media were 92.50± 2.96% positive for SM MHC. Immunofluorescent staining further confirmed that the BrdU+cells were 93.92± 8.62% positive for SM MHC, while CD34+ cells could be hardly detected (2.61± 0.82%). The dividing SM MHC+SMCs was captured by double labeling with anti-BrdU plus SM MHC. Flow cytometry analysis revealed that at either the active proliferating stage (day 5) or after confluency (day 9), the SM MHC+cells were overwhelming, occupying percentages of 85.16± 2.74% and 88.83 ± 3.60%, respectively. In contrast, the CD34+cells were only 1.82± 0.27% at day 5 and 2.12±0.29% by day 9. These suggested that the cell expansion in the standard SMC primary culture was mainly attributed to the media-isolated SM MHC+SMCs; rather than CD34+VRS/Pcs.Conclusions:1) The media-isolated SM MHC+SMCs were capable of dividing and proliferation.2) In traditional SMC primary culture, the fast cell expansion was mainly attributed to SM MHC+SMCs, the contribution of CD34+VRS/Pcs was very limited.Part 2:Induced SMC-differentiation of adventitial CD34+VRS/Pcs and its mechanismsObjectives:To investigate the potency of the adventitial CD34+VRS/Pcs to differentiate into the SMC and the related mechanism.Methods:The media-isolated and purified CD34+VRS/Pcs of SD rats were induced with PDGF-BB (20ng/ml) for 7 days in vitro. Immunofluorescent analysis of SM MHC and SM22a was performed before and after induction, with parallel-cultured untreated cells as control. The expression of myocardin, Elk-1, c-fos and CD34 were measured using qRT-PCR.Results:After 7 days of induction in 20ng/ml PDGF-BB, there were 75.79 ± 1.48% of the CD34+VRS/Pcs expressing SM22a, but only 5.57± 2.37% expressing SM MHC. Analysis of qRT-PCR showed that in their SMC-orientated differentiation, the CD34+VRS/Pcs up-regulated the expression of myocardin, and down-regulated Elk-1 and c-fos. The expression of the stem cell marker CD34 was also down regulated after induction.Conclusions:1) Adventitial CD34+VRS/Pcs have the potential of SMC-orientated differentiation in vitro; but most cells can only differentiate into less mature SM MHC- SMC-like cells, only a few of them can differentiate into mature SM MHC+ SMCs.2) The SMC-orientated differentiation of CD34+VRS/Pcs induced by PDGF-BB is achieved by changing the balance between myocardin and Elk-1.Part 3:Responses of adventitial CD34+VRS/Pcs and medial SMCs to carotid injuryObjectives:To investigate the roles of adventitial CD34+VRS/Pcs and medial SMCs in responses to carotid injury.Methods:Wire induced endothelial injury was made in the common carotid artery in the SD rats. HE staining, immunofluorescence staining, laser scanning STED ultra high-resolution microscopy and in vivo cell tracing analysis were performed to observe the responses and biological behaviors of the medial SMCs and adventitial CD34+VRS/Pcs in the process of neointimal formation after vascular injury.Results:H&E staining showed a gradual intimal thickening in the common carotid artery from days 7 to 28 after injury.In the immunofluorescent staining of CD34 at day 7 after injury, an accumulation of CD34+cells was observed in adventitia. But at the later time points (days 14 and 28), the density of CD34+cells was gradually decreased. During the whole course of the post-injury remodeling, CD34 signal could not be detected in-either subendothelial layer (the layer between endothelium and internal elastic lamina) of the neointima, or the media.Immunofluorescent staining of SM MHC and SM22a showed that before injury, only the medial SMCs could express SM MHC and SM22a. At day 7 post-injury, an increased number of SM22a+cells appeared in the adventitia, but no SM MHC+cells were detected. Vast majority of neointimal cells were SM22a+and SM MHC+, but the expression of SM MHC was relatively lower compared with medial SMCs. Then the percentages of SM22a+cells in adventitia gradually diminished at later time points (from days 14 to 28), meanwhile the expression of SM MHC in the neointimal cells gradually reached the level of the medial SMCs. A high magnification observation under confocal microscope showed that at day 7 post-injury, adventitial cells expressing SM22a were CD34+VRS/Pcs. STED super-resolution microscopy and Z-stack image reconstruction showed that at day 14 post-injury, a few of cells co-expressing both CD34 and SM22a appeared in the media closer to the external elastic lamina, suggesting the possibility of a migration of the differentiating VRS/Pcs (SM22a+SMC-like cells) from adventitia into the outer layer of media. This was further confirmed by in vivo cell tracing analysis in which very few of CM Dil-labeled CD34+appeared in the outer layer of the media close to the external elastic lamina, no CM DiI signals could be detected in the inner major part of the media and the neointima at day 14 post-injury.Conclusions:1) In the course of remodeling after rat carotid injury, the adventitial CD34+ VRS/Pcs were able to response to the injury by proliferating and differentiating into SMC-like cells, but these cells could migrate only to the outer layer of the media, they do not directly participate in neointimal formation. They may function to maintain homeostasis of the tunica media during remodeling.2) It is quite possible that the neointimal cells are mainly derived from the medial SMCs in our rat carotid injury model. The lower expression level of SM MHC in the early neointimal cells suggested that these cells had undergone a phenotype modulation.