The identification of MVSCs brings a new perspective on vascular remodeling and disease development. Our in vitro experiments suggest that proliferative/synthetic SMCs are derived from the differentiation of MVSCs instead of the de-differentiation of mature SMCs. In vivo experiments also demonstrate that MVSCs, rather than mature SMCs, repopulate the tunica media and form neointima after endothelial denudation injury. These results provide the first direct evidence to support the MVSC differentiation hypothesis and disprove the SMC de-differentiation theory. The fact that highly expandable and migratory MVSCs were not derived from mature SMCs suggests that the previously reported decrease of contractile markers in SMC culture in vitro or after vascular injury in vivo could be attributed to the rapid expansion of MVSCs and their spontaneous differentiation into immature SMCs. MVSCs not only proliferate and differentiate, but also become synthetic and secrete matrix proteins such as collagen I, collagen II and aggrecan. The multipotential of MVSC differentiation into SMCs, chondrogenic cells and other lineages offers a novel and reasonable explanation for the complex phenotypes of cells in the diseased vessel.
MVSCs at different stages of differentiation could also explain the heterogeneity of SMC in culture and in vivo
. The fact that MVSCs and their derivatives respond differently to vascular growth factors underscores the importance of characterizing the cell culture from blood vessels. In the literature, the differentiation stage of “SMCs” is usually not well characterized. For example, the cells positive for SMA, SM-22α and CNN1 are often treated as SMCs rather than immature SMCs or partially differentiated MVSCs. In many studies, the cells in culture may not be homogeneous at a specific differentiation stage, which may result in conflicting observations and explanation. Previous studies have identified a subpopulation of SMCs, termed “epithelioid” cells, from newborn or injured vessels 45–48
. The origin of these SMCs is not known, and the relationship of “epithelioid” cells with vascular stem cells has not been explored. It is very likely that “epithelioid” SMCs are derived from MVSCs. However, in adults, MVSCs can be isolated from both normal vessels and injured vessels, but “epithelioid” SMCs are only derived from injured vessels. It is possible that dormant MVSCs are a small population in normal vessels and may be overlooked.
In addition to proliferative/synthetic SMCs, MVSCs could differentiate into mature SMCs (SM-MHC+
) in vitro
and in vivo
. It is also worth noting that the extent of MVSC activation, proliferation and differentiation could be dependent on the extent of vascular injury and SMC damage 42
. However, a recent study using SM-MHC as a marker for lineage tracing demonstrates that neointima is populated by proliferative cells derived from mature SMCs at 3 weeks following vascular injury 49
. This apparent discrepancy has two possible explanations. Firstly, these proliferating cells could be derived from MVSCs after such a long time period. Indeed we have shown that newly differentiated SMCs (SM-MHC+
) may not have exited the cell cycle completely (Supplementary Fig. S11d-f
). In addition, at an earlier time point (e.g., within a week), we never detected proliferating cells positive for SM-MHC (). Secondly, the two transgenic mouse lines of SM-MHC-Cre used for the lineage tracing of SMCs were generated independently 30, 50
. We cannot exclude the possibility that a subtle difference in the promoter of SM-MHC in these two mouse lines exists. Although both mouse lines have been verified with extensive characterization and should be appropriate for tracing the fate of mature SMCs, further studies are needed to directly compare these mouse lines. Another study on the lineage tracing of SMCs shows that SMCs give rise to osteogenic and chondrogenic cells in arteries17
. However, SM22α instead of SM-MHC was used as the marker for lineage tracing in this study. Since SM22α is also expressed in MVSCs and their derivatives, this study may not distinguish the source of the cells between MVSCs and SMCs.
Another important finding is that MVSCs are the cell type in tunica media that can be activated by vascular injury to proliferate and participate in remodeling. Neointima cells have a large cell body, secrete extra-cellular matrix (ECM) and express lower levels of the smooth muscle-specific contractile proteins 51
, which is consistent with our findings on the changes of cell morphology and the expression of SMC markers during the spontaneous differentiation of MVSCs. In this study, we showed that MVSCs, instead of SMCs, are the major cell type in neointimal tissue as characterized by MVSC makers. Given the fact that some MVSCs in neointima still retain multipotency, it is reasonable to propose that aberrant activation, expansion and differentiation of MVSCs may contribute to not only SMC differentiation and cartilage formation but also many other aspects of vascular diseases such as fat and cholesterol metabolism, matrix remodeling and calcification. Since the rodent model of denudation injury does not result in significant fat and cholesterol accumulation and intima calcification, the possibility of MVSC differentiation into adipogenic and osteogenic cells in diseased vessels remains to be tested by using atherosclerosis models. The identification of MVSC in human arteries makes MVSC a potential therapeutic target of vascular diseases.
MVSCs in arteries and veins may have different developmental origins. Wnt1 lineage tracing experiments suggest that MVSCs in carotid arteries may be neural crest origin. In contrast, for blood vessels not developed from neural crest (e.g., jugular vein), MVSCs are negative for Wnt1. It is possible that these MVSCs are derived from other germ layers such as mesoderm. Nevertheless, MVSCs from carotid arteries and jugular veins have similar gene expression profile, suggesting that MVSCs in different vascular beds, regardless of their developmental origins, may share similar characteristics. In addition, MVSCs are different from NCSCs because MVSCs do not express some NCSC markers such as p75, HNK1, Slug or AP2 25, 52–54
To date, MVSCs are a unique precursor identified for adult MSCs and MSC-like cells, and MSC precursors similar to MVSCs likely exist in many other tissues. MVSCs express CD29 and CD44, two non-specific surface markers of MSCs and SMCs. However, there was no specific marker, especially no transcriptional factor marker, for MSCs. In this study, we have identified several transcriptional markers and cytoskeletal markers in MVSCs, such as Sox17, Sox10, nestin, NFM and S100β. In addition, we showed that MSC-like cells were also positive for some of these markers (except Sox17). These results provide insight into the characteristics of MVSCs and MSC-like cells, not only in blood vessels, but also in other tissue types.
This study supports a new hypothesis that MVSC activation and differentiation instead of SMC de-differentiation results in the proliferative and synthetic cells in the vascular wall, and that the aberrant activation and differentiation of MVSCs may have an important role in the development of vascular diseases. These findings provide unprecedented insight into the role of stem cells in vascular diseases and remodeling, which suggests that vascular diseases are stem cell diseases. These findings may have transformative impact on vascular biology and diseases, and may lead to new therapies by using MVSCs as a therapeutic target.