Neointimal smooth muscle cells can be readily distinguished from medial vascular smooth muscle cells by their ultrastructure, cytoskeletal features, growth pattern, response to a variety of growth factors and cytokines, and matrix synthesis and degradation. The Campbells have proposed that medial vascular smooth muscle cells are maintained in a contractile state and undergo "phenotypic modulation" in order to migrate, proliferate, and synthesize matrix following vascular injury. However, the phenotypic modulation theory is unable to explain why such a distinction persists in vivo following removal of inducing factors or in vitro even after several passages. Do neointimal cells represent a fundamentally different lineage of cells from medial vascular smooth muscle cells? If so, from where are they derived? On the basis of observation on the healing process of vascular grafts, it has been suggested that circulating mesenchymal progenitor cells do exist and are responsible for vascular healing. Are circulating mesenchymal progenitor cells simply detached viable vascular cells or are they released from the bone marrow?
In order to answer all these questions, a murine radiation chimeric model was established in our laboratory. Following lethal radiation, female recipient mice received either whole bone marrow cells or fluorescence-activated cell sorting isolated primitive hematopoietic stem cells (Rhl123/Höechst33342lo/spLin- c-kit+ Ly6A+) from congenic male mice . This approach allowed us to identify donor-derived cells by in situ hybridization with a mouse Y chromosome probe. The hypothesis that marrow-derived mesenchymal cells are involved in vascular pathology was tested in three different animal models:
1. neointimal formation following probe scratch denudation injury of the external common iliac artery;
2. organization of arterial thrombus following insertion of silk sutures to the left common carotid artery;
3. neoangiogenesis following deprivation of blood supply to the hind limb.
At 28 days following probe scratch denudation injury, the arterial lumen was obliterated by a cell-rich neointima. By in situ hybridization with a Y chromosome probe, we found some of the smooth muscle α actin-positive neointimal cells were derived from the donor. Additionally, a significant proportion of neointimal cells, although they were unable to be recognized as smooth muscle cells immunohistochemically, were also of donor origin. In fact, this subset of neointimal cells might represent the predominant type of cells in human atherosclerotic lesions which has been designated as "mesenchymal-appearing intimal cells" by previous researchers.
The involvement of donor-derived smooth muscle cells in the organization of arterial thrombus was observed in the second experiment. However, in mice receiving primitive hematopoietic stem cells only, we were unable to identify any donor-derived intimal cells within the organized arterial thrombus. This suggests that the donor-derived smooth muscle cells we described above are in fact derived from the bone marrow stromal component. Based on our observations, it does not seem likely that a common stem cell exists in adult mice for both the hematopoietic and marrow mesenchymal lineages. In mice which received whole marrow cells, we also found donor-derived endothelial cells were responsible for re-endothelialization following vascular injury. Although mouse bone marrow endothelial cells have not yet been fully characterized, it has been shown that human marrow endothelial cells are more susceptible to cytomegalovirus infection than vascular endothelial cells. Cytomegalovirus and other members of the herpesvirus family have close associations with both the initiation of atherosclerosis and restenosis. Our findings suggest marrow-derived endothelial cells might serve as a nidus for the initiation of atherosclerosis and also provide a putative answer as to the origin of activated endothelial cells following denudation injury.
At 28 days following deprivation of blood supply to the lower limb, there was a remarkable increase in blood vessels, proliferation of endomysial connective tissue, and degeneration of muscle bundles. In mice receiving primitive hematopoietic stem cells, we were unable to identify any donor-derived endothelial cells in ischemic calf muscle. Although a common ancestor cell, hemangioblast, has been proposed and is believed to be responsible for early hematopoiesis and vascular development, our data suggest that there are no hemangioblasts in adult mice.
In summary, these studies have demonstrated that medial vascular smooth muscle cells are not the only source of neointimal cells during vascular healing (neointima formation and organization of arterial thrombus). circulating bone marrow-derived progenitor cells may also contribute to the reparative process following vascular injury. Moreover, we also found that these marrow-derived mesenchymal cells (smooth muscle cells or endothelial cells) did not share a common progenitor with the hematopoietic lineage as previously believed. Instead, they are derived from a separate entity collectively being called the bone marrow mesenchymal (stromal) cells.