The Atherosclerotic Environment Accentuates Endochondral Ossification in Vessel-Derived Stem Cells: In-vitro and In-vivo Assessment
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Vascular calcification, far from being a passive degenerative process, is now perceived as an active, highly organized cell-controlled event. The presence of a number of stem progenitor niches and/or lineages in the vasculature has been established and their possible role in vascular calcification is under increasing investigation. Pericytes are purported to be involved in this process and are currently a poorly defined cell population. Although traditionally considered as supporting cells, more recently these cells have been proposed to play a more active role in the repair and pathogenesis of various vascular diseases. Pericytes have been shown to have a mesenchymal origin. Mesenchymal stem cells (MSCs) have been associated with a perivascular niche and have the ability to undergo osteogenic and chondrogenic differentiation. In this study, the hypothesis that a pericyte-like stem cell population, termed vessel-derived stem cells (VSCs) exists in the vessel wall was tested. Furthermore, it was proposed that in the presence of inflammatory cytokines seen in the atherosclerotic environment, these cells, along with circulating MSCs, contribute to the calcification of atherosclerotic plaque via endochondral ossification. VSCs from aortae of ApoE-/- atherosclerotic mice and background control C57BL/6 mice were isolated and characterized for differentiation potential and by screening for cell surface markers by flow cytometry and immunocytochemistry. MSCs from the bone marrow of these mice were also isolated and characterized in a similar fashion. When these cells were differentiated down the osteogenic lineage, there was an increase in calcium levels which was more pronounced in ApoE-/- VSCs. Differentiation to the chondrogenic lineage suggested that all the isolated cell populations have chondrogenic potential; however, ApoE-/- MSCs and VSCs showed more extensive deposition of glycosaminoglycan. Phenoytpic analysis demonstrated that both ApoE-/- and C57BL/6 VSCs were strongly positive for Sca-1 and CD44, positive for CD146 and negative for CD31 and CD34. Immunocytochemistry for the specific pericyte marker 3G5 revealed that a sub-population of VSCs expressed 3G5, demonstrating that cell populations isolated were of the hypothesized lineage and contained pericyte-like cells. In order to investigate the effect of proinflammatory cytokines associated with the atherosclerotic niche on chondrogenic differentiation in vitro, levels of gene expression for markers of chondrogenesis were monitored. All the cell populations were subjected to chondrogenesis for 21 days in the presence or absence of the pro-inflammatory cytokines IL-6, IL-1[Beta] or TNF-[Alpha]. At 21 days following IL-6 treatment, ApoE-/- VSCs showed increased levels of Sox9, fibromodulin, type II collagen, aggrecan and alkaline phosphatase indicating extensive differentiation along the chondrogenic lineage. On the contrary, IL-6 had a suppressive effect on C57BL/6 VSCs. For IL-[Beta] and TNF-[Alpha], results indicated that both cytokines suppressed chondrogenic markers in ApoE-/- and C57BL/6 VSCs at 21 days. To assess the ability of VSCs and MSCs from ApoE-/- and C57BL/6 mice to form bone, cells were seeded onto collagen glycosoaminoglycan scaffolds and chondrogenically primed in vitro followed by subcutaneous implantation in vivo for 8 weeks. From this study it was clear that endochondral ossification and calcification in atherosclerosis is a complex interplay of a number of factors, each contributing differently to the process. Histomorphological analysis showed that 5 out of 5 ApoE-/- MSCs and VSCs formed bone (with 77.5% and 58.4% overall bone formation, respectively) in C57BL/6 mice suggesting that the major contributing factor is the intrinsic capacity of MSCs and VSCs derived from atherosclerotic mice. In addition, the data indicated that 5 out of 5 ApoE-/- MSCs and 6 out of 6 ApoE-/- VSCs generated more mature bone in ApoE-/- mice (with 68.5% and 83.25% overall bone formation, respectively) than C57BL/6 MSCs (1 of 5) and VSCs (0 of 4). Thus, this study also demonstrated that the environment in which the pre-differentiated, or primed cells, are placed also plays a role; the host environment drives the implanted constructs to form bone or immature bone, bone marrow or calcified cartilage and promotes the infiltration of blood vessels and fat formation. This study supports the hypothesis that a progenitor cell population in the aorta vessel wall may contribute to vascular calcification via endochondral ossification in the atherosclerotic environment. However, recruitment of circulating MSCs derived from bone marrow to atherosclerotic plaque cannot be ruled out.