VSMC heterogeneity and tissue culture models

When VSMCs from the normal medial layer are cultured in vitro, they undergo phenotypic change and de-differentiate and proliferate to become

'synthetic' cells. These 'synthetic' cells have broad similarities to many intimal cells and have therefore been extensively used to model disease-associated VSMCs. However, recent evidence from studies of human vascular disease in vivo has clearly demonstrated that the definition of VSMCs as either 'contractile' or 'synthetic' is simplistic. Furthermore, using animal tissue culture systems, particularly rat and bovine VSMCs, it is possible to model multiple VSMC phenotypes in vitro [12, 13]. In adult bovine arteries, four distinct VSMC phenotypes can be recognised. These originate in different layers of the media and sub-intima. Cells cultured from specific layers retain distinct phenotypes as judged by morphology, smooth muscle marker expression and responsiveness to growth stimuli. Frid et al. concluded that these different cell types exist in the media to perform different functions (i.e. contraction or repair) [12]. Similarly, cloning of VSMCs from rat arteries identifies at least two distinct phenotypes; elongate and cobblestone, with the latter being almost exclusively present in the neointima after balloon injury-induced damage [13]. These cells are also more prevalent in cultures derived from neonatal vessels compared with adult vessels. Analysis of gene/protein expression in these cell cultures has led to the identification of specific gene markers such as cellular retinol-binding protein-1, which has distinct patterns of expression for each phenotype [14].

Although VSMC heterogeneity has also been observed in human blood vessels in vivo [15, 16], particularly between VSMCs occupying the sub-intimal layer and different layers within the media, tissue culture models of these differences are not as well characterized as those in other species. In vitro, human VSMCs can display different characteristics, including different cell shapes, cell sizes, protein expression and growth patterns [ 17]. For example, human VSMCs isolated from young arteries contain populations of cells with higher proliferative rates than VSMCs from adult arteries [18]. In addition, in a human VSMC culture model where 'contractile' VSMCs were established within collagen gels and compared to proliferating VSMCs, the cell surface protein CD9 was expressed in greater amounts in proliferative cells [19]. Furthermore, Bennett et al. found that human VSMCs derived from the vessel intima exhibit a higher natural rate of apoptosis in culture than cells derived from the normal vessel media [20]. Our group has also found that human arterial VSMCs cultured from normal media can exist in different forms judged by morphology, and can calcify in vitro (Figure 2, described in section 4, and ref 21). However, many of these morphologies do not appear to be stable in culture and specific gene markers of different human VSMC sub-populations in vitro have yet to be identified.

Figure 2. Human VSMC morphology. A. Typical spindle shaped VSMCs derived from aortic medial explants. B. Classic 'hill and valley' post-confluent growth of spindle shaped cells derived from aortic medial explants. C. Large, rounded senescent-type cells, some with long extending processes, derived from aortic medial explants. The insert is an enlargement of part of this culture which shows the morphology of this culture in greater detail. D. Mixed population of cells appearing as either spindle, elongate, large rounded or stellate. These cells were obtained by enzymatic dispersion of the aortic media. E. Mostly contact-inhibited morphology forming a cobblestone-like layer of VSMCs derived from aortic medial explants. If these VSMCs are allowed to remain as a monolayer, they gradually curl up as a sheet of cells and are difficult to trypsinize. Fugita et al (18) described this type ofVSMC morphology as similar to neonatal VSMCs. F. Mostly small, highly proliferative cells derived from an aortic medial explant (shown top right). G. Pericytes derived from placenta microvessels with a multi-cellular nodule. Note the prominent actin filaments in these cells. H. VSMCs, mostly of the spindle-shape, with a large multi-cellular nodule derived from aortic medial explants. (The bar in each figure represents approximately 10|xm).

Figure 2. Human VSMC morphology. A. Typical spindle shaped VSMCs derived from aortic medial explants. B. Classic 'hill and valley' post-confluent growth of spindle shaped cells derived from aortic medial explants. C. Large, rounded senescent-type cells, some with long extending processes, derived from aortic medial explants. The insert is an enlargement of part of this culture which shows the morphology of this culture in greater detail. D. Mixed population of cells appearing as either spindle, elongate, large rounded or stellate. These cells were obtained by enzymatic dispersion of the aortic media. E. Mostly contact-inhibited morphology forming a cobblestone-like layer of VSMCs derived from aortic medial explants. If these VSMCs are allowed to remain as a monolayer, they gradually curl up as a sheet of cells and are difficult to trypsinize. Fugita et al (18) described this type ofVSMC morphology as similar to neonatal VSMCs. F. Mostly small, highly proliferative cells derived from an aortic medial explant (shown top right). G. Pericytes derived from placenta microvessels with a multi-cellular nodule. Note the prominent actin filaments in these cells. H. VSMCs, mostly of the spindle-shape, with a large multi-cellular nodule derived from aortic medial explants. (The bar in each figure represents approximately 10|xm).

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