Differentiation of pericytes in culture is accompanied by changes in the extracellular matrix

We have previously reported that pericytes derived from retinal and brain microvessels aggregate into nodules soon after reaching confluence. Nodule formation involves a reorganization of the cells resulting in the presence of sparse cells, confluent monolayers, multilayers, sprouts, and nodules within the same culture dish. Extracellular calcification occurs only within the nodules, demonstrating that pericytes are capable of undergoing osteogenic differentiation in culture and that this differentiation is related to nodule formation. Using immunofluorescence we have now studied the distribution of laminin, type IV collagen, type X collagen, and tenascin in pericyte cultures during nodule formation. These matrix macromolecules were also identified by a combination of biochemical techniques, including Northern blot hybridization, immunoblotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecule that seems to be related to type X collagen was demonstrated by the presence of a pepsin-resistant, collagenase-sensitive polypeptide of molecular weight approximately 45 kDa. The production of laminin, type X-related collagen, and tenascin by pericytes has not been previously reported. Our results suggest that the synthesis or distribution or both of these molecules is dependent on the state of pericyte differentiation. The expression of laminin, type IV collagen, and type X-related collagen was maximal in multilayer areas, sprouts, and nodules. Tenascin appeared homogeneously distributed in monolayer and multilayer areas; when calcified nodules were present, the anti-tenascin serum preferentially decorated a discrete area circumscribing the nodules. Tenascin and type X collagen have been found transiently in vivo preceding calcification; their possible role in this process is not known. Our results also suggest an association between laminin, type IV collagen, and calcification. The in vitro experimental system described here may help to clarify the role of matrix macromolecules in the calcification process.