The accumulation of hyaluronan (HA) and the HA-binding proteoglycan versican around smooth muscle cells in lesions of atherosclerosis suggests that together these molecules play an important role in the events of atherogenesis. In this study we have examined the formation of HA- and versican-rich pericellular matrices by human aortic smooth muscle cells in vitro, using a particle-exclusion assay, and the role of the pericellular matrix in cell proliferation and migration. The structural dependence of the pericellular matrix on HA can be demonstrated by the complete removal of the matrix with Streptomyces hyaluronidase. The presence of versican in the pericellular matrix was confirmed immunocytochemically. By electron microscopy, the cell coat was seen as a tangled network of hyaluronidase-sensitive filaments decorated with ruthenium red-positive proteoglycan granules. Ninety percent of migrating cells in wounded cultures, and virtually all mitotic cells, displayed abundant HA- and versican-rich coats. Time-lapse video imaging revealed that HA- and versican-rich pericellular matrix formation is dynamic and rapid, and coordinated specifically with cell detachment and mitotic cell rounding. HA oligosaccharides, which inhibit the binding of HA to the cell surface and prevent pericellular matrix formation, significantly reduced proliferation and migration in response to platelet-derived growth factor, whereas larger HA fragments and high molecular weight HA had no effect. Treatment with HA oligosaccharides also led to changes in cell shape from a typical fusiform morphology to a more spread and flattened appearance. These data suggest that organization of HA- and versican-rich pericellular matrices may facilitate migration and mitosis by diminishing cell surface adhesivity and affecting cell shape through steric exclusion and the viscous properties of HA proteoglycan gels.

Abstract. SPARC (Secreted Protein Acidic and Richin Cysteine) is a Ca÷2-binding glycoprotein that is differentially associated with morphogenesis, remodeling, cellular migration, and proliferation. We show here that exogenous SPARC, added to cells in culture, was associated with profound changes in cell shape, caused rapid, partial detachment of a confluent monolayer, and inhibited spreading of newly plated cells.Bovine aortic endothelial cells, exposed to 2-40/zg SPARC/ml per 2 x 106 cells, exhibited a rounded morphology in a dose-dependent manner but remained attached to plastic or collagen-coated surfaces. These round cells synthesized protein, uniformly excluded trypan blue, and grew in aggregates after replating in media without SPARC. SPARC caused rounding of bovine endothelial cells, fibroblasts, and smooth muscle cells; however, the cell lines F9, PYS-2, and 3T3 were not affected. The activity of native SPARC was inhibited by heat denaturation and prior incubation with anti-SPARC IgG. The effect of SPARC on endothelial cells appeared to be independent of the rounding phenomenon produced by the peptide GRGDSP. Immunofluorescence localization of SPARC on endothelial cells showed preferential distribution at the leading edges of membranous extensions.SPARC bound Ca ÷2 in both amino-and carboxylterminal (EF-hand) domains and required this cation for maintenance of native structure. Solid-phase binding assays indicated a preferential affinity of native SPARC for several proteins comprising the extracellular matrix, including types III and V collagen, and thrombospondin. This binding was saturable, Ca +2 dependent, and inhibited by anti-SPARC IgG. Endothelial cells also failed to spread on a substrate of native type III collagen complexed with SPARC. We propose that SPARC is an extracellular modulator of Ca ÷2 and cation-sensitive proteins or proteinases, which facilitates changes in cellular shape and disengagement of cells from the extracellular matrix.

Cardiac muscle fibers consist of highly aligned cardiomyocytes containing myofibrils oriented parallel to the fiber axis, and successive cardiomyocytes are interconnected at their ends through specialized junctional complexes (intercalated disks). Cell culture studies of cardiac myofibrils and intercalated disks are complicated by the fact that cardiomyocytes become extremely flattened and exhibit disorganized myofibrils and diffuse intercellular junctions with neighboring cells. In this study we sought to direct the organization of cultured cardiomyocytes to more closely resemble that found in vivo. Lanes of laminin 5-50 microm wide were microcontact-printed onto nonadhesive (BSA-coated) surfaces. Adherent cardiomyocytes responded to the spatial constraints by forming elongated, rod-shaped cells whose myofibrils aligned parallel to the laminin lanes. Patterned cardiomyocytes displayed a striking, bipolar localization of the junction molecules N-cadherin and connexin43 that ultrastructurally resembled intercalated disks. When laminin lanes were widely spaced, each lane of cardiomyocytes beat independently, but with narrow-spacing cells bridged between lanes, yielding aligned fields of synchronously beating cardiomyocytes. Similar cardiomyocyte patterns were achieved on the biodegradable polymer PLGA, suggesting that patterned cardiomyocytes could be used in myocardial tissue engineering. Such highly patterned cultures could be used in cell biology and physiology studies, which require accurate reproduction of native myocardial architecture.

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Selected strains of vascular endothelial cells, grown as confluent monolayers on tissue culture plastic, generate flat networks of cellular cords that resemble beds of capillaries--a phenomenon referred to as "spontaneous angiogenesis in vitro". We have studied spontaneous angiogenic activity by a clonal population (clone A) of bovine aortic endothelial cells to identify processes that mediate the development of cellular networks. Confluent cultures of clone A endothelial cells synthesized type I collagen, a portion of which was incorporated into narrow, extracellular cables that formed a planar network beneath the cellular monolayer. The collagenous cables acted as a template for the development of cellular networks: flattened, polygonal cells of the monolayer that were in direct contact with the cables acquired spindle shapes, associated to form cellular cords, and became elevated above the monolayer. Networks of cables and cellular cords did not form in a strain of bovine aortic endothelial cells that did not synthesize type I collagen, or when traction forces generated by clone A endothelial cells were inhibited with cytochalasin D. In a model of cable development, tension applied by a confluent monolayer of endothelial cells reorganized a sheetlike substrate of malleable type I collagen into a network of cables via the formation and radial enlargement of perforations through the collagen sheet. Our results point to a general involvement of extracellular matrix templates in two-dimensional (planar) models of vascular development in vitro. For several reasons, planar models simulate invasive angiogenesis poorly. In contrast, planar models might offer insights into the growth and development of planar vascular systems in vivo.