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Abstract. Thrombospondin (TS) is an extracellular glycoprotein whose synthesis and secretion by vascular smooth muscle cells (SMC) is regulated by plateletderived growth factor. We have used a panel of five monoclonal antibodies against TS to determine an essential role for thrombospondin in the proliferation of cultured rat aortic SMC. All five monoclonal antibodies inhibited SMC growth in 3-d and extended cell number assays; the growth inhibition was specific for anti-TS IgG. The effects of one antibody (134.6) were examined in detail and were found to be reversable and dose dependent. Cells treated with D4.6 at 50 Ixg/ml (which resulted in a >60% reduction in cell number at day 8) were morphologically identical to control cells. D4.6-treated SMC were analyzed by flow cytofluorimetry and were found to be arrested in the G1 phase of the cell cycle. To determine a possible cellular site of action of TS in cell growth, SMC were examined by immunofluorescence using a polyclonal antibody against TS. TS was observed diffusely bound to the cell surface of serum-or platelet-derived growth factor-treated cells. The binding of TS to SMC was abolished in the presence of heparin, which prevents the binding of TS to cell surfaces and inhibits the growth of SMC. Monoclonal antibody D4.6, like hepatin, largely abolished cell surface staining of TS but had no detectable effect on the cellular distribution of fibronectin. These results were corroborated by metabolic labeling experiments. We conclude that cell surface-associated TS is functionally essential for the proliferation of vascular SMC, and that this requirement is temporally located in the G~ phase of the cell cycle. Agents that perturb the interaction of TS with the SMC surface, such as heparin, may inhibit SMC proliferation in this manner.

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory peptide that can inhibit or promote the proliferation of cultured vascular smooth muscle cells (SMCs), depending on cell density (Majack, R. A. 1987. J. Cell Biol. 105:465-471). In this study, we have examined the mechanisms underlying the growth-promoting effects of TGF-beta in confluent SMC cultures. In mitogenesis assays using confluent cells, TGF-beta was found to potentiate the stimulatory effects of serum, PDGF, and basic fibroblast growth factor (bFGF), and was shown to act individually as a mitogen for SMC. In gene and protein expression experiments, TGF-beta was found to regulate the expression of PDGF-A and thrombospondin, two potential mediators of SMC proliferative events. The induction of thrombospondin protein and mRNA was density-dependent, delayed relative to its induction by PDGF and, based on cycloheximide experiments, appeared to depend on the de novo synthesis of an intermediary protein (probably PDGF-A). The relationship between PDGF-A expression and TGF-beta-mediated mitogenesis was investigated, and it was determined that a PDGF-like activity (probably PDGF-A) was the biological mediator of the growth-stimulatory effects of TGF-beta on confluent SMC. The effects of purified homodimers of PDGF-A on SMC replication were investigated, and it was determined that PDGF-AA was mitogenic for cultured SMC, particularly when used in combination with other growth factors such as bFGF and PDGF-BB. The data suggest several molecular mechanisms that may account for the ability of TGF-beta to promote the growth of confluent SMC in culture.

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The nude mouse graft model for testing the hair-forming ability of selected cell populations has considerable potential for providing insights into factors that are important for hair follicle development and proper hair formation. We have developed a minimal component system consisting of immature hair follicle buds from newborn pigmented C57BL/6 mice and adenovirus E1A-immortalized rat vibrissa dermal papilla cells. Hair follicle buds contribute to formation of hairless skin when grafted alone or with Swiss 3T3 cells, but produce densely haired skin when grafted with a fresh dermal cell preparation. The fresh dermal cell preparation represents the single cell fraction after hair follicles have been removed from a collagenase digest of newborn mouse dermis. It provides dermal papilla cells, fibroblasts, and possibly other important growth factor-producing cell types. Rat vibrissa dermal papilla cells supported dense hair growth at early passage in culture but progressively lost this potential during repeated passage in culture. Of 19 E1A-immortalized, clonally derived rat vibrissa dermal papilla cell lines, the four most positive clones supported hair growth to the extent of approximately 200 to 300 hairs per 1-2 cm2 graft area. The remaining clones were moderately positive (five clones), weakly positive (three clones), or negative (seven clones). Swiss 3T3 cells prevented contraction of the graft area but did not appear to affect the number of hairs in the graft site produced by dermal papilla cells plus hair follicle buds alone. The relatively low hair density (estimated 1-5% of normal) resulting from grafts of hair follicle buds with the most positive of the immortalized dermal papilla cell clones compared to fresh dermal cells suggests that optimal reconstitution of hair growth requires some function of dermal papilla cells partially lost during the immortalization process and possibly the contribution of other cell types present in the fresh dermal cell preparation, which is not supplied by the Swiss 3T3 cells. The current graft system, comprising hair follicle buds and immortalized dermal papilla cell clones, provides an assay for positive or negative influences on hair growth exerted by added selected cell types, growth factors, or other substances. Characterization of the phenotype of the dermal papilla cell lines, which differ in their ability to support hair growth when grafted with hair follicle buds, may provide insight into specific dermal papilla cell properties important for their function in this system.