Using an in vitro coculture system to mimic the interactions between the cells of the vessel wall, we have previously shown that pericytes and smooth muscle cells (SMC) inhibit the growth of capillary endothelial cells (EC). We have undertaken studies to determine the mechanism of this inhibition. Using conditioned media and affinity-purified antibodies to transforming growth factor (3 (TGF-I3), we now demonstrate that activated TGF-13 produced in these cocultures mediates EC growth inhibition. No inhibitory activity was detected when media conditioned by individual cultures of EC, SMC, or pericytes were examined for their effect on EC growth. In contrast, media conditioned by cocultures of EC-SMC and EC-pericytes inhibited EC proliferation to the same degree as the coculture itself. Immunoadsorption of coculturederived conditioned media with antibodies to TGF-(3 eliminated the inhibitory activity. Acid activation of serum-free media conditioned by any of the cells cultured alone yielded inhibitory activity, whereas activation of coculture conditioned media did not increase its inhibitory activity. Addition of anti-TGF-fi neutralizing antibodies to cocultures blocked the pericyte-mediated EC growth inhibition. These results indicate that latent TGF-fi is produced by these cells and it is activated by a mechanism that requires contact between the two cell types.All capillaries of the vascular network consist of two cell types, endothelial cells (EC) and pericytes. Interactions between EC and cells of the vessel wall [smooth muscle cells (SMC) and pericytes] are known to be important in the maintenance of vascular integrity (1), including growth control. Cases of pathological neovascularization such as diabetic retinopathy are associated with pericyte loss (2). In addition, tissues with the slowest EC turnover appear to have the highest extent of EC coverage by pericytes (3). These observations suggest a role for the pericyte in the control of capillary growth. Using an in vitro coculture system, we have shown that pericytes inhibit EC proliferation in a contactdependent manner (4). We now demonstrate that the elaboration of the activated form of transforming growth factor f3 (TGF-,B) by these cocultures is responsible for this pericyteinduced EC growth inhibition. TGF-,3 has been reported to stimulate or inhibit growth and differentiation in vitro, depending on the cell type examined (5). Since nearly all cultured cells examined secrete TGF-,B in an inactive (latent) form (6), the physiological relevance of TGF-,B as a growth modulator appears to lie in the regulation of its activation. There is a single report of the production of activated TGF-,B by human breast cancer cells in vitro (7) and no reports of the production of an activated form of TGF-P by nontransformed cells in vitro. EC-pericyte cocultures employed in these studies provide a nontumorigenic in vitro system to produce activated TGF-,/. Furthermore, the similarity between the interactions that we observe in these cocultures (4) and those th...

Pericytes have been suggested to play a role in regulation of vessel stability; one mechanism for this stabilization may be via pericyte-derived vascular endothelial growth factor (VEGF). To test the hypothesis that differentiation of mesenchymal cells to pericytes/smooth muscle cells (SMC) is accompanied by VEGF expression, we used endothelial cell (EC) and mesenchymal cell cocultures to model cell-cell interactions that occur during vessel development. Coculture of EC and 10T1/2 cells, multipotent mesenchymal cells, led to induction of VEGF expression by 10T1/2 cells. Increased VEGF expression was dependent on contact between EC-10T1/2 and was mediated by transforming growth factorbeta (TGFbeta). A majority of VEGF produced in coculture was cell- and/or matrix-associated. Treatment of cells with high salt, protamine, heparin, or suramin released significant VEGF, suggesting that heparan sulfate proteoglycan might be sequestering some of the VEGF. Inhibition of VEGF in cocultures led to a 75% increase in EC apoptosis, indicating that EC survival in cocultures is dependent on 10T1/2-derived VEGF. VEGF gene expression in developing retinal vasculature was observed in pericytes contacting newly formed microvessels. Our observations indicate that differentiated pericytes produce VEGF that may act in a juxtacrine/paracrine manner as a survival and/or stabilizing factor for EC in microvessels.

—Embryological data suggest that endothelial cells (ECs) direct the recruitment and differentiation of mural cell precursors. We have developed in vitro coculture systems to model some of these events and have shown that ECs direct the migration of undifferentiated mesenchymal cells (10T1/2 cells) and induce their differentiation toward a smooth muscle cell/pericyte lineage. The present study was undertaken to investigate cell proliferation in these cocultures. ECs and 10T1/2 cells were cocultured in an underagarose assay in the absence of contact. There was a 2-fold increase in bromodeoxyuridine labeling of 10T1/2 cells in response to ECs, which was completely inhibited by the inclusion of neutralizing antiserum against platelet-derived growth factor (PDGF)-B. Antisera against PDGF-A, basic fibroblast growth factor, or transforming growth factor (TGF)-β had no effect on EC-stimulated 10T1/2 cell proliferation. EC proliferation was not influenced by coculture with 10T1/2 cells in the absence of contact. The cells were then cocultured so that contact was permitted. Double labeling and fluorescence-activated cell sorter analysis revealed that ECs and 10T1/2 cells were growth-inhibited by 43% and 47%, respectively. Conditioned media from contacting EC-10T1/2 cell cocultures inhibited the growth of both cell types by 61% and 48%, respectively. Although we have previously shown a role for TGF-β in coculture-induced mural cell differentiation, growth inhibition resulting from contacting cocultures or conditioned media was not suppressed by the presence of neutralizing antiserum against TGF-β. Furthermore, the decreased proliferation of 10T1/2 cells in the direct cocultures could not be attributed to downregulation of the PDGF-B in ECs or the PDGF receptor-β in the 10T1/2 cells. Our data suggest that modulation of proliferation occurs during EC recruitment of mesenchymal cells and that heterotypic cell-cell contact and soluble factors play a role in growth control during vessel assembly.

Acidic and basic fibroblast growth factors (aFGF, bFGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor type beta (TGF-beta) are well-characterized growth regulators. Though there are reports of the effects of some of these factors on vascular cells, variability in the purity of growth factor preparations and differing cell culture conditions make comparison among the results difficult. Thus, a study employing homogeneous preparations of recombinant growth factors on well-characterized cell populations was conducted. Each of the factors was tested for its effect on the proliferation of cells derived from large and small blood vessels: aortic and capillary endothelial cells (EC), smooth muscle cells (SMC) and pericytes. Of the five growth factors only bFGF stimulated the proliferation of both large vessel and microvessel EC; aFGF was mitogenic for microvessel but not large vessel EC. Neither PDGF (AA, BB or AB) nor EGF had any effect on EC growth. TGF-beta was a potent inhibitor of both aortic and capillary EC proliferation with half maximal inhibition at concentrations as low as 0.05 and 0.25 ng/ml for microvessel and aortic EC, respectively. The FGFs were potent mitogens for the mural cells, SMC and pericytes. Likewise, PDGF was stimulatory for SMC and pericytes. The BB homodimer yielded the greatest degree of growth stimulation for both pericytes and SMC. In the case of SMC the AA homodimer stimulated small but significant growth and the effect of AB was intermediate to that of the homodimeric forms. On the other hand, neither the AA homodimer nor the heterodimer stimulated any significant increase in pericyte number. EGF was moderately mitogenic for both types of mural cells, reaching maximum stimulation at 100 pg/ml of EGF. TGF-beta was inhibitory for SMC but not for pericyte proliferation. These data indicate both quantitative and qualitative differences between the responses of large and small vessel EC to the various growth factors and distinct differences in the responsiveness of SMC and pericytes to PDGF and TGF-beta.