RESUMO
Morphological studies of developing capillaries and observations of alterations in capillaries associated with pathologic neovascularization indicate that pericytes may act as suppressors of endothelial cell (EC) growth. We have developed systems that enable us to investigate this possibility in vitro. Two models were used: a co-culture system that allowed direct contact between pericytes and ECs and a co-culture system that prevented physical contact but allowed diffusion of soluble factors. For these studies, co-cultures were established between bovine capillary ECs and the following growth-arrested cells (hereafter referred to as modulating cells): pericytes, smooth muscle cells (SMCs), fibroblasts, epithelial cells, and 3T3 cells. The modulating cell type was growth arrested by treatment with mitomycin C before co-culture with ECs. In experiments where cells were co-cultured directly, the effect of co-culture on EC growth was determined by comparing the mean number of cells in the co-cultures to the mean for each cell type (EC and modulating cell) cultured separately. Since pericytes and other modulating cells were growth arrested, any cell number change in co-cultures was due to EC growth. In the co-cultures, pericytes inhibited all EC proliferation throughout the 14-d time course; similar levels of EC inhibition were observed in SMC-EC co-cultures. Co-culture of ECs with fibroblasts, epithelial cells, and 3T3 cells significantly stimulated EC growth over the same time course (30-192% as compared to EC cultured alone). To determine if cell contact was required for inhibition, cells were co-cultured using Millicell chambers (Millipore Corp., Bedford, MA), which separated the cell types by 1-2 mm but allowed the exchange of diffusible materials. There was no inhibition of EC proliferation by pericytes or SMCs in this co-culture system. The influence of the cell ratios on observed inhibition was assessed by co-culturing the cells at EC/pericyte ratios of 1:1, 2:1, 5:1, 10:1, and 20:1. Comparable levels of EC inhibition were observed at ratios from 1:1 to 10:1. When the cells were co-cultured at a ratio of 20 ECs to 1 pericyte, inhibition of EC growth at 3 d was similar to that observed at other ratios. However, at higher ratios, the inhibition diminished so that by the end of the time course the co-cultured ECs were growing at the same rate as the controls. These results suggest that pericytes and SMCs can modulate EC growth by a mechanism that requires contact or proximity. We postulate that similar interactions may operate to modulate vascular growth in vivo.
Assuntos
Endotélio Vascular/citologia , Músculo Liso Vascular/citologia , Retina/citologia , Córtex Suprarrenal/irrigação sanguínea , Animais , Aorta/citologia , Capilares/citologia , Bovinos , Divisão Celular , Células Cultivadas , Células EpiteliaisAssuntos
DNA/biossíntese , Fibroblastos/metabolismo , Heparina/farmacologia , Músculo Liso Vascular/citologia , Miocárdio/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Coração/efeitos dos fármacos , Heparitina Sulfato/farmacologia , Imunoensaio , Masculino , CoelhosRESUMO
Capillary formation has been correlated with changes in basement membrane-associated glycosaminoglycans (GAGs). During capillary growth when endothelial cells (EC) undergo extensive proliferation and migration and pericytes are scarce, hyaluronic acid (HA) levels are elevated. Upon capillary maturation when EC migration and proliferation cease and pericytes appear, the dominant GAG is heparan sulfate (HS). To investigate the potential role of GAGs in the angiogenic process, we studied the effect of HA, heparin, chondroitin sulfate, and dermatan sulfate on the attachment and proliferation of vascular wall cells in vitro. Cell attachment was studied by determining the number of cells attached to GAG-treated substrates. Whereas HA inhibited the attachment of both pericytes and smooth muscle cells (SMC) by nearly 80% after 8 hr, it enhanced capillary EC attachment by more than 30%. Retinal pigment epithelial cells and dermal fibroblasts were employed as controls and none of the GAGs examined significantly altered the attachment of these cells. The effect of GAGs on cell proliferation was determined by the addition of soluble GAGs to cells cultured for the time required for three population doublings. Heparin addition resulted in a dose-dependent inhibition of both pericyte and SMC proliferation with maximal inhibition of 50% at 100 micrograms/ml, whereas this concentration of heparin moderately enhanced capillary EC proliferation. These effects were not observed for any other cell type or with any other GAG and indicate that GAGs have cell-specific effects on the attachment and proliferation of cells of the vascular wall.
Assuntos
Glicosaminoglicanos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Animais , Aorta/citologia , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Capilares/citologia , Bovinos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio/citologia , Heparina/efeitos adversos , Heparina/farmacologia , Ácido Hialurônico/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Ratos , TrítioRESUMO
We studied histamine metabolism, i.e., histidine decarboxylase (HD)-mediated synthesis and histaminase-mediated catabolism, in relation to intracellular histamine content in both aortic endothelial and subjacent smooth muscle cells of control and diabetic rats. Diabetes was induced by a single jugular vein injection of streptozotocin (55 mg/kg in acidified saline, pH 4.5), and animals were held for either 2 or 4 weeks following overt manifestation of diabetes. An additional 4-week diabetic group received insulin (Iletin NPH, 10 U per 24 hour) during the last week. With respect to control values, the histamine content of aortic endothelial cells increased 138%, HD activity increased 250%, and histaminase activity decreased 50% over the 4-week period. In subjacent smooth muscle cells, the histamine content increased in excess of 150%, HD activity increased more than 300%, and histaminase activity decreased in excess of 30%. Insulin treatment for the last week resulted in complete reversal of all these changes. These results support the concept that a large vessel response similar to the microcirculatory prolonged phase of inflammation occurs in experimental diabetes, a change similar to that occurring in experimental atherosclerosis. They also indicate that both synthetic and catabolic changes occur in histamine metabolism under these conditions, changes that alter arterial wall histamine pools, and suggest that insulin administration under conditions of experimental diabetes may modulate aortic histamine metabolism and the resultant intraaortic histamine pools.
Assuntos
Aorta/metabolismo , Diabetes Mellitus Experimental/metabolismo , Histamina/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Arteriosclerose/metabolismo , Endotélio/metabolismo , Histidina Descarboxilase/metabolismo , Masculino , Músculo Liso Vascular/metabolismo , Ratos , Ratos EndogâmicosRESUMO
We studied rat aortic endothelial and smooth muscle cell de novo histamine synthesis mediated by histidine decarboxylase (HD) and the effects of its inhibition by alpha-hydrazinohistidine on the intracellular histamine content and intraaortic albumin accumulation in streptozotocin-induced diabetes. Diabetes was induced by a single jugular vein injection of streptozotocin (60 mg/kg, pH 4.5, ether anesthesia), with animals held 4 weeks following the overt manifestation of diabetes. Additional diabetic and nondiabetic rats received alpha-hydrazinohistidine (25 mg/kg, i.p. every 12 hours) during the last week; this had no effect on the severity of diabetes in any animal receiving streptozotocin. Data indicate that the aortic endothelial (EC) HD activity was increased more than 130% in the untreated diabetic group but was similar to control values in the diabetic group receiving alpha-hydrazinohistidine; similarily, the EC histamine content from diabetic aortas increased 127% over control values, but in EC from diabetic animals receiving alpha-hydrazinohistidine it was comparable to control values. Similar trends were observed for the subjacent aortic smooth muscle. In untreated diabetic animals the aortic 125I-albumin mass transfer rate was increased 60% over control values, while in diabetic animals receiving alpha-hydrazinohistidine the 125I-albumin mass transfer rate was essentially identical to controls. These data indicate that in streptozotocin diabetes there is an expansion of the inducible aortic histamine pool, and that this expansion is intimately related to the increased aortic albumin accumulation.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Histamina/metabolismo , Músculo Liso Vascular/metabolismo , Soroalbumina Radioiodada/metabolismo , Animais , Aorta Torácica/metabolismo , Transporte Biológico , Diabetes Mellitus Experimental/patologia , Endotélio/metabolismo , Endotélio/patologia , Histidina Descarboxilase/metabolismo , Masculino , Músculo Liso Vascular/patologia , Ratos , Ratos EndogâmicosRESUMO
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 beta (TGF-beta), we now demonstrate that activated TGF-beta 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 coculture-derived conditioned media with antibodies to TGF-beta 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-beta neutralizing antibodies to cocultures blocked the pericyte-mediated EC growth inhibition. These results indicate that latent TGF-beta is produced by these cells and it is activated by a mechanism that requires contact between the two cell types.