Influence of Endothelium on Cultured Vascular Smooth Muscle Cell Proliferation
Abstract The endothelium exerts a large influence on the underlying vascular smooth muscle, not only by the release of both contracting and relaxing factors but also by its ability to synthesize a large number of molecules that influence vascular smooth muscle growth. In addition to well-characterized growth promoters or growth inhibitors, some endothelium-derived factors, originally described as vasoactive compounds, seem to possess growth-regulatory properties. The vasoconstrictor endothelin-1 elicited a dose-dependent increase of cultured vascular smooth muscle cell DNA synthesis with a maximal effect of 57±14% over basal levels, whereas vasodilators such as prostacyclin, sodium nitroprusside, and 8-bromoguanosine 3′:5′-cyclic monophosphate reduced DNA synthesis by 19±5%, 22±2%, and 31±3%, respectively. Medium conditioned by cultured bovine aortic endothelial cells markedly stimulated both DNA synthesis and proliferation of smooth muscle cells. When medium was conditioned in the presence of the endothelin-converting enzyme inhibitor phosphoramidon, the mitogenic effect was significantly reduced, thus indicating a role for endothelin in the stimulation of smooth muscle cell growth by endothelial cells. However, when both cell types were maintained in a coculture system, a 13±2% decrease of DNA synthesis was observed in smooth muscle cultures. The addition of the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester, the cyclooxygenase inhibitor indomethacin, or both during the coculture period did not revert the antiproliferative effect of endothelial cells in coculture, thereby indicating it is not likely due to these unstable endothelium-derived vasorelaxant molecules.
The endothelium is a key organ in the control of vascular homeostasis.1 At the smooth muscle level, endothelial cells (EC) regulate vascular tone by releasing both vasoconstrictor and vasorelaxing substances in a coordinated manner.2 3 In addition, the endothelium may also act as a modulator of vascular smooth muscle cell (VSMC) growth. Indeed, EC have been shown to produce a large number of growth factors for VSMC, such as platelet-derived growth factor, basic fibroblast growth factor, and insulin-like growth factor–1,4 5 as well as growth-inhibitory molecules, such as heparin/heparan sulfate.6 In addition, some evidence indicates that the vasoactive agents of endothelial origin possess growth-regulatory properties for VSMC. In this way, vasoconstrictors such as endothelin-1 (ET-1) have been shown to promote VSMC growth,7 8 whereas vasorelaxant molecules such as nitric oxide (NO) or prostacyclin (PGI2) have been described as growth inhibitors.9 10
In the present study, we examined the influence of endothelium-derived vasoactive factors on VSMC proliferation, using cell cultures to avoid the possible interferences with other components of the vessel wall. The influence of vasoactive compounds on growth parameters was also analyzed when VSMC were exposed to medium conditioned by EC or maintained in a coculture system with EC.
VSMC were obtained from femoral arteries of 20-week-old male Sprague-Dawley rats by enzymatic disaggregation, as previously described.11 Cells were routinely cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (Gibco), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B (Sigma Chemical Co) and were passaged by trypsinization when confluent. VSMC were used for experiments between passages 5 and 18. EC cultures were obtained from bovine aortas. In brief, after each vessel was longitudinally opened, the lumen was cleaned with PBS and then incubated at room temperature with a PBS solution containing 2 mg/mL collagenase (type II; Sigma) for 10 minutes. Afterwards, the solution was collected and centrifuged, and the resulting bovine aortic endothelial cell (BAEC) pellet was resuspended and cultured in the same medium as VSMC. Confluent cultures presented a typical cobblestone pattern. BAEC between the 5th and 15th passages were used.
Conditioned Medium and Coculture
To obtain conditioned medium, BAEC were grown to confluency on 75-cm2 culture flasks (Nunc) and then growth-arrested by replacement of culture medium by serum-free DMEM containing 0.1% bovine serum albumin (Sigma) and antibiotics (basal medium) for 24 hours. Afterwards, BAEC were incubated with fresh basal medium for 48 hours. The supernatant was then collected and stored at −20°C until it was used. For experiments, different concentrations of conditioned medium were tested by diluting the stock medium (100% vol/vol) with basal medium. For coculture experiments, BAEC were grown to confluency onto 24-well plate inserts (Transwell, Costar), growth-arrested for 24 hours, and then inserted into wells containing confluent serum-depleted VSMC. Both cell types were cocultured in basal medium for 24 hours. VSMC DNA synthesis was determined during the coculture period, as described below. Coculture and conditioned-medium experiments were performed in parallel by use of the same respective VSMC and EC passages.
DNA Synthesis and Cell Number
To determine DNA synthesis, confluent VSMC cultures were 24-hour–growth arrested and then incubated with basal medium containing [3H]thymidine (0.5 μCi/mL, 50 to 60 mCi/mmol, Amersham), and the different compounds were tested for 24 hours. DNA synthesis was determined as the uptake of trichloroacetic acid–insoluble [3H]thymidine into cell cultures, as previously described.11 Cell number was determined by the method of Gillies et al.12 VSMC were seeded onto 24-well plates (Nunc) at a concentration of 5×104 cells per well and allowed to grow for 2 days in the presence of 10% fetal calf serum or different concentrations of BAEC-conditioned medium. Afterwards, VSMC were stained with crystal violet, a basic dye that stains cell nuclei, and the resulting color was measured by absorbance at 595 nm. A standard curve was carried out to establish the relationship between optic units and cell number determined by hemocytometer counting (r=.96).
Drugs and Statistical Analysis
Iloprost was from Schering. Except where noted, all drugs used were from Sigma. Results are expressed as mean±SEM. The statistical analysis was evaluated by Student’s t test for unpaired data and by one-way ANOVA, with a level of significance of P<.05.
Effect of Vasoactive Compounds on VSMC Growth
When ET-1 (100 pmol/L to 1 μmol/L) was exogenously added to VSMC cultures, there was a concentration-dependent increase in DNA synthesis, with a threshold concentration of 1 nmol/L and maximal effect of 57±14% (Figure⇓; ANOVA, P<.05). To assess the effect of NO on VSMC DNA synthesis, cell cultures were treated with the NO donor sodium nitroprusside (SNP) (1 nmol/L to 100 μmol/L). SNP exerted a biphasic action on [3H]thymidine uptake; ie, at low concentrations (1 nmol/L to 1 μmol/L), it reduced [3H]thymidine uptake, whereas at higher concentrations, it promoted DNA synthesis (Figure⇓). The use of SNP concentrations above 100 μmol/L exerted marked cytotoxic effects on VSMC cultures. Additionally, VSMC were treated with the NO-derived second messenger analogue 8-bromo-guanosine 3′:5′-cyclic monophosphate (1 nmol/L to 1 mmol/L). This compound evoked a concentration-dependent reduction of [3H]thymidine uptake, which was significant at 10 nmol/L, with a maximal effect of 31±3% inhibition over basal uptake (Figure⇓; ANOVA, P<.05). The stable analogue of PGI2, iloprost (1 nmol/L to 1 μmol/L), elicited a 22±2% inhibition of DNA synthesis at the concentration of 10 nmol/L (Figure⇓; ANOVA, P<.05).
Effect of BAEC-Conditioned Medium
When VSMC were treated with different concentrations of BAEC-conditioned medium (100%, 60%, 30%, and 15%), there was a marked increase of DNA synthesis and cell proliferation (Table 1⇓). To assess the possible implication of ET-1 on this mitogenic effect, medium was conditioned in the presence of the endothelin-converting enzyme inhibitor phosphoramidon (10 μmol/L). This treatment reduced DNA synthesis by 37±1% when conditioned medium was used at a concentration of 15% (Table 1⇓). Phosphoramidon had no effect by itself on VSMC proliferation (data not shown).
Effect of Coculture on Sprague-Dawley VSMC DNA Synthesis
Coculture of VSMC with BAEC under serum-free conditions resulted in a 13±2% reduction of basal [3H]thymidine uptake into Sprague-Dawley cultures (Table 2⇓). To study the possible role of endothelial NO and/or PGI2 in the reduction of DNA synthesis, BAEC and VSMC were cocultured in the presence of the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (L-NAME) (10 μmol/L), the cyclooxygenase inhibitor indomethacin (10 μmol/L), or both. These compounds had no effects by themselves on basal [3H]thymidine uptake in VSMC cultures. When added in coculture conditions, the inhibitors failed to revert the reduction of DNA synthesis exerted by BAEC (Table 2⇓).
The endothelium possesses the ability to synthesize a large number of substances that may modulate VSMC growth.4 5 The addition of BAEC-conditioned medium to VSMC cultures strongly promoted proliferation, thereby demonstrating the ability of EC to synthesize mitogens for VSMC. Several growth factors have been previously identified in the supernatant of cultured EC,5 13 which may explain the proliferative effect of conditioned medium. In addition to growth factors, the endothelial vasoconstrictor ET-1 may also be involved. Indeed, ET-1 acted as a proliferative agent when exogenously added to cultured VSMC, as previously described by others.7 8 Furthermore, in some experiments, the medium was conditioned in the presence of the endothelin-converting enzyme inhibitor phosphoramidon, which prevents ET-1 secretion.14 This treatment reduced the conditioned medium–induced DNA synthesis, thus indicating that not only growth factors but also vasoactive compounds may participate in the endothelial modulation of the vessel structure.
To further study the endothelial regulation of smooth muscle growth, a coculture system was established. The aim of this experimental approach was to more closely mimic in vivo conditions, allowing the exchange of diffusible compounds between both cell types. In these experimental conditions, the net effect of endothelium was the inhibition of VSMC DNA synthesis, the opposite of the effect seen with the conditioned medium. In fact, this result is more consistent with that observed in entire vessels, in which the endothelium is an agent that counteracts the effect of growth promoters. Indeed, when mature vessels are denuded of EC, the normally quiescent VSMC exhibit a marked increase in their proliferation rate.15
The different effects observed with conditioned medium and coculture may derive from interactions between cocultured VSMC and EC. Indeed, it has been shown that the production of ET-1 by EC is markedly reduced when EC are exposed to medium conditioned by VSMC.16 17 This could contribute to a shift in the balance between the different endothelium-derived growth regulatory molecules toward an antiproliferative action. Furthermore, the possible effects of molecules with short half-lives may be lost in conditioned medium. In the coculture system, unstable compounds such as NO or PGI2 could reach VSMC and therefore exert antiproliferative effects. Indeed, when exogenously added, iloprost and 8-bromo-guanosine 3′:5′-cyclic monophosphate had the ability to reduce VSMC DNA synthesis. SNP had similar effects at low doses, but at higher doses it stimulated DNA synthesis, possibly because of side effects not related to NO.18 The participation of these unstable vasodilators on the antiproliferative effect of EC in coculture was tested by inhibiting their respective synthetic pathways. However, such treatments did not modify the EC-induced effects, thereby indicating a rather limited antimitogenic role, if any, for NO and PGI2, at least in the present experimental conditions. Although other authors have reported a clear antiproliferative role for endothelium-derived vasorelaxant molecules,19 our results suggest that other substances such as heparinoids6 or other as yet unidentified molecules20 may be more relevant in the endothelium-derived growth inhibitory activity.
In summary, EC may synthesize both growth promoters and growth inhibitors for VSMC, including vasoactive compounds, with a net balance toward proliferation or quiescence, depending on the environmental conditions. In mature vessels, the endothelium helps to maintain VSMC quiescence, but pathological alterations might lead to an impaired balance between the different growth-regulatory substances of endothelial origin, therefore favoring excessive VSMC proliferation, as observed in cardiovascular diseases such as atherosclerosis or hypertension.
This work was supported by grants from FISS (93/0916E), DGICYT (FAR 91/0205 and PM 92-0037), and Bayer España. It was integrated into the EEC Concerted Action TRANSGENEUR.
Reprint requests to Dr Carlos F. Sánchez-Ferrer, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, c/Arzobispo Morcillo, 4, 28029-Madrid, Spain.
Rubanyi GM. The role of endothelium in cardiovascular homeostasis and diseases. J Cardiovasc Pharmacol. 1993;8:344-348.
Dzau VJ, Gibbons GH. Endothelium and growth factors in vascular remodeling in hypertension. Hypertension. 1991;18(suppl III):III-115-III-121.
Castellot JJ, Addonizio ML, Rosenberg RD, Karnowsky MJ. Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle growth. J Cell Biol. 1981;90:372-379.
Bobik A, Grooms A, Millar JA, Mitchell A, Grinpunkel S. Growth factor activity of endothelin on vascular smooth muscle. Am J Physiol. 1990;258:C408-C415.
Janakidevi K, Fisher MA, Del Vecchio PJ, Tiruppathi C, Figge J, Malik AB. Endothelin-1 stimulates DNA synthesis and proliferation of pulmonary artery smooth muscle cells. Am J Physiol. 1992;263:C1295-C1301.
Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest. 1989;83:1774-1777.
Peiró C, De Sagarra MR, Redondo J, Sánchez-Ferrer CF, Marín J. Vascular smooth muscle proliferation in hypertensive transgenic rats. J Cardiovasc Pharmacol. 1992;20(suppl 12):S128-S131.
Dicorleto PE, Bowen-Pope DF. Cultured endothelial cells produce a platelet-derived growth factor-like protein. Proc Natl Acad Sci U S A. 1983;80:1919-1923.
Schwartz SM, Heimark RL, Majesky MW. Developmental mechanisms underlying pathology of arteries. Physiol Rev. 1990;70:1177-1209.
Stewart DJ, Langleben D, Cernacek P, Cianflone K. Endothelin release is inhibited by coculture of endothelial cells with cells of vascular media. Am J Physiol. 1990;259:H1928-H1932.
Cade C, Ilozoue V, Rubanyi GM, Parker-Botelho LG. Smooth muscle cell factor responsible for decreasing big endothelin and endothelin produced by cultured endothelial cells. J Cardiovasc Pharmacol. 1991;17(suppl 7):71-75.
Feelisch M. The biochemical pathways of nitric oxide formation from vasodilators: appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solution. J Cardiovasc Pharmacol. 1991;17(suppl 3):S25-S33.
Scott-Burden T, Vanhoutte PM. The endothelium as a regulator of vascular smooth muscle proliferation. Circulation. 1993;87(suppl V):V-51-V-55.