Proliferation Of Aortic Smooth Muscle Cells Research Articles

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: study of strain dependency and the role of protein kinase A and C signaling pathways.

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0-7% center; 7-24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7-24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0-7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 microM Rp-cAMP) or PKC (with 5-20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses.

Intimal hyperplasia following vascular injury is not inhibited by an antisense thrombin receptor oligodeoxynucleotide.

Thrombin is a multifunctional serine protease with central functions in hemostasis, but demonstration of its role in the initiation and maintenance of cell proliferation which occurs following vascular injury is still lacking. To determine the role played by thrombin and its receptor in neointimal accumulation of smooth muscle cells in a rabbit carotid artery model, we have used an 18 mer antisense phosphorothioate oligonucleotide (ODN) directed against the translation initiation region of the human thrombin receptor gene. The antisense ODN inhibited in a dose-dependent manner thrombin- or thrombin receptor activating peptide-induced human aortic smooth muscle cell proliferation. The growth-inhibitory effect of thrombin receptor antisense ODN was preventable by an excess of sense oligomer and specific for thrombin. The suppression of growth was accompanied by a marked decrease of the level of thrombin receptor expression as evidenced by [125I]-thrombin binding to smooth muscle cells. Under the same experimental conditions, the corresponding sense ODN was inactive. The effect of the antisense ODN on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to endothelial injury was then investigated. The topical application of the antisense (500 microg/artery) but not the sense ODN dissolved in F127 pluronic gel around the injured artery resulted, 2 weeks after the application, in a dramatic reduction of the expression of the thrombin receptor mRNA and protein levels as determined by in situ hybridization and immunohistochemistry. However, intimal smooth muscle cell accumulation as estimated by an intimal to medial cross-sectional area ratio was reduced only by 2.7% (vs. 10.3% for the sense ODN), whereas r-hirudin (200 microg/kg/day, s.c.), a potent direct thrombin inhibitor significantly reduced the formation of neointima in denuded carotid arteries (35.4% inhibition, P = 0.03).

Minimally modified low density lipoproteins induce aortic smooth muscle cell proliferation via the activation of mitogen activated protein kinase.

We have investigated the effects of modifying LDL by Cu++ and various hemoglobin preparations on aortic smooth muscle cell proliferation and on the activation of mitogen activated protein kinase. We found that at very low concentrations (10 microg/ml), LDL modified by all of the above agonists markedly stimulated cell proliferation (5-10 fold). This was accompanied by a 2-3 fold stimulation in mitogen activated protein kinase (MAPK) activity. We conclude that modification of LDL under situations that are closer to those found in vivo (i.e. hypoxic conditions), may involve the activation of MAPK as a common biochemical mechanism of action. This in turn, contributes to aortic smooth muscle cell proliferation.

Accumulation of glycosphingolipids in human atherosclerotic plaque and unaffected aorta tissues

We have measured the levels of glycosphingolipids and the activity of glycosphingolipid glycosyltransferases in human aortic intima and media from patients who died of atherosclerosis. The effects of lactosylceramide (LacCer) and glucosylceramide (GlcCer) from plaque intima on smooth muscle cell proliferation were assessed. When the GlcCer data was expressed as (micrograms GlcCer/mg cholesterol and/mg total phospholipid, a 28-fold and 7-fold increase in plaque intima compared to normal intima was observed. Similarly, the level of LacCer was elevated 5-fold and 4-fold, respectively, compared to unaffected intima. The activity of UDP-GlcCer: ceramide beta 1-->4 glucosyltransferase (GlcT-1) was similar in unaffected tissue, fatty streaks, and plaques. However, the activity of UDP-galactose: GlcCer, beta 1-->4 galactosyltransferase (GalT-2) activity was moderately higher in plaque than in unaffected tissue. LacCer, but not GlcCer derived from plaque intima exerted a approximately 2.8-fold increase in the proliferation of human aortic smooth muscle cells grown in tissue culture compared to control presumably due to a marked increase in LacCer molecular species containing C16:0, C22:1, and C24:0 fatty acids in plaque intima compared to control. In sum, our findings provide an interesting and novel pathogenic mechanism of lactosylceramide mediated plaque formation via stimulation of aortic smooth muscle cell proliferation.

Inhibition of Collagen Synthesis, Smooth Muscle Cell Proliferation, and Injury-Induced Intimal Hyperplasia by Halofuginone

Proliferation of vascular smooth muscle cells (SMCs) and accumulation of extracellular matrix (ECM) components within the arterial wall in response to local injury are important etiologic factors in vascular proliferative disorders such as arteriosclerosis and restenosis after angioplasty. Fibrillar and nonfibrillar collagens are major constituents of the ECM that modulate cell shape and proliferative responses and thereby contribute to the pathogenesis of intimal hyperplasia. Halofuginone, an anticoccidial quinoazolinone derivative, inhibits collagen type I gene expression. We investigated the effect of halofuginone on (1) proliferation of bovine aortic endothelial cells and SMCs derived from the same specimen and maintained in vitro, (2) ECM deposition and collagen type I synthesis and gene expression, and (3) injury-induced intimal hyperplasia in vivo. DNA synthesis and proliferation of vascular SMCs in response to serum or basic fibroblast growth factor were abrogated in the presence of as little as 0.1 microgram/mL halofuginone; this inhibition was reversible upon removal of the compound. Under the same conditions, halofuginone exerted a relatively small antiproliferative effect on the respective vascular endothelial cells. Halofuginone also inhibited the synthesis and deposition of ECM components by vascular SMCs as indicated both by a substantial reduction in the amount of sulfated proteoglycans and collagen type I synthesis and gene expression. Local administration of halofuginone in the rabbit ear model of crush injury-induced arterial intimal hyperplasia resulted in a 50% reduction in intimal thickening as measured by a morphometric analysis of the neointima/media ratio. The differential inhibitory effect of halofuginone on vascular SMCs versus endothelial cells, its inhibition of ECM deposition and collagen type I synthesis, and its ability to attenuate injury-induced intimal hyperplasia may place halofuginone alone or in combination with other antiproliferative compounds as a potential candidate for prevention of arterial stenosis and accelerated atherosclerosis.

Macrophage Products Inhibit Human Aortic Smooth Muscle Cell Proliferation and Alter 1α(I) Procollagen Expression

While the role of the foam cell in early atherogenesis has been well characterized, much less is known about the interaction between infiltrating macrophages and medial smooth muscle cells (SMC) in chronic atherosclerosis. Our purpose was to determine the effects of soluble macrophage mediators on normal human aortic SMC proliferation and matrix expression. Human aortic SMC in subconfluent culture were exposed to supernatants from activated lipopolysaccharide (LPS) and nonactivated macrophages. SMC proliferation and type-I procollagen expression were determined. Both activated and nonactivated macrophage supernatants exhibited a potent growth inhibitory effect which became apparent at 48 hours. While nonactivated macrophage supernatant had no effect on procollagen expression, activated supernatant inhibited its expression. (33%; p < 0.05) These findings are consistent with the loss of medial SMC and matrix proteins associated with chronic atherosclerosis.

Rapamycin inhibits vascular smooth muscle cell migration.

Abnormal vascular smooth muscle cell (SMC) proliferation and migration contribute to the development of restenosis after percutaneous transluminal coronary angioplasty and accelerated arteriopathy after cardiac transplantation. Previously, we reported that the macrolide antibiotic rapamycin, but not the related compound FK506, inhibits both human and rat aortic SMC proliferation in vitro by inhibiting cell cycle-dependent kinases and delaying phosphorylation of retinoblastoma protein (Marx, S.O., T. Jayaraman, L.O. Go, and A.R. Marks. 1995. Circ. Res. 362:801). In the present study the effects of rapamycin on SMC migration were assayed in vitro using a modified Boyden chamber and in vivo using a porcine aortic SMC explant model. Pretreatment with rapamycin (2 ng/ml) for 48 h inhibited PDGF-induced migration (PDGF BB homodimer; 20 ng/ml) in cultured rat and human SMC (n = 10; P < 0.0001), whereas FK506 had no significant effect on migration. Rapamycin administered orally (1 mg/kg per d for 7 d) significantly inhibited porcine aortic SMC migration compared with control (n = 15; P < 0.0001). Thus, in addition to being a potent immunosuppressant and antiproliferative, rapamycin also inhibits SMC migration.

Smooth Muscle Cell Cycle and Proliferation: RELATIONSHIP BETWEEN CALCIUM INFLUX AND SARCO-ENDOPLASMIC RETICULUM Ca2+ATPase REGULATION

The role of Ca2+ influx in the regulation of the sarco-endoplasmic reticulum Ca2+ATPases (SERCA) associated with intracellular Ca2+ pools was investigated during smooth muscle cell (SMC) proliferation induced by platelet-derived growth factor (PDGF). We first defined that the previously described up-regulation of the SERCA2a isoform found in vascular SMC after a 24-h stimulation with PDGF (Magnier, C. , Papp, B., Corvazier, E., Bredoux, R., Wuytack, F., Eggermont, F., Maclouf, J., and Enouf, J. (1992) J. Biol. Chem. 267, 15808-15815) was precisely associated with SMC entry into S phase as it appeared linked with [3H]thymidine incorporation. This was further confirmed by testing the effect of transforming growth factor-beta1, which inhibited both aortic SMC proliferation associated with G1 cell cycle arrest and PDGF-induced SERCA2a up-stimulation. Then, we tested the role of Ca2+ influx by using SR 33805, a new Ca2+ channel blocker, which was characterized with regard to the voltage Ca2+ channel blocker nifedipine and the capacitative entry Ca2+ blocker SKF 96365. SR 33805 was found to be the most potent inhibitor of both PDGF-induced SMC proliferation and the associated rise in intracellular Ca2+ concentration with IC50 values of 0.2 +/- 0.1 and 0.31 +/- 0. 04 microM, respectively. Finally, by examining in parallel both SERCA2a and SERCA2b isoforms, in terms of activity and expression, we could determine that PDGF-induced stimulation of total SERCA activity (detected by formation of the phosphorylated intermediate, E approximately P) and of SERCA2a expression (Western blotting) were abolished when extracellular Ca2+ entry was prevented by SR 33805. This study demonstrates that SERCA2a up-regulation is: 1) related to the G1/S transition step of cell cycle and 2) dependent on Ca2+ entry during PDGF-induced SMC proliferation.

Vascular endothelial growth factor and heparin in a biologic glue promotes human aortic endothelial cell proliferation with aortic smooth muscle cell inhibition

Incomplete luminal endothelialization may contribute to small diameter vascular graft failure. Vascular endothelial growth factor (VEGF) can be used to stimulate endothelialization without provoking smooth muscle cell (SMC) proliferation. Heparin and VEGF in a fibrin glue (FG) were investigated for their ability to promote selective human aortic endothelial cell (HAEC) proliferation and human aortic smooth muscle cell (HASMC) inhibition. HAECs and HASMCs were seeded on FG containing VEGF (2.5, 10, 30, 100 ng/ml) or VEGF and heparin (5, 50, 500 units/ml). Proliferation assays were performed with tritiated thymidine on days 1 and 3. Results were analyzed by ANOVA, with p < or = 0.05 significant. HAEC proliferation on FG with 10, 30, and 100 ng/ml VEGF was significantly greater than FG alone at days 1 and 3. The addition of 50 units/ml heparin to VEGF significantly increased HAEC proliferation to greater than FG with VEGF alone at day 1. Human aortic SMC proliferation was not stimulated by the addition of VEGF. The addition of 5, 50, and 500 units/ml heparin significantly inhibited HASMC proliferation regardless of VEGF concentration. VEGF at 10 ng/ml combined with heparin at 50 units/ml exhibited maximal stimulation of HAECs with inhibition of HASMCs. VEGF and heparin in a biologic glue may improve patency by selectively promoting HAEC proliferation without HASMC growth on synthetic vascular bypass grafts.

Inhibition of smooth muscle cell proliferation and migration in vitro by antisense oligonucleotide to c-myb

Smooth muscle cell (SMC) migration and proliferation are prominent features of intimal hyperplasia. Previous studies have shown that inhibition of c-myb inhibits arterial SMC proliferation. Our goal was to evaluate the effect of an antisense oligonucleotide targeted to c-myb on the proliferation and migration of SMC explanted from synthetic vascular grafts. SMCs were enzymatically removed from aortas and Dacron grafts explanted from dogs (n = 5). For proliferation studies, quiescent SMCs were incubated with either 0.0, 0.5, 5.0, or 10.0 microM antisense (GTGTCGGGGTCTCCGGGC) or sense (GCCCGGAGACCCCGACAC) oligonucleotides to c-myb. Proliferation was measured after 24 hours by incorporation of [3H]thymidine. Migration was assessed 24 hours after a razor injury. Antisense to c-myb consistently inhibited proliferation and migration of both native aortic and graft SMCs in a dose-dependent fashion. At a concentration of 10 microM antisense oligonucleotide, aortic and graft SMC proliferation rates were 32% +/- 20% and 56% +/- 9% of control samples, respectively. At 25 microM antisense, the number of migrating aortic and graft SMCs decreased to 41.9% +/- 26.8% and 51.9% +/- 34.1% of control samples, respectively. Our results suggest that antisense oligonucleotides to c-myb may be useful in the inhibition of SMC proliferation and migration associated with development of intimal hyperplasia.

Smooth Muscle Cell–Derived Adenosine Inhibits Cell Growth

Several endogenous factors generated within the vessel wall have been implicated in contributing to the vascular remodeling process associated with hypertension and atherosclerosis. Furthermore, substances generated by smooth muscle cells (SMCs) are known to regulate SMC proliferation in an autocrine fashion. Adenosine is a vasodilator synthesized by SMCs, and exogenous adenosine inhibits SMC proliferation. However, whether adenosine produced endogenously has antimitogenic effects is not known. Hence, we evaluated the effects of SMC-derived adenosine on 2.5% fetal calf serum-induced proliferation of rat aortic SMCs. SMC proliferation was assayed by measurement of DNA synthesis ([3H]thymidine incorporation) and cell counting. To determine the effects of endogenous adenosine on SMC proliferation, we stimulated growth-arrested SMCs with 2.5% fetal calf serum in the presence and absence of modulators of adenosine levels, including (1) erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA; inhibits adenosine deaminase), (2) dipyridamole (blocks adenosine transport and inhibits phosphodiesterase), (3) dipyridamole plus EHNA, and (4) adenosine with or without EHNA. [3H]Thymidine incorporation and cell number were measured after 24 and 96 hours, respectively. EHNA and dipyridamole inhibited both FCS-induced DNA synthesis and cell proliferation in a concentration-dependent manner. Furthermore, extracellular (in medium) adenosine levels were significantly increased when cultured cells were treated with EHNA, and the inhibitory effects of dipyridamole as well as exogenous adenosine were enhanced in the presence of EHNA. Additionally, the inhibitory effects of dipyridamole and EHNA on DNA synthesis were significantly reduced in the presence of KF17837, an A2 adenosine receptor antagonist. These results indicate that SMC-derived adenosine can inhibit SMC proliferation. Hence, it is possible that a defect in localized adenosine synthesis within the vessel wall could contribute to vascular thickening and neointima formation.

Oxidized low density lipoprotein stimulates aortic smooth muscle cell proliferation.

We have investigated the effects of oxidized low density lipoproteins (Ox-LDL) on aortic smooth muscle cell (SMC) proliferation and the biosynthesis of glycosphingolipids. We found that Ox-LDL exerted a concentration, time, and temperature dependent alteration of cell proliferation and the biosynthesis of lactosylceramide. At low concentrations (5-10 micrograms/ml medium) Ox-LDL stimulated cell proliferation measured by an increase in the incorporation of [3H]-thymidine in cells and the synthesis of lactosylceramide, but not glucosylceramide synthesis. Oxidized LDL exerted a threefold increase in the incorporation of [3H]-galactose and [3H]-serine in lactosylceramide. The activity of lactosylceramide synthetase; UDP-galactose glucosylceramide beta 1 --> 4 galactosyltransferase (GalT-2), but not glucosylceramide synthetase (GlcT-1) was stimulated by Ox-LDL. On the other hand, LDL suppressed the activity of GalT-2 in these cells. When cells were preincubated with antibody against Ox-LDL or GalT-2 it compromised the Ox-LDL mediated stimulated in cell proliferation and GalT-2 activity. Similarly, D-PDMP an inhibitor of GalT-2 compromised the Ox-LDL mediated effects in cells. In contrast, L-PDMP further stimulated the Ox-LDL mediated cell proliferation and GalT-2 activity. However, preincubation of cells with preimmune rabbit serum IgG failed to abrogate Ox-LDL mediated stimulation in cell proliferation and GalT-2 activity. In sum, we found that Ox-LDL stimulated aortic smooth muscle cell proliferation in culture. This effect resulted from Ox-LDL mediated activation of GalT-2 that produced lactosylceramide. Lactosylceramide in turn, contributed to cell proliferation. Such correlations are supportive of the notion that GalT-2 action mediates the signal transduction of Ox-LDL contributing to cell proliferation.

Design, synthesis, and evaluation of mitomycin-tethered phosphorothioate oligodeoxynucleotides.

Mitomycin C (1) is the prototypical bioreductive alkylating agent. Studies have shown that mitomycin C and its derivatives selectively alkylate guanine residues within di- and trinucleotide DNA sequences. This investigation sought to improve the selective DNA bonding properties of the mitomycins by coupling them with antisense oligodeoxynucleotides. Two procedures were developed that allowed the attachment of a phosphorothioate oligodeoxynucleotide containing a hexylamino spacer at the 5' terminus with a C(10)-activated mitomycin. In the first procedure, decarbamoylation of 1 (NaOCH3/ benzene) afforded 10-decarbamoylmitomycin C (10), which was treated with either dimethyl sulfate or methylthiochloroformate and base to yield 10-decarbamoylporfiromycin (11) and N(1a)-[(methylthio)-carbonyl]-10-decarbamoylmitomycin C (12), respectively. Activation of the C(10) site in 11 and 12 with 1,1'-carbonyldiimidazole or with 1,1'-thiocarbonyldiimidazole provided the N(1a)-substituted mitomycin 10-decarbamoyl-10-O-carbonylimidazoles (5, 7) and 10-decarbamoyl-10-O-thiocarbonylimidazoles (6, 8), respectively. Compounds 5-8 were reacted with glycine methyl ester hydrochloride (17) and base in both methylene chloride and aqueous buffered solutions to determine the ease and efficiency in which these C(10)-activated mitomycin derivatives coupled to amines. It was found that 5-8 all reacted with 17 in methylene chloride to give the coupled products 18-21 but that improved amine coupling yields in water were observed for the 10-decarbamoyl-10-O-thiocarbonylimidazoles 6 and 8 as compared with the 10-decarbamoyl-10-O-carbonylimidazoles 5 and 7. This finding led to the coupling of the phosphorothioate oligodeoxynucleotide, H2N(CH2)6-P(S)(OH)-GGCCCCGTG-GTGGCTCCAT (22) to 8. Compound 22 complemented a 19-base sequence in the translation initiation region of the human A-raf-1 gene. Use of excess 8 (28 equiv) with 22 gave only a 36% yield of the coupled product 23, which proved difficult to separate from 22. In the second procedure, phosphorothioate oligodexynucleotides that contained a hexylamino spacer at the 5'termini were coupled to 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 was prepared in four steps from 11. Mesylation (methanesulfonyl chloride/pyridine) of 11 gave the C(10) mesylate 13, which was then treated with NaN3 (dimethylformamide, 90 degrees C) to give 10-des(carbamoyloxy)-10-azidoporfiromycin (14). Catalytic reduction (PtO2, H2) of 14 in pyridine afforded C(10) amine 15. Treatment of 15 with di-2-pyridyl thionocarbonate provided the desired 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 readily coupled with 17 and base in both methylene chloride and aqueous buffered solutions to give 25. Use of the 5'hexylaminophosphorothioate oligodeoxynucleotides 32-35 in place of 17 gave the conjugated adducts 28-31, respectively, in a 12% to near-quantitative yield. The products were purified by semipreparative HPLC. Antisense agents 28-31 were designed to target a 30-base-long region from the coding region of the human FGFR1 gene. One adduct, 29, reduced the number of FGFR1 receptors in human aortic smooth cells for bFGF on the cell surface, which suggested down-regulation of FGFR1 gene expression. Further, 29 inhibited cultured human aortic smooth muscle cell proliferation and was less cytotoxic than porfiromycin (2). The biological assay data suggest that the phosphorothioate oligodexynucleotide porfiromycin conjugates may be more target selective and less toxic than either mitomycin or porfiromycin and thus be promising therapeutic agents.

Simultaneous Bindings of uPA and Latent TGF-.BETA. for the Activation of Latent TGF-.BETA. in Homotypic Smooth Muscle Cells.

Latent transforming growth factor-β (TGF-β) is activated when endothelial cells (ECs) and smooth muscle cells (SMCs) are in contact. This activation requires plasminogen activator (PA) activity on ECs and the binding of latent TGF-β to SMCs. Since SMCs are not always in contact with ECs in the vascular wall, a mechanism for the activation of latent TGE-β in homotypic SMCs needs to be elucidated. A low-dose of TGF-β1 (10∼100 pg/ml) stimulated the proliferation of porcine aortic SMCs (PASMCs), while a high-dose of TGF-β1 (1,000 pg/ml) inhibited proliferation. Exogenous urokinase-type PA (uPA) enhanced the proliferation of PASMCs, and the stimulatory effect of uPA was comparable with that of low-dose TGF-β1. The effect of uPA on the proliferation of PASMCs was blocked by a co-administration of either neutralizing anti-TGF-β antibody or α2 plasmin inhibitor, suggesting that exogenous uPA generates active TGF-β via plasmin activity. A binding experiment using 125I-uPA showed that PASMCs expressed specific receptors for uPA. The 2 hr incubation of PASMCs with uPA increased the cell-associated uPA activity. When PASMCs were washed after incubation under the same conditions, the stimulatory effect of uPA on the proliferation of PASMCs persisted. Thus, receptor-bound uPA has been found to be involved in this reaction. The stimulatory effect of uPA on the proliferation of PASMCs was abrogated by the monoclonal antibody against latency-associated peptide, which inhibits the binding of latent TGF-β to PASMCs. These results indicate that the simultaneous bindings of uPA and latent TGF-β generates active TGF-β in homotypic SMCs.

Mechanisms of vascular preservation by a novel NO donor following rat carotid artery intimal injury.

We studied the effects of a novel organic nitric oxide (NO) donor, 4-hydroxymethyl-furazan-3-carboxylic acid-2-oxide (CAS-1609), in a rat carotid artery intimal injury model. The NO donor, CAS-1609, or its non-NO-donating control compound, 4-hydroxymethyl-furazan-3-carboxylic acid (C-93-4845), was infused intravenously at 30 micrograms/day. Seven days after injury, carotid artery rings contracted only 56 +/- 6 mg to NG-nitro-L-arginine methyl ester in C-93-4845-treated rats, compared with 120 +/- 17 mg in CAS-1609-treated rats (P < 0.02), indicating a preservation of endogenous NO release. Improved responses to the endothelium-dependent dilator, acetylcholine, also occurred in injured arteries treated with CAS-1609. Morphometric analysis of injured carotid arteries given the inactive compound showed marked intimal thickening with an intimal-to-medial ratio (I/M) of 0.76 +/- 0.02, compared with a significantly lower I/M of 0.32 +/- 0.04 (P < 0.01) in injured carotid arteries given CAS-1609. Additionally, CAS-1609 was found to have a concentration-dependent stimulatory effect on cultured rat aortic endothelial cell proliferation (P < 0.01) but and inhibitory effect on platelet-derived growth factor-BB (10 ng/ml)-stimulated rat aortic smooth muscle cell proliferation (P < 0.01). This is the first study to demonstrate that NO plays a dual role in vascular cell proliferation, stimulating endothelial cells but inhibiting smooth muscle cell proliferation. This dual effect of NO on cell proliferation is associated with an in vivo reduction in neointimal thickening and an acceleration of endothelial recovery determined by both anatomic and functional methods.

T lymphocytes affect smooth muscle cell phenotype and proliferation.

The effects of rabbit T lymphocytes on rabbit aortic smooth muscle cell (SMC) phenotype and proliferation were investigated in vitro. SMCs seeded at confluent density in primary culture had a volume fraction of myofilaments (Vvmyo) of 49.8 +/- 2.6% after 3 days of culture, not significantly different from that of freshly dispersed cells (Vvmyo, 54.1 +/- 2.1%). Sister cultures of SMCs to which Concanavalin A-activated T lymphocytes or T lymphocyte-conditioned medium was added had significantly lower Vvmyo (35.5 +/- 2.2% and 31.6 +/- 2.3%, respectively) at the same time point. We have previously shown that a decrease in Vvmyo could be induced by the heparan sulfate-degrading activity of living macrophages and by commercial preparations of heparinase. While activated T lymphocytes also completely degraded heparan sulfate-rich 35S-labeled extracellular matrix (an effect inhibited by the addition of 10 micrograms/mL heparin), no heparanase-like activity was detected in T lymphocyte-conditioned medium, indicating that for this cell type SMC phenotypic change is induced by a different mechanism. Incubation of the T lymphocyte-derived cytokine interferon gamma (IFN-gamma) with freshly isolated rat SMCs caused a significant reduction in Vvmyo at day 2 in primary culture from 54.3 +/- 2.1% (control) to 35.4 +/- 3.0%. Furthermore, a neutralizing antibody specific for IFN-gamma removed the effect of T lymphocytes and medium conditioned by them, thus positively identifying IFN-gamma as the T lymphocyte factor responsible for this activity. T lymphocyte-conditioned medium was mitogenic for passaged (low Vvmyo) SMCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-activating factor (PAF) is not an essential component of the cascade leading to smooth muscle cell proliferation following vascular injury

In order to evaluate the relative importance platelet-activating factor (PAF) in the proliferative process leading to restenosis, the effect of SR 27417, a novel highly potent PAF receptor antagonist, on PAF-induced rabbit aortic smooth muscle cell (SMC) proliferation and intimal hyperplasia in rabbit carotid arteries subjected to air-drying endothelial injury was investigated. When added to low concentrations of foetal calf serum, PAF showed a dose-dependent mitogenic effect with regard to rabbit arterial SMC. SR 27417 inhibited PAF-induced SMC growth (IC 50 = 2.4 ± 0.4 nM) but remained without effect on the mitogenic effect of foetal calf serum. A 16 day treatment of SR 27417 (10 mg/kg per day, p.o.) abrogated PAF-induced platelet aggregation ex vivo but did not affect the development of intimal thickening, therefore showing that PAT is not an essential component of the cascade leading to-restenosis following vascular injury.

Inhibition of rabbit aortic smooth muscle cell proliferation by selective inhibitors of protein kinase C.

1. We studied the effect of two structurally-related, selective inhibitors of protein kinase C, Ro 31-8220 and Ro 31-7549, on the reinitiation of proliferation in quiescent first passage rabbit aortic smooth muscle cells in response to (a) the direct activator of protein kinase C, phorbol dibutyrate (PDBu), (b) platelet-derived growth factor (PDGF), (c) a combination of PDGF and 5-hydroxytryptamine (5-HT) or (d) serum. 2. Ro 31-8220 and Ro 31-7549 concentration-dependently inhibited proliferation in response to each mitogen. The inhibitory potency (IC50) of Ro 31-8220 and Ro 31-7549, respectively, was similar against proliferation induced by PDBu (0.55 and 1.1 microM), PDGF (0.6 and 0.9 microM), PDGF and 5-HT (0.68 and 1.1 microM), although slightly less against serum (1.7 and 5 microM). The effects of the protein kinase C inhibitors on proliferation could not be ascribed to cytotoxicity. Neither Ro 31-8220 nor Ro 31-7549 (0.3-3 microM) inhibited PDGF receptor tyrosine phosphorylation. 3. The results show that Ro 31-8220 and Ro 31-7549 are potent inhibitors of smooth muscle cell proliferation in response to a direct activator of protein kinase C, the defined growth factors, PDGF and 5-HT, and the complex mixture of mitogens in serum. Protein kinase C activation thus appears to be an important growth transducing mechanism for each of these agents.

Apoptosis (programmed cell death) in arteries of the neonatal lamb.

We have examined whether cell death contributes to postnatal remodeling of arteries in lambs. First, abdominal aortic smooth muscle cell proliferation rates fell from 2.87 +/- 0.08% per day at 3 days of age to 1.75 +/- 0.15% per day at 21 days. These proliferation rates would yield a 50% increase in DNA content in the absence of cell death. No increase in DNA content was observed (P < .05 for predicted versus measured accumulation); therefore, significant cell death was inferred. The same analysis did not indicate high cell-death rates in the carotid, renal, or iliac arteries; however, cell death was detected in situ by end-labeling partially degraded DNA with terminal deoxynucleotidyl transferase or by nuclear labeling with propidium iodide, a fluorescent dye that permeates only nonviable cells. Nuclei were labeled in all arteries, although labeling was most frequent in the abdominal aorta, a vessel that regresses substantially after birth. Cell death was apoptotic because DNA extracted from arteries and end-labeled with [32P]dCTP produced a series of low molecular weight bands (DNA ladder) on an agarose gel, a hallmark of apoptosis. The ladder was strong for neonatal abdominal aorta but weak for other arteries. Only weak laddering was observed for fetal abdominal aortas in late gestation, confirming that high apoptosis rates in this vessel were initiated after birth. Intense DNA ladders and frequent in situ labeling indicated high rates of apoptosis in the postnatal intra-abdominal umbilical artery, another vessel that regresses after birth. We conclude that apoptosis contributes to postpartum arterial remodeling. This contribution is greatest in arteries that regress after birth.

The effect of lovastatin on proliferation of cultured rat mesangial and aortic smooth muscle cells.

In order to investigate the anti-proliferative effect of 3-hydroxy-3-methylglutaryl coenzyme. A reductase inhibitor, we evaluated the effects of lovastatin on DNA replication and the proliferation of rat mesangial and aortic smooth muscle cells, both of which were mesenchymal origin cells. Proliferations were determined by measuring [3H]thymidine uptake, and counting the number of cells. Growth-arrested mesangial and aortic smooth muscle cells were exposed to platelet-derived growth factor (PDGF), endothelin (ET) and angiotensin II (Ang II) to stimulate mitogenesis. All agents exhibited dose-dependent stimulation of [3H] thymidine uptake. PDGF was more potent than the others. Ang II increased [3H] thymidine uptake without demonstrable mitogenic activity. Lovastatin inhibited PDGF (10 ng/ml in mesangial cell, 25 ng/ml in smooth muscle cell)-, ET (10(-7)M)- and Ang II (10(-7)M)-induced [3H] thymidine uptake significantly in a dose-dependent manner in both cells. The increase of cell number in response to PDGF and ET treatment were also inhibited at 10 microM of lovastatin. The inhibitory effect of lovastatin was largely overcome in the presence of exogenous mevalonate at 200 microM, with 75.5% restoration from lovastatin-induced inhibition on PDGF-induced [3H] thymidine uptake in mesangial cells (77.8% in aortic smooth muscle cells). However, the addition of cholesterol did not prevent inhibition by lovastatin. In conclusion, lovastatin had an inhibitory effect on mesangial and aortic smooth muscle cell proliferation, and mevalonate was essential for DNA replication in both types of cells. Lovastatin may reduce glomerular and atherosclerotic injury through an anti-proliferative effect on mesangial and vascular smooth muscle cells, in addition to lowering circulating lipids.