Rat Smooth Muscle Cell Proliferation Research Articles

PCSK9 Induces Rat Smooth Muscle Cell Proliferation and Counteracts the Pleiotropic Effects of Simvastatin.

Human atherosclerotic plaque contains smooth muscle cells (SMCs) negative for the contractile phenotype (α-smooth muscle actin) but positive for proprotein convertase subtilisin/kexin type 9 (PCSK9). Thus, we generated rat SMCs which overexpressed human PCSK9 (SMCsPCSK9) with the aim of investigating the role of PCSK9 in the phenotype of SMCs. PCSK9 overexpression in SMCsPCSK9 led to a significant downregulation of the low-density lipoprotein receptor (Ldlr) as well as transgelin (Sm22α), a marker of the contractile phenotype. The cell proliferation rate of SMCsPCSK9 was significantly faster than that of the control SMCs (SMCspuro). Interestingly, overexpression of PCSK9 did not impact the migratory capacity of SMCs in response to 10% FCS, as determined by Boyden’s chamber assay. Expression and activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) was significantly increased in the presence of PCSK9, both in SMCPCSK9 and after treatment with recombinant PCSK9. The transcriptional activity of sterol regulatory element-binding protein (SREBP) was also increased in the presence of PSCK9, suggesting a direct role of PCSK9 in the control of SRE-responsive genes, like HMGCR. We also observed that cholesterol biosynthesis is elevated in SMCPCSK9, potentially explaining the increased proliferation observed in these cells. Finally, concentration-dependent experiments with simvastatin demonstrated that SMCsPCSK9 were partially resistant to the antiproliferative and antimigratory effect of this drug. Taken together, these data further support a direct role of PCSK9 in proliferation, migration, and phenotypic changes in SMCs—pivotal features of atherosclerotic plaque development. We also provide new evidence on the role of PCSK9 in the pharmacological response to statins—gold standard lipid-lowering drugs with pleiotropic action.

Tanshinone II A attenuates vascular remodeling through klf4 mediated smooth muscle cell phenotypic switching

The aim of this study is to investigate the therapeutic role of Tanshinone II A, a key integrant from salvia miltiorrhiza, against pathological vascular remodeling. Completed ligation of mouse left common carotid arteries animal model and rat smooth muscle cells used to investigate the role of Tanshinone II A in regulating pathological vascular remodeling through hematoxylin and eosin staining, immunohistochemistry staining, immunofluorescence staining, adenovirus infection, real time PCR and western blotting. Our data demonstrated that Tanshinone II A treatment suppresses vascular injury-induced neointima formation. In vitro studies on rat smooth muscle cell indicated that Tanshinone II A treatment attenuates PDGF-BB induced cell growth, and promotes smooth muscle cell differentiated marker genes expression that induced by rapamycin treatment. Tanshinone II A treatment significant inhibits rat smooth muscle cell proliferation and migration. Tanshinone II A promotes KLF4 expression during smooth muscle phenotypic switching. Overexpression of KLF4 exacerbates Tanshinone II A mediated smooth muscle cell growth inhibition. Tanshinone II A plays a pivotal role in regulating pathological vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching. This study demonstrated that Tanshinone II A is a potential therapeutic agent for vascular diseases.

Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation

Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K+ channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention.

Differential effect of zoledronic acid on human vascular smooth muscle cells

IntroductionThe activation of human vascular smooth muscle cell proliferation, adhesion and migration is essential for intimal hyperplasia formation. These experiments were designed to test whether zoledronic acid (ZA) would modulate indices of human smooth muscle cell activation, exert differential effects on proliferating versus quiescent cells, and determine whether these effects were dependent on GTPase binding proteins prenylation. ZA was chosen for testing in these experiments because it is clinically used in humans with cancer, and has been shown to modulate rat smooth muscle cell proliferation and migration. MethodsHuman aortic smooth muscle cells (HASMC) were cultured under either proliferating or growth arrest (quiescent) conditions in the presence or absence of ZA for 48 hours, whereupon the effect of ZA on HASMC proliferation, cellular viability, metabolic activity, and membrane integrity were compared. In addition, the effect of ZA on adhesion and migration were assessed in proliferating cells. The effect of increased concentration of ZA on the mevalonate pathway and genomic/cellular stress related poly-adenosine diphosphate ribose polymerase enzyme activity were assessed using the relative prenylation of Rap-1A/B protein and the formation of poly adenosine diphosphate-ribosylated protein, respectively. ResultsThere was a dose dependent inhibition of cellular proliferation, adhesion and migration following ZA treatment. ZA treatment decreased indices of cellular viability and significantly increased membrane injury in proliferating versus quiescent cells. This was correlated with the appearance of unprenylated Rap-1A protein and dose dependent down regulation of activity. ConclusionsThese data suggest that ZA is effective in inhibiting HASMC proliferation, adhesion, and migration, which coincide with the appearance of unprenylated RAP-1A/B protein, thereby suggesting that the mevalonate pathway may play a role in the inhibition of HASMC activation.

Synthesis, Characterization, and Paclitaxel Release from a Biodegradable, Elastomeric, Poly(ester urethane)urea Bearing Phosphorylcholine Groups for Reduced Thrombogenicity

Biodegradable polymers with high elasticity, low thrombogenicity, and drug loading capacity continue to be pursued for vascular engineering applications, including vascular grafts and stents. A biodegradable elastomeric polyurethane was designed as a candidate material for use as a drug-eluting stent coating, such that it was nonthrombogenic and could provide antiproliferative drug release to inhibit smooth muscle cell proliferation. A phosphorylcholine containing poly(ester urethane) urea (PEUU-PC) was synthesized by grafting aminated phosphorylcholine onto backbone carboxyl groups of a polyurethane (PEUU-COOH) synthesized from a soft segment blend of polycaprolactone and dimethylolpropionic acid, a hard segment of diisocyanatobutane and a putrescine chain extender. Poly(ester urethane) urea (PEUU) from a soft segment of polycaprolactone alone was employed as a control material. All of the synthesized polyurethanes showed high distensibility (>600%) and tensile strengths in the 20-35 MPa range. PEUU-PC experienced greater degradation than PEUU or PEUU-COOH in either a saline or lipase enzyme solution. PEUU-PC also exhibited markedly inhibited ovine blood platelet deposition compared with PEUU-COOH and PEUU. Paclitaxel loaded in all of the polymers during solvent casting continued to release for 5 d after a burst release in a 10% ethanol/PBS solution, which was utilized to increase the solubility of the releasate. Rat smooth muscle cell proliferation was significantly inhibited in 1 wk cell culture when releasate from the paclitaxel-loaded films was present. Based on these results, the synthesized PEUU-PC has promising functionality for use as a nonthrombogenic, drug eluting coating on metallic vascular stents and grafts.

Oral administration of the thrombin receptor antagonist E5555 (atopaxar) attenuates intimal thickening following balloon injury in rats

Thrombin is a powerful agonist for a variety of cellular responses including platelet aggregation and vascular smooth muscle cell (SMC) proliferation. These actions are mediated by a thrombin receptor known as protease-activated receptor-1 (PAR-1). Recently we discovered that 1-(3- tert-butyl-4-methoxy-5-morpholinophenyl)-2-(5,6-diethoxy-7-fluoro-1-imino-1,3-dihydro-2 H-isoindol-2-yl)ethanone hydrobromide (E5555, atopaxar) is a potent and selective thrombin receptor antagonist. This study characterized the pharmacological effects of E5555 on SMC proliferation in vitro and in a rat model of intimal thickening after balloon injury in vivo. E5555 selectively inhibited rat aortic SMC proliferation induced by thrombin and thrombin receptor-activating peptide (TRAP) with half maximal inhibitory concentration (IC 50) values of 0.16 and 0.038 μM, respectively. E5555 did not inhibit rat SMC proliferation induced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at concentrations up to 1 μM. In addition, E5555 inhibited human aortic SMC proliferation induced by thrombin at concentrations of 0.3 and 3 units/ml with IC 50 values of 0.028 and 0.079 μM, respectively, whereas it did not affect bFGF-induced proliferation at concentrations up to 1 μM. Repeated oral administration of 30 mg/kg E5555 (once daily for 16 days) significantly reduced neointimal formation in the balloon-injured rat arterial model. These results suggested that a PAR-1 antagonist could be effective for treating restenosis following vascular intervention in addition to preventing thrombus formation. E5555 could thus have therapeutic potential for restenosis and chronic atherothrombotic disease.

The IκB Kinase Inhibitor Nuclear Factor-κB Essential Modulator–Binding Domain Peptide for Inhibition of Injury-Induced Neointimal Formation

The activation of nuclear factor-κB (NF-κB) is a crucial step in the arterial wall's response to injury. The identification and characterization of the NF-κB essential modulator-binding domain (NBD) peptide, which can block the activation of the IκB kinase complex, have provided an opportunity to selectively abrogate the inflammation-induced activation of NF-κB. The aim of the present study was to evaluate the effect of the NBD peptide on neointimal formation. In the rat carotid artery balloon angioplasty model, local treatment with the NBD peptide (300 μg/site) significantly reduced the number of proliferating cells at day 7 (by 40%; P<0.01) and reduced injury-induced neointimal formation (by 50%; P<0.01) at day 14. These effects were associated with a significant reduction of NF-κB activation and monocyte chemotactic protein-1 expression in the carotid arteries of rats treated with the peptide. In addition, the NBD peptide (0.01 to 1 μmol/L) reduced rat smooth muscle cell proliferation, migration, and invasion in vitro. Similar results were observed in apolipoprotein E(-/-) mice in which the NBD peptide (150 μg/site) reduced wire-induced neointimal formation at day 28 (by 47%; P<0.01). The NBD peptide reduces neointimal formation and smooth muscle cell proliferation/migration, both effects associated with the inhibition of NF-κB activation.

E0098 HIF-1α, SDF-1α and VEGF gene expression affected by HIF-1α siRNA in MSCs

ObjectiveHIF-1a, SDF-1a and VEGF gene expression affected by HIF-1a siRNA in MSCs.MethodsBone marrow mesenchymal stem cells were cultured in vitro (3–5 generation,), inverted microscope was used to observed morphology, flow...

A small diameter, fibrous vascular conduit generated from a poly(ester urethane)urea and phospholipid polymer blend

The thrombotic and hyperplastic limitations associated with synthetic small diameter vascular grafts have generated sustained interest in finding a tissue engineering solution for autologous vascular segment generation in situ. One approach is to place a biodegradable scaffold at the site that would provide acute mechanical support while vascular tissue develops. To generate a scaffold that possessed both non-thrombogenic character and mechanical properties appropriate for vascular tissue, a biodegradable poly(ester urethane)urea (PEUU) and non-thrombogenic bioinspired phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-methacryloyloxyethyl butylurethane) (PMBU) were blended at PMBU weight fractions of 0–15% and electrospun to create fibrous scaffolds. The composite scaffolds were flexible with breaking strains exceeding 300%, tensile strengths of 7–10 MPa and compliances of 2.9–4.4 × 10 −4 mmHg −1. In vitro platelet deposition on the scaffold surfaces significantly decreased with increasing PMBU content. Rat smooth muscle cell proliferation was also inhibited on PEUU/PMBU blended scaffolds with greater inhibition at higher PMBU content. Fibrous vascular conduits (1.3 mm inner diameter) implanted in the rat abdominal aorta for 8 weeks showed greater patency for grafts with 15% PMBU blending versus PEUU without PMBU (67% versus 40%). A thin neo-intimal layer with endothelial coverage and good anastomotic tissue integration was seen for the PEUU/PMBU vascular grafts. These results are encouraging for further evaluation of this technique in larger diameter applications for longer implant periods.

Fluvastatin Synergistically Improves the Antiproliferative Effect of Everolimus on Rat Smooth Muscle Cells by Altering p27Kip1/Cyclin E Expression

Multiple intracellular signaling pathways stimulate quiescent smooth muscle cells (SMCs) to exit from G(0) and re-enter the cell cycle. Thus, a combination of two drugs with different mechanisms of action may represent a suitable approach to control SMC proliferation, a prominent feature of in-stent restenosis. In the present study, we investigated the effect of everolimus, a mammalian target of rapamycin inhibitor, in combination with fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on proliferation of rat SMCs. The antiproliferative action of everolimus was amplified by 2.5-fold by the addition of subliminal concentrations of fluvastatin (5 x 10(-7) M), lowering the IC(50) value from 2.5 x 10(-9) to 1.0 x 10(-9) M. The increased antiproliferative effect of everolimus by fluvastatin was prevented in the presence of mevalonate, farnesol, or geranylgeraniol, suggesting the involvement of prenylated proteins. Cell cycle analysis and [3H]thymidine incorporation assay demonstrated that the two drugs synergistically interfered with the progression of G(1) phase. In particular, the drug combination significantly up-regulated p27(Kip1) levels by 47.0%, suppressed cyclin E by 43.0%, and it reduced retinoblastoma (Rb) hyperphosphorylation by 79.0%, compared with everolimus alone. Retroviral overexpression of cyclin E conferred a significant resistance of rat SMCs to the antiproliferative action of the drug combination, measured by cell counting, [3H]thymidine incorporation, and cell cycle analysis, with higher levels of hyperphosphorylated form of Rb. Taken together, these results demonstrated that everolimus acts synergistically with fluvastatin to inhibit SMC proliferation by altering the expression of cyclin E and p27(kip1), which affects Rb phosphorylation and leads to G(1) phase arrest.

722 POSTER Antitumor activity and pharmacokinetics of a novel PEGylated irinotecan in irinotecan-resistant colorectal tumours implanted in mice

A number of proteins post-translationally modified by the covalent attachment of mevalonate-derived isoprene groups farnesol (FOH) or geranylgeraniol (GGOH), play a role in cell proliferation. For this reason, protein farnesyltransferase (PFTase) and protein geranylgeranyltransferases (PGGTases) I and II have gained attention as novel targets for the development of antiproliferative agents. Monoterpenes [limonene, perillic acid (PA) and its derivatives] have been shown to inhibit cell growth and protein prenylation in cancer cells. In the present study, we evaluated the effect of S(−) PA on diploid rat aorta smooth muscle cell (SMC) proliferation as related to protein prenylation. S(−) PA (1–3.5 mM) decreased, in a concentration-dependent manner, rat SMC proliferation as evaluated by cell counting and DNA synthesis. Morphological criteria and flow cytometry analysis excluded the induction of apoptosis as a potential antiproliferative mechanism of S(−) PA on SMC and confirmed a block of the cell cycle progression in G0/G1 phase. The antiproliferative effect of S(−) PA could not be prevented by the addition of mevalonate, FOH, and GGOH to the culture medium and was independent of cholesterol biosynthesis. Densitometric analysis of fluorographed gels, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell lysates, further supported that S(−) PA (1–3.5 mM), under the same experimental conditions, concentration-dependently inhibited FOH (up to 70%) and GGOH (up to 70%) incorporation into cellular proteins. We provide evidence that S(−) PA affects protein prenylation, an effect that may contribute to its inhibition of SMC proliferation.

Isothiazole dioxide derivative 6n inhibits vascular smooth muscle cell proliferation and protein farnesylation

Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4–C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC 50 ranging from 8.56 to 1015 μM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC 50 = 61.4 μM) to those required to inhibit [ 3H]-farnesol incorporation into cellular proteins (−44.1% at 100 μM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [ 3H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.

Elucidating the Inhibitory Mechanisms of Magnolol on Rat Smooth Muscle Cell Proliferation

The pathological mechanism of percutaneous transluminal coronary angioplasty-induced restenosis has been attributed to outgrowth of vascular smooth muscle cells. Pretreatment with antioxidants has been shown to reduce restenosis. Magnolol, an active compound of Magnolia officinalis, has exhibited approximately 1,000 times more potent antioxidant effects than alpha-tocopherol. In this study, we demonstrate, using cytometric analysis, an approximate 61% reduction of smooth muscle cells progressing to the S-phase by 0.05 mg/ml of magnolol. A BrdU incorporation assay also showed a significant reduction (73%) of DNA synthesis using 0.05 mg/ml of magnolol. The protein level of the proliferating cell nuclear antigen was suppressed by approximately 48% using 0.05 mg/ml of magnolol. This was in agreement with the promoter activity of nuclear factor-kappa B, which was also attenuated by 0.05 mg/ml of magnolol. Since receptor interacting protein and caspase-3 protein expression levels were both increased by magnolol in a dose-dependent manner, the apoptotic pathway may mediate the inhibition of cell growth. Our finding that malondialdehyde formation was significantly inhibited by 0.05 mg/ml of magnolol further supported the antioxidant effect of magnolol. These studies suggest that magnolol might be a potential pharmacological reagent in preventing balloon injury-induced restenosis.

Modulation of cyclin D1 and early growth response factor‐1 gene expression in interleukin‐1β‐treated rat smooth muscle cells by n‐6 and n‐3 polyunsaturated fatty acids

The proliferation of smooth muscle cells (SMC) is a key event in the development of atherosclerosis. In addition to growth factors or cytokines, we have shown previously that n-3 polyunsaturated fatty acids (PUFAs) act in opposition to n-6 PUFAs by modulating various steps of the inflammatory process. We have investigated the molecular mechanisms by which the incorporation of the n-6 PUFA, arachidonic acid, increases the proliferation of rat SMC treated with interleukin-1beta, while the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), elicit no mitogenic response. Incorporation of EPA or DHA into SMC, which are then activated by interleukin-1beta to mimic inflammation, decreases promoter activity of the cyclin D1 gene and phosphorylation of the retinoblastoma protein. Together, our data demonstrate that n-3 effects are dependent on the Ras/Raf-1/extracellular signal regulated kinase (ERK)/mitogen-activated protein kinase pathway, and that down-regulation of the cyclin D1 promoter activity is mediated by the specific binding of the early growth response factor-1. Finally, we have shown that the incorporation of EPA and DHA also increased the concentration of caveolin-1 and caveolin-3 in caveolae, which correlated with n-3 PUFA inhibition of SMC proliferation through the mitogen-activated protein kinase pathway. We provide evidence indicating that, in contrast to n-6 PUFAs, n-3 PUFAs exert antiproliferative effects on SMC through the mitogen-activated protein kinase/ERK pathway.

Intraperitoneal infusion of homocysteine increases intimal hyperplasia in balloon-injured rat carotid arteries

Hyperhomocysteinemia is a significant risk factor in atherosclerosis and thrombosis. However, its role in the development of intimal hyperplasia after arterial reconstructive procedures remains uncertain. We therefore studied the effect of homocysteine on intimal hyperplasia in a rat model of carotid artery balloon injury. Twenty-four Spraque–Dawley rats were divided into three groups: control (saline infusion), and low dose (0.14 mg/day) and high dose (0.71 mg/day) homocysteine delivered continuously via osmotic pumps implanted intraperitoneally. All animals underwent left common carotid artery balloon denudation with sacrifice after 14 days. Plasma homocysteine levels, intimal hyperplasia, and cell proliferation of rat carotid arteries were determined. In vitro rat smooth muscle cell (SMC) proliferation with homocysteine treatment was also performed. Plasma homocysteine levels at sacrifice were 1.80±0.35, 2.65±0.05 and 3.50±0.22 μM in three groups, respectively. Intimal hyperplasia developed in all balloon-injured arteries in both control and homocysteine-treated animals. The intimal area and intima/media area ratio were increased by 92% ( P<0.05) and 105% ( P<0.05), respectively, in the high dose-homocysteine-treated animals as compared to the control animals. Homocysteine (high dose) also significantly promoted the intimal cell proliferation (bromodeoxyuridine incorporation) by 2.2-fold as compared to controls. Furthermore, homocysteine treatment in the cell culture study showed a concentration-dependent increase of rat SMC proliferation. These data demonstrate that the continuous intraperitoneal administration of homocysteine significantly increases intimal hyperplasia and SMC proliferation after carotid artery balloon injury in the rat as well as in vitro SMC proliferation. This study suggests that, following arterial reconstructive procedures, elevated plasma homocysteine may increase the complications of clinical restenoses that are associated with intimal hyperplasia.

Effect of S(−) perillic acid on protein prenylation and arterial smooth muscle cell proliferation ☆ 2

A number of proteins post-translationally modified by the covalent attachment of mevalonate-derived isoprene groups farnesol (FOH) or geranylgeraniol (GGOH), play a role in cell proliferation. For this reason, protein farnesyltransferase (PFTase) and protein geranylgeranyltransferases (PGGTases) I and II have gained attention as novel targets for the development of antiproliferative agents. Monoterpenes [limonene, perillic acid (PA) and its derivatives] have been shown to inhibit cell growth and protein prenylation in cancer cells. In the present study, we evaluated the effect of S(−) PA on diploid rat aorta smooth muscle cell (SMC) proliferation as related to protein prenylation. S(−) PA (1–3.5 mM) decreased, in a concentration-dependent manner, rat SMC proliferation as evaluated by cell counting and DNA synthesis. Morphological criteria and flow cytometry analysis excluded the induction of apoptosis as a potential antiproliferative mechanism of S(−) PA on SMC and confirmed a block of the cell cycle progression in G 0/G 1 phase. The antiproliferative effect of S(−) PA could not be prevented by the addition of mevalonate, FOH, and GGOH to the culture medium and was independent of cholesterol biosynthesis. Densitometric analysis of fluorographed gels, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell lysates, further supported that S(−) PA (1–3.5 mM), under the same experimental conditions, concentration-dependently inhibited FOH (up to 70%) and GGOH (up to 70%) incorporation into cellular proteins. We provide evidence that S(−) PA affects protein prenylation, an effect that may contribute to its inhibition of SMC proliferation.

Inhibition of PDGF-stimulated rat smooth muscle cell proliferation by MNA 279 and MNA 715

Inhibition of PDGF-stimulated rat smooth muscle cell proliferation by MNA 279 and MNA 715

Picotamide, an antithromboxane agent, inhibits the migration and proliferation of arterial myocytes

Picotamide is an antiplatelet drug with a peculiar dual mechanism of action: it inhibits thromboxane A 2 synthase and antagonizes the pharmacological responses mediated by thromboxane A 2 receptor. We investigated the in vitro effect of picotamide on smooth muscle cell migration and proliferation. Picotamide (1–500 μM) decreased human and rat smooth muscle cell proliferation, evaluated as cell number, in a concentration-dependent and reversible manner. Picotamide inhibited DNA synthesis induced by fetal calf serum (10%), platelet-derived growth factor (PDGF-BB (20 ng/ml)), epidermal growth factor (EGF (1 nM)) and (15 S)-hydroxy-11,9-(epoxymethano)prosta-5 Z,13 E-dienoic acid (U46619 (10 μM, thromboxane A 2 receptor agonist)). Co-incubation of U46619 together with EGF or PDGF-BB resulted in a marked amplification of [ 3 H ]thymidine incorporation that was completely reversed by picotamide. The drug also inhibited smooth muscle cell migration induced by fibrinogen (600 μg/ml) or PDGF-BB (20 ng/ml) in a concentration-dependent manner. The ability of picotamide to interfere with myocyte migration and proliferation confers, at least in vitro, a pharmacological interest on the compound in atherogenesis.

Antibody to thrombin receptor inhibits neointimal smooth muscle cell accumulation without causing inhibition of platelet aggregation or altering hemostatic parameters after angioplasty in rat.

An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH2-terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor-signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter-induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (P<0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (P<0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG-treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury.

Effect of cyclosporin A on rat smooth-muscle cell proliferation.

Cyclosporin A (CsA) is a potent immunosuppressive agent that has significantly improved graft survival in organ- and bone-marrow-transplant recipients. However, in the context of graft transplantation, CsA has been suggested to potentiate vascular disease by stimulating smooth-muscle cell (SMC) proliferation. As previous studies on the effect of CsA on smooth-muscle proliferation have afforded conflicting results, we conducted an in vitro study of the effect of two concentrations of CsA--10(-6) M (corresponding to the maximal concentration in patients) and 10(-7) M (corresponding to trough concentrations)--on cultured rat SMC proliferation, as assessed by [3H]thymidine incorporation into DNA and measuring cell number by a colorimetric method based on the quantitative staining of cell nuclei. In the presence of 0.5% fetal calf serum (FCS), 10(-6) M CsA induced an increase in [3H]thymidine incorporation into DNA (from 614.44 +/- 67.76 to 1,472.6 +/- 177.63 cpm/well; p < 0.05) with no increase in the number of cells. A cytotoxic effect for this dose was ruled out owing to the absence of significant levels of lactate dehydrogenase (LDH) activity in the supernatant. CsA, 10(-7) M, induced an increase in both [3H]thymidine incorporation into DNA (from 614.44 +/- 67.76 to 1,220.91 +/- 145.59 cpm/well) and cell number (82.49 +/- 6.16 to 165.79 +/- 10.48 cells x 10[3]; p < 0.05). In the presence of 10% FCS, the highest CsA concentration increased [3H]thymidine incorporation to 2,115.91 +/- 224.06 cpm/well, with no significant changes in cell number. However, the lowest CsA concentration increased both [3H]thymidine incorporation (to 3.752.58 +/- 525.06 cpm/well) and cell number (to 181.27 +/- 14.2 cells x 10[3]). These findings suggest that the proliferative effect of CsA on SMCs is variable and that it depends on the concentration of the drug, in support of the discordant results reported previously.