Rat Arterial Smooth Muscle Cell Research Articles

THE EFFECT OF SERUM FACTORS FROM PATIENTS WITH/WITHOUT ESSENTIAL HYPERTENSIOIN ON MEMBRANE RECEPTORS FROM RAT CEREBRAL CORTEX VASCULAR AND CARDIAC MUSCLE CELLS

Essential Hypertension (EHT)is one of the main risk factors for Alzheimer's disease(AD). The aim of our study was to point out experimental data on the potential role of some serum factors from blood of patients with (EHT) in determining some changes in the ionic flow and neuromediators uptake by the rat cerebral cortex, cardiac and arterial smooth muscle cell receptors with a view to estimate the relevance of these factors in the development of cardiovascular pathology and (AD) and the possible therapeutic management of these agents under study. Our study has been conducted on 34 patients :17 aged 35–86 with EHT (10 men and 7 women) and 17 normotensive subjects (aged 30–84 years old) hospitalized in NIGG Ana Aslan. The radioactive incubation experiments of rat cerebral cortex, heart and aorta have been done according with the published technique. The uptake of 45CaCl2,3H Serotonine,3H Cortisol and 3H Norepinephrine by animal tissues in the presence of serum from (EHT) and normotensive patients has been assayed using a Beta Berthold Scintilator Counter. Our data have pointed out a a significant increase in 45Ca uptake by cerebral cortex incubated with serum from (EHT) patients while in rat heart and aorta there is a decrease in 45Ca uptake. When incubated with serum from (EHT) patients in the presence of 3H Norepinephrine, the uptake is increased in cerebral cortex and heart and decreased in aorta. In samples incubated with serum from hypertensive patients, the uptake of 3H Serotonine was significantly decreased in rat brain cortex, heart and aorta. in comparison with biological samples incubated with serum from normothensive patients. Regarding 3H Cortisol uptake, a significant increase in brain cortex and aorta has been recorded in the presence of serum from hypertensive patients while in rat heart there was a decrease in 3H Cortisol uptake. According to our experimental results serum factors present in the blood of (EHT) patients are responsible for changes in receptor state behaviour concerning the uptake of 45Ca,3H SErotonine,3H Noradrenaline and 3H Cortisol in rat cerebral cortex as well as in heart and aorta which may have a significance in the onset of (AD) in (EHT) patients.

Perivascular delivery of resolvin D1 inhibits neointimal hyperplasia in a rat model of arterial injury

Lipid mediators derived from omega-3 polyunsaturated fatty acids such as resolvin D1 (RvD1) accelerate the resolution of inflammation and have potential as vascular therapeutics. The objective of this study was to evaluate local perivascular delivery of RvD1 as a means to attenuate neointimal hyperplasia in a rat model of arterial injury. Smooth muscle cells were harvested from rat aortas to study the effects of RvD1 on rat arterial vascular smoothmuscle cell responses invitro, with focus on inflammation, proliferation, migration, cytoskeletal changes, and cytotoxicity. The safety and efficacy of perivascular delivery of RvD1 through thin biodegradable three-layered poly(lactic-co-glycolic acid) wraps or 25% Pluronic F127 gels were studied in a rat model of carotid angioplasty. Atotal of 200ng of RvD1 was loaded into each construct for perivascular delivery after injury. Morphometric and histologic analyses were performed 3 and 14days after injury. RvD1 attenuated rat arterial vascular smooth muscle cell inflammatory pathways, proliferation, migration, and mitogen-induced cytoskeletal changes invitro, without evidence of cytotoxicity. RvD1-loaded wraps reduced neointimal formation after carotid angioplasty by 59% vs no-wrap controls (P= .001) and by 45% vs vehicle-wrap controls (P= .002). RvD1-loaded Pluronic gels similarly reduced neointimal formation by 49% vs no-gel controls (P= .02) and by 52% vs vehicle-gel controls (P= .02). No group was associated with infection, thrombosis, or negative vessel remodeling. Wraps were found to be easier to apply than gel constructs. Ki67 proliferation index was significantly lower in RvD1-loaded wrap-treated arteries compared with both no-wrap and vehicle-wrap controls at both 3 and 14days after injury (65% vs no-wrap group and 70% vs vehicle-wrap group at day 3, 49% vs both control groups at day 14; P< .05). Similarly, oxidative stress (30%and 29%; P< .05) and nuclear factor κB activation (42% and 45%; P< .05) were significantly lower in the RvD1-loaded wrap group compared with both no-wrap and vehicle-wrap controls at 3days after injury. Local perivascular delivery of RvD1 attenuates formation of neointimal hyperplasia without associated toxicity in a rat model of carotid angioplasty. This effect is likely due to attenuation of inflammatory pathways as well as decreased arterial smooth muscle cell proliferation and migration.

The Role of Piezo1 in Regulating Cytosolic Calcium Level in Aortic Smooth Muscle Cells in Rats

IntroductionPiezo1 proteins are large integral proteins with 24‐39 transmembrane domains. Piezos are expressed in several tissues. Piezo ion channel family is evolutionarily conserved in mechanotransduction. We examined the expression level of Piezo1 in rat aortic smooth muscle cells (ASMCs) and determined stretch induced cytosolic calcium rise in these cells.MethodmRNA and protein expressions of piezo 1 and 2 proteins in rat tissues were detected by RT‐PCR and western blot study, respectively. Stretch‐induced cytosolic calcium elevation in RASMC was determined by confocal microscopy.ResultsIn rats, Piezo1 mRNA and protein was highly expressed in aorta, aortic smooth muscle cells and mesenteric artery, but low in cerebral artery, nodose ganglion and petrosal ganglion. Stretch‐induced [Ca2+]i elevation was found to be increased in Piezo1 over‐expressing rat ASMCs compared with native rat ASMCs.ConclusionThe results indicated that Piezo1 may play a functional role in stretch induced [Ca2+]i elevation in rat arterial smooth muscle cell.

Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

Conclusion: Sustained release of gene vectors is possible through reversible immobilization of adenovirus vectors on bare-metal surfaces of vascular stents. Summary: Polymer-coated stents facilitate local drug delivery to the vasculature and have proven efficacious in preventing in-stent restenosis. There are, however, concerns about the inflammatory effects of polymer coatings and late outcomes of drug-eluting stents. The authors investigated whether adenoviral vectors could be delivered from bare metal surfaces of stents using a synthetic complex for reversible vector binding. Three components of a gene vector-binding complex were synthesized: (1) a polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethylenimine (PEI) with pyridyldithio groups for amplification of attachments sites [PEI (PVT)], and (3) a bifunctional amine-reactive and thiol-reactive cross-linker with labile ester bond (HL). The HL-modified adenovirus attached to PABT/PEI(PDT)-treated steel surfaces and demonstrated in vitro sustained release for 30 days. They also demonstrated localized green fluorescent protein expression in rat arterial smooth muscle cell cultures. This expression was not sensitive to inhibition by neutralizing antiadenovirus antibodies or inactivation after storage at 37°C. In rat carotid studies, steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated site-specific arterial adenovirus green fluorescent protein expression and adenovirus-luciferase transgene activity by optical imaging. Adenovirus encoding inducible nitric oxide synthase delivered by a carotid stent resulted in significant inhibition of restenosis. Comment: This study investigated local delivery of gene therapy from bare-metal stent surfaces using reversible chemical attachment of vectors to the bare-metal stents. Adenovirus vectors on the surfaces of the stents demonstrated >1-month release kinetics and site-specific transduction of target cell types in vitro and in vivo in this rat model. Deployment of stents configured with 109 adenovirus vectors encoded for inducible nitric oxide synthase resulted in significant reduction of in-stent restenosis. It appears that the concept of gene-eluting stents is valid and may provide a mechanism of local and systemic delivery of gene products by the vasculature.

Local Delivery of Gene Vectors From Bare-Metal Stents by Use of a Biodegradable Synthetic Complex Inhibits In-Stent Restenosis in Rat Carotid Arteries

Local drug delivery from polymer-coated stents has demonstrated efficacy for preventing in-stent restenosis; however, both the inflammatory effects of polymer coatings and concerns about late outcomes of drug-eluting stent use indicate the need to investigate innovative approaches, such as combining localized gene therapy with stent angioplasty. Thus, we investigated the hypothesis that adenoviral vectors (Ad) could be delivered from the bare-metal surfaces of stents with a synthetic complex for reversible vector binding. We synthesized the 3 components of a gene vector binding complex: (1) A polyallylamine bisphosphonate with latent thiol groups (PABT), (2) a polyethyleneimine (PEI) with pyridyldithio groups for amplification of attachment sites [PEI(PDT)], and (3) a bifunctional (amine- and thiol-reactive) cross-linker with a labile ester bond (HL). HL-modified Ad attached to PABT/PEI(PDT)-treated steel surfaces demonstrated both sustained release in vitro over 30 days and localized green fluorescent protein expression in rat arterial smooth muscle cell cultures, which were not sensitive to either inhibition by neutralizing anti-Ad antibodies or inactivation after storage at 37 degrees C. In rat carotid studies, deployment of steel stents configured with PABT/PEI(PDT)/HL-tethered adenoviral vectors demonstrated both site-specific arterial Ad(GFP) expression and adenovirus-luciferase transgene activity per optical imaging. Rat carotid stent delivery of adenovirus encoding inducible nitric oxide synthase resulted in significant inhibition of restenosis. Reversible immobilization of adenovirus vectors on the bare-metal surfaces of endovascular stents via a synthetic complex represents an efficient, tunable method for sustained release of gene vectors to the vasculature.

UROKINASE‐INDUCED SMOOTH MUSCLE CELL MIGRATION REQUIRES PKC‐alpha AND PKC‐delta ISOFORMS

Purpose: To determine a role for protein kinase C in urokinase (uPA)-induced smooth muscle cell (SMC) migration, and to examine specific kinase pathways that may act downstream of PKC to influence the response of SMCs to uPA. Background: Vascular SMC migration is an important component of the development of intimal hyperplasia. During tissue remodeling, uPA is one of the key serine proteases involved and stimulates SMC migration in vitro. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of uPA (1–100 nM) with and without general PKC inhibitor, GF109203X (GFX, 1–10 nM), PKC-alpha inhibitor, Goe6976 (1μM) and PKC-delta inhibitor, rottlerin (3μM). Western blotting was performed for phosphorylated and total MEK1/2 and ERK1/2 after stimulation with uPA (10nM) with and without PKC inhibitors. Results: uPA stimulated migration of SMCs in both the linear wound assay (p<0.01) and in the Boyden chamber (p<0.01). PKC-alpha and PKC-delta were the abundant isozymes in the VSMC. Inhibition of PKC with GFX resulted in a concentration dependent decrease in migration in response to uPA. Specific inhibition of PKC-alpha and PKC-delta resulted in ~ 30% decrease in migration (p<0.05). uPA stimulated phosphorylation of MEK1/2 and ERK1/2 (2-fold increase over control for both, p<0.05). These responses were inhibited by the presence of GFX and were dependent on both PKC-alpha and PKC-delta (p<0.01). Conclusions: uPA-induced SMC migration is PKC dependent and is due to their regulation of MEK1/2 and ERK1/2. Both PKC-alpha and PKC-delta isoforms are involved. Understanding the basic mechanisms of cell migration will allow therapeutic intervention for restenosis.

UROKINASE‐INDUCED SMOOTH MUSCLE CELL MIGRATION INVOLVES THE MAMMALIAN TARGET OF RAPAMYCIN

Background: Vascular SMC migration is an important component of the development of intimal hyperplasia. During tissue remodeling, uPA is one of the key serine proteases involved and stimulates SMC migration in vitro. Rapamycin is an antifungal and immunosuppressant that inhibits mTOR, which regulates p70S6 kinase (p70S6K) activity. Purpose: To determine a role for the mammalian target of rapamycin (mTOR) in urokinase (uPA)-induced smooth muscle cell (SMC) migration, and to examine specific kinase pathways that may act downstream of mTOR to influence the response of SMCs to uPA. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of uPA (1–100 nM) with and without rapamycin (1–10 nM). Additional studies were performed in the presence of the PI3-Kinase inhibitor LY294002 (10μM). Western blotting was performed for phosphorylated and total mTOR and p70S6K after stimulation with uPA (10nM) with and without rapamycin. MMP-2 activity determined by zymography. Results: uPA stimulated migration of SMCs in both the linear wound assay (p<0.01) and in the Boyden chamber (p<0.01); these response were inhibited completely by rapamycin in a concentration dependent manner. uPA stimulated phosphorylation of mTOR and p70S6K (2-fold increase over control for both, p<0.05). These responses were inhibited by the presence of the PI3-K inhibitor (p<0.01). Rapamycin completely inhibited both mTOR and p70S6K phosphorylation. uPA induced expression and activity of MMP-2 was markedly inhibited by the presence of rapamycin Conclusions: uPA-induced SMC migration is mTOR dependent. Activation of mTOR and p70S6K is PI3-K dependent. Inhibition of mTOR and p70S6K resulted in a decrease in MMP-2 expression and activity.

MDM2: A Novel Mineralocorticoid-Responsive Gene Involved in Aldosterone-Induced Human Vascular Structural Remodeling

Aldosterone has been demonstrated to play an important role in the pathogenesis of various cardiovascular diseases. Vascular structural remodeling, including vascular smooth muscle cell (VSMC) proliferation, has been also reported in small resistance arteries of patients with primary aldosteronism. Therefore, in this study, we examined whether genes involved in the regulation of the cell cycle were induced by aldosterone alone in cultured human VSMCs and in human small resistance arteries. Results of these studies eventually demonstrated that MDM2, one of the genes involved in anti-apoptosis and cell growth, was markedly increased in mineralocorticoid receptor (MR)-positive VSMCs by aldosterone in all microarray, reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence analyses. In addition, an analysis using small interfering RNA demonstrated that this gene product was involved in cell proliferation of VSMCs induced by aldosterone. Eplerenone, a specific MR antagonist, inhibited this gene induction by aldosterone in VSMCs. MDM2 protein was also more abundant in VSMCs of small resistance arteries in patients with primary aldosteronism compared with a control population. MDM2 is therefore considered one of the mineralocorticoid-responsive genes that regulates cell proliferation of VSMCs induced by MR-mediated aldosterone stimulation, possibly playing an important role in aldosterone-induced vascular structural remodeling.

Urokinase-induced smooth muscle cell migration requires PKC-alpha and PKC-delta isoforms

Purpose: To determine a role for protein kinase C in urokinase (uPA)-induced smooth muscle cell (SMC) migration, and to examine specific kinase pathways that may act downstream of PKC to influence the response of SMCs to uPA. Background: Vascular SMC migration is an important component of the development of intimal hyperplasia. During tissue remodeling, uPA is one of the key serine proteases involved and stimulates SMC migration in vitro. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of uPA (1-100 nM) with and without general PKC inhibitor, GF109203X (GFX, 1-10 nM), PKC-alpha inhibitor, Goe6976 (1μM) and PKC-delta inhibitor, rottlerin (3μM).

Sphingosine-1-phosphate–induced smooth muscle cell migration involves the mammalian target of rapamycin

Vascular smooth muscle cell (SMC) migration is an important component of the development of intimal hyperplasia. Sphingosine-1-phosphate (S-1-P) is a lipid released from activated platelets with numerous cellular effects including the stimulation of SMC migration in vitro. We examined the role of the mammalian target of rapamycin and ribosomal p70S6 kinase (p70S6K) in S-1-P-induced SMC migration . Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P (0.01 to 100 micromol/L) with and without rapamycin (10 nmol/L). Western blotting was performed for phosphorylated and total p70S6K, ERK1/2, and p38(MAPK) after stimulation with S-1-P (0.1 micromol/L), with and without rapamycin pretreatment. Phosphorylation of p70S6K was also assayed after S-1-P treatment in the presence and absence of inhibitors of PI3 kinase (wortmannin, WN, and LY294002, LY), Akt (AktI), p38(MAPK) (SB203580), and MEK1 (PD98059). S-1-P stimulated migration of SMCs in both linear wound and Boyden chamber assays compared to control (P < .05); these responses were inhibited by rapamycin to below the level of control (P < .05 vs S-1-P alone for both assays) in a dose-dependent manner (inhibitory concentration of 50%, 10 nmol/L). S-1-P stimulated phosphorylation of ERK1/2, p38(MAPK), and p70S6K, which peaked at 5 minutes for ERK1/2 and p38(MAPK) and10 minutes for p70S6K (2-fold increase over control for each, P < .05). Rapamycin prevented the phosphorylation of p70S6K at the Thr 389 site (which correlates with enzyme activity), reduced ERK1/2 phosphorylation, but had no effect on the Thr 421/Ser 424 site or on p38(MAPK) phosphorylation. Wortmannin and LY294002 inhibited phosphorylation of the Thr 389 site of p70S6K. AktI and SB203580 had no effect on p70S6K, whereas PD98059 had a marginal effect. S-1-P-induced SMC migration was completely inhibited by rapamycin, indicating that the p70S6K pathway is involved. This mechanism likely involves modulation of the ERK1/2 pathway. S-1-P stimulates phosphorylation of p70S6K in a MEK1-dependent, PI3 kinase-dependent, but Akt-independent manner.

Sphingosine-1-phosphate induced smooth muscle cell migration requires src activation

Abstract Introduction: The object of this study is to examine the role of src signaling axis during smooth muscle cell (SMC) migration in response to sphingosine-1-phosphate (S-1-P). S-1-P is a bioactive sphingolipid released from activated platelets stimulates migration of SMC in vitro through Gi G-proteins and both MAPK and p70S6 kinase activation. Activation of the src signaling axis regulates multiple cellular functions (transcription and translation) during cell migration. Methods: Rat arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P in the presence and absence of the src inhibitor (PP2, 10μM). Western blotting was performed for MEK1/2 and ERK 1/2, MKK3/6 and p38(MAPK), and PI3K/p70S6 kinase phosphorylation after stimulation with S-1-P in the presence and absence of the src inhibitor. Results: S-1-P produced concentration-dependent cell migration in both assays and induced time dependent MEK1/2 and ERK1/2, MKK3/6 and p38(MAPK), and PI3K and p70S6 kinase phosphorylation. Cell migraton was blocked in both assays by src inhibition. PP2 inhibited MKK3/6 and p38(MAPK) but not MEK1/2, ERK1/2 or p70S6kinase activation. S-1-P mediated activation of PI3K was marginally reduced by src inhibition. Conclusions: src mediated p38(MAPK) activation is important in S-1-P induced SMC migrationand ocurs in part in part through PI3K activation. While ERK/2 and p70S6 kinase are involved in migration due to S-1-P, src is not a crucial upstream kinase in the activation of these pathways.

Mechanism for aldosterone potentiation of angiotensin II-stimulated rat arterial smooth muscle cell proliferation.

After earlier studies in which secretion of aldosterone was demonstrated to be important in rat arterial smooth muscle cell (RASMC) proliferation in vitro, the presence of both 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene transcription were shown in these cells by real-time reverse transcription-polymerase chain reaction (RT-PCR). In proliferation studies, tritiated thymidine incorporation into RASMC and RASMC cell number were both significantly increased by angiotensin II (Ang II) (10(-7) mol/L) compared with controls (P<0.01), but this effect was inhibited by the 3beta-hydroxysteroid-dehydrogenase inhibitor trilostane (10(-6) mol/L and 10(-5) mol/L, P<0.05). Aldosterone alone added to RASMC did not significantly change tritiated thymidine incorporation when compared with controls, but the Ang II-induced increase was significantly enhanced by aldosterone at 10(-10) mol/L and 10(-8) mol/L (P<0.05). Neither corticosterone nor 18-hydroxydeoxycorticosterone had any such potentiating effect. RT-PCR analysis and real-time quantitative RT-PCR revealed an increase of Ang II type-1 (AT1) receptor mRNA in RASMC treated by aldosterone (10(-8) mol/L) compared with untreated controls, and this was correlated with a small but significant increase in AT1 receptor protein (P<0.05), as assessed by immunoblotting analysis. These data confirm that steroid production by RASMC is critical in the response to Ang II, and the data support the view that aldosterone specifically is required for the full proliferative response to Ang II in RASMC. One way it may act is by modulating the expression and functions of the AT1 receptor.

125. Formulation and In Vitro Properties of Adenovirus-Polylactide Nanoparticle Affinity-Based and Magnetic Composites

Top of pageAbstract Background Adenoviral vectors have shown promise as a tool for gene delivery-based therapeutic applications. Their use is however limited by the reduced efficacy and systemic adverse reactions resulting from the inability to effectively localize and provide the sustained presence of the vector in the target tissue, while minimizing its escape from the delivery site. To address these problems we investigated the affinity immobilization of adenovirus (AdV) on a biodegradable nanoparticulate platform. Additionally we explored the possibility of enhancing the gene expression by rendering the composite magnetically responsive. Methods Two polylactide-based nanoparticle (NP) formulations were formed by the nanoprecipitation and emulsification-solvent evaporation methods. The NP surface modification with an anionic thiol-reactive derivative of polyallylamine was accomplished photochemically by a brief exposure to long-wave UV light. After separation from unreacted polymer NP were coated with a thiolated form of D1 domain of the Coxsackie-AdV receptor and finally incubated with GFP-encoding AdV suspension in presence of 5% bovine serum albumin. Magnetic NP were formed by inclusion of iron oxide nanocrystals in the polymeric matrix. The particle core and the virus were stained with green BODIPY and red Cy3 dyes, respectively, for fluorimetric studies. AdV-NP binding efficacy was evaluated by measuring the residual fluorescence of unbound virus following magnetic separation of AdV-NP composite. The formulation uptake and efficacy were studied on rat arterial smooth muscle cell culture. The intracellular localization was determined on live cells as a function of incubation time and particle size. The effect of magnetic force on gene expression was assessed by measuring GFP fluorescence in cell lysates using free AdV as a control. Results NP sized 160 and 360 nm were surface-modified with a thiol-reactive polymer having a particle stabilizing effect due to its negative charge. The colloidal stability of the NP was not adversely affected by their subsequent modification with D1 protein and AdV. Five and 20 min photoactivation resulted in an equally high NP capacity (~92%) for AdV binding, whereas unspecifically bound AdV fraction was consistently below 25% in the studied concentration range. Smaller NP exhibited a 1.2-fold higher in vitro cellular uptake and a more rapid elimination than that of larger NP; about 14% of the 360 nm NP initially taken up were found in the cells after 48 hr, while 160 nm NP were undetectable at this timepoint. GFP expression mediated by AdV-particle composite was 2.4 and 10.2-fold higher than that of free AdV when incubated with or without magnet, respectively. The magnetic field exposure resulted in a 4.7-fold increase in gene expression for the composite formulation, having no effect on the free AdV treated cells. Conclusions Immobilization of AdV on a magnetic polymer-based NP platform resulted in a substantial increase in the efficacy of gene transfer in vitro. Rendering this formulation magnetically-responsive provides a potential means for optimizing its biodistribution and efficacy in vivo, and makes this novel gene delivery system an interesting candidate for therapeutic applications.

Nucleotide Receptors Involved in UTP-Induced Rat Arterial Smooth Muscle Cell Migration

Many factors have been shown to be involved in the development of hyperplasic lesions of vessels, but the role of extracellular nucleotides remains largely unknown. The presence of P2Y and P2X nucleotide receptors on arterial endothelial and smooth muscle cells suggests a potential role for nucleotides in the vessel pathophysiology. Although the role of P2X in physiology of vessels is well documented, that of P2Y is not completely understood. We recently demonstrated that extracellular nucleotides, and particularly UTP, induced migration of cultured arterial smooth muscle cells (ASMCs). This migration is dependent on osteopontin expression and involves the Rho and mitogen-activated protein (MAP) kinase pathways. An important question is to determine the specific role of the different P2Y receptors of rat ASMCs in the UTP-induced migration process. Therefore, we first quantified mRNA levels of P2Y(2), P2Y(4), and P2Y(6) nucleotide receptors in cultured rat ASMCs by a competitive RT-PCR approach and demonstrated that P2Y(2) is the most highly expressed among these receptors potentially involved in the UTP-mediated response. In addition to UTP, UDP also induced ASMC migration even when UTP regeneration was inhibited, suggesting the involvement of UDP receptor P2Y(6). Moreover, suramin, a specific antagonist of rat P2Y(2) receptor, acted as an inhibitor of UTP-induced migration. Taken together, these results suggest a prominent role for the UTP receptor, P2Y(2), and for the UDP receptor, P2Y(6), in UTP-induced rat ASMC migration.

Influence of some phospholipase A2and cytochrome P450 inhibitors on rat arterial smooth muscle K+currents

The hyperpolarizing factor that is liberated by vascular endothelial cells in response to various agonists, and known to induce relaxation by opening of smooth muscle K+ channels, has been suggested to be a product of cytochrome P450 dependent arachidonic acid metabolism. In this study, the direct influence of two phospholipase A2 inhibitors and of five structurally and mechanistically different cytochrome P450 inhibitors on K+ currents in freshly isolated vascular smooth muscle cells from the rat aorta was investigated. On stepping the cell membrane potential from -70 mV to a series of depolarized test potentials, a noisy outward current developed at test potentials > +10 mV, which showed no appreciable inactivation during the voltage pulse. It was largely abolished by 3 mM external tetraethylammonium chloride (TEA), suggesting that it predominantly consisted of current through large-conductance Ca(2+)-activated K+ channels. The phospholipase A2 inhibitor quinacrine considerably inhibited this TEA-sensitive current, while 4-bromophenacylbromide exerted no effect. The cytochrome P450 inhibitors proadifen and miconazole reversibly decreased the amplitude of I(K), while clotrimazole and 1-aminobenzotriazole had no effect. Conversely, 17-octadecynoic acid increased whole-cell I(K). These results show that some phospholipase A2 and cytochrome P450 inhibitors may interfere with K+ channel activation in the rat arterial smooth muscle cell. The relevance of these findings to studies on the involvement of cytochrome P450 dependent metabolism in the generation of the endothelium-derived hyperpolarizing factor in intact arteries is discussed.

Heparinase III limits rat arterial smooth muscle cell proliferation in vitro and in vivo

Heparinase III degrades heparan sulfate proteoglycans, which are co-receptors for growth factors that stimulate arterial proliferation. We assessed the ability of locally-delivered heparinase III to limit medial vascular smooth muscle cell proliferation induced by balloon catheter injury in rat carotid arteries. Whereas vehicle-treated arteries showed 12% of smooth muscle cells proliferating after 2 days, heparinase III (0.022–5.7 mg/kg) treated arteries showed 0.8–4%. Chemically-inactivated heparinase III did not limit proliferation. In isolated rat A10 vascular smooth muscle cells, heparinase III (1 IU/ml) inhibited both PDGF-BB and bFGF mediated increases in proliferation and migration. These results suggest that heparinase III can limit proliferation by affecting heparan sulfate proteoglycan binding growth factors following arterial injury.

Mitogen-activated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation.

Smooth muscle cell proliferation and migration is important in arteriosclerosis. In this process, cytokines and growth factors are upregulated and bind to their respective receptors, which in turn stimulate mitogen-activated protein (MAP) kinases. MAP kinases then relay signals to the nucleus that activate quiescent smooth muscle cells. Phosphatases downregulate MAP kinases. We investigated the role of a dual-specificity tyrosine phosphatase, MAP kinase phosphatase-1 (MKP-1), in smooth muscle cell proliferation. MKP-1 expression was high in arterial tissue by Northern analysis, and MKP-1 message was detected mainly in the arterial smooth muscle layer by in situ hybridization. After balloon injury of the rat carotid artery, expression of MKP-1 decreased greatly, whereas that of MAP kinases, especially p44 MAP kinase, increased. The time course of the reduction in MKP-1 message correlated with increased tyrosine phosphorylation and elevated p44 MAP kinase enzymatic activity. In rat arterial smooth muscle cells overexpressing MKP-1, growth was arrested in the G1 phase and entry into the S phase was blocked. A reduction in MKP-1 expression may contribute in part to proliferation of smooth muscle cells after vascular injury, possibly through a decrease in dephosphorylation of p44 MAP kinase.

Phenotypic heterogeneity of rat arterial smooth muscle cell clones: Implications for the development of experiment intimal thickening

Pathological proliferation of human airway smooth muscle cells (HASMCs) causes hyperplasia in chronic lung diseases. Signaling pathways that link airway inflammation to HASMC proliferation might provide therapeutic targets for the prevention of airway remodeling and chronic lung diseases. Endothelin-1 (ET-1) signals via endothelin-A- and B-receptors (ETAR, ETBR) to perpetuate HASMC-associated and TNFα-dependent inflammatory processes. Hypothesis: endothelin receptor antagonists (ERAs) suppress HASMC proliferation induced by inflammatory cytokines. HASMCs were stimulated ex vivo with cytokines in the presence or absence of ERAs (ETAR-specific/selective: BQ123, ambrisentan; ETBR-specific: BQ788; non-selective: bosentan, macitentan, ACT-132577) or cytokine-blocking antibodies. Cell counts, DNA-synthesis (BrdU-incorporation assay), cytokine production (ELISA) and ETBR expression (whole-genome microarray data, western blot) were analyzed. ET-1-induced HASMC proliferation and DNA-synthesis were reduced by protein kinase inhibitors and ETAR-specific/selective ERAs but not by BQ788. TNFα-induced HASMC proliferation and DNA-synthesis were reduced by all ERAs. TNFα induced ET-1 and ETBR expression. TNFα- and ET-1-induced GM-CSF releases were both reduced by BQ123 and BQ788. TNFα- and ET-1-induced IL-6 releases were both reduced by BQ123 but not by BQ788. Combined but not single blockade of GM-CSF-receptor-α-chain and IL-6 reduced TNFα- and ET-1-induced HASMC proliferation and DNA-synthesis. Combined but not single treatment with GM-CSF and IL-6 induced HASMC proliferation and DNA-synthesis in the presence of ET-1. In conclusion, TNFα induces HASMC proliferation via ET-1/GM-CSF/IL-6. ETBR requires up-regulation by TNFα to mediate ET-1 effects on HASMC proliferation. This signaling cascade links airway inflammation to HASMC-associated remodeling processes and is sensitive to ERAs. Therefore, ERAs could prevent inflammation-induced airway smooth muscle hyperplasia.

Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening.

It is well accepted that smooth muscle cells (SMCs) cultured from normal rat arterial media have different morphological and biological features compared with SMCs cultured from experimental intimal thickening (IT) 15 days after endothelial injury. It is not known, however, whether the phenotypic modulation producing IT cells occurs in any medial SMCs or only in a particular SMC subpopulation. To distinguish among these possibilities, the phenotypic features of SMC clones derived from normal adult media and the IT 15 days after endothelial lesion were analyzed according to morphological appearance, replicative activity in the presence and absence of fetal calf serum, and [3H]thymidine incorporation and motile activity; these features were compared with those of the respective SMC parental populations. Two categories of SMC clones predominated: spindle clones, with morphological features similar to those of the parental population from the normal media, and epithelioid clones, with morphological features similar to those of the IT parental population. Both categories were present among clones produced from normal media and IT; however, spindle was more common among normal media clones, and epithelioid, among IT clones. The behavior in vitro was distinct for each category of clones and did not depend on their origin. Our results are compatible with the possibility that the SMC population of IT in vivo derives mainly from SMCs belonging to the category exhibiting epithelioid features in vitro.