Transfection Efficiency and Cytotoxicity of Nonviral Gene Transfer Reagents in Human Smooth Muscle and Endothelial Cells

PURPOSE: Evaluation of a nonviral transfection reagent with respect to efficient gene transfer into primary human vascular cells. METHODS: Complexes consisting of seven commercially available transfection reagents (DAC-30, DC-30, Lipofectin, LipofectAMINE PLUS, Effectene, FuGene 6 and Superfect) and EGFP encoding plasmid DNA were studied. The in vitro transfection efficiency and cytotoxicity in human aorta smooth muscle cells (HASMCs) and endothelial cells (HAECs) and rat smooth muscle cells (A-10 SMCs) were assayed in the presence of serum using flow cytometric analysis and ATP-quantitation assay, respectively. RESULTS: Human primary cells were transfected less efficiently compared to the rat smooth muscle cell line. Transfection efficiency depended on the type of reagent, the reagent/DNA ratio, and, most importantly, on the cell type used. Determination of cytotoxicity showed that the effects of transfection on cell viability did not significantly differ from one another depending on the cell type. The exception to this was Superfect, which obviously reduced cell viability in all cell types. CONCLUSIONS: Our experiments showed that DAC-30 is the preferred transfection reagent for HASMCs and HAECs, exhibiting an improved efficiency combined with an acceptable cytotoxicity. Therefore, it might offer a therapeutic option for the treatment of cardiovascular disease and prove suitable for further drug development.     

Gene expression profiles in response to the activation of adrenoceptors in A7r5 aortic smooth muscle cells

1. Vascular adrenoceptors play an important role in vascular physiology and pathophysiology, such as hypertension, atherosclerosis and restenosis after angioplasty. To define the changes in the ene expression in vascular smooth muscle cells in response to the activation of alpha1- or beta-adrenoceptors, a DNA microarray was used. 2. First, the existence of alpha1- and beta-adrenoceptors in A7r5 aortic smooth muscle cells was confirmed by radioligand binding. Then, the inhibitory effects of phenylephrine (an alpha1-adrenoceptor agonist) and isoproterenol (a beta-adrenoceptor agonist) on the proliferation of A7r5 cells were determined by [3H]-thymidine incorporation. 3. The A7r5 cells were treated with 10 micromol/L phenylephrine or 1 micromol/L isoproterenol for 24 h and changes in gene expression were detected with the DNA microarray. Only 14 and 20 genes were identified after treatment of cells with phenylephrine and isoproterenol, respectively, and most genes displayed decreased expression. The changed genes could be grouped into five major functional categories: cell signalling/communication, cell structure/motility, cell/organism defence, gene/protein expression and metabolism. The gene expression profile in response to the activation of alpha1-adrenoceptors was very different from that following activation of beta-adrenoceptors. Interestingly, many phenylephrine-responsive genes were associated with metabolism, whereas many isoproterenol-responsive genes encoded cell signalling and structure proteins. This means that adrenoceptors may modulate multiple aspects of biological function in vascular smooth muscle cells. 4. Collectively, the activation of both alpha1-adrenoceptors (with phenylephrine) and beta-adrenoceptors (with isoproterenol) inhibited the proliferation of A7r5 cells, but microarray data revealed that the mechanisms may be different: the activation of alpha1-adrenoceptors could induce the expression of metabolic genes, resulting in the inhibition of proliferation, whereas activation of beta-adrenoceptors altered the expression of genes that encoded cell signalling and structure proteins to inhibit cell proliferation.     

Expression and characterization of the human alpha 2B-adrenoceptor in a vascular smooth muscle cell line

A vascular smooth muscle cell line stably expressing the human alpha 2B-adrenoceptor at a density of 1.5 pmol/mg membrane protein was generated by transfection of rat A7r5 cells. [35S]GTPgammaS binding experiments and [3H]thymidine incorporation experiments indicated that the expressed receptors were functional, had the expected pharmacological characteristics and efficiently stimulated smooth muscle cell proliferation. Confocal fluorescence microscopy was used to visualize alpha2B-adrenoceptors in A7r5-alpha 2B cells and indicated that the receptors were mainly localized in the plasma membrane. The expression of the smooth muscle-specific marker alpha-actin was similar in transfected A7r5-alpha 2B cells and in non-transfected A7r5 wild-type cells. The generated A7r5-alpha 2B cell line will be a useful tool for studying the function and regulation of alpha 2B-adrenoceptors in vascular smooth muscle cells.     

Relaxation of pig coronary arteries by new and potent cGMP analogs that selectively activate type I alpha, compared with type I beta, cGMP-dependent protein kinase.

Smooth muscle preparations of human aorta or pig coronary arteries contain nearly equal amounts of cGMP-dependent protein kinase isozymes (cGMP kinase I alpha and I beta). In order to understand the roles of these isozymes in relaxing vascular smooth muscle, several new cGMP analogs were synthesized and tested for potencies in activating each enzyme and in relaxing pig coronary arteries. Analogs modified with a derivatized phenylthio group at the 8-position were as much as 72-fold more potent in activating purified cGMP kinase I alpha than cGMP kinase I beta. Electron-donating substituents, such as hydroxy, amino, and methoxy, on the phenyl ring enhanced the potencies of these analogs in activating cGMP kinase I alpha. The most potent of these cGMP analogs [8-(4-hydroxyphenylthio)-cGMP] was 17 times more potent (EC50 = 1.1 microM) as a muscle relaxant than the most efficacious analog tested previously. Among derivatives with an 8-halo group, 8-iodo-cGMP was the most potent compound (Ka = 9 nM for I alpha and 122 nM for I beta) for both I alpha and I beta. Analogs modified at the 1,N2-position or at both the 1,N2-and 8-positions of cGMP were highly potent for activating both isozymes. Within this group, 8-I-beta-phenyl-1,N2-etheno-cGMP had Ka values of 22 nM and 17 nM for cGMP kinase I alpha and I beta, respectively, whereas the Ka values of cGMP were 110 nM and 250 nM for the two isozymes. 8-I-beta-phenyl-1,N2-etheno-cGMP was the most potent muscle relaxant tested, with EC50 of 0.4 microM. For all cGMP analogs tested, there was a positive correlation between potency for activation of cGMP kinase I alpha and that for relaxation of pig coronary arteries. Assuming that the kinase assay conditions yielded a cyclic nucleotide specificity similar to that which would exist in intact cells, it was concluded that the cGMP kinase I alpha isozyme mediates the relaxation of pig coronary artery smooth muscle caused by cGMP elevation. However, an additional role for cGMP kinase I beta in the relaxation process could not be ruled out.     

The induction of nitric oxide synthase activity is inhibited by TGF-beta 1, PDGFAB and PDGFBB in vascular smooth muscle cells.

The effect of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor (PDGF) was investigated on the induction of nitric oxide synthase activity caused by interleukin-1 beta in cultured smooth muscle cells from rat aorta. TGF-beta 1, PDGFAB and PDGFBB but not PDGFAA inhibited in a concentration-dependent manner the production of nitrite, an oxidation product of nitric oxide, evoked by interleukin-1 beta. The growth factors alone did not stimulate the release of nitrite. The addition of interleukin-1 beta-treated smooth muscle cells to suspensions of indomethacin-treated human washed platelets inhibited the aggregation evoked by thrombin whereas no effect was observed with untreated cells. Platelet aggregation was not inhibited by smooth muscle cells that had been pretreated with interleukin-1 beta in combination with either TGF-beta 1, PDGFAB or PDGFBB but not with PDGFAA. These observations demonstrate that platelet-derived products such as TGF-beta and PDGFs inhibit the induction of nitric oxide synthase activity in vascular smooth muscle cells.     

Vascular calcium channels and high blood pressure: pathophysiology and therapeutic implications

Long-lasting Ca(2+) (Ca(L)) channels of the Ca(v)1.2 gene family are heteromultimeric structures that are minimally composed of a pore-forming alpha(1C) subunit and regulatory beta and alpha(2)delta subunits in vascular smooth muscle cells. The Ca(L) channels are the primary pathways for voltage-gated Ca(2+) influx that trigger excitation-contraction coupling in small resistance vessels. Notably, vascular smooth muscle cells of hypertensive rats show an increased expression of Ca(L) channel alpha(1C) subunits, which is associated with elevated Ca(2+) influx and the development of abnormal arterial tone. Indeed, blood pressure per se appears to promote Ca(L) channel expression in small arteries, and even short-term rises in pressure may alter channel expression. Membrane depolarization has been shown to be one stimulus associated with elevated blood pressure that promotes Ca(L) channel expression at the plasma membrane. Future studies to define the molecular processes that regulate Ca(L) channel expression in vascular smooth muscle cells will provide a rational basis for designing antihypertensive therapies to normalize Ca(L) channel expression and the development of anomalous vascular tone in hypertensive pathologies.     

alpha(v)beta(3) Integrin antagonists as inhibitors of bone resorption

The alpha(v)beta(3) integrin is a non-covalent, heterodimeric, cell-surface protein that is expressed with varying density on numerous cell types, including osteoclasts, vascular smooth muscle cells, endothelial cells and a variety of tumour cells. Functionally, alpha(v)beta(3) mediates a diverse range of biological events including the adhesion of osteoclasts to bone matrix, smooth muscle cell migration and angiogenesis. Specifically, there has been significant attention focused on the preparation of inhibitors of alpha(v)beta(3) for use as inhibitors of bone resorption, in recognition of the medical need for improved prevention and treatment of osteoporosis. Herein, we summarise the pertinent chemistry and biological advances in the medicinal design and biological evaluation of peptide and small molecule alpha(v)beta(3) antagonists as inhibitors of bone resorption.     

Direct demonstration of beta1- and evidence against beta2- and beta3-adrenoceptors, in smooth muscle cells of rat small mesenteric arteries

1 Recent evidence supports additional subtypes of vasodilator beta-adrenoceptor (beta-AR) besides the 'classical' beta(2). The aim of this study was to investigate the distribution of beta-ARs in the wall of rat mesenteric resistance artery (MRA), to establish the relative roles of beta-ARs in smooth muscle and other cell types in mediating vasodilatation and to analyse this in relation to the functional pharmacology. 2 We first examined the vasodilator beta-AR subtype using 'subtype-selective' agonists against the, commonly employed, phenylephrine-induced tone. Concentration-related relaxation was produced by isoprenaline (pEC(50): 7.70+/-0.1) (beta(1) and beta(2)). Salbutamol (beta(2)), BRL 37344 (beta(3)) and CGP 12177 (atypical beta) caused relaxation but were 144, 100 and 263 times less potent than isoprenaline; the 'beta(3)-adrenoceptor agonist' CL 316243 was ineffective. 3 In arteries precontracted with 5-HT or U 46619, isoprenaline produced concentration-related relaxation but salbutamol, BRL 37344, CGP 12177 and CL 316243 did not. SR 59230A, CGP 12177 and BRL 37344 caused a parallel rightward shift in the concentration-response curve to phenylephrine indicating competitive alpha(1)-AR antagonism, explaining the false-positive 'vasodilator' action against phenylephrine-induced tone. Endothelial denudation but not L-NAME slightly attenuated isoprenaline-mediated vasodilatation in phenylephrine and U 46619 precontracted MRA. 4 The beta-AR fluorescent ligand BODIPY TMR-CGP 12177 behaved as an irreversible beta(1)-AR antagonist in MRA and bound to the surface and inside vascular smooth muscle cells in intact vascular wall. Beta-ARs in smooth muscle cells were observed in a perinuclear location, consistent with the location of Golgi and endoplasmic reticulum. 5 Binding of BODIPY TMR-CGP 12177 was inhibited by BAAM (1 microM) in all three vascular tunics, confirming the presence of beta-ARs in adventitia, media and intima. Binding in adventitia was observed in both neuronal and non-neuronal cell types. Lack of co-localisation with a fluorescent ligand for alpha-ARs confirms the selectivity of BODIPY TMR-CGP 12177 for beta-ARs over alpha-ARs. 6 Our results support the presence of functional vasodilator beta(1)-ARs and show that they are mainly located in smooth muscle cells. Furthermore, we have demonstrated, for the first time, the usefulness of BODIPY TMR-CGP 12177 for identifying beta-AR distribution in the 'living' vascular wall.     

Interaction between the 90-kDa heat shock protein and soluble guanylyl cyclase: physiological significance and mapping of the domains mediating binding

The 90-kDa heat shock protein (hsp90) regulates the stability and function of many client proteins, including members of the NO-cGMP signaling pathway. Soluble guanylyl cyclase (sGC), an NO receptor, was recently reported to be an hsp90-interacting partner. In the present study, we show that hsp90 binds to both subunits of the most common sGC form (alpha(1)beta(1)) when these are expressed individually but only interacts with beta(1) in the heterodimeric form of the enzyme. Characterization of the region of hsp90 required to bind each subunit in immunoprecipitation experiments revealed that residues 310 to 456 of hsp90 interact with the sGC subunits. The region of beta(1) responsible for binding to hsp90beta was mapped using in vitro binding assays and immunoprecipitation experiments and was found to lie in the regulatory domain. The physiological importance of the hsp90/sGC interaction was investigated by treating rat smooth muscle cells with the hsp90 inhibitors radicicol and geldanamycin (GA) and determining both sGC activity and protein levels. Long-term (24 or 48 h) inhibition of hsp90 resulted in a strong decrease of both alpha(1) and beta(1) protein levels and sGC activity. Moreover, incubation of smooth muscle cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) blocked the GA-induced down-regulation of sGC. We conclude that the N-terminal region of the beta(1) subunit mediates binding of the heterodimeric form of sGC to hsp90 and that this interaction involves the M domain of hsp90. Hsp90 binding to sGC regulates the pool of active enzymes by affecting the protein levels of the two subunits.     

Statins regulate alpha2beta1-integrin expression and collagen I-dependent functions in human vascular smooth muscle cells

HMG-CoA reductase inhibitors have direct vascular effects that contribute to plaque stability. In the current study, the authors demonstrate that the HMG-CoA reductase inhibitors atorvastatin and pravastatin augment the adhesion of human (HSMCs) and rat aortic smooth muscle cells (RASMCs) to collagen I via induction of alpha2beta1-integrin receptors. Atorvastatin (0.1 microM ) increased the adhesion of HSMCs to collagen I up to 2-fold (p < 0.01) and pravastatin (1.0 microM ) up to 1.8-fold (p < 0.01) after treatment of at least 24 h. This increase in adhesion was concentration dependent and was observed for treatment periods from 16 to 72 h. Inhibition of isoprenoid synthesis with mevalonate and geranyl-geraniol prevented the statin-induced effect on human and rat smooth muscle cells. Flow cytometry revealed an increased expression of alpha2- and beta1-integrins after treatment with atorvastatin (0.1 microM ) at 24 and 48 h. Atorvastatin increased levels of beta1-integrin mRNA after 12- and 24-h treatment in HSMCs, which was inhibited by mevalonate. Furthermore, atorvastatin (0.1 microM ) and pravastatin (1.0 microM ) inhibited chemotaxis of HSMCs on collagen I, which was also reversed by mevalonate treatment. In contrast, inhibition of beta1-integrins with a specific antibody nearly doubled (p < 0.01) the rate of chemotaxis. These data indicate that the chemotactic activity in HSMCs is inhibited in part by up-regulation of alpha2beta1-integrin receptors. The current study indicates that HMG-CoA reductase inhibitors increase cell-matrix interaction with collagen I via induction of alpha2beta1-integrins and increased adhesion to collagen I.     

Glibenclamide inhibits thromboxane-mediated vasoconstriction by thromboxane receptor blockade

Because sulfonylureas, such as glibenclamide, are used to treat Type 2 diabetes and because this disease is associated with various cardiovascular complications that may be mediated by thromboxane (TX), this study was designed to characterize the role of glibenclamide on TX-mediated contractions in isolated ring segments of bovine coronary arteries and rabbit aortas. A series of TXA(2) analogs [9,11 Dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha) (U46619), [1S-(1alpha, 2beta(5Z),3alpha(1E, 3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), carbocyclic TXA(2) (CTA(2)) and 9,11-dideoxy-9alpha,11alpha-epoxymethano prostaglandin F(2alpha) (U44069)], endothelin and phenylephrine contracted both types of blood vessels. Glibenclamide (10 microM) inhibited the contraction to each of the TX agonists but had no effect on endothelin- or phenylephrine-induced contractions. We hypothesized that this effect was due to a direct effect to block the vascular smooth muscle cell TX receptor. Receptor binding studies were performed in rabbit vascular smooth muscle cells and indicated that glibenclamide (10 microM) inhibited (125)I-BOP binding by more than 80%. The inhibition constants or K(i) for glibenclamide was 0.53 microM. These studies provide the first evidence that the ability of glibenclamide to inhibit TX-mediated contractions occurs independent of the vascular K(ATP) channel and is, instead, mediated by the blockade of the vascular TX receptor.     

Cloning, cell-type specificity, and regulatory function of the mouse alpha(1B)-adrenergic receptor promoter

The functionality of a 3422-base pair promoter fragment from the mouse alpha(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expressing BC(3)H1, NB41A3, and DDT(1)MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known alpha(1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that alpha(1)ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be alpha(1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis-acting elements, RAT-1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation of that CAT reporter when induced by challenge with forskolin and/or hypoxic conditions.     

The effect of cold storage on the sensitivity to alpha and beta agonists in the isolated rabbit kidney.

Rabbit kidneys were perfused at 25 degrees C, and the effect of alpha and beta agonists was studied, before and after 24 hr of cold storage. Vascular and in vitro functional parameters were evaluated. We concluded that cold storage impairs partially the vascular alpha receptor. We could not find beta receptors under these conditions. An electron microscope analysis of these kidneys has shown some degree of autolysis of the tubular cells and good preservation of the vascular smooth muscle cells and membranes. These results are very important for kidney preservation before transplantation.     

RGS2 is a mediator of nitric oxide action on blood pressure and vasoconstrictor signaling

The nitric oxide (NO)-cGMP pathway regulates vascular tone and blood pressure by mechanisms that are incompletely understood. RGS2, a GTPase-activating protein for Gqalpha that is critical for blood pressure homeostasis, has been suggested to serve as an effector of the NO-cGMP pathway that promotes vascular relaxation based on studies of aortic rings in vitro. To test this hypothesis and its relevance to blood pressure control, we determined whether RGS2 functions as an NO effector in smooth muscle of the resistance vasculature. We report that 1) the ability of the NO donor sodium nitroprusside to reduce blood pressure is impaired in RGS2-/- mice, 2) vasopressin-triggered Ca2+ transients are augmented in smooth muscle cells from resistance arteries of RGS2-/- mice, and 3) cGMP analogs fail to inhibit vasopressin-triggered Ca2+ transients in smooth muscle cells from resistance arteries of RGS2-/- mice even though cGMP-dependent protein kinase (PKG)1alpha and PKG1beta are expressed and activated normally. These results indicated that the NO-cGMP pathway uses RGS2 as a novel downstream effector to promote vascular relaxation by attenuating vasoconstrictor-triggered Ca2+ signaling in vascular smooth muscle cells. Genetic or epigenetic impairment of this mechanism may contribute to the development of hypertension, and augmenting it pharmacologically may provide a novel means of treating this disease.     

Alpha 1-adrenergic stimulation and beta 2-adrenergic inhibition of DNA synthesis in vascular smooth muscle cells

Effects of catecholamines on DNA synthesis in vascular smooth muscle cells (VSMC) were investigated in a chemically defined medium that included insulin, transferrin, and sodium selenite. Smooth muscle-rich preparation was obtained from rat aortic media and VSMC were further purified by cell cloning. A clone that was positive for smooth muscle actin and was negative for the coagulation factor VIII was used in this study. The fetal calf serum-induced proliferation was enhanced by alpha-adrenergic and inhibited by beta-adrenergic stimulation. When cells of low passages were used, dose-response curves for norepinephrine were biphasic; when cells were subconfluent, norepinephrine stimulated DNA synthesis at as low as 1 nM and was apparently ineffective at more than 100 nM. When cells were confluent, the effect of norepinephrine was inhibitory at lower concentrations (less than 1 nM) and stimulatory at relatively higher concentrations. Cells of higher passages exhibited only inhibitory effects of the amine. Stimulatory and inhibitory effects on DNA synthesis were mediated through alpha 1- and beta 2-adrenergic receptors, respectively. Thus, the alpha 1-agonist phenylephrine was more potent than the alpha 2-agonist clonidine in stimulating DNA synthesis. An alpha 1-adrenergic antagonist, prazosin, was more effective than the alpha 2-adrenergic antagonist yohimbine in antagonizing the stimulatory effect of norepinephrine. beta-Adrenergic agonists inhibited DNA synthesis with IC50 values in the nanomolar range; the rank order of potency of agonists was isoproterenol greater than salbutamol greater than or equal to (-)-epinephrine much greater than (-)-norepinephrine, consistent with beta 2-receptor specificity. (+)-Epinephrine or (+)-norepinephrine, the stereoisomers of the catecholamines, were ineffective. The inhibitory effects of norepinephrine were reversed by beta-adrenergic antagonists, with the rank order of potency of pindolol greater than butoxamine greater than atenolol, consistent with beta 2-receptor specificity. The dose-response curves of norepinephrine, therefore, seemed to be determined by a balance between alpha 1-receptor-mediated stimulation and beta 2-receptor-mediated inhibition of DNA synthesis. Minimum time required for exhibiting alpha 1-adrenergic or beta 2-adrenergic effects was between 6 and 15 hr, suggesting that the G0 or G1 phase of the cell cycle might be the site of action. These results show that catecholamines dually modulate DNA synthesis in VSMC through specific adrenergic receptors.     

Regulation of the expression of soluble guanylyl cyclase by reactive oxygen species

BACKGROUND AND PURPOSE: Superoxide anions produced during vascular disease scavenge nitric oxide (NO), thereby reducing its biological activity. The aim of the present study was to investigate whether reactive oxygen species (ROS) have a direct effect on soluble guanylyl cyclase (sGC) subunit levels and function and to ascertain the mechanism(s) involved. EXPERIMENTAL APPROACH: Rat aortic smooth muscle cells (RASM) or freshly isolated vessels were exposed to reactive oxygen species (ROS)-generating agents and sGC subunit expression was determined at the mRNA and/or protein level. cGMP accumulation was also determined in RASM exposed to ROS. KEY RESULTS: Incubation of smooth muscle cells with H(2)O(2), xanthine/xanthine oxidase (X/XO) or menadione sodium bisulphite (MSB) significantly decreased protein levels of alpha1 and beta1 subunits of sGC and reduced SNP-induced cGMP formation. Similarly, sGC expression was reduced in freshly isolated vessels exposed to ROS-generating agents. The ROS-triggered inhibition of alpha1 and beta1 levels was not blocked by proteasome inhibitors, suggesting that decreased sGC protein was not due to protein degradation through this pathway. Real time RT-PCR analysis demonstrated a 68% reduction in steady state mRNA levels for the alpha1 subunit following exposure to H(2)O(2). In addition, alpha1 promoter-driven luciferase activity in RASM decreased by 60% after H(2)O(2) treatment. CONCLUSION AND IMPLICATIONS: We conclude that oxidative stress triggers a decrease in sGC expression and activity that results from reduced sGC steady state mRNA levels. Altered sGC expression is expected to contribute to the changes in vascular tone and remodeling observed in diseases associated with ROS overproduction.     

Inhibition of cultured bovine aortic smooth muscle cell proliferation by colominic acid

Colominic acid (CA) is an alpha2,8-linked polymer of sialic acid, originally isolated from capsular Escherichia coli K1. Since inhibition of arterial smooth muscle cell hyperplasia is one of the effective strategies to prevent atherosclerosis, we investigated the effect of CA, purified as an alpha2,8-linked homopolymer of N-acetylneuraminic acid, on the proliferation of bovine aortic smooth muscle cells in culture. The results demonstrate that CA inhibits the proliferation of the cells without nonspecific cell damage. Sulfation did not modify the inhibitory effect of CA. Specifically, the inhibitory effect of sulfated CA was almost equal to that of CA in vascular smooth muscle cell proliferation. On the other hand, it was suggested that the inhibition of the proliferation by CA is in a degree similar to that by heparin but weaker than that by sodium/calcium-spirulans, known sulfated polysaccharides as the potent inhibitor of vascular smooth muscle cells. The present data suggest that CA with or without sulfate groups can be an origin of beneficial agents that prevents atherosclerosis through a moderate inhibition of arterial smooth muscle cell proliferation.