Role of atrial natriuretic peptide in the suppression of lysophosphatydic acid-induced rat aortic smooth muscle (RASM) cell growth

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. In the present study we investigated the possible role of atrial natriuretic peptide (ANP), a hormone affecting cardiovascular homeostasis and inducing antimitogenic effects in different cell types, on LPA-induced cell growth and reactive oxygen species (ROS) production in rat aortic smooth muscle (RASM) cells. Both LPA effects on cell growth and levels of ROS were totally abrogated by physiological concentrations of ANP, without modifying the overexpression of LPA-receptors. These effects were also affected by cell pretreatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Moreover, the LPA-induced activation of Akt, a downstream target of PI3K, was completely inhibited by physiological concentrations of ANP, which were also able to inhibit p42/p44 phosphorylation. Taken together, our data suggest that PI3K may represent an important step in the LPA signal transduction pathway responsible for ROS generation and DNA synthesis in RASM cells. At same time, the enzyme could also represent an essential target for the antiproliferative effects of ANP.     

Interaction of atrial natriuretic polypeptide and angiotensin II on protooncogene expression and vascular cell growth.

Recent evidence suggests that vasoconstrictive substances, including angiotensin II (Ang II), may function as a vascular smooth muscle growth promoting substance and may contribute to vascular hypertrophy in hypertension. Atrial natriuretic polypeptide (ANP) is known to be a physiological antagonist to Ang II in blood pressure and fluid homeostasis. Moreover, we have demonstrated that ANP can attenuate Ang II's action on vascular hypertrophy. In this study, we investigated the potential molecular mechanisms for the interaction of ANP and Ang II on vascular cell growth. Ang II dose-dependently induced RNA synthesis in post confluent cultured rat aortic smooth muscle (RASM) cells. ANP (10(-7) M) inhibited the hypertrophic effect of Ang II at the concentration of 10(-10) - 10(-8) M) but exerted no effect on the action of higher doses (10(-7) - 10(-6) M) of Ang II. Ang II (10(9) - 10(-8) M) and a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA, 10(-8) M) rapidly induced c-fos as well as c-Jun and Jun-B mRNA expression in RASM cells. ANP (10(-7) M) itself had no apparent effect on the expression of these protooncogenes. Furthermore, ANP did not inhibit the induction of these protooncogenes by Ang II or PMA. Paradoxically, ANP (10(-7) M) significantly enhanced c-fos mRNA expression induced by Ang II and PMA. However, the chloramphenicol acetyl transferase (CAT) assay using a CAT expression vector containing the AP-1 binding element showed that ANP had no effect on the basal and PMA-stimulated AP-1 activity in transfected RASM cells. We conclude, therefore, that the inhibitory effect of ANP on the growth of vascular smooth muscle cells in vitro does not occur through the regulation of these protooncogene expressions.     

Control of smooth muscle cell proliferation in vascular disease

PURPOSE OF REVIEW: Smooth muscle cell proliferation has previously been regarded as a central feature in vascular disease. The role of this process has recently been substantially re-evaluated, and we have reconsidered the functional importance of smooth muscle cell proliferation, the origin of proliferating smooth muscle cells in lesions, and the mechanisms whereby smooth muscle cell proliferation is controlled. In this review, we summarize recent progress in the understanding of smooth muscle cell proliferation, with a particular focus on how interactions between the extracellular matrix, smooth muscle cells, and mitogens control critical steps in this process. RECENT FINDINGS: Irrespective of the origin of smooth muscle cells in vascular lesions, fundamental interactions between the extracellular matrix and cell surface integrins are necessary in order to initiate a proliferative response in a quiescent smooth muscle cell, in a similar manner to any non-malignant cell. These interactions trigger intracellular signaling and cell cycle entry, which facilitate cell cycle progression and proliferation by mitogens. In addition, extracellular matrix interactions may also control the availability and activity of growth factors such as heparin-binding mitogens, which can be sequestered by heparan sulfate containing extracellular matrix components and regulate smooth muscle cell proliferation. SUMMARY: New insights into mechanisms whereby the extracellular matrix takes part in the control of smooth muscle cell proliferation suggest a number of putative targets for future therapies that can be applied to increase plaque stability, prevent the clinical consequences of atherosclerosis and improve outcomes after interventional procedures and organ transplantation.     

Atrial natriuretic factor inhibits proliferation of vascular smooth muscle cells stimulated by platelet-derived growth factor

The effect of atrial natriuretic factor (Isoleucine-ANF 101-126) on basal and platelet-derived growth factor (PDGF)-stimulated proliferation of rat aortic vascular smooth muscle cells (VSMC) was assessed by microscopy and measurement of incorporation of tritiated thymidine by cells cultured with or without addition of PDGF in the presence of various concentrations (10(-8)-10(-6) molar) of ANF. ANF had little effect on proliferation of cells grown in media supplemented with 2% fetal calf serum (FCS) alone but exhibited clear dose-related inhibition of PDGF-stimulated thymidine incorporation.     

Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells

Pulmonary hypertension is characterized by vascular remodeling involving smooth muscle cell proliferation and migration. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators, and the inhibition of aortic smooth muscle cell (ASMC) proliferation by NO has been documented, but less is known about the effects of CGRP. The mechanism by which overexpression of CGRP inhibits proliferation in pulmonary artery smooth muscle cells (PASMC) and ASMC following in vitro transfection by the gene coding for prepro-CGRP was investigated. Increased expression of p53 is known to stimulate p21, which inhibits G(1) cyclin/cdk complexes, thereby inhibiting cell proliferation. We hypothesize that p53 and p21 are involved in the growth inhibitory effect of CGRP. In this study, CGRP was shown to inhibit ASMC and PASMC proliferation. In PASMC transfected with CGRP and exposed to a PKA inhibitor (PKAi), cell proliferation was restored. p53 and p21 expression increased in CGRP-treated cells but decreased in cells treated with CGRP and PKAi. PASMC treated with CGRP and a PKG inhibitor (PKGi) recovered from inhibition of proliferation induced by CGRP. ASMC treated with CGRP and then PKAi or PKGi recovered only when exposed to the PKAi and not PKGi. Although CGRP is thought to act through a cAMP-dependent pathway, cGMP involvement in the response to CGRP has been reported. It is concluded that p53 plays a role in CGRP-induced inhibition of cell proliferation and cAMP/PKA appears to mediate this effect in ASMC and PASMC, whereas cGMP appears to be involved in PASMC proliferation.     

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.     

Distinct atrial natriuretic factor receptor sites on cultured bovine aortic smooth muscle and endothelial cells

Cultured bovine aortic smooth muscle and endothelial cells each display distinct specific binding sites for radiolabeled atrial natriuretic peptide (ANF). 125I-pro-rANF (103-126)I binding to both cell types is rapid, reversible and competitive. Scatchard plots of the binding data show Bmax values of 5.5 and 0.1 - 2.1 X 10(5) sites/cell and Kd values of 2.1 and 0.3 nM for smooth muscle and endothelial cells, respectively. In addition, ANF elevates levels of cGMP substantially in both cell types at concentrations of ANF close to its Kd and Ki for binding. Sodium nitroprusside, however, has essentially no effect on cGMP levels in either cell type. These results show that distinct functionally active receptor sites for ANF exist on both vascular smooth muscle and endothelial cells.     

Antibodies against a putative heparin receptor slow cell proliferation and decrease MAPK activation in vascular smooth muscle cells

Heparin has long been known to slow the growth of vascular smooth muscle cells. However, the mechanism(s) by which heparin acts has yet to be resolved. The identification of a putative heparin receptor in endothelial cells with antibodies that blocked heparin binding to the cells provided the means to further examine the possible involvement of a heparin receptor in smooth muscle cell responses to heparin. Immunoprecipitation of a smooth muscle cell protein with the anti-heparin receptor antibodies provided evidence that the protein was present in smooth muscle cells. Experiments with the anti-heparin receptor antibodies indicate that the antibodies can mimic heparin in decreasing PDGF induced thymidine and BrdU incorporation. The anti-heparin receptor antibodies were also found to decrease MAPK activity levels after activation similarly to heparin. These results support the identification of a heparin receptor and its role in heparin effects on vascular smooth muscle cell growth.     

Effects of hypoxia on rat airway smooth muscle cell proliferation.

Although it is well known that hypoxemia induces pulmonary vasoconstriction and vascular remodeling, due to the proliferation of both vascular smooth muscle cells and fibroblasts, the effects of hypoxemia on airway smooth muscle cells are not well characterized. The present study was designed to assess the in vitro effects of hypoxia (1 or 3% O(2)) on rat airway smooth muscle cell growth and response to mitogens (PDGF and 5-HT). Cell growth was assessed by cell counting and cell cycle analysis. Compared with normoxia (21% O(2)), there was a 42.2% increase in the rate of proliferation of cells exposed to 3% O(2) (72 h, P = 0.006), as well as an enhanced response to PDGF (13.9% increase; P = 0.023) and to 5-HT (17.2% increase; P = 0.039). Exposure to 1% O(2) (72 h) decreased cell proliferation by 21.0% (P = 0.017) and reduced the increase in cell proliferation induced by PGDF and 5-HT by 16.2 and 15.7%, respectively (P = 0.019 and P = 0.011). A significant inhibition in hypoxia-induced cell proliferation was observed after the administration of bisindolylmaleimide GF-109203X (a specific PKC inhibitor) or downregulation of PKC with PMA. Pretreatment with GF-109203X decreased proliferation by 21.5% (P = 0.004) and PMA by 31.5% (P = 0.005). These results show that hypoxia induces airway smooth muscle cell proliferation, which is at least partially dependent on PKC activation. They suggest that hypoxia could contribute to airway remodeling in patients suffering from chronic, severe respiratory diseases.     

Molecular mechanisms of cholesterol or homocysteine effect in the development of atherosclerosis: Role of vitamin E

The development of atherosclerosis is a multifactorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidized low density lipoprotein. Some epidemiological studies have shown an association between high dietary intake and high serum concentrations of vitamin E and lower rates of ischemic heart disease. Cell culture studies have shown that alpha-tocopherol brings about inhibition of smooth muscle cell proliferation. This takes place via inhibition of protein kinase C activity. alpha-Tocopherol also inhibits low density lipoprotein induced smooth muscle cell proliferation and protein kinase C activity. The following animal studies showed that vitamin E protects development of cholesterol induced atherosclerosis by inhibiting protein kinase C activity in smooth muscle cells in vivo. Elevated plasma levels of homocysteine have been identified as an important and independent risk factor for cerebral, coronary and peripheral atherosclerosis. However the mechanisms by which homocysteine promotes atherosclerotic plaque formation are not clearly defined. Earlier reports have been suggested that homocysteine exert its effect via H2O2 produced during its metabolism. To evaluate the contribution of homocysteine in the pathogenesis of vascular diseases, we examined whether the homocysteine effect on vascular smooth muscle cell growth is mediated by H2O2. We show that homocysteine induces DNA synthesis and proliferation of vascular smooth muscle cells in the presence of peroxide scavenging enzyme, catalase. Our data suggest that homocysteine induces smooth muscle cell growth through the activation of an H2O2 independent pathway and accelerate the progression of atherosclerosis. The results indicate a cellular mechanism for the atherogenicity of cholesterol or homocysteine and protective role of vitamin E in the development of atherosclerosis.     

Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle

Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.     

Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation

Hyperglycemia leads to vascular disease specific to diabetes mellitus. This pathology, which results from abnormal proliferation of smooth muscle cells in arterial walls, may lead to cataract, renal failure, and atherosclerosis. The hexosamine biosynthetic pathway is exquisitely responsive to glucose concentration and plays an important role in glucose-induced insulin resistance. UDP-GlcNAc: polypeptide O-N-acetylglucosaminyltransferase (O-GlcNAc transferase; OGTase) catalyzes the O-linked attachment of single GlcNAc moieties to serine and threonine residues on many cytosolic or nuclear proteins. Polyclonal antibody against OGTase was used to examine the expression of OGTase in rat aorta and aortic smooth muscle (RASM) cells. OGTase enzymatic activity and expression at the mRNA and protein levels were determined in RASM cells cultured at normal (5 mM) and at high (20 mM) glucose concentrations. OGTase mRNA and protein are expressed in both endothelial cells and smooth muscle cells in the aorta of normal rats. In both cell types, the nucleus is intensely stained, while the cytoplasm stains diffusely. Immunoelectron microscopy shows that OGTase is localized to euchromatin and around the myofilaments of smooth muscle cells. In RASM cells grown in 5 mM glucose, OGTase is also located mainly in the nucleus. Hyperglycemic RASM cells also display a relative increase in OGTase's p78 subunit and an overall increase protein and activity for OGTase. Biochemical analyses show that hyperglycemia qualitatively and quantitatively alters the glycosylation or expression of many O-GlcNAc-modified proteins in the nucleus. These results suggest that the abnormal O-GlcNAc modification of intracellular proteins may be involved in glucose toxicity to vascular tissues.     

Characterization of the phosphorylation state of natriuretic peptide receptor-C

Many internalized receptors are known to be phosphorylated within their cytoplasmic domain. Natriuretic peptide receptor-C (NPR-C) is a covalent homodimer primarily involved in the internalization of bound ligand resulting in tissue uptake and degradation of natriuretic peptides. In this report, we have investigated the phosphorylation state of NPR-C receptors present at high level in rat aortic smooth muscle cells (RASM).³² P labeled cells, NPR-C purification and phosphoamino acid analysis clearly demonstrate that NPR-C exists as a phosphoprotein in RASM cells and that phosphorylation occurs exclusively on serine residues. Transient expression of bovine NPR-C in Cos-P cells of kidney origin confirmed that phosphorylation occurs within the cytoplasmic domain of the receptor. These results provide the first evidence for NPR-C phosphorylation as well as a model for future studies of its role in altering receptor function.     

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.     

Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor.

To study thrombin's receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell thrombin receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human thrombin receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human thrombin receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell thrombin receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human thrombin receptor. Although the putative thrombin cleavage and binding sites are present, there are significant differences between the rat and human receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this receptor. The steady state thrombin receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell thrombin receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in thrombin receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a thrombin receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the thrombin receptor, and 3) the mRNA level for this receptor is regulated under growth-stimulating conditions in vitro.     

Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells

The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein.     

Differential antimitogenic effectiveness of atrial natriuretic peptides in primary versus subcultured rat aortic smooth muscle cells: Relationship to expression of ANF-C receptors

Previous studies have shown that atrial natriuretic peptides inhibit mitogenesis in subcultured aortic smooth muscle cells by a mechanism that appears to be mediated via the C-type or “clearance” receptor. In the current study, we have compared the antimitogenic effect of these peptides in serum-stimulated primary aortic smooth muscle cell cultures and in subcultured cells. A series of atrial peptides, including rANF_99–126, rANF_103–126, and rANF_103–125, were only poorly antimitogenic in serum-stimulated primary cultures, whereas des[Cys¹⁰⁵, Cys¹²¹] rANF_104–126 which binds selectively to the ANF-C receptors had no antimitogenic activity. In contrast, in subcultured cells (between subcultures 3 and 25), rANF_99–126, rANF_103–126, rANF_103–126, Cys¹¹⁶rANF_102–116, and des[Cys¹⁰⁵, Cys¹²¹]rANF_104–126 inhibited serum-induced [³H]thymidine incorporation (IC₅₀ in the range of 10–50 nM), with maximal inhibition of 40–70%. The lack of antimitogenic activity in primary cultures did not appear to be related to the lack of cGMP elevation elicited by atrial peptides or to an inherent insensitivity to the action of antimitogens, because primary cultures were responsive to the cGMP-elevating effect of atrial peptides and the cells were more rather than less sensitive to the antimitogenic effect of the nitric-oxide-vasodilator, SNAP, as compared to subcultured cells. Analysis of the affinity and binding capacity of freshly isolated aortic membranes, and primary or secondary cultures for [¹²⁵I]rANF_99–126, revealed that the number of ANF receptors increased by tenfold, following subculture. Moreover, subcultured cells contained receptors with increased binding affinity for peptide analogues selective for the ANF-C-type type receptor. Covalent cross-linking studies with (¹²⁵I)rANF_99–126 confirmed that membranes prepared from fresh aortae predominantly expressed the ANF-A/guanylate cyclase receptor, whereas in subcultured cells the predominantly cross-linked protein was the ANF-C-type receptor, with receptors in primary cultures occupying an intermediate position. These results suggest that the binding and antimitogenic activity of atrial peptides in aortic smooth muscle cells depends on the phenotypic state of these cells. Moreover, the increased antimitogenic potency of atrial peptides in secondary cultures may reflect increased expression of the ANF-C-type receptors. © 1993 Wiley-Liss, Inc.