Quiescent Smooth Muscle Cells Research Articles

Expression of nonmuscle myosin heavy chain-B isoform in the vessel wall of porcine coronary arteries after balloon angioplasty.

Nonmuscle myosin heavy chain-B isoform (NMMHC-B) is expressed by proliferating vascular smooth muscle cells (SMCs), and its expression in primary lesions has been proposed to be predictive of restenosis after atherectomy. The present study was designed to study the time-course expression of NMMHC-B after angioplasty of porcine coronary arteries by in situ hybridization and immunohistochemistry. Domestic juvenile swine underwent percutaneous transluminal coronary angioplasty (PTCA) of the left anterior descending and circumflex coronary arteries with standard clinical angioplasty catheters. To identify proliferating cells, 5'-bromo-2'-deoxyuridine (BrdU) was administered and detected by immunohistochemistry on serial sections. Vessels were examined at 3, 7, and 14 days after balloon angioplasty, and uninjured coronary vessels were used as controls. Normal arteries showed hybridization to 35S-labeled NMMHC-B riboprobes localized mainly in the medial layer. NMMHC-B expression in the adventitia was markedly increased 3 days after balloon angioplasty. Seven and 14 days after injury, NMMHC-B mRNA-containing cells were localized in the adventitia and neointima at the arterial injury site. Cell proliferation, as indicated by BrdU staining, colocalized with NMMHC-B mRNA expression 3 and 7 days after angioplasty. These data indicate that cells proliferating in the adventitia and neointima express NMMHC-B; however, its expression is not limited to the proliferative state, since NMMHC-B mRNA was also found in quiescent SMCs of normal coronary arteries and in nonproliferating adventitial and neointimal cells 14 days after angioplasty.

The expression of transforming growth factor-beta s and TGF-beta receptor mRNA and protein and the effect of TGF-beta s on human myometrial smooth muscle cells in vitro.

In this study we investigated the expression of transforming growth factor-beta (TGF-beta) isoform and TGF-beta receptor mRNA and protein, and the effect of TGF-beta 1-3 on the rate of DNA synthesis and proliferation of human myometrial smooth muscle cells in vitro. To determine these, we utilized primary cultures of myometrial smooth muscle cells, standard and competitive quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, enzyme-linked immunoassay, radioreceptor assay, [3H] thymidine incorporation and cell proliferation assay. Standard RT-PCR and immunocytochemistry revealed that myometrial smooth muscle cells express TGF beta 1-3 and TGF-beta type I-III receptor (TGF-beta R) mRNA and protein. Quantitative RT-PCR, using an external synthetic RNA standard, indicated that the cells express 10 copies/cell of TGF-beta 1 and TGF-beta 2, less than one copy/cell of TGF-beta 3 and TGF-beta type IR, three copies/cell of type IIIR, and > 200 copies/cell, of TGF-beta type IIR mRNA. The cells also synthesized and released TGF-beta 1 at the rate of 7.8 +/- 0.7 ng/10(6) cells, of which 1.4 +/- 0.2 ng/10(6) cells was in an active form. The rate of [3H] thymidine incorporation or proliferation of subconfluent quiescent smooth muscle cells was not altered by TGF-beta s (0.1-10 ng/ml) under serum-free conditions, nor in the presence of 10% fetal bovine serum (FBS). TGF-beta 1-3 at 0.25-0.5 ng/ml in the presence of 2% FBS, which induces half maximal stimulation of these cells, stimulated the rate (P < 0.05), whereas at higher doses it reduced the rate of [3H]-thymidine incorporation compared to the controls. The effect of TGF-beta was partially reversible using neutralizing antibodies specific to TGF-beta 1, TGF-beta 2 (10 micrograms/ml) or TGF-beta 3 (3-6 micrograms/ml). TGF-beta s had no significant effect on cell proliferation determined by cell counting. The data indicate that human myometrial smooth muscle cells express the necessary components of the TGF-beta system, suggesting an autocrine/paracrine role for TGF-beta s in myometrium.

Angiotensin II Stimulates Phosphorylation of the Translational Repressor 4E-binding Protein 1 by a Mitogen-activated Protein Kinase-independent Mechanism

To investigate the molecular basis of the hypertrophic action of angiotensin II (AII) in vascular smooth muscle cells (SMC), we have examined the ability of the hormone to regulate the function of the translational repressor 4E-binding protein 1 (4E-BP1). Addition of AII to quiescent aortic SMC potently increased the phosphorylation of 4E-BP1 as revealed by a decreased electrophoretic mobility and an increased phosphate content of the protein. The stimulation of 4E-BP1 phosphorylation was maximal at 15 min and persisted up to 120 min. Results from affinity chromatography on m7GTP-agarose demonstrated that AII-induced phosphorylation of 4E-BP1 promotes its dissociation from eIF4E in target cells. Further characterization of 4E-BP1 phosphorylation by phosphoamino acid analysis and phosphopeptide mapping revealed that 4E-BP1 is phosphorylated on eight distinct peptides containing serine and threonine residues in AII-treated cells. The combination of results obtained from kinetics experiments, phosphopeptide analysis of in vitro and in vivo phosphorylated 4E-BP1, and pharmacological studies with the MAP kinase kinase inhibitor PD 98059 provided strong evidence that the MAP kinases ERK1/ERK2 are not involved in the regulation of 4E-BP1 phosphorylation in aortic SMC. Together, our results demonstrate that AII treatment of vascular SMC leads to hyperphosphorylation of the translational regulator 4E-BP1 and to its dissociation from eIF4E by a MAP kinase-independent mechanism.

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.

Dietary Lipid Source Alters Murine Macrophage/Vascular Smooth Muscle Cell Interactions in Vitro

This study was conducted to compare the impact of dietary lipids on the ability of macrophages to modulate vascular smooth muscle cell (SMC) DNA synthesis in vitro. C57BL/6 female mice were fed six different diets (6 mice/diet) containing 10% fat from corn oil (CO), borage oil (BO), primrose oil (PO), fish-corn oil mix (FC, 9:1, w/w), fish-borage oil mix (FB, 1:3, w/w), or fish-primrose oil mix (FP, 1:3, w/w) for 2 wk. Peritoneal macrophages were isolated from these mice, stimulated with zymosan or vehicle, and subsequently co-cultured with naive mouse aortic SMC in the presence of 3H-thymidine to measure SMC DNA synthesis. In this co-culture system, macrophages were seeded on 25-mm culture inserts (upper chamber) and SMC were seeded on 35-mm culture dishes (lower chamber). The two cell types were separated by a semipermeable membrane with a 30-kD cut-off. When quiescent SMC were co-cultured with macrophages, only the PO and FP diet groups had significantly (P < 0.05) lower SMC DNA synthesis compared with the control CO group whose diet contained no γ-linolenic acid (GLA) or (n-3) polyunsaturated fatty acids (PUFA). In contrast, when cycling SMC were co-cultured with dietmodulated macrophages, all dietary groups except for those fed FC had significantly lower (P < 0.05) SMC DNA synthesis relative to the CO group. Although the level of GLA in PO and BO diets was different (11.5 and 22.3 g/100 g fatty acids, respectively), these treatments exerted comparable inhibitory effects on SMC DNA synthesis. The FP treatment consistently exhibited the lowest SMC DNA synthetic profile among the six dietary groups irrespective of SMC growth conditions. These data suggest that BO and PO alone or in combination with fish oil influence macrophage/smooth muscle cell interactions in a manner consistent with favorable modulation of the atherogenic process.

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

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

B-Myb Expression in Vascular Smooth Muscle Cells Occurs in a Cell Cycle-dependent Fashion and Down-regulates Promoter Activity of Type I Collagen Genes

The members of the Myb family of transcription factors are defined by homology in the DNA-binding domain; all bind the Myb-binding site (MBS) sequence (YG(A/G)C(A/C/G)GTT(G/A)). Here we report that cultured bovine vascular smooth muscle cells (SMCs) express B-myb. Levels of B-myb RNA found in exponential growth were reduced dramatically in serum-deprived quiescent SMCs; B-myb mRNA levels increased in the cell cycle during the late G1 to S phase transition following restimulation with serum, epidermal growth factor, or phorbol ester plus insulin-like growth factor-1. Changes in the rate of B-myb gene transcription could account for part of the observed increase following serum addition. Treatment of SMC cultures with actinomycin D indicated a >4-h half-life for B-myb mRNA during the S phase of the cell cycle. Cotransfection of either a bovine or human B-myb expression vector down-regulated the activity of a multimerized MBS element-driven reporter construct in SMCs. Putative MBS elements were detected upstream of the promoters of the two chains of type I collagen, which we have found to be expressed inversely with growth state of the SMC (Kindy, M. S., Chang, C.-J., and Sonenshein, G. E. (1988) J. Biol. Chem. 263, 11426-11430). In cotransfection experiments, B-myb expression down-regulated the promoter activity of alpha1(I) and alpha2(I) collagen constructs an average of 92 and 82%, respectively. Thus, B-myb represents a potential link in the observed inverse relationship between collagen gene expression and growth of vascular SMCs.

Stimulation of endothelial cells by doses of basic FGF-saporin that are lethal to smooth muscle cells.

Basic fibroblast growth factor (FGF) receptors are up-regulated in proliferating (vs. quiescent) aortic smooth muscle cells, according to the results of recent studies. This up-regulation allows the ribosome inactivator saporin (if linked to basic FGF) to enter and kill proliferating, but not quiescent smooth muscle cells in vitro and in vivo. The authors now report that endothelial cells exhibit a different response. In 10% serum, FGF-SAP (0.1-1 nM) stimulates protein synthesis and cell division in subconfluent endothelial cells, but inhibits protein synthesis and cell division in subconfluent smooth muscle cells. Endothelial cells were inhibited at 10 nM FGF-SAP. A stimulatory response was seen in smooth muscle cells only at 0.1 nM FGF-SAP, and only after serum deprivation. Both cell types were resistant to FGF-SAP at high cell density. These responses correlated with FGF receptor density, which was sixfold higher in smooth muscle than endothelial cells and twice as high in serum-free smooth muscle cells as in serum-deprived smooth muscle cells. Moreover, a dose of FGFSAP that inhibited neointimal smooth muscle accumulation after balloon injury did not inhibit reendothelialization. Thus, there is a dose range at which FGF-SAP has unique properties that may make it useful in the treatment of vascular injury.

Biointeractive polymers and tissue engineered blood vessels

The regulation of endothelial cell (EC) and smooth muscle cell (SMC) proliferation following vascular interventions is critical to clinical efficacy. Our laboratory has developed a method of impregnating biomaterials with suspensions containing bioactive proteins resulting in the capability of differentially modulating EC and SMC growth in vitro and in vivo following implantation. We have previously reported that 60 μ internodal distance ePTFE grafts impregnated with fibrin glue (FG) containing FGF-1 and heparin develop confluent endothelialization with transiently increased EC and SMC proliferation after 4 weeks in dogs. Thoraco-abdominal implants after 20 weeks were developed significantly thicker (139 μ) inner capsules in response to the FGF. To minimize SMC proliferation we studied the effects of FGF-1, heparin, and thrombin concentrations on SMC growth in vitro. FG caused a 182% increase ( P < 0.001) in DNA synthesis. Heparin within FG diminished this effect in a dose-dependant manner, with complete inhibition of FG-induced growth at 500 U ml −1 (versus FG alone, P < 0.001). FGF-1 within FG without heparin had no effect, but together, FGF-1 caused a dose-dependant growth increase while increasing heparin concentrations initially increased and then decreased proliferation. FGF-1 and heparin in the medium of quiescent SMCs had similar effects. Only thrombin concentrations >3.2 U ml −1 stimulated SMC growth and this stimulation was blocked by heparin. A synergism between FGF and heparin on EC proliferation was also found but without EC growth inhibition in response to higher concentrations of heparin. It is thus possible to modulate the relative proliferative activity of ECs versus SMCs by altering the FGF:heparin ratio. This same system may be useful with other proteins to induce other local affects by the applied protein or systemic affects following release of that protein.

Transforming growth factor beta 1 inhibits fetal lamb ductus arteriosus smooth muscle cell migration.

Anatomical closure of the ductus arteriosus (DA) requires normally quiescent smooth muscle cells (SMC) to migrate out of the muscle media into the subendothelial space, forming intimal mounds that eventually coalesce to occlude the vessel's lumen. Transforming growth factor-beta 1 (TGF beta 1), a potent modulator of vascular SMC migration, is found in the wall of the closing DA. We examined the effect of TGF beta 1 on the migration of fetal lamb DA-SMC. Although TGF beta 1 has been shown to be a chemoattractant for other mesenchymal cells, it had no chemotactic effect on DA-SMC; furthermore, TGF beta 1 did not enhance the migration of DA-SMC (as has been reported for aortic SMC). Rather, incubating DA-SMC with TGF beta 1 for 22 h decreased the rate of migration of SMC on extracellular matrix substrata composed of fibronectin, vitronectin, laminin, and collagen I and IV. Exposure of DA-SMC to TGF beta 1 was associated with an increase in the formation of focal adhesion plaques (tight associations between the cells' surface and extracellular matrix). DA-SMC use integrin receptors to attach to and migrate on extracellular matrix components. The decrease in DA-SMC migration was not associated with a significant change in the profile of integrin receptors expressed by the cell. TGF beta 1 had little effect on overall DA-SMC integrin expression, except for a modest increase in the fibronectin receptor (alpha 5 beta 1 integrin). Rather, the decrease in migration and changes in cell morphology were associated with an increased ability of integrin receptors to associate with the cytoskeleton. TGF beta 1 appears to anchor the cell's cytoskeleton to the extracellular matrix, making the cells more adherent and less capable of migrating.

Interference with Protein Kinase C-Related Signal Transduction in Vascular Smooth Muscle Cells by Benzo[a]pyrene

Elucidation of the mechanisms involved in the deregulation of vascular smooth muscle cell (SMC) growth and differentiation during the course of atherogenesis and the putative role of toxic injury in this process have been a subject of considerable interest in recent years, In this regard, we have recently shown that in vitro exposure of vascular (aortic) SMCs to benzo[a]pyrene (BaP), an atherogenic polycyclic aromatic hydrocarbon, initially delays cell cycle progression and inhibits cell proliferation and then causes permanent modulation to a highly proliferative state, To define the molecular basis of this response, we have examined critical components of the protein kinase C (PKC) signal transduction system upon exposure to BaP, Marked inhibition of serum-stimulated inositol phospholipid turnover was observed in growth-arrested SMC cultures challenged with 30 μM BaP for 24 h and then stimulated with 10% fetal bovine serum for 120 or 1800 s, Benzo[a]pyrene inhibited PKC-mediated phosphorylation of exogenous and endogenous proteins in the cytosolic and particulate fraction of cycling, as well as quiescent cultures. The PKC inhibitory response was observed as early as 0.5 h following BaP treatment and maintained for at least 5 days. Exposure of quiescent SMCs to 30 μM BaP inhibited the ability of serum to induce c-fos mRNA expression and decreased AP-1 binding to a 12-O-tetradecanoyl phorbol-13-acetate responsive element, Inhibition of PKC-related signal transduction was not due to generalized interference with cell cycle events since peak: expression of the c-myc and c-Ha-ras protooncogenes following serum stimulation of quiescent cultures was unchanged, or slightly enhanced, by 30 μM BaP. Collectively, these data suggest that the ability of BaP to modulate growth and differentiation programs in vascular SMCs involves early interference with PKC-related mitogenic signal transduction.

914-69 Expression of α5β1Integrin in Human Vascular Smooth Muscle Cells: A Potential Mediator for Vascular Remodeling

Integrins are heterodimeric ( α β ) cell surface proteins that promote attachment to the extracellular matrix. Their role in vascular lesion formation is unknown. The α 5 β 1 integrin can, however, interact with fibronectin which has been implicated in lesion formation following vascular injury and can promote vascular smooth muscle cell (SMC) activation. Therefore, we tested the hypothesis that SMC activation is functionally linked to α 5 β 1 integrin expression. To determine which integrins are expressed on human SMCs, cells were cultivated from internal mammary artery (IMA) and surface-labeled with 125 I. Lysates were immunoprecipitated using a panel of subtype-specific antiintegrin antibodies. This identified 5 β 1 integrins: α 1 β 1 and α 1 β 1 integrins, which were expressed at relatively low levels, and α 3 β 1 , α 5 β 1 and α 6 β 1 integrins which were abundantly expressed. The cellular location of α 5 β 1 integrin was studied by confocal microscopy of SMCs seeded on to fibronectin. This revealed discrete linear accummulations of the α 5 integrin subunit on the bottom surface of the cell, consistent with focal adhesion plaques. The role of the α 5 β 1 integrin in mediating an interaction with fibronectin was evaluated by adhesion assay. SMCs adhered fully to afibronectin-coated substrate within 30 min. In contrast, when SMCs were incubated with an anti- α 5 integrin antibody, adherence was reduced by 65% (p &lt; 0.05). Finally, to assess integrin expression in quiescent SMCs, sections of human IMA were studied by immunofluorescence microscopy. In contrast to the abundant expression in activated SMCs proliferating in vitro, quiescent SMCs in the arterial media stained weakly for α 5 integrin. The α 5 β 1 integrin is selectively expressed in human SMCs when activated and mediates a functional interaction with fibronectin. This receptor therefore may be a critical modulator of SMC behavior following vessel injury.

Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells.

Action potentials activate voltage-dependent calcium channels and attendant increases in cytosolic calcium concentration ([Ca2+]i) in many excitable cells. The role of these channels in the regulation of [Ca2+]i in nonspiking cells that do not depolarize to membrane potentials sufficient to activate a substantial fraction of the available current is less clear. Measurements of the peak activation and steady-state inactivation of L-type calcium currents have predicted the existence of a noninactivating current window over a voltage range where channel inactivation is incomplete. The degree to which such small currents might regulate [Ca2+]i, however, has not been established. Here we demonstrate a "calcium window" in nondialyzed, quiescent smooth muscle cells over a small voltage range near the resting membrane potential. Sustained depolarizations in this voltage range, but not to more positive potentials, resulted in sustained rises in calcium, despite the fact that macroscopic inward currents were < 2 pA. The calcium window corresponded well with the predicted window current determined under the same conditions; the peak of the calcium window occurred at -30 mV, with steady-state rises in [Ca2+]i in some cells at -50 mV. Steady-state rises in [Ca2+]i following depolarization were completely blocked by nisoldipine and were augmented and shifted to more negative potentials by BAY K8644. Voltage-dependent calcium channels thus regulate steady-state calcium levels in nonspiking cells over a voltage range where macroscopic currents are only barely detectable. This voltage range is bounded at negative potentials by calcium channel activation and at more positive potentials by channel inactivation.

Molecular Cloning and Localization of the Human GAX Gene to 7p21

The GAX homeobox gene is expressed in the cardiovascular tissues of the adult rat, including heart, lung, kidney, and blood vessels. In the vasculature it is specifically expressed in quiescent smooth muscle cells, but its expression is rapidly down-regulated when these cells are stimulated to proliferate with mitogens. Since vascular smooth muscle cell proliferation is important in the pathology of blood vessel disorders, the human GAX gene was isolated and characterized. The human GAX cDNA was obtained by an anchored-PCR approach using cDNA templates from cardiovascular tissues and amplification primers designed from sequence information of the rat GAX cDNA and the homeodomain-containing exon of the human GAX gene. The human and rat GAX gene coding sequences are 98% conserved at the amino acid level and 83% conserved at the nucleotide level. Similar to rat, the human homolog contains a CAX trinucleotide repeat N-terminal to the homeodomain that encodes for a stretch of 17 consecutive histidine or glutamine residues. The human GAX locus was mapped by fluorescence in situ hybridization to the short arm of chromosome 7 at band p21. The human cDNA sequence will be useful for analyses of GAX gene expression in cardiovascular diseases.

Platelet-induced vascular smooth muscle cell proliferation is modulated by the growth amplification factors serotonin and adenosine diphosphate.

Platelet-mediated mechanisms have been implicated in intimal lesion formation following vascular injury. Although the participation of peptide growth factors has been suspected in this process, little has been known about the possible mitogenic role of other platelet factors that are released at sites of vascular injury. We tested the hypothesis that platelet products, which are not peptide growth factors, are important modulators of the platelet-induced smooth muscle cell (SMC) proliferative response by acting as growth amplification factors. In these studies, cell proliferation was assessed by [3H]thymidine incorporation, flow cytometry, and direct cell counting. We examined the potential mitogenicity of several platelet products, including serotonin, ADP, norepinephrine, histamine, platelet-activating factor, the thromboxane A2 mimetic U46619, and bradykinin. Of the platelet products tested, serotonin and ADP induced a synergistic response with peptide growth factors. This synergy was greatest at low growth-factor concentrations. Addition of nonaggregated platelets to quiescent SMC cultures strongly stimulated cell proliferation. Since the addition of suramin to platelet-treated cultures markedly inhibited SMC proliferation, peptide growth factors are most likely the primary mitogens mediating this response. However, platelet-induced proliferation was also markedly reduced by the serotonin antagonists ketanserin and LY53857 (44%), and by the ADP antagonist apyrase (35%). Therefore, serotonin and ADP contribute significantly, in synergy with peptide growth factors, to the platelet-induced SMC proliferative response. We propose that in vivo serotonin and ADP act as amplification factors for SMC proliferation at sites of vascular injury.

Response of atherosclerotic intimal smooth muscle cells to epidermal growth factor in vitro.

Increased proliferation of intimal smooth muscle cells (SMCs) plays an important role in the early phase of atherogenesis. To investigate growth mechanisms of these cells, we used intimal SMCs from rabbits fed an atherogenic diet and examined the sequential events that may facilitate induction of intimal SMC proliferation as well as the possible effects of growth-promoting factors secreted by these cells. In serum-free medium, epidermal growth factor (EGF) stimulated [3H]thymidine uptake by quiescent intimal SMCs at a rate six times higher than quiescent medial SMCs. There was no significant difference between the two cell types in terms of the number of specific EGF receptor per cell, the dissociation constant of EGF, and the time course of EGF binding and internalization. Furthermore, in both types of cells, c-fos, c-jun, and c-myc mRNAs were induced after 1, 1, and 4 hours of EGF treatment, respectively, whereas they required 8 hours of contact with EGF to induce proliferation. Growth response of medical SMCs to EGF was greatly enhanced when rabbit serum, deficient in lipoproteins and free of platelet-derived growth factor, was added to the medium. Moreover, EGF induced a twofold to fourfold increase in DNA synthesis in medial SMCs cocultured with intimal SMCs compared with medial SMCs incubated alone. Likewise, DNA synthesis of medial SMCs grown in medium conditioned by intimal SMCs was six times higher than that observed in medium conditioned by medical SMCs. Adding EGF to the medium conditioned by intimal SMCs increased their DNA synthesis even further.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cyclooxygenase-2 expression in aortic smooth muscle cells.

Activation of the gene for inducible cyclooxygenase (cyclooxygenase-2 [Cox-2], prostaglandin endoperoxide synthase) is an early response to injury in vascular smooth muscle cells. We used in vitro and in vivo models to demonstrate that activation of quiescent smooth muscle cells by mitogens leads to a rapid, short-term rise in mRNA for Cox-2, followed by synthesis of new Cox-2 enzyme protein and a marked increase in prostaglandin production that depends on new enzyme synthesis. Moreover, the Cox-2 mRNA response observed in smooth muscle cells differs in both time and degree, depending on the particular mitogenic stimulus. Serum, platelet-derived growth factor, epidermal growth factor, and thrombin are strong inducers of Cox-2 mRNA, whereas acidic and basic fibroblast growth factors and interleukin-1 alpha are weak inducers. In contrast to the transient activation of Cox-2 in vitro after introduction of a mitogenic stimulus, the Cox-2 response after in vivo vascular injury extends over many days and may represent protracted cellular activation. During induction of Cox-2 message and protein, expression of constitutive cyclooxygenase (Cox-1) remains unchanged, however. These data suggest a pathophysiological role for Cox-2 in the early modulation of vascular responses to injury.

Induction of cyclooxygenase-2 in rat vascular smooth muscle cells in vitro and in vivo.

Prostaglandins are synthesized from arachidonic acid by the rate-limiting enzyme cyclooxygenase (prostaglandin G/H synthase). Active cyclooxygenase is encoded by two distinct and independently regulated genes, termed cyclooxygenase-1 (cox1) and cyclooxygenase-2 (cox2). In this investigation, we examined the expression of cox1 and cox2 mRNA in rat aorta following balloon deendothelialization (BDE) in vivo and in rat aortic smooth muscle cells (SMC) after serum stimulation in vitro. Two h after BDE, rat aortic cox2 mRNA levels increased greater than 50-fold relative to the lowest detectable levels on days 2 and 14. No message was detectable in non-BDE control rat aortas. Similar to the results found in vivo, cultured SMC exhibited a greater than 45-fold increase in cox2 mRNA levels after a 2-h exposure to serum. This increase was transient because cox2 levels declined at 4 and 8 h. In contrast, minimal changes in cox1 mRNA levels were observed after BDE or serum treatments. Increased levels of cox2 mRNA and corresponding protein synthesis led to an accumulation of total cyclooxygenase protein, which remained elevated 24 h after serum stimulation. Serum-treated SMC also generated greater amounts of cyclooxygenase-dependent metabolites than quiescent SMC as evidenced by marked increases in prostaglandin E2 content in conditioned media. This increase is associated with a 2.5-3.0-fold increased rate of arachidonic acid conversion to prostaglandin E2. Our data indicate that injury and serum stimulation differentially regulate mRNA and protein expression of two distinct cox genes in vascular SMC in vivo and in vitro. The findings suggest that the prostanoid responses after vascular injury are, in part, mediated by acute increases in cox2 mRNA and cyclooxygenase-2 protein.

Persistence of the fetal pattern of tropoelastin gene expression in severe neonatal bovine pulmonary hypertension.

Neonatal hypoxic pulmonary hypertension causes increases and spatial changes in tropoelastin expression in pulmonary arteries. However, it is not clear if this is due to recruitment of quiescent smooth muscle cells (SMC) into an elastin-producing phenotype or persistence of the fetal pattern of tropoelastin gene expression. We evaluated the distribution and relative concentration of tropoelastin mRNA in intralobar pulmonary arteries from late gestation fetuses and in animals exposed to hypobaric hypoxia (430 mmHg) from birth for 1, 3, 7, or 14 d, as well as in age-matched and adult room air-breathing controls. In situ hybridization demonstrated that tropoelastin mRNA was distributed throughout the entire radius of the pulmonary vessel wall in the fetus and newborn calf. By 15 d of age, only cells in the inner third of the media expressed tropoelastin mRNA, and by adulthood no tropoelastin mRNA was detected in the vessel wall. These findings demonstrated that tropoelastin expression shuts off in a spatially specific pattern, moving from the abluminal to the luminal side of the medial in the neonatal pulmonary artery when pressures and resistance are falling. In the aorta of 15-d-old calves, tropoelastin mRNA expression was seen equally throughout the media, indicating tissue-specific regulation of elastin in the neonatal period. In contrast, intralobar pulmonary arteries from calves exposed to hypoxia, which prevented the normal postnatal decline in pulmonary artery pressure, maintained the fetal pattern and levels of tropoelastin mRNA expression at all time points. Thus, rather than causing a recruitment of SMC into an elastin-producing phenotype, neonatal pulmonary hypertension caused a persistence of the fetal pattern of tropoelastin expression in medial SMC. Cell-free translation showed that the same tropoelastin isoforms were made by mRNA from control and hypertensive calves and, unlike the ligamentum nuchae, did not change during the transition from fetal to neonatal life. We conclude that pulmonary hypertension in the neonate perturbs the normal postpartum repression of tropoelastin expression resulting in a persistence of the fetal spacial and isoform patterns of tropoelastin gene expression.

PDGF-like Growth Factor from SV40-Transformed Smooth Muscle Cells Promotes Growth in an Autocrine or Paracrine Manner

We investigated the growth characteristics of smooth muscle cells transformed by oridefective simian virus 40 plasmid DNA (TSMCs). TSMCs demonstrated a significantly higher proliferative activity when compared with nontransformed smooth muscle cells (SMCs). The SMCs were not able to proliferate in media with 5% plasma-derived serum (PDS) instead of fetal bovine serum, while TSMCs grew under this condition. When the DNA synthesis of quiescent SMCs was reinitiated by the addition of serum or platelet-derived growth factor (PDGF), the amount of [3H]thymidine incorporation into DNA varied with the concentrations of serum or PDGF and decreased on type V collagen compared with the other collagen substrates. In contrast, the TSMCs showed no differences in the amount of [3H]thymidine incorporation into DNA among the various concentrations of serum or PDGF and different types of collagen. When quiescent SMCs were cocultured with TSMCs, they showed a remarkable increase in the amount of [3H]thymidine incorporation into DNA, which was most intensively inhibited by type V collagen. These results suggest that TSMCs can promote the growth of TSMCs and SMCs in either an autocrine or paracrine manner and that they proliferate independently of the collagen matrices. Trapidil, a pyrimidine derivative that may inhibit the action of PDGF, decreased the number and cAMC levels of TSMCs in the media with 5% PDS in a concentration-dependent manner. The addition of polyclonal anti-PDGF-B antibody induced up to 59.5% inhibition of enhancement of DNA synthesis of SMCs cocultured with TSMCs. The level of cyclic adenosine monophosphate (cAMP) increased in the TSMCs compared with the SMCs. There was, however, no difference in the cAMP levels of either cells between the various types of collagen. Thus, the release of PDGF-like growth factor and an accumulation of cellular cAMP may be related to an increased DNA synthesis of TSMCs.