The small leucine-rich repeat proteoglycans in tissue repair and atherosclerosis.

Proteoglycans consist of a protein core with one or more covalently attached glycosaminoglycan (GAG) side chains and have multiple roles in the initiation and progression of atherosclerosis. Here we discuss the potential and known functions of a group of small leucine-rich repeat proteoglycans (SLRPs) in atherosclerosis. We focus on five SLRPs, decorin, biglycan, lumican, fibromodulin and PRELP, because these have been detected in atherosclerotic plaques or demonstrated to have a role in animal models of atherosclerosis. Decorin and biglycan are modified post-translationally by substitution with chondroitin/dermatan sulphate GAGs, whereas lumican, fibromodulin and PRELP have keratan sulphate side chains, and the core proteins have leucine-rich repeat (LRR) motifs that are characteristic of the LRR superfamily. The chondroitin/dermatan sulphate GAG side chains have been implicated in lipid retention in atherosclerosis. The core proteins are discussed here in the context of (i) interactions with collagens and their implications in tissue integrity, fibrosis and wound repair and (ii) interactions with growth factors, cytokines, pathogen-associated molecular patterns and cell surface receptors that impact normal physiology and disease processes such as inflammation, innate immune responses and wound healing (i.e. processes that are all important in plaque development and progression). Thus, studies of these SLRPs in the context of wound healing are providing clues about their functions in early stages of atherosclerosis to plaque vulnerability and cardiovascular disease at later stages. Understanding of signal transduction pathways regulated by the core protein interactions is leading to novel roles and therapeutic potential for these proteins in wound repair and atherosclerosis.

Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia.

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

Extracellular matrix components in atherosclerotic arteries of Apo E/LDL receptor deficient mice: an immunohistochemical study.

During accelerated vascular remodeling such as in atherosclerosis, the composition of the extracellular matrix becomes altered. The matrix components of the diseased artery influence cellular processes such as adhesion, migration and proliferation. Furthermore, in atherosclerosis, the inability of the cells within the lesion to produce a mechanically stable matrix may lead to plaque rupture. In this immunohistochemical study of atherosclerotic mice aorta, we have reviewed the presence of ECM components with roles in maintaining tissue structure and function. These components include osteopontin and COMP as well as the leucine rich repeats proteins decorin, PRELP, and fibromodulin. Immunohistochemistry demonstrated presence of osteopontin, COMP, decorin, PRELP and fibromodulin in lesion areas of ApoE/LDLr deficient mice. Some advanced lesions exhibited areas of cartilage-like morphology and were shown to represent cartilage by their content of the cartilage specific proteins collagen II and aggrecan. The results suggest that cartilage-associated cell/collagen binding ECM proteins may be involved in the pathogenesis of atherosclerosis.

Injury-induced osteopontin gene expression in rat arterial smooth muscle cells is dependent on mitogen-activated protein kinases ERK1/ERK2.

Previous work shows that osteopontin has a role during matrix reorganization after tissue injury including vascular conditions such as atherosclerosis and restenosis following angioplasty. In vitro, osteopontin promotes activities such as adhesion and migration but the mechanisms that regulate the expression of this matrix protein remain essentially unknown. This study examined if the ERK signaling pathway is involved in injury-induced osteopontin expression in cultured rat aortic smooth muscle cells. Northern and Western blotting demonstrated a marked activation of osteopontin expression in response to injury. Treating the cells with PD98059, a specific MEK1 inhibitor, prior to injury, blocked this upregulation. MEK1 phosphorylates ERK1/ERK2, which belong to the family of mitogen-activated protein kinases. We conclude that ERK1/ERK2 are involved in the regulation of osteopontin expression in cultured vascular smooth muscle cells.

Smooth muscle cell response to mechanical injury involves intracellular calcium release and ERK1/ERK2 phosphorylation.

We have investigated possible signaling pathways coupled to injury-induced ERK1/2 activation and the subsequent initiation of vascular rat smooth muscle cell migration and proliferation. Aortic smooth muscle cells were cultured to confluency and subjected to in vitro injury under serum-free conditions. In fluo-4-loaded cells, injury induced a rapid wave of intracellular Ca(2+) release that propagated about 200 microm in radius from the injured zone, reached a peak in about 20 s, and subsided to the baseline within 2 min. The wave was abolished by prior treatment with the sarcoplasmic reticulum ATPase inhibitor thapsigargin, but not by omission of extracellular Ca(2+). ERK1/2 activation reached a peak at 10 min after injury and was inhibited by the MEK1 inhibitor PD98059, as well as by thapsigargin, fluphenazine, genistein, and the Src inhibitor PP2. These inhibitors also reduced [(3)H]thymidine incorporation and migration of cells into the injured area determined at 48 h after injury. These results show that mechanical injury to vascular smooth muscle cells induces a Ca(2+) wave which is dependent on intracellular Ca(2+) release. Furthermore, the injury activates ERK1/2 phosphorylation as well as cell migration and replication.

7beta-hydroxycholesterol induces Ca(2+) oscillations, MAP kinase activation and apoptosis in human aortic smooth muscle cells.

In the present study, we characterize the early cytotoxic effects of 7beta-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7beta-hydroxycholesterol induced Ca(2+) oscillations with a frequency of approximately 0.3-0.4 min(-1). A few hours later, thapsigargin-sensitive Ca(2+) pools were depleted, indicating that 7beta-hydroxycholesterol perturbs intracellular Ca(2+) homeostasis. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (but not JNK) were activated within 5 min after addition of 7beta-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7beta-hydroxycholesterol and cholesterol-5alpha,6alpha-epoxide, as determined by TUNEL staining. Addition of TNFalpha (10 ng/ml) and IFNgamma (20 ng/ml) enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7beta-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4-8 h as determined by [35S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the ER and Golgi membranes. The Ca(2+) and ERK signals preceded the ultrastructural changes induced by 7beta-hydroxycholesterol.

The synthetic metalloproteinase inhibitor batimastat suppresses injury-induced phosphorylation of MAP kinase ERK1/ERK2 and phenotypic modification of arterial smooth muscle cells in vitro.

Smooth muscle cell (SMC) migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. The extracellular matrix has important functions in modulating SMC structure and function, but less is known about the role of the matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors. The present study investigates the effects of the synthetic MMP inhibitor batimastat (BB94) on vascular SMCs. As experimental model, rat aortic smooth muscle cells in primary and secondary cultures were employed. Electron microscopy was used to investigate the effects of BB94 on the overall phenotypic properties of the cells. Induction of DNA synthesis and migration was studied by thymidine autoradiography and counting of cells moving into an injured zone. Gelatin zymography was used for the detection of BB94-mediated inhibition of injury-induced MMP activity. Phosphorylation of the mitogen-activated protein kinases ERK1/ERK2, two potential mediators of the injury-induced activation of the cells, was measured by Western blotting. The results show that BB94 restrained the phenotypic modulation of vascular SMCs in primary cultures and suppressed injury-induced DNA synthesis and migration. Moreover, the upregulation of ERK1/ERK2 phosphorylation in injured secondary cultures and in cells treated with bFGF was markedly reduced by BB94, whereas TIMP-2 lacked a clear effect. Our data suggest that BB94 inhibits injury-induced activation of vascular SMCs by acting on MMPs as well as other targets.

Antisense oligonucleotides to stromelysin mRNA inhibit injury-induced proliferation of arterial smooth muscle cells.

Smooth muscle cell migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. To make this possible, the smooth muscle cell has to change from a contractile to an activated repair cell with capacity to synthesize DNA and extracellular matrix components. There is now considerable evidence that the extracellular matrix has important functions in modulating the phenotypic properties of smooth muscle cells, but less is known about the role of the matrix metalloproteinases. The present study investigates the role of stromelysin in the modulation of rat aortic smooth muscle cell morphology and function following mechanical injury in vitro and in vivo. Antisense mRNA oligonucleotides were used to investigate the role of stromelysin expression in injury-induced phenotypic modulation and the subsequent migration and proliferation of vascular smooth muscle cells. Cultured rat aortic smooth muscle cells and balloon-injured rat carotid arteries were used as experimental models. Light- and electron microscopy were used to follow changes in smooth muscle cell phenotype and lesion formation and incorporation of 3H-thymidine to detect DNA synthesis. Injury-induced DNA synthesis and migration in vitro were inhibited by 72% and 36%, respectively, by adding stromelysin antisense oligonucleotides to the medium prior to injury. In primary cultures, 67% of the smooth muscle cells treated with stromelysin antisense were retained in a contractile phenotype as judged by analysis of cell fine structure, compared to 15% untreated cells and 40% in cells treated with mismatched oligonucleotides. Examination of the carotid arteries one week after balloon injury likewise demonstrated a larger fraction of contractile cells in the inner parts of the media in vessels treated with antisense oligonucleotides compared to those treated with mismatched oligonucleotides. The neointima was also distinctly thinner in antisense-treated than in mismatched-treated and control arteries at this time. These findings indicate that stromelysin mRNA antisense oligonucleotides inhibited phenotypic modulation of rat arterial smooth muscle cells and so caused a decrease in migration and proliferation and neointima formation in response to vessel wall injury.

Lipoprotein-like phospholipid particles inhibit the smooth muscle cell cytotoxicity of lysophosphatidylcholine and platelet-activating factor.

Oxidation of LDL is associated with degradation of phosphatidylcholine into platelet-activating factor (PAF)-like phospholipids and lysophosphatidylcholine (LPC). Exposure of cultured human smooth muscle cells to PAF and LPC in a concentration of 25 micromol/L was found to result in complete cell death, as assessed by the MTT cytotoxicity assay and cell counting. Addition of 50 microg/mL apolipoprotein A-I- and apolipoprotein A-I(Milano)-containing phospholipid particles completely inhibited this cytotoxicity. Phospholipid complexes alone were almost as effective, whereas free apolipoprotein A-I(Milano) and albumin were without effect, suggesting that the effect was phospholipid dependent. Experiments using [14C]LPC demonstrated that apolipoprotein A-I- and apolipoprotein A-I(Milano)-containing phospholipid particles effectively bind LPC. The results show that HDL-like phospholipid particles effectively inhibit the toxic effect of phospholipids and other lipid-soluble factors. The ability of HDL to inhibit the proinflammatory and toxic effects of phospholipids generated during oxidation of LDL may be responsible for part of the antiatherogenic properties of HDL.

Immunization with homologous oxidized low density lipoprotein reduces neointimal formation after balloon injury in hypercholesterolemic rabbits.

OBJECTIVES: In this study we tested the hypothesis that immunization with homologous oxidized low density lipoprotein (oxLDL) would inhibit the neointimal response to balloon injury in hypercholesterolemic rabbits. BACKGROUND: Immunization with homologous oxLDL has been shown to markedly reduce aortic atherosclerosis in LDL receptor-deficient as well as cholesterol-fed rabbits; however, the effect of this strategy on the balloon injury-induced neointimal lesion is unknown. METHODS: New Zealand White rabbits were immunized with 280 microg of homologous native LDL (n = 5), copper-oxidized LDL (n = 5) or phosphate buffer as control (n = 5) and fed a 1% cholesterol diet. Rabbits were reimmunized after 3 weeks, and balloon injury of the right ileofemoral artery was performed 1 week later. Four weeks after balloon injury, rabbits were killed, and the neointimal lesion area was measured by computerized morphometry after perfusion fixation of the arteries. Circulating antibodies against oxLDL were measured by enzyme-linked immunosorbent assay. RESULTS: In comparison with the control animals, those immunized with oxLDL had a 58% reduction in the neointimal area (0.53 +/- 0.13 vs. 1.27 +/- 0.26 mm2; p = 0.01). The group immunized with native LDL had a 19% reduction in the neointimal area compared with the control group (p = NS). Circulating cholesterol levels and antibody titers against oxLDL were comparable in the three groups. There was a trend toward reduced immunoreactivity for T cells and oxLDL in the neointima of oxLDL-immunized animals. CONCLUSIONS: Hypercholesterolemic rabbits immunized with homologous oxLDL have a markedly reduced neointimal area after balloon injury despite severe hypercholesterolemia. Together with previous work, these data suggest that an immunization strategy (vaccination) against atherosclerosis and restenosis warrants further investigation.

Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture.

OBJECTIVES: After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a sympathetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and then in vitro system has been established as a useful model in which to study the control of SMC differentiation. The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture. METHODS: SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin-collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy. RESULTS: The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days. Especially in the serum-free groups, there was also early activation of the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2). In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in collagen type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern. Among the matrix receptors investigated, the beta 1 integrin subunit showed a high and persistent expression, whereas the alpha 5 and alpha 1 integrin subunits showed lower and more variable mRNA level. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF alpha-receptor gene were noted. CONCLUSION: It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.

Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta.

In experimental models of atherosclerosis, activation of smooth muscle cell (SMC) migration from the media to the intima is preceded by intimal accumulation of inflammatory cells, suggesting that cytokines may be involved in this process. The present study demonstrates that tumor necrosis factor-alpha (TNF-alpha) regulates cytoskeletal organization of SMCs by inducing depolymerization of actin stress fibers and dispersion of vinculin from sites of focal adhesion and stimulates the migration of cultured human SMCs in a dose-dependent manner. Moreover, TNF-alpha induces rapid activation of the c-ets-1 gene, which codes a transcription factor known to regulate enzymes important for matrix degradation during cell migration. Balloon catheter injury of the rat femoral artery resulted in medial expression of TNF-alpha within 6 hours. This expression appeared to be localized to SMCs and remained elevated until SMCs began to migrate into the intima 7 days after injury. These findings demonstrate that TNF-alpha has a stimulatory effect on SMC migration and suggest that TNF-alpha may be involved in the intimal recruitment of SMCs during plaque formation.

Effect of immunization with homologous LDL and oxidized LDL on early atherosclerosis in hypercholesterolemic rabbits.

Although the existence of an immune response against modified lipoproteins in atherosclerosis has been observed in experimental animals as well as in humans, the precise pathophysiological relevance of these findings remains unclear. In this study we determined the effect of an immunization with homologous LDL and copper-oxidized LDL on the formation of atherosclerotic plaque in hypercholesterolemic rabbits. Immunizations were performed at the start of a cholesterol-rich diet and 3 weeks later. After 16 weeks, antibodies against oxidized LDL had developed in rabbits given hypercholesterolemic diet alone, but the titers were increased by twofold in rabbits immunized with oxidized LDL as well as in rabbits immunized with LDL, suggesting that the LDL had also become oxidized during the preparation and/or immunization procedure. Immunization with LDL and oxidized LDL reduced atherosclerotic lesions in the proximal aorta by 74% (P < .05) and 48% (P = NS), respectively. The cellular composition of the lesions was not affected by the immunizations. These results support the hypothesis that an immune response against modified LDL has a protective effect against the development of early atherosclerotic lesions.

Regulated expression of the ets-1 transcription factor in vascular smooth muscle cells in vivo and in vitro.

Ets-1 regulates the transcription of several genes encoding extracellular matrix proteins (ie, osteopontin and tenascin) as well as enzymes involved in degradation and remodeling of the extracellular matrix (ie, stromelysin and urokinase plasminogen activator). In the present study, we investigated the regulation of c-ets-1 in cultured rat vascular smooth muscle cells as well as in the arterial wall after balloon injury in vivo. Serum-starved smooth muscle cells exposed to serum for various time points express a major c-ets-1 mRNA transcript of 5.3 kb and minor bands of 4.0 and 2.5 kb with a peak at 2 hours after stimulation. These effects were concentration dependent. Western blotting revealed an increase in 55- and 40-kD immunoreactive ets-1 proteins in cells treated with serum for 2 hours, and binding to an oligonucleotide containing the ets-1 consensus cis-acting motif was demonstrated by electrophoretic mobility shift assay. Ets-1 mRNA abundance was induced with a peak at 2 hours after stimulation with platelet-derived growth factor-BB and with angiotensin II. There was a distinct increase of ets-1 immunoreactivity in the inner layer of the media 2 hours after balloon catheter injury of rat arteries, which declined after 6 hours and returned to the basal level 1 day after vessel wall damage. Arterial c-ets-1 mRNA content was induced with an identical time course. These findings suggest that c-ets-1 may be of importance in the mitogenic signaling pathway of smooth muscle cells grown in culture. In addition, ets-1 may play a role in the activation of smooth muscle cells in vivo after mechanical injury of the vessel wall. Because the ets-1 transcription factor activates the gene expression of a number of mRNA species involved in matrix deposition and degradation, these data are compatible with a role for ets-1 in vascular remodeling and/or cell migration.

Autocrine induction of DNA synthesis by mechanical injury of cultured smooth muscle cells. Potential role of FGF and PDGF.

To determine whether replication of arterial smooth muscle cells (SMCs) in response to mechanical injury would occur in the absence of serum and other cells, we created an in vitro model in which confluent, growth-arrested cultures of rat SMCs were injured by gentle pressure of a soft plastic tube and then kept in serum-free medium for up to 4 days. Replication of SMCs in and around the injury, as measured by tritiated thymidine incorporation, was noted within 24 hours and peaked at 48 hours after injury, whereas noninjured cells remained quiescent. An increased expression of platelet-derived growth factor (PDGF) A mRNA, noted 6 hours after injury, was followed by an increased PDGF AA immunoreactivity in SMCs in and around the zone of injury at 24 and 48 hours after injury. A PDGF A chain antisense oligonucleotide inhibited 87.0 +/- 4.0% (P < .005) of SMC replication in the injury zone, whereas the corresponding sense oligonucleotide reduced SMC replication by only 37.2%. An antibody to fibroblast growth factor (FGF) almost completely inhibited SMC replication in the injured zone, whereas an antibody to PDGF AA was without effect. Incubation of SMCs with FGF increased PDGF A mRNA levels in SMCs, and 5 mumol/L PDGF A antisense oligonucleotides reduced FGF-induced SMC replication by 62%. Taken together, these results demonstrate that injured rat SMCs in culture release FGF that activates DNA synthesis of neighboring SMCs both by a direct mechanism and by stimulating the production of PDGF AA.

Inhibitors of ADP-ribosylation suppress phenotypic modulation and proliferation of smooth muscle cells cultured from rat aorta.

The effects of hexamethylenebisacetamide (HMBA), an inhibitor of poly-ADP-ribosylation, and meta-iodobenzylguanidine (MIBG), an inhibitor of mono-ADP-ribosylation, on the phenotypic properties and proliferation of cultured rat aortic smooth muscle cells were studied using a combination of structural and chemical methods. The results show that HMBA and MIBG both slowed down the transition of the cells from a contractile to a synthetic phenotype in primary culture. While the control cells rapidly lost most of their myofilaments and built up an extensive endoplasmic reticulum and Golgi complex, a conspicuous fraction of the drug-treated cells retained a characteristic smooth muscle morphology for at least 6 days. Moreover, most of the treated cells remained positive for smooth muscle alpha-actin and desmin throughout this period. In contrast, the drugs lacked distinct effects on cell morphology and cytoskeletal organization in secondary cultures. Nevertheless, they strongly inhibited serum-stimulated cell growth both in primary and secondary cultures. The ability of serum-starved cells to synthesize DNA after exposure to platelet-derived growth factor or serum was also restrained. Notably, the drugs could be added several hours after the mitogens without loss of effect, suggesting that they did not prevent the entrance into but rather the progression through the cell cycle. Accordingly, the expression of early response genes like c-fos, c-jun and c-myc was not blocked by the drugs. On the other hand, HMBA reduced the expression of transcripts for smooth muscle alpha-actin, type IV collagenase, collagen type I, and osteopontin both in primary and secondary cultures. Weaker and more variable effects were obtained with MIBG. Taken together, the findings support the notion that poly- and mono-ADP-ribosylation of proteins are involved in the control of smooth muscle cell differentiation and growth.

Recombinant apolipoprotein A-I Milano reduces intimal thickening after balloon injury in hypercholesterolemic rabbits.

BACKGROUND: Several epidemiological studies have shown an inverse relation between high-density lipoprotein (HDL) cholesterol levels and coronary heart disease. Recently, observational studies have suggested a similar inverse relation between HDL and restenosis after coronary balloon angioplasty. Despite these observations, it is unclear whether this inverse relation reflects a direct vascular protective effect of HDL or apolipoprotein (apo) A-I, the major apolipoprotein component of HDL. Therefore, to determine whether HDL directly influences neointima formation, we investigated the effect of recombinant apo A-I Milano (apo A-I M), a mutant of human apo A-I with Arg-173 to Cys substitution, on intimal thickening after balloon injury in cholesterol-fed rabbits. METHODS AND RESULTS: Cholesterol feeding was initiated 18 days before injury and continued until the time of death. Eight rabbits received intravenous injections of 40 mg of apo A-I M linked to a phospholipid carrier on alternate days, beginning 5 days before and continuing for 5 days after balloon injury of femoral and iliac arteries. Eight rabbits received the carrier alone, and four received neither apo A-I M nor the carrier. Three weeks after balloon injury, apo A-I M-treated rabbits had significantly reduced intimal thickness compared with the two control groups (mean +/- SD): 0.49 +/- 0.29 versus 1.14 +/- 0.38 mm2 and 1.69 +/- 0.43 mm2, P < .002 by ANOVA). The intima-to-media ratio was also significantly reduced by apo A-I M (0.7 +/- 0.2 versus 1.5 +/- 0.5 and 2.1 +/- 0.1, P < .002 by ANOVA) compared with the two controls. The fraction of intimal lesion covered by macrophages, as identified by immunohistochemistry using macrophage-specific monoclonal antibody, was significantly less in apo A-I M-treated rabbits compared with carrier-treated animals (25.3 +/- 17% versus 59.4 +/- 12.3%, P < .005). Aortic cholesterol content, measured in an additional 10 rabbits, did not differ significantly between apo A-I M-treated animals (n = 5) and carrier-treated controls (n = 5). CONCLUSIONS: Apo A-I M significantly reduced intimal thickening and macrophage content after balloon injury in cholesterol-fed rabbits without a change in arterial total cholesterol content. Although the precise mechanism of action remains to be defined, these findings are consistent with a direct vascular effect of apo A-I, which could have potential therapeutic implications.

Cyclic AMP, early response gene expression, and DNA synthesis in rat smooth muscle cells.

Smooth muscle cells (SMC) isolated from neonatal and adult rats differ markedly in their growth characteristics. The growth of neonatal cells is mainly due to autocrine stimulation, whereas the growth of adult SMC is dependent upon addition of exogenous mitogens. Increasing intracellular cyclic AMP (cAMP) levels effectively inhibits DNA synthesis in adult cells, but is essentially without effect on the rate of DNA synthesis in neonatal cells. In the present study we investigated whether this difference in cAMP sensitivity is due to an effect of cAMP on early response genes. The results show that increasing intracellular levels of cAMP by exposing the cells to the synthetic adenosine analogue N-ethyl-carboxamido adenosine (NECA) results in an accumulation of c-jun and c-fos mRNA in both cell types. NECA also lowered c-myc mRNA levels in neonatal cells, whereas it marginally increased the presence of c-myc mRNA in adult cells. Exposure to NECA also resulted in a limited increase in alpha-actin mRNA levels. NECA did not inhibit DNA synthesis or growth of adult SMC actively proliferating in the presence of 10% serum, suggesting that cAMP interferes with processes taking place during the early G1 phase or in the entry of growth-arrested cells into the G1 phase of the SMC cell cycle. It is concluded that the growth-inhibitory effect of NECA is unlikely to be due to actions of cAMP on early response genes. However, it cannot be completely excluded that an increased synthesis of jun/fos transcription factors may induce the transcription of other, growth-suppressing genes in the cells.

Fibronectin and the basement membrane components laminin and collagen type IV influence the phenotypic properties of subcultured rat aortic smooth muscle cells differently.

A substrate of the extracellular matrix protein fibronectin has previously been found to promote the modulation of freshly isolated rat aortic smooth muscle cells from a contractile to a synthetic phenotype early in primary culture. In contrast, substrates of the basement membrane proteins laminin and collagen type IV were found to retain the cells in a contractile phenotype. Here, we have studied whether rat aortic smooth muscle cells tht have already adopted a synthetic phenotype are also affected differently by these proteins. For this sake, subcultured cells were detached with trypsin, seeded on substrates of either fibronectin or laminin plus collagen type IV, and incubated in a serum-free medium for one to three days. RNA blot and immunoblot analyses indicated that cells grown on laminin plus collagen type IV expressed smooth muscle alpha-actin transcripts and protein at higher levels than cells grown on fibronectin. Moreover, immunocytochemical and electron-microscopic analyses revealed that cells positively stained for smooth muscle alpha-actin and cells with a cytoplasm dominated by large microfilament bundles were more numerous on laminin plus collagen type IV than on fibronectin. Finally, thymidine autoradiography showed that the DNA synthetic response to stimulation with platelet-derived growth factor or serum was weaker in cells grown on laminin plus collagen type IV than in cells grown on fibronectin. These findings confirm the notion that a substrate of laminin and collagen type IV stimulates the in vitro expression of differentiated smooth muscle traits at a higher level than does a substrate of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous activation of c-myc expression and DNA synthesis in serum-starved neonatal rat smooth muscle cells.

Earlier studies have shown that smooth muscle cells (SMC) from arteries of neonatal and adult rats differ markedly in their in vitro growth characteristics. Since some of these differences may be relevant to the proliferation of SMC in atherosclerotic plaques we examined the expression of three proto-oncogenes (c-fos, c-jun, and c-myc) and an SMC-specific differentiation marker (alpha-actin) in cultured SMC. In presence of serum cultured adult SMC contained lower levels of alpha-actin mRNA than neonatal cells. In neonatal cells serum-starvation resulted in a distinct increase in both c-myc and alpha-actin mRNA levels, whereas the expression of these genes appeared to be unaffected in adult cells. Transfer of adult SMC proliferating in the presence of fetal calf serum to serum-free medium for 48 h almost completely inhibited DNA synthesis, whereas transfer of neonatal SMC to serum-free medium reduced DNA synthesis only to about 50%. Serum-starved adult and neonatal SMC did not contain c-fos or c-jun transcripts, but in both cell types serum-stimulation resulted in a comparable increase in the expression of both genes. The present results demonstrate clear differences in the mechanisms regulating gene expression in adult and neonatal SMC.