Is smooth muscle growth in primate arteries regulated by endothelial nitric oxide synthase?

PURPOSE: We investigated whether control of constitutive endothelial cell nitric oxide synthase (cNOS) and nitric oxide (NO) by changes in shear stress might be important for the regulation of smooth muscle cell (SMC) growth and vascular diameter. METHODS: Bilateral femoral arteriovenous fistulas were placed in baboons to increase the blood flow in the external iliac arteries. At 2 months, the fistula was ligated on one side to restore normal flow (flow switch). RESULTS: In response to flow switch and a decrease in shear stress, iliac artery lumenal area decreased and SMC proliferation was induced. A decline in NO production, cNOS messenger RNA (mRNA), and protein were associated with these biological effects. In a subset of animals with iliac arteries under high flow, infusion of N(omega)-nitro-L-arginine, an inhibitor of cNOS, did not induce proliferation. CONCLUSION: Shear stress can regulate cNOS, vasoconstriction, and SMC proliferation. A decrease in nitric oxide may be necessary, but is not sufficient to induce SMC proliferation in response to a decrease in blood flow.

Acute reductions in blood flow and shear stress induce platelet-derived growth factor-A expression in baboon prosthetic grafts.

Abrupt reductions in fluid shear stress induce subendothelial smooth muscle cells (SMCs) to proliferate in experimental prosthetic grafts. Platelet-derived growth factor (PDGF), an important SMC mitogen, is expressed by cultured endothelial cells and modulated by shear stress. We hypothesized that this growth factor would be modulated by changes in shear stress in vivo. Bilateral aortoiliac prosthetic grafts were implanted into five baboons. High flow was generated by construction of femoral arteriovenous fistulas on both sides. Two months later, one of the fistulas was ligated, reducing shear stress in the upstream graft by 78 +/- 6%. Four days after fistula ligation, all grafts were removed and analyzed. As previously reported, SMC proliferation in low-flow grafts exceeded that in high-flow grafts, although the neointimal area was similar. mRNA levels for PDGF-A were significantly increased in low-flow grafts compared with high-flow grafts. In situ hybridization and immunohistochemical studies localized the increased PDGF-A mRNA and protein to the luminal endothelium and subjacent SMCs. Abrupt reductions in blood flow and fluid shear stress may induce accelerated neointimal thickening by a PDGF-A-mediated mechanism, since endothelial expression of this gene is temporally and anatomically associated with neointimal SMC proliferation.

Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid artery.

PURPOSE: Although matrix metalloproteinase (MMP) expression has been correlated with proliferation and migration of various tumor cells, the relation between MMP expression and smooth muscle cell (SMC) proliferation and migration has not been established. METHODS: We measured MMP expression (gelatin, casein, and elastin zymography) by vascular wall cells in balloon-injured carotid artery during the period of medial SMC proliferation, migration of SMC from the media to the intima, and subsequent intimal SMC proliferation. RESULTS: The 72 and 64-kd gelatinases (presumably 72 kd type IV collagenase or MMP 2) were constitutively expressed in normal carotid arteries, and the activated (59 and 54 kd) forms of this enzyme were increased at 5 days when SMCs start to migrate. A 92 kd gelatinase (presumably 92 kd type IV collagenase or MMP 9) was increased at 24 hours, when SMCs entered the growth cycle, and decreased thereafter. A low-molecular-weight metalloproteinase with elastolytic activity was present in the adventitia, and the activity was increased at 5 days after surgery. CONCLUSIONS: These results suggest that the 72 kd and 92 kd gelatinases may be involved in basement membrane and matrix degradation in the media in relation to SMC proliferation and migration, whereas the low-molecular-weight metalloproteinase may have a role in elastin turnover in the adventitia.

Heparin decreases activator protein-1 binding to DNA in part by posttranslational modification of Jun B.

Heparin is a potent inhibitor of the proliferation and migration of vascular smooth muscle cells. This agent selectively inhibits the transcription of tissue-type plasminogen activator and interstitial collagenase, probably by decreasing the binding of activator protein-1 (AP-1) to phorbol ester-responsive elements in the promoters of these genes. Decreased AP-1 binding is not due to a direct inhibition by heparin, since heparinase digestion of nuclear extracts prepared from heparin-treated smooth muscle cells does not restore AP-1 binding activity. Treatment of cells with heparin suppresses the expression of Jun B, one of the components of AP-1. The major effect of heparin is at the level of posttranslational modification of Jun B. Results from pulse-chase labeling experiments show that the newly synthesized Jun B is rapidly converted to a higher-molecular-weight form and that conversion is suppressed by heparin. Evidence is presented suggesting that the heparin-inhibited event is phosphorylation of Jun B.

Mechanisms of inhibition by heparin of vascular smooth muscle cell proliferation and migration.

Heparin, an inhibitor of vascular smooth muscle cell proliferation and migration, affects a number of other cell functions. These effects include inhibition of growth factor binding, deposition of matrix proteins and gene expression. Various mechanisms have been proposed and, yet, how heparin works as an inhibitor remains unclear. We have postulated that heparin inhibits smooth muscle cell growth and migration by suppressing the expression of matrix-degrading enzymes such as plasminogen activators and interstitial collagenase. The molecular mechanism of heparin's inhibitory action on these proteases is currently under investigation.

Heparin inhibits the expression of tissue-type plasminogen activator by smooth muscle cells in injured rat carotid artery.

Smooth muscle cells (SMCs) in balloon-injured rat carotid artery express tissue-type plasminogen activator (t-PA) at a time when they are migrating from the media to the intima. Since heparin inhibits SMC migration and intimal thickening, we have examined the possibility that heparin might also inhibit t-PA expression. Heparin (nonanticoagulant fraction; molecular weight, approximately 6,000) was administered by continuous intravenous infusion (1.0 mg/kg per hour) to Sprague-Dawley rats subjected to balloon injury of the left common carotid artery. At various times up to 14 days after injury, plasminogen activator expression was analyzed by zymography, plasmin generation, enzyme-linked immunosorbent assay, Northern blotting, and in situ hybridization. This dose of heparin inhibited SMC accumulation at 14 days by 60%. Both urokinase plasminogen activator (u-PA) and t-PA activity increased in injured arteries and reached a maximum at 7 days. Heparin treatment decreased t-PA, but not u-PA, activity. Total t-PA protein was decreased by treatment with heparin but not chondroitin sulfate, and the decrease in t-PA protein was associated with decreased t-PA mRNA in the media. These results in the injured rat carotid artery agree with our earlier observations that heparin inhibits t-PA gene expression in cultured baboon aortic SMCs. They also provide support for the hypothesis that heparin interferes with the expression of certain proteases required for SMC migration and proliferation.

Heparin inhibits collagenase gene expression mediated by phorbol ester-responsive element in primate arterial smooth muscle cells.

Heparin is a potent inhibitor of arterial smooth muscle cell (SMC) migration and proliferation in vivo and in vitro. We propose that heparin affects these SMC functions by interfering with either the expression or the activity of secreted proteases required for cell movement. We have reported that heparin selectively inhibits the expression of tissue-type plasminogen activator in SMCs during mitogenesis. In this study we show that the gene expression of another kind of protease, interstitial collagenase, is induced by fetal bovine serum and is also suppressed by heparin. The inhibitory effect on the induced collagenase mRNA is specific to heparin-like molecules and does not depend on the anticoagulant activity of heparin. The induction of the collagenase gene depends on the protein kinase C pathway, since it can be induced by phorbol esters such as phorbol 12-myristate 13-acetate and blocked by inhibitors such as H-7 and staurosporine. In transient transfection assays with chloramphenicol acetyltransferase constructs containing the phorbol ester-responsive element introduced into baboon SMCs, heparin inhibits transcription induced by serum or phorbol 12-myristate 13-acetate. These results support the conclusion that, in primate SMCs, interstitial collagenase gene transcription mediated by the phorbol ester-responsive element is blocked by heparin.

Heparin selectively inhibits the transcription of tissue-type plasminogen activator in primate arterial smooth muscle cells during mitogenesis.

How heparin inhibits vascular smooth muscle cell proliferation and migration has not been established. We have investigated the hypothesis that heparin inhibits vascular smooth muscle cell proliferation and migration by interfering with the expression and activity of proteases such as plasminogen activators. In an in vitro mitogenesis model, tissue-type plasminogen activator (tPA) mRNA and protein increase in baboon smooth muscle cells stimulated with fetal bovine serum or phorbol esters. Heparin inhibits smooth muscle cell proliferation and suppresses the induction of tPA mRNA and protein while it has little effect on the mRNA of urokinase-type plasminogen activator, plasminogen activator inhibitor type I, and a number of genes that are also modulated by serum and phorbol esters. The inhibitory effect on tPA mRNA is specific to heparin-like molecules and does not depend on the anticoagulation activity of heparin. The increase in tPA mRNA is due to increased transcription, which is suppressed by heparin. The induction of tPA by serum and phorbol esters is diminished by protein kinase C inhibitors such as H7 or staurosporine and by protein kinase C depletion. Since heparin suppresses the induction of the tPA gene by phorbol esters, these results suggest that heparin may interfere with the protein kinase C pathway.

Platelet-derived growth factor activity and mRNA expression in healing vascular grafts in baboons. Association in vivo of platelet-derived growth factor mRNA and protein with cellular proliferation.

In a baboon graft model of arterial intimal thickening, smooth muscle cells (SMC) have been observed to proliferate underneath an intact monolayer of endothelium and in the absence of platelet adherence. Because platelets are not present and therefore cannot be a major source of growth stimulus, we have proposed that the vascular wall cells in the graft intima express mitogens and regulate SMC proliferation. To test this hypothesis, we assayed the grafts for mitogenic activity and expression of growth factor genes. Segments of healing graft and of normal artery, when perfused ex vivo, released mitogenic activity into the perfusate. The graft released more mitogen than the normal arterial segment, and some of the activity was inhibitable with an antibody to human platelet-derived growth factor (PDGF). In addition, Northern analysis of total RNA demonstrated higher expression of PDGF-A chain mRNA in the graft intima compared to normal artery. PDGF-B chain mRNA was barely detectable in both tissues. PDGF mRNA levels within the graft interstices were not measured. In situ hybridization of 7.5- or 12-wk grafts indicated that some luminal endothelial cells and adjacent intimal SMC contained PDGF-A chain mRNA. By thymidine autoradiography, intimal SMC were observed to be proliferating in the inner third of the intima. These data demonstrate a difference in the pattern of PDGF transcript expression and luminal perfusate activity in graft as compared with control arteries. The association of intimal smooth muscle cell proliferation with intimal PDGF mRNA expression and release of PDGF-like protein supports the hypothesis that factors from cells that have grown into the graft or populated its surface rather than platelets may regulate intimal smooth muscle cell proliferation in this model.

Intimal lesion formation in rat carotid arteries after endothelial denudation in absence of medial injury.

Injury of an artery by passage of a balloon catheter causes both endothelial denudation and medial damage and produces a marked smooth muscle cell (SMC) proliferative response. In this study, the endothelium from rat carotid arteries was removed by use of a rotating loop of 5/0 monofilament suture (gentle denudation technique), which did not cause any detectable damage to the underlying medial cells but did cause platelet adherence. Expression of platelet-derived growth factor (PDGF) A-chain and PDGF receptor mRNA was comparable to that seen in ballooned carotids, but the medial SMC proliferative response to gentle denudation was markedly reduced when compared to that observed after balloon denudation (1.4% vs. 13.6%). Intimal lesions were only observed in those zones that remained denuded for more than 7 days. These results demonstrate that a denuding injury with no medial trauma is sufficient to induce intimal lesions and that the significantly higher proliferation seen in ballooned vessels might reflect a response of the medial cells to trauma that occurred during denudation.

Smooth muscle cells express urokinase during mitogenesis and tissue-type plasminogen activator during migration in injured rat carotid artery.

Although the level of plasminogen activator (PA) expression has been correlated with cellular proliferation and migration in vitro, this relation has not been established in tissue undergoing repair. In a rat model of arterial injury, we have measured the expression of PAs by vascular smooth muscle cells (SMCs) during entry into the growth cycle (0-24 hours) and subsequent migration from the media to the intima (starting at approximately 4 days). In normal rat carotid, low levels of urokinase-type PA (uPA) and tissue-type PA (tPA) are present; after removal of the endothelium, only uPA is detected in the media. uPA activity in extracts of carotid arteries increases and reaches a maximum between 16 and 24 hours after injury; uPA mRNA increases steadily and is maximal at 7 days. tPA activity appears at 3 days and is maximal at 7 days; tPA mRNA is present in normal vessels and reaches a maximum by 7 days. Most of the tPA in the media is associated with SMC and not with regenerating endothelium. Furthermore, tPA is present in the media before the SMCs migrate into the intima. These results demonstrate that PA expression by vascular SMCs is differentially regulated, with uPA present during mitogenesis and tPA during migration.

Growth factor gene expression by intimal cells in healing polytetrafluoroethylene grafts.

Vascular smooth muscle cell proliferation resulting in intimal hyperplasia is a major cause of late graft failure. In baboons, healing 60 microns internodal distance polytetrafluoroethylene vascular grafts form an intima composed of proliferating smooth muscle cells with a luminal lining of endothelium. The presence of intimal smooth muscle cell proliferation underneath an intact endothelium, without platelet adherence, suggests that intimal cells rather than platelets may provide the growth factors regulating the smooth muscle cell proliferation. This idea is supported by the observation that, when segments of graft and artery are excised and perfused ex vivo, there is greater mitogenic activity present in the graft perfusate compared to artery perfusate. Two factors expressed by vascular wall cells and known to influence smooth muscle cell growth in vitro are platelet-derived growth factor and transforming growth factor-beta 1. The expression of these growth factors was measured by Northern blot analysis of total ribonucleic acid extracted from thoracic aorta and the intima of 6-week thoracoabdominal polytetrafluoroethylene grafts, and from smooth muscle cell cultured from the aorta and polytetrafluoroethylene graft. Growth of the cultured smooth muscle cell was arrested in serum-free conditions for 3 days and then stimulated with 10% fetal calf serum. Twenty-four hours later, the smooth muscle cells were harvested. Probing the blots for platelet-derived growth factor-A, platelet-derived growth factor-B, and transforming growth factor-beta 1 messenger ribonucleic acid revealed that in vivo, the graft intima expressed more platelet-derived growth factor-A than the aorta.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between anemia and cholesterol metabolism in 'sex-linked anemic' (gene symbol, sla) mouse.

The relationship between hypocholesterolemia and anemia has been recognized in humans. However, no metabolic studies in humans have been reported, nor has an animal model been developed to investigate the effects of anemia on cholesterol metabolism. We have identified an animal model, the 'sex-linked anemic' (gene symbol, sla) mouse, characterized by iron deficiency anemia, to study the relationship between anemia and cholesterol metabolism. Results from our studies showed that the serum cholesterol was significantly lower in anemic male SLA mice compared to non-anemic littermates. The lower serum cholesterol observed in anemic SLA mice was related to a decreased in vivo hepatic cholesterol synthesis. However, the decreased hepatic cholesterol synthesis in anemic SLA mice was not due to a block at the primary regulatory site, the hydroxymethylglutaryl-CoA reductase, nor at one of the secondary regulatory sites: the acetoacetyl-CoA thiolase and hydroxymethylglutaryl-CoA synthase.

Use of a quality-control plasma sample to decrease interassay variation in radioimmunoassays of apolipoprotein A-I.

The apolipoprotein A-I (apo A-I) radioimmunoassay established in our laboratory involves use of purified apo A-I as the primary standard for quantifying apo A-I in plasma and a pooled plasma (quality-control sample) as a secondary standard to decrease interassay variation. The measured values for apo A-I increased over time as the labeled antigen degraded. We observed these results in two separate studies: (a) apo A-I in plasma from 16 subjects was measured 12 times during four months, and (b) apo A-I in a single pooled plasma was measured 48 times during 10 months. We show that use of a quality-control plasma sample as a secondary standard decreased interassay variation, which was, in part, ascribable to degradation of the labeled antigen.

A rapid apolipoprotein E radioimmunoassay using solid-phase staphylococcus protein. Use of pooled plasma as a secondary standard.

A rapid apolipoprotein E (apo E) radioimmunoassay, which requires a total of 24 hour incubation as compared to the usual 3-5 days, has been developed in our laboratory. Solid phase staphylococcus protein A was used to separate bound and unbound labeled antigen. Use of a pooled plasma (quality control sample) as a secondary standard to reduce interassay variation was also described.