Active site-blocked factor IXa prevents intravascular thrombus formation in the coronary vasculature without inhibiting extravascular coagulation in a canine thrombosis model.

To assess the contribution of Factor IX/IXa, to intravascular thrombosis, a canine coronary thrombosis model was studied. Thrombus formation was initiated by applying current to a needle in the circumflex coronary artery. When 50% occlusion of the vessel developed, the current was stopped and animals received an intravenous bolus of either saline, bovine glutamyl-glycyl-arginyl-Factor IXa (IXai), a competitive inhibitor of Factor IXa assembly into the intrinsic Factor X activation complex, bovine Factor IX, or heparin. Animals receiving saline or Factor IX developed coronary occlusion due to a fibrin/platelet thrombus in 70 +/- 11 min. In contrast, infusion of IXai prevented thrombus formation completely (greater than 180 min) at doses of 460 and 300 micrograms/kg, and partially blocked thrombus formation at 150 micrograms/kg. IXai attenuated the accumulation of 125I-fibrinogen/fibrin at the site of the thrombus by approximately 67% (P less than 0.001) and resulted in approximately 26% decrease in serotonin release from platelets in coronary sinus (P less than 0.05). Hemostatic variables in animals receiving IXai, remained within normal limits. Animals given heparin in a concentration sufficient to prevent occlusive thrombosis had markedly increased bleeding, whereas heparin levels that maintained extravascular hemostasis did not prevent intracoronary thrombosis. This suggests that Factor IX/IXa can contribute to thrombus formation, and that inhibition of IXa participation in the clotting mechanism blocks intravascular thrombosis without impairing extravascular hemostasis.

Lysophosphatidylcholine and reactive oxygen species mediate the synergistic effect of mildly oxidized LDL with serotonin on vascular smooth muscle cell proliferation.

BACKGROUND: Mild oxidation of LDL enhances its atherogenic potential and induces a synergistic interaction with serotonin (5HT) on vascular smooth muscle cell (VSMC) proliferation. Because of its complex chemical nature, the mitogenic components of mildly oxidized LDL (moxLDL) remain unclear. METHODS AND RESULTS: We examined both the effects of lysophosphatidylcholine (LPC) and hydrogen peroxide (H(2)O(2)), a donor of reactive oxygen species, as major components of moxLDL and their interactions with 5HT on VSMC proliferation. Growth-arrested VSMCs were incubated with different concentrations of moxLDL, LPC, H(2)O(2), or LPC with H(2)O(2) in the absence or presence of 5HT. DNA synthesis in VSMCs was examined by [(3)H]thymidine incorporation. MoxLDL, LPC, H(2)O(2), and 5HT stimulated DNA synthesis in a dose-dependent manner. MoxLDL had a maximal stimulatory effect at a concentration of 5 microg/mL (211%), LPC at 15 micromol/L (156%), H(2)O(2) at 5 micromol/L (179%), and 5HT at 50 micromol/L (205%). Added together, moxLDL (50 ng/mL) and 5HT (50 micromol/L) synergistically increased DNA synthesis (443%). Coincubation of LPC (1 micromol/L) with H(2)O(2) (0.5 micromol/L) and 5HT (5 micromol/L) resulted in a synergistic increase in DNA synthesis (439%), which was nearly equal to that of moxLDL with 5HT (443%). The combined effects of LPC, H(2)O(2), and 5HT on DNA synthesis were completely reversed by the combined use of an antioxidant, N:-acetylcysteine (400 micromol/L) or butylated hydroxytoluene (20 micromol/L), with a 5HT(2) receptor antagonist, LY281067 (10 microg/mL). CONCLUSIONS: Our results suggest that both LPC and reactive oxygen species may contribute to the mitogenic effect of moxLDL on VSMCs and its synergistic effect with 5HT.

Synergistic effect of urotensin II with mildly oxidized LDL on DNA synthesis in vascular smooth muscle cells.

BACKGROUND: The urotensin II (UII) found in coronary atheroma is the most potent vasoconstrictor known to date. Mildly oxidized LDL (moxLDL) contributes to atherogenesis and plaque formation. We assessed the effect of UII and its interaction with moxLDL and the oxidative components of moxLDL on vascular smooth muscle cell (VSMC) proliferation. Methods and Results-Growth-arrested VSMCs were incubated in serum-free medium with different concentrations of LDL, moxLDL, oxLDL, hydrogen peroxide, lysophosphatidylcholine, or 4-hydroxy-2-nonenal, with or without UII. [(3)H]Thymidine incorporation into DNA was measured as an index of VSMC proliferation. UII stimulated [(3)H]thymidine incorporation in a dose-dependent manner, with a maximal effect at a concentration of 50 nmol/L (161%). Low concentrations of UII potentiated the mitogenic effect of LDL (108% to 242%), oxLDL (129% to 302%), moxLDL (120% to 337%), hydrogen peroxide (177% to 226%), lysophosphatidylcholine (115% to 332%), and 4-hydroxy-2-nonenal (142% to 299%). The synergistic interaction between UII and moxLDL was partially inhibited by anti-Gq/11alpha antibody, the epidermal growth factor receptor tyrosine kinase inhibitor erbstatin A (10 micromol/L), and the intracellular free radical scavenger N-acetylcysteine (400 micromol/L) and was completely inhibited by the c-Src tyrosine kinase inhibitor radicicol (10 micromol/L), the protein kinase C (PKC) inhibitor Ro31-8220 (0.1 micromol/L), and the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 micromol/L). CONCLUSIONS: Our results suggest that UII acts synergistically with moxLDL in inducing VSMC proliferation via the c-Src/PKC/MAPK pathway, which may explain the relatively rapid progression of atherosclerosis in patients with hypertension and hypercholesterolemia.

Effects of dopamine beta-hydroxylase inhibition with nepicastat on the progression of left ventricular dysfunction and remodeling in dogs with chronic heart failure.

BACKGROUND: Inhibition of dopamine beta-hydroxylase (DBH) results in a decrease in norepinephrine synthesis. The present study was a randomized, blinded, placebo-controlled investigation of the long-term effects of therapy with the DBH inhibitor nepicastat (NCT) on the progression of left ventricular (LV) dysfunction and remodeling in dogs with chronic heart failure (HF). METHODS AND RESULTS: Moderate HF (LV ejection fraction [LVEF] 30% to 40%) was produced in 30 dogs by intracoronary microembolization. Dogs were randomized to low-dose NCT (0.5 mg/kg twice daily, n=7) (L-NCT), high-dose NCT (2 mg/kg twice daily, n=7) (H-NCT), L-NCT plus enalapril (10 mg twice daily, n=8) (L-NCT+ENA), or placebo (PL, n=8). Transmyocardial (coronary sinus-arterial) plasma norepinephrine (tNEPI), LVEF, end-systolic volume, and end-diastolic volume were measured before and 3 months after initiating therapy. tNEPI levels were higher in PL compared with NL (86+/-20 versus 13+/-14 pg/mL, P:<0.01). L-NCT alone and L-NCT+ENA reduced tNEPI toward normal (28+/-4 and 39+/-17 pg/mL respectively), whereas HD-NCT reduced tNEPI to below normal levels (3+/-10 pg/mL). In PL dogs, LVEF decreased but was unchanged with L-NCT and increased with L-NCT+ENA. L-NCT and L-NCT+ENA prevented progressive LV remodeling, as evidenced by lack of ongoing increase in end-diastolic volume and end-systolic volume, whereas H-NCT did not CONCLUSIONS: In dogs with HF, therapy with L-NCT prevented progressive LV dysfunction and remodeling. The addition of ENA to L-NCT afforded a greater increase in LV systolic function. NCT at doses that normalize tNEPI may be useful in the treatment of chronic HF.

Vascular smooth muscle proliferation: synergistic interaction between serotonin and low density lipoproteins.

OBJECTIVES: The purpose of this study was to examine whether low density lipoproteins (LDLs) or mildly oxidized LDL (mox-LDL) are mitogens for vascular smooth muscle cells (VSMCs) and whether they can act synergistically with serotonin (5HT), a known mitogen for VSMC, in potentiating the proliferative effect of 5HT on VSMC. BACKGROUND: Whether LDL or mox-LDL has a mitogenic effect on VSMC has been controversial. It is possible that LDL may not be mitogenic to VSMC but modification of LDL may confer mitogenic properties on LDL. A known mitogen for VSMC is 5HT that is released by aggregating platelets at sites of atherosclerotic changes or endothelial dysfunction. It is possible that LDL may interact with 5HT to enhance VSMC proliferation induced by 5HT. METHODS: Growth arrested primary VSMCs were incubated with different concentrations of LDL or mox-LDL for 24 h followed by incubation with 5HT for another 24 h (mild oxidation of LDL was achieved by incubating LDL with Cu++ which increased the thiobarbituric acid product formation without a change in electrophoretic mobility). The increase in cell number or the amount of 3H-thymidine incorporated into the DNA was then measured. RESULTS: Low density lipoprotein and mox-LDL induced significant VSMC proliferation by themselves and this effect was potentiated by 5HT. The 5HT2 receptor antagonist (LY281067) and pertussis toxin reversed only the proliferative effect of 5HT. Polyinosinic acid (poly-I), an inhibitor of scavenger receptors, did not inhibit the proliferative effect of LDL or mox-LDL or their synergistic interaction with 5HT. CONCLUSIONS: These results suggest that LDL and mox-LDL act synergistically with 5HT in inducing VSMC proliferation. The synergistic interaction could be blocked by LY281067 and pertussis toxin but not by poly-I acid.

Hemodynamic and cardiac effects of the selective T-type and L-type calcium channel blocking agent mibefradil in patients with varying degrees of left ventricular systolic dysfunction.

OBJECTIVES: This study sought to assess the hemodynamic and cardiac effects of two dose levels of mibefradil in patients with varying degrees of ischemic left ventricular dysfunction. BACKGROUND: Mibefradil is a new, selective T-type and L-type calcium channel blocking agent. Because L-type channel blockade may depress myocardial performance, an invasive hemodynamic study was performed to assess the safety of this agent. METHODS: We performed an open label study, examining the effects of two intravenous doses of mibefradil, selected to produce plasma levels comparable to those measured after oral administration of 50 mg (dose 1: 400 ng/ml) or 100 mg (dose 2: 800 ng/ml) of the drug. Variables studied included the indexes of left ventricular function and neurohormone levels. Patients were stratified according to ejection fraction (EF) (> or = 40%, n = 26; < 40%, n = 24) and the presence (n = 15) or absence (n = 35) of heart failure. RESULTS: In patients with preserved systolic function, dose 1 had no clinically significant hemodynamic effects, but dose 2 decreased mean aortic pressure and systemic vascular resistance (-8.5 mm Hg, -12%, both p < 0.01) and also reduced end-systolic stress and volume, thus improving EF (52% to 58%, p < 0.01). Heart rate tended to decrease. In patients with depressed EF, heart rate decreased significantly with both doses. The effects of dose 1 mimicked those observed after dose 2 in patients with preserved EF. Dose 2 (plasma levels 1,052 +/- 284 ng/ml) still decreased left ventricular systolic wall stress and improved EF (24.0% to 28.5%, p < 0.05) but also significantly depressed the maximal first derivative of left ventricular pressure. Examination of individual pressure-volume loops in two patients with heart failure showed a clear rightward shift of the loop despite a decrease in systolic pressure, suggesting negative inotropy. Neurohormone levels were unchanged at both dose levels and in all subgroups. CONCLUSIONS: Intravenous mibefradil was well tolerated and produced an overall favorable cardiovascular response. However, high plasma concentrations might produce myocardial depression in patients with heart failure, and caution should be exerted in this setting.

Comparative neurohormonal responses in patients with preserved and impaired left ventricular ejection fraction: results of the Studies of Left Ventricular Dysfunction (SOLVD) Registry. The SOLVD Investigators.

OBJECTIVES: The aim of this study was to determine the differences in neurohumoral responses between patients with pulmonary congestion with and without impaired left ventricular ejection fraction. BACKGROUND: Previous studies have established the presence of neurohumoral activation in patients with congestive heart failure. It is not known whether the activation of these neurohumoral mechanisms is related to the impairment in systolic contractility. METHODS: The 898 patients recruited into the Studies of Left Ventricular Dysfunction (SOLVD) Registry substudy were examined to identify those patients with pulmonary congestion on chest X-ray film who had either impaired (< or = 45%, group I) or preserved (> 45%, group II) left ventricular ejection fraction. Plasma norepinephrine, plasma renin activity, arginine vasopressin and atrial natriuretic peptide levels were measured in these two groups of patients and compared with values in matched control subjects. RESULTS: Distribution of the New York Heart Association symptom classification was the same in the two groups of patients. Compared with control subjects, patients in group II with pulmonary congestion and preserved ejection fraction had no activation of the neurohumoral mechanisms, except for a small but statistically significant increase in arginine vasopressin and plasma renin activity. Compared with patients in group II, those in group I with pulmonary congestion and impaired ejection fraction had significant increases in plasma norepinephrine (p < 0.002), plasma renin activity (p < 0.02) and atrial natriuretic peptide levels (p < 0.0007). When we controlled for baseline differences between groups I and II, the between-group differences in plasma norepinephrine (p < 0.02) and atrial natriuretic peptide (p < 0.002) remained significant. However, plasma renin activity was not significantly different between groups I and II. When the effects of diuretic agents and angiotensin-converting enzyme inhibitors were adjusted, patients with lower ejection fraction were found to have significantly higher plasma norepinephrine and atrial natriuretic peptide levels. CONCLUSIONS: The results point to the importance of the decrease in left ventricular ejection fraction as one of the mechanisms for activation of neurohormones in patients with heart failure.

Relation of neurohumoral activation to clinical variables and degree of ventricular dysfunction: a report from the Registry of Studies of Left Ventricular Dysfunction. SOLVD Investigators.

OBJECTIVES: This study examined the relation between neurohumoral activation and severity of left ventricular dysfunction and congestive heart failure in a broad group of patients with depressed left ventricular function who were not recruited on the basis of eligibility for a therapeutic trial. BACKGROUND: Previous studies have established the presence of neurohumoral activation in patients with severe congestive heart failure. It is not known whether the activation of these neurohumoral mechanisms is related to an impairment in left ventricular function. METHODS: From the 6,273 patients recruited into the Studies of Left Ventricular Dysfunction Registry (SOLVD), a subgroup of 859 patients were randomly selected, and their plasma norepinephrine, plasma renin activity, arginine vasopressin and atrial natriuretic peptide levels were correlated with clinical findings, New York Heart Association functional class, left ventricular ejection fraction and drug use. RESULTS: There was a weak but significant correlation between ejection fraction and an increase in plasma norepinephrine (rho = -0.18, p < 0.0001), plasma renin activity (rho = -0.24, p < 0.0001) and arginine vasopressin (rho = -0.12, p < 0.003). The only exception was atrial natriuretic peptide, which showed the best correlation to ejection fraction (rho = -0.37, p < 0.0001). Deterioration in functional class was associated more with increases in atrial natriuretic peptide (p = 0.0003) and plasma renin activity (p = 0.0003) and less with an increase in plasma norepinephrine. Of the clinical variables, elevated jugular venous pressure and third heart sound (S3) gallop were significantly associated with increased levels of plasma norepinephrine, plasma renin activity and atrial natriuretic peptide. We then compared the relation of neurohormones with clinical signs, functional status, ejection fraction and drug therapy and controlled for mutual interactive effects. After adjustment, a decrease in ejection fraction was still significantly related to an increase in plasma norepinephrine, plasma renin activity and atrial natriuretic peptide. In contrast, only a difference between functional classes I and III/IV was associated with an increase in plasma renin activity and atrial natriuretic peptide levels. CONCLUSIONS: Neurohumoral activation in patients with heart failure is related to severity of left ventricular functional depression, and this relation is independent of functional class or concomitant drug therapy.

Seasonal Variations in Rubber Biosynthesis, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, and Rubber Transferase Activities in Parthenium argentatum in the Chihuahuan Desert.

The rubber content and the activities of enzymes in the polyisoprenoid pathway in Parthenium argentatum (guayule) were examined throughout the growing season in field plots in the Chihuahuan Desert. The rubber content of the plants was low in July and August and slowly increased until October. From October to December there was a rapid increase in rubber formation (per plant) from 589.0 mg to 4438.0 mg. The percentage of rubber in the plants increased from 0.7% (mg/g dry weight) in August and 1.27% in October to 5.5% in December. The rapid increase in rubber formation may result from exposing the plants to low temperatures of 5 to 7[deg]C. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was 21.1 nmol mevalonic acid (MVA) h-1 g-1 fresh weight in the bark of the lower stems in June during seedling growth and decreased to 5.1 nmol MVA h-1g-1 fresh weight in July and 2.9 nmol MVA h-1 g-1 fresh weight in September. From October to December, the activity increased from 5.0 to 29.9 nmol MVA h-1 g-1 fresh weight. The activity of rubber transferase was 65.5 nmol isopentenyl pyrophosphate (IPP) h-1 g-1fresh weight in the bark in September and increased to 357.5 nmol IPP h-1 g-1 fresh weight in December. The rapid increase in the activities of HMGR and rubber transferase coincided with the rapid increase in rubber formation. The activities of MVA kinase and IPP isomerase did not significantly increase in the fall and winter. A tomato HMGR-1 cDNA probe containing a highly conserved C-terminal region of HMGR genes hybridized at low stringency with several bands on blots of HindIII-digested genomic DNA from guayule. In northern blots with the HMGR-1 cDNA probe at low stringency, HMGR mRNA was high in June and November, corresponding to periods of high HMGR activity during seedling growth and rapid increase in rubber formation. The seasonal variations in rubber formation and HMGR mRNA, HMGR activity, and rubber transferase activity may be due to low temperature stimulation in the fall and winter months.

Differential induction of 3-hydroxy-3-methylglutaryl CoA reductase in two cotton species following inoculation with Verticillium.

Gossypium barbadense cottons are typically more resistant to wilt pathogens than are Gossypium hirsutum cultivars. Both species make terpenoid phytoalexins in response to infection, implicating isoprenoid biosynthesis as a factor in resistance. Conserved regions in plant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the first enzyme in the terpene biosynthesis pathway, were used to design polymerase chain reaction primers for cloning a fragment of a cotton HMGR gene. The clone was used as a probe on Northern blots to show that induction of HMGR mRNA following introduction of Verticillium dahliae spores into the vascular system is much more rapid in Seabrook Sea Island, a restant G. barbadense cotton, than it is in Rowden, a susceptible G. hirsutum. The amount of HMGR mRNA returned to near control levels in 4 days in the former variety but continued to accumulate in the latter. Specific enzyme activity of HMGR also increased more rapidly in stele extracts of Seabrook Sea Island than in Rowden.

Chronic dietary tyrosine supplements do not affect mild essential hypertension.

The blood pressure and plasma norepinephrine response to oral tyrosine, the precursor of norepinephrine, supplementation (2.5 g t.i.d.) of regular meals was examined in 13 untreated patients with mild essential hypertension. Using a randomized double-blind crossover design, each 2-week treatment was followed by a 2-week supplement-free interval. Supine and standing blood pressure and plasma norepinephrine levels were measured at the beginning and end of each 2-week treatment. Plasma tyrosine levels increased (p less than 0.001) from 71.2 +/- 8.0 nM/ml at baseline to 152.8 +/- 17.4 nM/ml 2 hours after the tyrosine supplement. Blood pressure under control conditions was 144 +/- 3 Hg systolic, 91 +/- 2 mm Hg diastolic (109 +/- 2 mm Hg mean) after 30 minutes in the supine position and 148 +/- 4 mm Hg systolic, 102 +/- 3 mm Hg diastolic (117 +/- 3 mm Hg mean) after 5 minutes of standing. Plasma norepinephrine levels were 191 +/- 18 pg/ml in the supine subjects and 390 +/- 33 pg/ml in the standing subjects. No difference in systolic, diastolic, or mean blood pressure, heart rate, or plasma norepinephrine levels were seen between the beginning and end of each period or between groups. Individual changes in blood pressure showed no correlation with individual changes in norepinephrine levels. These results indicate that the addition of a tyrosine supplement to the usual diet of mild hypertensive subjects has no beneficial effect on blood pressure.

The influence of oral tyrosine and tryptophan feeding on plasma catecholamines in man.

We investigated the effects of oral tyrosine (7.5 g/day) and oral tryptophan (3 g/day) feeding with regular meals in normal male volunteers using a double-blind cross-over design with a "run in" period for acclimatization. Oral tyrosine feeding significantly decreased both free and conjugated plasma norepinephrine concentrations while oral tryptophan feeding did not have such an effect. Since alpha adrenergic stimulation in certain areas of the CNS has been shown to decrease peripheral sympathetic tone, we postulate that dietary tyrosine supplementation in man causes an increase in brain catecholaminergic activity which in turn leads to a decrease in peripheral sympathetic activity as evidenced by the decrease in plasma catecholamines.

Endothelial cells regulate the proliferation of monocytes in vitro.

Monocytes (MPhis) are among the first cells to accumulate in early atherosclerotic lesions and generally are believed to be incapable of proliferation. However, recent studies indicate that the number of MPhis in atherosclerotic lesion may increase due to induction of local proliferation. Since proliferation of hematopoietic lineage cells is strongly influenced by interaction with neighboring cell types, we examined the ability of vascular endothelial cells (EC), smooth muscle cells or fibroblasts to stimulate MPhi proliferation. In this study, we show that only when seeded at high densities MPhis could proliferate in culture. However, when contact co-cultured with EC, MPhis proliferated at a higher rate (260% on day 6) than those cultured alone or co-cultured with smooth muscle cells or fibroblasts. Endothelial cells could stimulate the proliferation of MPhis even at non-proliferating densities. Only EC that were growth arrested or in lag phase could induce MPhi proliferation, whereas those in the exponential proliferating phase were non-stimulatory. Conditioned medium prepared from EC in growth arrested or lag phase failed to stimulate MPhi proliferation. Similarly physical separation of MPhis from EC also resulted in no proliferation. These results suggest that EC induced MPhi proliferation is contact dependent and no soluble factors are involved in this induction. This EC induced MPhi proliferation may have a profound effect on the rate of progression of atherosclerosis.

Serotonin fails to induce proliferation of endothelial cells preloaded with eicosapentaenoic acid and docosahexaenoic acid.

Diets rich in fish oils are associated with a reduced risk of cardiovascular disease including reduction of atherosclerosis and restenosis. We examined the effect of omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), major components of fish oils, on serotonin (5HT) stimulated vascular endothelial cells proliferation as a possible mechanism for this vascular protective effect. In this study we demonstrate that 5HT, a known mitogen for vascular endothelial cells, failed to stimulate proliferation of endothelial cells pre-incubated with EPA and DHA. This inhibitory effect was specific for omega-3 fatty acids only and not shared by other fatty acids like oleic acid (monounsaturated) or arachidonic acid (polyunsaturated) or palmitic acid (saturated). When endothelial cells were exposed to EPA and DHA in the ratio present in fish oils, EPA and DHA were shown to act synergistically in inhibiting the proliferative effect of 5HT. These results suggests that one of the mechanisms by which fish oils may confer vascular protective effect is by making the endothelial cells less responsive to mitogenic stimuli of growth factors such as 5HT that are released by aggregating platelets at sites of vascular injury. This inhibition of endothelial cell proliferation may account for the clinically observed effects of fish oil in attenuating the progression of atherosclerotic changes or neointimal proliferation following vascular injury.

Vascular smooth muscle cells preloaded with eicosapentaenoic acid and docosahexaenoic acid fail to respond to serotonin stimulation.

Epidemiological, animal and clinical studies indicate that n-3 fatty acids may benefit individuals with known history of cardiovascular disease or at risk of developing it. Though there is indirect evidence to suggest that the beneficial effects of n-3 fatty acids may be because of their ability to inhibit smooth muscle cell (SMC) proliferation, there are no studies that have examined this hypothesis. In this study, the mitogenic effect of serotonin (5HT) and platelet derived growth factor (PDGF), known mitogens for vascular SMC, on aortic SMCs preloaded with eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) is examined. 5HT and PDGF could only partially stimulate proliferation of SMC that were preloaded with EPA or DHA as compared to the control cells. gamma-Linolenic acid (LA) and oleic acid (OA) did not block the 5HT or PDGF induced 3[H]thymidine incorporation suggesting that the anti-proliferative effect was specific to n-3 fatty acids only. Further, when EPA and DHA were combined in the ratio they are present in fishoils, there was a synergistic interaction in inhibiting the proliferation of SMC. Further, SMC grown in the presence of EPA or DHA, when stimulated with 5HT, failed to show an increase in 5HT(2) receptor mRNA. One of the potential mechanism by which fish oils may prevent the development of atherosclerosis or restenosis could be inhibition of the mitogen induced SMC proliferation. Combination of EPA with DHA is likely to be more beneficial.

Lipid peroxidation product 4-hydroxy-2-nonenal acts synergistically with serotonin in inducing vascular smooth muscle cell proliferation.

Formation of an atherosclerotic lesion is in part mediated by inflammatory and oxidative mechanisms including lipid peroxidation. To characterize the potential role of lipid peroxidation products in atherogenesis, we assessed the effect of 4-hydroxy-2-nonenal (HNE), a component of oxidatively modified lipids on vascular smooth muscle cells (VSMCs) proliferation, and its interaction with serotonin (5-hydroxytryptamine, 5-HT), a known mitogen for VSMCs. Growth-arrested rabbit VSMCs were incubated with different concentrations of HNE in the absence or presence of 5-HT. VSMCs proliferation was examined by increases in [3H]thymidine incorporation into DNA and cell number. HNE and 5-HT stimulated DNA synthesis in a dose-dependent manner. HNE had a maximal proliferative effect at a concentration of 1 microM (143% of the control) and 5-HT at 50 microM (211%). When added together, low concentrations of HNE (0.1 microM) and 5-HT (5 microM) synergistically induced DNA synthesis (273%). These effects on DNA synthesis were paralleled by an increase in cell number. A 5-HT2 receptor antagonist LY 281067 (10 microg/ml) and pertussis toxin (10 ng/ml) inhibited the mitogenic effect of 5-HT only. Protein tyrosine kinase inhibitor erbstatin A (10 microM) completely inhibited the mitogenic effect of HNE and partially that of 5-HT and the combined effect of HNE+5-HT. Protein kinase C inhibitor Ro 31-8220 (0.1 microM) completely inhibited mitogenic effects of both HNE and 5-HT, and also the combined effect of HNE+5-HT. The synergistic effect of HNE+5-HT on DNA synthesis was completely reversed by the combined use of LY 281067 (10 microg/ml) and antioxidants N-acetylcysteine (400 microM), vitamin C (200 microM), or vitamin E (20 microM). Our results suggest that HNE acts synergistically with 5-HT in inducing VSMCs proliferation. Combined use of both antiplatelet and antioxidant therapies may be useful for the prevention of VSMCs proliferative disorders associated with atherosclerosis and restenosis after angioplasty.

Serotonin potentiates angiotensin II--induced vascular smooth muscle cell proliferation.

Vascular smooth muscle cell (VSMC) proliferation is a key feature in the development of atherosclerosis and restenosis after angioplasty, which can occur in response to many different humoral and mechanical stimuli. We investigated the growth promoting activities of two potent vasoactive substances, angiotensin II (Ang II) and serotonin (5-HT), on cultured rabbit VSMCs. Growth-arrested VSMCs were incubated with serum-free medium containing different concentrations of Ang II in the presence or absence of 5-HT. [3H]thymidine incorporation into VSMC DNA was measured as an index of cell proliferation. Ang II and 5-HT stimulated DNA synthesis in a dose-dependent manner with a maximal effect at 1.75 microM for Ang II (202%) and 50 microM for 5-HT (205%). When added together, low concentrations of Ang II (1 microM) and 5-HT (5 microM) synergistically induced DNA synthesis (363%). Candesartan (1 microM), an AT(1) receptor antagonist, but not PD 123319 (1 microM), an AT(2) receptor antagonist, inhibited the mitogenic effect on Ang II and its interaction with 5-HT. Sarpogrelate (10 microM), a 5-HT(2A) receptor antagonist, and pertussis toxin (10 ng/ml) inhibited the mitogenic effect of 5-HT and its interaction with Ang II. The protein kinase C inhibitor Ro 31-8220 (0.1 microM), the Raf-1 inhibitor radicicol (10 microM), and the MAPK kinase inhibitor PD 098059 (10 microM) abolished mitogenic effects of Ang II and 5-HT, and also their synergistic interaction. The JAK2 inhibitor AG 490 (10 microM) had only a minimal inhibitory effect of Ang II-induced DNA synthesis but significantly inhibited the interaction of Ang II with 5-HT. The synergistic effect on Ang II (1 microM) with 5-HT (5 microM) on DNA synthesis was completely reversed by the combined use of both candesartan (1 microM) and sarpogrelate (10 microM). Our results suggest that Ang II and 5-HT exert a synergistic interaction on VSMC proliferation via AT(1) and 5-HT(2A) receptors. The activation of MAPK and JAK/STAT pathways may explain the synergistic interaction between Ang II and 5-HT.

Hyperlipemic-very low density lipoprotein, intermediate density lipoprotein and low density lipoprotein act synergistically with serotonin on vascular smooth muscle cell proliferation.

BACKGROUND: Previous studies have shown that very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) from hyperlipidemic plasma are more atherogenic than those from normal plasma. Since platelet aggregation at sites of atherosclerotic injury exposes the cells to high concentrations of serotonin (5HT), a known mitogen for vascular smooth muscle cells (VSMCs), it was examined whether VLDL, IDL or LDL from plasma of 1% cholesterol-fed rabbits can potentiate the mitogenic effect of 5HT on VSMC. METHODS: Growth arrested primary aortic VSMC in 1st or 2nd passage were incubated with different concentrations of VLDL, IDL or LDL in the presence or absence of pertusis toxin (PTX) for 24 h followed by incubation with 5HT for 24 h. The amount of [3H]thymidine incorporated into the DNA as well as the increase in cell number was measured. RESULTS: Either VLDL, IDL or LDL at a concentration of 60 microg/ml induced proliferation of VSMC by themselves (196, 137 or 122% increase in [3H]thymidine incorporation, or 122, 119 or 122% increase in cell number, respectively when compared to the control, P<0.05). This effect on DNA synthesis was markedly potentiated by 50 microM 5HT to 465, 714 and 1369%, respectively. PTX reversed the mitogenic effect of 5HT, but not that of VLDL, IDL or LDL. CONCLUSION: These results suggest that even low concentration of VLDL, IDL or LDL from hypercholesterolemic plasma may significantly potentiate the mitogenic effect of 5HT, that is released by aggregating platelets at sites of vascular damage.

Angiotensin II and serotonin potentiate endothelin-1-induced vascular smooth muscle cell proliferation.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation induced by various growth factors has been implicated in a wide variety of pathological processes, including hypertension, atherosclerosis and restenosis after angioplasty. OBJECTIVES: To investigate the interactions among well-known potent vasoconstrictor substances, endothelin-1 (ET-1), angiotensin II (Ang II), and serotonin (5-HT), on VSMC proliferation. METHODS: Growth-arrested rabbit VSMCs were incubated with different concentrations of ET-1 in the absence or presence of Ang II, 5-HT, or both. VSMC proliferation was examined by increases in incorporation of [3H]thymidine into DNA and in cell number. RESULTS: ET-1, Ang II and 5-HT stimulated DNA synthesis in a dose-dependent manner. ET-1 had a maximal effect at a concentration of 0.5 micromol/l (259% of control), Ang II at 1 micromol/l (173%), and 5-HT at 50 micromol/l (205%). When added together, ET-1 (0.1 micromol/l) and Ang II (1 micromol/l) synergistically induced DNA synthesis (341%). When the vasoconstrictors were tested in combination, even non-mitogenic concentrations of ET-1 (0.01 nmol/l) potentiated 5-HT (5 micromol/l)-induced DNA synthesis (404%). Co-incubation of ET-1 (0.01 micromol/l) with Ang II (1 micromol/l) and 5-HT (5 micromol/l) synergistically induced DNA synthesis (566%). These effects on DNA synthesis were paralleled by an increase in cell number. The ETA/B non-selective receptor antagonist, TAK044 (1 micromol/l) and the ETA receptor antagonist, BQ123 (1 micromol/l), but not the ETB receptor antagonist, BQ788 (1 micromol/l), inhibited the mitogenic effect of ET-1 and its interaction with Ang II or 5-HT. In addition, TAK044 (1 micromol/l) or BQ123 (1 micromol/l) along with the angiotensin II type 1 (AT1) receptor antagonist, candesartan (1 micromol/l), the 5-HT2A receptor antagonist, sarpogrelate (10 micromol/l), or both, inhibited the interactions of ET-1 with Ang II or 5-HT. CONCLUSIONS: Our results suggest that Ang II and 5-HT could potentiate ET-1-induced VSMC proliferation. Inhibition of ETA, AT1, and 5-HT2A may be effective in the treatment of VSMC proliferative disorders associated with hypertension, atherosclerosis and restenosis after angioplasty.

Mildly oxidized low-density lipoprotein acts synergistically with angiotensin II in inducing vascular smooth muscle cell proliferation.

OBJECTIVES: Considerable attention has been focused on both mildly oxidized low-density lipoprotein (mox-LDL) and highly oxidized LDL (ox-LDL) as important risk factors for cardiovascular disease. Further, angiotensin II (Ang II) appears to play a crucial role in the development of hypertension and atherosclerosis. We assessed the effect of oxidatively modified LDL and its major oxidative components, i.e., hydrogen peroxide (H2O2), lysophosphatidylcholine (LPC), and 4-hydroxy-2-nonenal (HNE) and their interaction with Ang II on vascular smooth muscle cell (VSMC) DNA synthesis. METHODS: Growth-arrested rabbit VSMCs were incubated in serum-free medium with different concentrations of native LDL, mox-LDL, ox-LDL, H2O2, LPC, or HNE with or without Ang II. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. RESULTS: Ang II stimulated DNA synthesis in a dose-dependent manner with a maximal effect at a concentration of 1 micromol/l (173%). Ang II (0.5 micromol/l) amplified the effect of native LDL at 500 ng/ml, ox-LDL at 100 ng/ml, and mox-LDL at 50 ng/ml on DNA synthesis (108 to 234%, 124 to 399%, 129 to 433%, respectively). H2O2 had a maximal effect at a concentration of 5 micromol/l (177%), LPC at 15 micromol/l (156%), and HNE at 0.5 micromol/l (137%). Low concentrations of H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) also acted synergisitically with Ang II (0.5 micromol/l) in inducing DNA synthesis to 308, 304, or 238%, respectively. Synergistic interactions of Ang II (0.5 micromol/l) with mox-LDL, ox-LDL (both 50 ng/ml), H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) on DNA synthesis were completely reversed by the combined use of probucol (10 micromol/l), a potent antioxidant and candesartan (0.1 micromol/l), an AT1 receptor antagonist. CONCLUSIONS: Our results suggest that mox-LDL, ox-LDL, and their major components H2O2, LPC, and HNE act synergistically with Ang II in inducing VSMC DNA synthesis. A combination of antioxidants with AT1 receptor blockade may be effective in the treatment of VSMC proliferative disorders associated with hypertension and atherosclerosis.