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.

Synergistic effect of urotensin II with serotonin on vascular smooth muscle cell proliferation.

BACKGROUND: Urotensin II (U-II), the most potent vasoconstrictor, and serotonin (5-HT) are known to play an important role in pulmonary hypertension. However, little is known about the effect of U-II and its interaction with 5-HT on vascular smooth muscle cell (VSMC) proliferation. OBJECTIVE: We assessed the interaction between U-II and 5-HT in inducing VSMC proliferation. METHODS: Growth-arrested rabbit VSMCs were incubated in serum-free medium with different concentrations of U-II and 5-HT. VSMC proliferation was examined by the increase in [3H]thymidine incorporation into DNA and cell number. RESULTS: U-II or 5-HT induced [3H]thymidine incorporation in a dose-dependent manner with a maximal effect at a concentration of 50 nmol/l (161%) or 50 micromol/l (205%), respectively. When added together, low concentrations of U-II (50 nmol/l) and 5-HT (1 micromol/l) interacted synergistically in inducing [3H]thymidine incorporation (382%). These effects on [3H]thymidine incorporation were paralleled by an increase in cell number. The G-protein inactivator GDP-beta-S (100 micromol/l), protein kinase C (PKC) inhibitor Ro31-8220 (0.1 micromol/l), Src family tyrosine kinase inhibitor PP2 (1 micromol/l), and mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 micromol/l) inhibited the mitogenic effects of U-II and 5-HT and also their interaction in inducing [3H]thymidine incorporation. CONCLUSION: Our results suggest that U-II and 5-HT may induce the synergistic interaction in inducing VSMC proliferation via a G-protein-coupled receptor/PKC/Src tyrosine kinase/MAPK pathway, thus contributing to the relatively rapid development of atherosclerosis in hypertensive vascular disease.

Monocyte chemotactic protein 1 amplifies serotonin-induced vascular smooth muscle cell proliferation.

Monocyte chemotactic protein 1 (MCP-1), which is synthesized by vascular cells, is a chemoattractant for monocytes and has been implicated in a wide range of acute and chronic inflammatory processes characterized by monocyte infiltration, including atherosclerosis. However, it is unclear whether MCP-1 is able to modulate vascular smooth muscle cell (VSMC) proliferation. We assessed the effect of MCP-1 on VSMC proliferation and its interaction with serotonin (5-HT), a mitogen for VSMCs. Growth-arrested VSMCs were stimulated with different concentrations of MCP-1 (25-200 ng/ml) and 5-HT (5 and 50 microM) in serum-free medium. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. 5-HT at concentrations of 5 and 50 microM significantly stimulated DNA synthesis by 1.8- and 2.1-fold over the control value, respectively (p < 0.0001). However, MCP-1 at the concentrations tested did not have any significant effect on DNA synthesis. Even though MCP-1 (50 ng/ml) by itself is not mitogenic, when added to 5-HT, it significantly amplified the mitogenic effect of 5-HT compared with that of 5-HT alone (p < 0.0001). The 5-HT2A receptor antagonist sarpogrelate (10 microM) and its major metabolite M-1 (0.1 microM), pertussis toxin (10 ng/ml), Src family protein tyrosine kinase (PTK) inhibitor PP2 (1 microM), protein kinase C (PKC) inhibitor Ro31-8220 (0.1 microM) and mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 microM) significantly inhibited the mitogenic effect of 5-HT and its interaction with MCP-1. Anti-MCP-1 antibody (2 microg/ml) and the Janus kinase 2 (JAK2) inhibitor AG490 (10 microM) significantly inhibited the interaction of MCP-1 with 5-HT. Further, the amplified mitogenic effect of 5-HT with MCP-1 was completely reversed by the combined use of sarpogrelate with anti-MCP-1 antibody. Our results suggest that MCP-1 amplifies the mitogenic effect of 5-HT on VSMCs. The mitogenic effect of 5-HT may be mediated by the G protein-Src family PTK-PKC-MAPK pathway. The activation of the JAK2/signal transducer and activator of transcription 3 pathway by MCP-1 in addition to the MAPK pathway by 5-HT may explain the potentiating effect of MCP-1 on 5-HT-induced mitogenesis.

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.

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.

A peptide analogue of thrombin receptor-activating peptide inhibits thrombin and thrombin-receptor-activating peptide-induced vascular smooth muscle cell proliferation.

The serine protease thrombin, in addition to its pivotal role in the coagulation cascade, plays an important role in the development of atherosclerosis and restenosis by inducing smooth cell proliferation. Thrombin exerts its cellular effects mainly by cleaving its own receptor, leaving a new NH2-terminus that can act as a tethered ligand to activate the thrombin receptor. Peptides derived from the new NH2-terminus are able to fully activate thrombin receptor and mimic cellular effects of thrombin. Peptides with structural similarities to the tethered ligand have been tested for their ability to prevent thrombin- and tethered ligand-induced platelet aggregation and thrombus formation. We synthesized a peptide with multiple alanine substitutions in both critical and noncritical residues of tethered ligand that specifically inhibited platelet aggregation induced by thrombin and thrombin receptor-activating peptide and prevented thrombus formation in a rabbit thrombosis model. In the present study we demonstrate that this peptide inhibited only thrombin- and tethered ligand-induced human vascular smooth muscle cell proliferation as determined by (3H)-thymidine incorporation and has no effect on platelet-derived growth factor and serum-induced smooth muscle cell proliferation. The inhibitory effect of this peptide is dependent on the concentration of the antagonist used and length of preincubation time. The possible mechanism by which this peptide exerts its inhibitory effect may by desensitizing the thrombin receptor. The results of the present study suggest that apart from being antithrombotic, tethered ligand antagonist peptides can also act as antiatherosclerotic or antirestenotic agents.

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.

The cyclization of farnesyl diphosphate and nerolidyl diphosphate by a purified recombinant delta-cadinene synthase.

The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate (FDP), to sesquiterpene phytoalexins in cotton (Gossypium barbadense) plants is catalyzed by delta-cadinene (CDN) synthase. CDN is the precursor of desoxyhemigossypol and hemigossypol defense sesquiterpenes. In this paper we have studied the mechanism for the cyclization of FDP and the putative intermediate, nerolidyl diphosphate, to CDN. A purified recombinant CDN synthase (CDN1-C1) expressed in Escherichia coli from CDN1-C1 cDNA isolated from Gossypium arboreum cyclizes (1RS)-[1-2H](E, E)-FDP to >98% [5-2H]and [11-2H]CDN. Enzyme reaction mixtures cyclize (3RS)-[4,4,13,13,13-2H5]-nerolidyl diphosphate to 62.1% [8,8,15,15,15-2H5]-CDN, 15.8% [6,6,15,15,15-2H5]-alpha-bisabolol, 8.1% [6,6,15,15,15-2H5]-(beta)-bisabolene, 9.8% [4,4,13,13-2H4]-(E)-beta-farnesene, and 4.2% unknowns. Competitive studies show that (3R)-nerolidyl diphosphate is the active enantiomer of (3RS)-nerolidyl diphosphate that cyclized to CDN. The kcat/Km values demonstrate that the synthase uses (E,E)-FDP as effectively as (3R)-nerolidyl diphosphate in the formation of CDN. Cyclization studies with (3R)-nerolidyl diphosphate show that the formation of CDN, (E)-beta-farnesene, and beta-bisabolene are enzyme dependent, but the formation of alpha-bisabolol in the reaction mixtures was a Mg2+-dependent solvolysis of nerolidyl diphosphate. Enzyme mechanisms are proposed for the formation of CDN from (E,E)-FDP and for the formation of CDN, (E)-beta-farnesene, and beta-bisabolene from (3RS)-nerolidyl diphosphate. The primary structures of cotton CDN synthase and tobacco epi-aristolochene synthase show 48% identity, suggesting similar three-dimensional structures. We used the SWISS-MODEL to test this. The two enzymes have the same overall structure consisting of two alpha-helical domains and epi-aristolochene synthase is a good model for the structure of CDN synthase. Several amino acids in the primary structures of both synthases superimpose. The amino acids having catalytic roles in epi-aristochene synthase are substituted in the CDN synthase and may be related to differences in catalytic properties.

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.

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.

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.

Interactions of the 5-hydroxytryptamine 3 antagonist class of antiemetic drugs with human cardiac ion channels.

Administration of the 5-hydroxytryptamine 3 receptor class of antiemetic agents has been associated with prolongation in the QRS, JT, and QT intervals of the ECG. To explore the mechanisms underlying these findings, we examined the effects of granisetron, ondansetron, dolasetron, and the active metabolite of dolasetron MDL 74,156 on the cloned human cardiac Na(+) channel hH1 and the human cardiac K(+) channel HERG and the slow delayed rectifier K(+) channel KvLQT1/minK. Using patch-clamp electrophysiology we found that all of the drugs blocked Na(+) channels in a frequency-dependent manner. At a frequency of 3 Hz, the IC(50) values for block of Na(+) current measured 2.6, 88.5, 38.0, and 8.5 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Block was relieved by strong hyperpolarizing potentials, suggesting a possible interaction with an inactivated channel state. Recovery from inactivation was impaired at -80 mV compared with -100 mV, and the fractional recovery was impaired by drug in a concentration-dependent manner. IC(50) values for block of the HERG cardiac K(+) channel measured 3.73, 0.81, 5.95, and 12.1 microM for granisetron, ondansetron, dolasetron, and MDL 74,156, respectively. Ondansetron (3 microM) also slowed decay of HERG tail currents. In contrast, none of these drugs (10 microM) produced greater than 30% block of the slow delayed rectifier K(+) channel KvLQT1/minK. We concluded that the antiemetic agents tested in this study block human cardiac Na(+) channels probably by interacting with the inactivated state. This may lead to clinically relevant Na(+) channel blockade, especially when high heart rates or depolarized/ischemic tissue is present. The submicromolar affinity of ondansetron for the HERG K(+) channel likely underlies the prolongation of cardiac repolarization reported for this drug.

Inhibition of arterial thrombosis by a peptide ligand of the thrombin receptor.

Thrombin plays an important role in promoting arterial thrombosis by platelet activation and by catalyzing fibrin formation. Use of thrombin inhibitors that block both the platelet-activating and fibrin formation properties of thrombin are associated with hemostasis. This problem might be overcome by developing agents that block only the platelet-activating property of thrombin. Because the platelet-activating property of thrombin is mediated by the thrombin receptor, antagonists of the thrombin receptor might be efficacious and potentially safer with regard to bleeding complications. We investigated whether a peptide ligand (AFLARAA) of the thrombin receptor that blocked alpha-thrombin and thrombin receptor activating peptide-induced platelet aggregation could inhibit thrombosis. A partially occlusive thrombus was generated by application of electric current in rabbit carotid artery. In control animals, the artery was completely occluded within 42+/-12 min after the current was discontinued. When the thrombin receptor activating peptide antagonist was given (100 micromol/kg as an IV bolus followed by 900 micromol/kg infusion for a period of 180 min) starting at the time the current was stopped, blood flow remained patent throughout the infusion period and for an additional 60 min after the infusion was stopped. The antithrombotic effect of the antagonist peptide was not associated with increased bleeding tendency, as judged by the amount of blood adsorbed by a gauze pad placed in a surgical incision extending to the muscle tissue and by a standard template bleeding time. These results indicate that thrombin receptor antagonist peptides can be used as antithrombotic agents.

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.

Safe and effective management of hypertension with fixed-dose combination therapy: focus on losartan plus hydrochlorothiazide.

Treatment of hypertensive patients with fixed-dose combination therapy consisting of losartan and hydrochlorothiazide (HCTZ) has several potential benefits over monotherapy with each of the individual components: more effective blood pressure control, a reduction in the likelihood of adverse effects, and facilitation of patients staying on therapy due to a simple once-daily regimen. Losartan plus HCTZ fixed-dose combination therapy lowers blood pressure in mild to moderate or severe hypertensive patients to a level comparable with other classes of antihypertensive drugs in combination with HCTZ. Fixed-dose combination therapy with losartan plus HCTZ is therefore an excellent choice for hypertensive patients in whom combination therapy is necessary to achieve additional blood pressure reductions.

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.

RAR gamma agonists inhibit proliferation of vascular smooth muscle cells.

The multifactorial and unpredictable nature of human restenosis will probably necessitate interventional strategies that target multiple processes involved in neointimal proliferation. Retinoids represent a growing class of pleiotropic biologic response modifiers with demonstrable efficacy in managing several pathologic conditions pertaining to neointimal proliferation. However, retinoid treatment is associated with a high incidence of adverse effects. The action of all-trans-retinoic acid is mediated by two families of nuclear receptors, RARs and RXRs, each containing three isoforms alpha, beta, and gamma. Because synthetic retinoids that are receptor and function specific have been shown to differ from each other by several orders of magnitude in their potencies and are associated with limited adverse effects, we examined the effect of synthetic retinoids on serum- and serotonin-induced vascular smooth muscle cell (VSMC) proliferation. Naturally occurring retinoids were used as controls. All-trans-retinoic acid at nanomolar concentrations inhibited smooth muscle cell proliferation. In this study, we report that RAR gamma subgroup-specific agonists are the most potent inhibitors of serum and serotonin VSMC proliferation, as compared with other RAR pan-agonists and naturally occurring retinoids tested. Our results indicate that RAR gamma subgroup-specific agonists should be assessed further in in vivo models of neointimal proliferation.

Synergistic interaction between thromboxane A2 and mildly oxidized low density lipoproteins on vascular smooth muscle cell proliferation.

Low density lipoprotein (LDL) and mildly oxidized low density lipoprotein (mox-LDL) are known mitogens for vascular smooth muscle cell (VSMC). Since aggregating platelets at sites of atherosclerotic injury release thromboxane A2(TXA2), a known mitogen for VSMC, we examined whether TXA2 can act synergistically with mox-LDL or its oxidative components in inducing VSMC proliferation. Growth arrested primary aortic rabbit VSMCs in 1st or 2nd passage were incubated with different concentrations of LDL or mox-LDL or lysophosphatidylcholine (LPC) or H2O2 or 4-hydroxy-2-nonenel (HNE) for 24 h followed by incubation with TXA2 mimetic U46619 for another 24 h. The amount of 3[H]-thymidine incorporated into the DNA was measured. Both LDL and mox-LDL at a concentration of 120 microg/ml induced proliferation of VSMC (168% or 184% respectively) when compared to the control. U46619 induced VSMC proliferation was observed at a concentration of 5 microm/L. As compared to native LDL, the mitogenic effect of mox-LDL on VSMC proliferation was markedly potentiated by U46619 to 301% or 316% at 0.5 or 5 microm/L U46619 respectively. LPC, H2O2 and HNE induced DNA synthesis was also marked by enhanced by U46619. These results suggest that even low concentration of TXA2 released from aggregating platelets may potentiate the mitogenic effect of mox-LDL at sites of vascular damage. The mitogenic effect of mox-LDL may be mediated via its oxidation products LPC, H2O2 (reactive oxygen species donor), and HNE.

Modulation of endothelial cell proliferation by retinoid x receptor agonists.

One feature that contraindicates the wide therapeutic use of natural retinoids is their adverse effects during systemic use and the lack of receptor selectivity. In contrast, synthetic retinoids are distinguishable from each other on the basis of their partial or exclusive preference in binding and activation of selective retinoid receptors. We examined the inhibitory activities of natural and synthetic retinoids for their ability to reverse basic fibroblast growth factor-induced endothelial cell proliferation. Both the naturally occurring retinoids at nanomolar concentrations reversed basic fibroblast growth factor-induced endothelial cell proliferation. Among the synthetic retinoids tested, retinoic acid receptor/retinoid x receptor pan-agonist AGN 191659 [(E)-5-[2-(5,6,7, 8-tetrahydro-3, 5,5,8,8-pentamethyl-2-naphtyl) propen-1-yl]-2-thiophenecarboxylic acid] and retinoid x receptor pan-agonist AGN 191701 [(E)-2-[2-(5,6,7,8-tetrahydro-3, 5,5,8, 8-pentamethyl-2-naphthyl) propen-1-yl]-4-thiophenecarboxylic acid] at nanomolar concentrations reversed the basic fibroblast growth factor-induced endothelial cell proliferation. Since none of the retinoic acid receptor agonists tested had any effect, the inhibitory effect of AGN 191659 could be attributed to its retinoid x receptor receptor activity. These results suggest that retinoid x receptor agonists may be more selective anti-angiogenic agents due to their ability to inhibit endothelial cell proliferation.

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.