Rajbabu Pakala

Effect of Serotonin, Thromboxane A2, and Specific Receptor Antagonists on Vascular Smooth Muscle Cell Proliferation

BACKGROUND Restenosis is a major complication that limits the long-term efficacy of coronary angioplasty. Migration and proliferation of activated medial smooth muscle cells (SMCs) is considered an important mechanism in this process. Because at sites of vascular injury, aggregating platelets release both serotonin (5-HT) and thromboxane A2 (TXA2), we examined whether 5-HT and TXA2 can induce SMC proliferation and whether there is synergistic interaction between these two mediators. METHODS AND RESULTS The mitogenic effects of 5-HT and TXA2 either alone or in combination was examined in serum-free medium on canine aortic SMCs by [3H]thymidine incorporation into DNA and by cell counting. 5-HT induced SMC proliferation at a concentration of 100 nmol/L, whereas the effect of TXA2 (U46619, a stable TXA2 mimetic) on inducing proliferation of SMCs was observed at a concentration of 100 nmol/L. When these two mediators were added together, there was a synergistic interaction on inducing SMC proliferation even at subthreshold concentrations. The mitogenic effect of 5-HT and its synergistic interaction with TXA2 on SMC proliferation was abolished by a 5-HT2 receptor antagonist, LY281067, without affecting the contribution of TXA2. Similarly, the TXA2 synthase inhibitor/receptor antagonist ridogrel abolished the mitogenic effect of TXA2 and the interaction between 5-HT and TXA2 without affecting the response to 5-HT. When LY281067 and ridogrel were used together, they abolished the mitogenic effects of 5-HT and TXA2. CONCLUSIONS At sites of vascular injury, platelet-induced SMC proliferation may also be modulated by nonpeptide growth mediators. A combination of a 5-HT2 receptor antagonist and TXA2 synthase inhibitor/receptor may be useful for attenuation of restenosis after angioplasty.

New Variant of Human Tissue Plasminogen Activator (TPA) With Enhanced Efficacy and Lower Incidence of Bleeding Compared With Recombinant Human TPA

BACKGROUND The thrombolytic properties of a new variant of tissue plasminogen activator (TPA) (T103N, N117Q, KHRR 296-299 AAAA, or TNK-TPA) with longer plasma half-life, greater fibrin specificity, and increased resistance to inhibition by plasminogen activator inhibitor (PAI-1) were investigated in a rabbit thrombosed carotid artery model. METHODS AND RESULTS After 60 minutes of arterial occlusion, TPA (1.5, 3.0, 6.0, or 9.0 mg/kg as a front-loaded IV infusion for 90 minutes; n = 22) or TNK-TPA (0.38, 0.75, or 1.5 mg/kg as IV bolus; n = 16) was administered. Blood flow through the artery was monitored for an additional 120 minutes. Bleeding was assessed by weighing the amount of blood absorbed in a gauze pad placed in a subcutaneous muscular incision. Recanalization rates and duration of recanalization were dose dependent. The doses that produced > 80% recanalization rates with the longest duration of recanalization were 9.0 mg/kg for TPA and 1.5 mg/kg for TNK-TPA. At these doses, time to reperfusion (mean +/- SEM) was significantly faster (11 +/- 2 versus 23 +/- 7 minutes) and duration of recanalization longer (77 +/- 9 versus 51 +/- 18 minutes) for TNK-TPA compared with TPA (P < .025). Weights of the residual thrombi of the TPA group were greater than those of the TNK-TPA group (P = .004). Concentrations of fibrinogen, plasminogen, and alpha 2-antiplasmin at 120 minutes were significantly higher for TNK-TPA-treated animals compared with TPA-treated animals (P < .001). ANOVA of the blood loss data determined that there were significant differences between thrombolytic agents but not between doses. After correction for saline controls, total blood loss for pooled doses of TPA and TNK-TPA was 82 +/- 6 mg and 40 +/- 4 mg, respectively (P < .01). CONCLUSIONS From these data, we conclude that TNK-TPA, given as a bolus, produces faster and more complete recanalization of occluded arteries in a rabbit experimental model compared with TPA, without increasing systemic plasmin generation or peripheral bleeding. In addition, we observed that TNK-TPA, unlike TPA, did not potentiate collagen-induced aggregation of platelets obtained from human plasma. This lack of effect on platelet aggregation by TNK-TPA potentially could be associated with a decreased risk of reocclusion after successful thrombolysis.

Platelet Microparticles Promote Platelet Interaction With Subendothelial Matrix in a Glycoprotein IIb/IIIa–Dependent Mechanism

BACKGROUND Platelets, on activation, release vesicular particles called platelet microparticles. Despite their procoagulant activity, their functional role in platelet-vessel wall interactions is not known. METHODS AND RESULTS We examined the binding of microparticles to vessel wall components in vitro and in vivo. Microparticles bound to fibrinogen-, fibronectin-, and collagen-coated surfaces. Compared with activated platelets, we observed minimal binding of microparticles to vitronectin and von Willebrand factor. The glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors abciximab and eptifibatide (Integrilin) inhibited the binding to fibrinogen and fibronectin but had minimal effect on binding to collagen. Furthermore, monoclonal antibodies to GP Ib or anionic phospholipid-binding proteins (beta2-glycoprotein I or annexin V) had no effect in these interactions. Microparticles did not bind to monolayers of resting or stimulated human umbilical vein endothelial cells (HUVECs), even in the presence of fibrinogen or von Willebrand factor. However, under similar conditions, microparticles bound to extracellular matrix produced by cultured HUVECs. Abciximab inhibited this interaction by approximately 50%. In a rabbit model of arterial endothelial injury, the infusion of 51Cr-labeled microparticles resulted in a 3- to 5-fold increase of microparticle adhesion to the injured site compared with the uninjured site (P<0.05%). Furthermore, activated platelets bound to surface-immobilized microparticles in a GP IIb/IIIa-dependent mechanism. This binding increased in the presence of fibrinogen by approximately 30%. CONCLUSIONS Platelet microparticles bind to subendothelial matrix in vitro and in vivo and can act as a substrate for further platelet binding. This interaction may play a significant role in platelet adhesion to the site of endothelial injury.

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 µM) in serum-free medium. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. 5-HT at concentrations of 5 and 50 µM 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 µM) and its major metabolite M-1 (0.1 µM), pertussis toxin (10 ng/ml), Src family protein tyrosine kinase (PTK) inhibitor PP2 (1 µM), protein kinase C (PKC) inhibitor Ro31-8220 (0.1 µM) and mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 µM) significantly inhibited the mitogenic effect of 5-HT and its interaction with MCP-1. Anti-MCP-1 antibody (2 µg/ml) and the Janus kinase 2 (JAK2) inhibitor AG490 (10 µM) 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.

Induction of endothelial cell proliferation by angiogenic factors released by activated monocytes

INTRODUCTION Cell-cell interaction is an essential component of atherosclerotic plaque development. Activated monocytes appear to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the production of various growth factors that induce proliferation of different cell types that are involved in the plaque development. Using serum free co-culture method, we determined the effect of monocytes on endothelial cell proliferation. METHODS Endothelial cell proliferation is determined by the amount of [3H]thymidine incorporated in to the DNA. Basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) levels in the conditioned medium were determined by ELISA. RESULTS Conditioned medium from unactivated monocytes partially inhibited endothelial cell proliferation, whereas conditioned medium from activated monocytes promoted endothelial cell proliferation. The mitogenic effect of conditioned medium derived from activated monocytes is due to the presence of b-FGF, VEGF and IL-8. Neutralizing antibodies against b-FGF, VEGF and IL-8 partially reversed the mitogenic effect of conditioned medium derived from activated monocytes. When b-FGF, VEGF and IL-8 were immunoprecipitated from conditioned medium derived from activated monocytes, it is less mitogenic to endothelial cells. CONCLUSION Activated monocytes may play an important role in the development of atherosclerotic plaque by producing endothelial cell growth factors.

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.

Effect of serotonin and throm☐ane A2 on endothelial cell proliferation: Effect of specific receptor antagonists

Platelet aggregation at sites of vascular injury releases both peptide growth factors and vasoactive compounds. Although significant attention has been focused on peptide growth factors, very little is known about the mitogenic effect of vasoactive compounds. We evaluated the effect of serotonin (5-HT) and thromboxane A2 (TXA2) mimetic U46619 alone and in combination on aortic endothelial cells. Stimulation of endothelial cells by 5-HT resulted in an increase in tritiated thymidine uptake and an increase in cell number, whereas U46619 did not have any significant effect. However, when endothelial cells were exposed to both compounds, U46619 potentiated the mitogenic effect of 5-HT on endothelial cells. When endothelial cells were preincubated with LY281067 (a 5-HT2 receptor antagonist) or ridogrel (a combined TXA2 synthase inhibitor and receptor antagonist), LY281067 blocked the mitogenic effect of 5-HT and ridogrel blocked the potentiating effect of U46619 on 5-HT2-induced tritiated thymidine incorporation. When endothelial cells were preincubated with both antagonists, the effects of both 5-HT and U46619 were blocked. Recent studies have indicated that regenerating endothelial cells at sites of vascular injury may release growth factors for vascular smooth muscle cells, leading to smooth muscle cell proliferation and development of neointima. This study suggests that the combined use of 5-HT and TXA2 receptor antagonists may inhibit the growth of endothelial cells at sites of vascular injury and attenuate the formation of neointima.

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.

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 (H2O2), 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, H2O2, or LPC with H2O2 in the absence or presence of 5HT. DNA synthesis in VSMCs was examined by [3H]thymidine incorporation. MoxLDL, LPC, H2O2, and 5HT stimulated DNA synthesis in a dose-dependent manner. MoxLDL had a maximal stimulatory effect at a concentration of 5 &mgr;g/mL (211%), LPC at 15 &mgr;mol/L (156%), H2O2 at 5 &mgr;mol/L (179%), and 5HT at 50 &mgr;mol/L (205%). Added together, moxLDL (50 ng/mL) and 5HT (50 &mgr;mol/L) synergistically increased DNA synthesis (443%). Coincubation of LPC (1 &mgr;mol/L) with H2O2 (0.5 &mgr;mol/L) and 5HT (5 &mgr;mol/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, H2O2, and 5HT on DNA synthesis were completely reversed by the combined use of an antioxidant, N-acetylcysteine (400 &mgr;mol/L) or butylated hydroxytoluene (20 &mgr;mol/L), with a 5HT2 receptor antagonist, LY281067 (10 &mgr;g/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.

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.