Yong-Ri Jin

Antiplatelet Activity of Epigallocatechin Gallate Is Mediated by the Inhibition of PLCγ2 Phosphorylation, Elevation of PGD2 Production, and Maintaining Calcium-ATPase Activity

We have previously reported that green tea catechins displayed a potent antithrombotic effect by inhibition of platelet aggregation. In the present study, the antiplatelet and antithrombotic activities of epigallocatechin gallate (EGCG), the major catechin derived from green tea, were extensively investigated. EGCG inhibited arterial thrombus formation and U46619-, collagen-, and arachidonic acid (AA)-induced washed rabbit platelet aggregation in a concentration-dependent manner, with IC50 values of 61 ± 3, 85 ± 4, and 99 ± 4 μM, respectively. In line with the inhibition of collagen-induced platelet aggregation, EGCG revealed blocking of the collagen-mediated phospholipase (PL) Cγ2 and protein tyrosine phosphorylation, and it caused concentration-dependent decreases of cytosolic calcium mobilization, AA liberation, and serotonin secretion. In addition, the platelet aggregation, intracellular Ca2+ mobilization, and protein tyrosine phosphorylation induced by thapsigargin, a Ca2+-ATPase pump inhibitor, were completely blocked by EGCG. Contrary to the inhibition of AA-induced platelet aggregation, EGCG failed to inhibit cyclooxygenase and thromboxane (TX) A2 synthase activities, but it concentration-dependently elevated AA-mediated PGD2 formation. In contrast, epigallocatechin (EGC), a structural analogue of EGCG lacking a galloyl group in the 3′ position, slightly inhibited collagen-stimulated cytosolic calcium mobilization, but failed to affect other signal transductions as did EGCG in activated platelets and arterial thrombus formation. These results suggest that antiplatelet activity of EGCG may be attributable to its modulation of multiple cellular targets, such as inhibitions of PLCγ2, protein tyrosine phosphorylation and AA liberation, and elevation of cellular PGD2 levels, as well as maintaining Ca2+-ATPase activity, which may underlie its beneficial effect on the atherothrombotic diseases.

Inhibitory effects of J78, a newly synthesized 1,4-naphthoquinone derivative, on experimental thrombosis and platelet aggregation

Several compounds with the backbone of 1,4-naphthoquinone chemical structure have been reported to display antiplatelet and antithrombotic activities, indicating that this congener compound may be a new source in the antithrombotic drug development. In the present study, the possible antiplatelet activity and antithrombotic efficacy of J78 (2-chloro-3-[2′-bromo, 4′-fluoro- phenyl]-amino-8-hydroxy-1,4-naphthoquinone), a newly synthesized 1,4-naphthoquinone derivative, were examined. Orally administered J78 (50, 100 mg/kg) dose dependently protected mice against the collagen + epinephrine-induced thromboembolic death. Orally administered J78 also significantly inhibited the ADP- and collagen-induced rat platelet aggregation ex vivo, with inhibition values of 44 and 40%, respectively. J78 inhibited the collagen-, arachidonic acid- and thrombin-induced human platelet aggregation concentration dependently in vitro, with IC50 values of 7.8 ± 0.4, 10.1 ± 0.4 and 18.4 ± 2.0 µmol/l, respectively. It was also active in inhibiting Ca2+ ionophore, A23187-induced platelet aggregation, suggesting that J78 may have an inhibitory effect on Ca2+ mobilization. J78, however, did not alter coagulation parameters such as activated partial thromboplastin time and prothrombin time in human plasma. Taken together, these results suggest that J78 may be a promising antithrombotic agent, and its antithrombotic activity may be due to antiplatelet rather than anticoagulation activity.

Antiplatelet activity of β-carboline alkaloids from Perganum harmala : A possible mechanism through inhibiting PLCγ2 phosphorylation

Beta-carboline alkaloids including harmalol, harmaline, norharmane, harmol, harmine and harmane are important constituents of the medicinal plant, Perganum harmala L. (Zygophylaceae), which has been used in traditional medicine. In the present study, the antiplatelet activities of six beta-carboline alkaloid compounds were investigated in vitro. At a concentration of 200 microM, these compounds have no effect on arachidonic acid (AA)-, thrombin- and U46619 (a thromboxane A2 mimic)-stimulated platelet aggregation. On the contrary, it was revealed that collagen-induced platelet aggregation could be inhibited by these compounds with different potencies (harmane and harmine were most potent, harmol had medium potency, and harmol, norharmane, harmalol and harmaline had a weak, non significant effect), indicating a selective inhibition on collagen-mediated platelet activation. Consistently, further study revealed that collagen-mediated phospholipase (PL) Cgamma2 and protein tyrosine phosphorylation, cytosolic calcium mobilization and arachidonic acid liberation were completely inhibited by harmane and harmine in a concentration-dependent manner, while the other compounds were only partially or not effective at all. Taken together, these results indicate that three of these six beta-carboline alkaloids can selectively affect collagen-induced platelet aggregation with different potencies; in particular, harmane and harmine were most potent, and their antiplatelet activities may be mediated by inhibiting PLCgamma2 and protein tyrosine phosphorylation with sequential suppression of cytosolic calcium mobilization and arachidonic acid liberation, indicating that harmane and harmine have a potential to be developed as a novel agent for atherothrombotic diseases.

Hesperetin, a bioflavonoid, inhibits rat aortic vascular smooth muscle cells proliferation by arresting cell cycle.

Diet can be one of the most important factors that influence risks for cardiovascular diseases. Hesperetin, a flavonoid present in grapefruits and oranges, is one candidate that may benefit the cardiovascular system. In this study, we have investigated the effect of hesperetin on the platelet‐derived growth factor (PDGF)‐BB‐induced proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Hesperetin significantly inhibited 50 ng/ml PDGF‐BB‐induced rat aortic VSMCs proliferation and [3H]‐thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 µM. In accordance with these findings, hesperetin revealed blocking of the PDGF‐BB‐inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Western blot showed that hesperetin inhibited not only phosphorylation of retinoblastoma protein (pRb) and expressions of cyclin A, cyclin D, cyclin E, cyclin‐dependent kinase 2 (CDK2) as well as proliferating cell nuclear antigen (PCNA) protein, but also downregulation of cyclin‐dependent kinase inhibitor (CKI) p27kip1, while did not affect CKI p21cip1, p16INK4, p53, and CDK4 expressions as well as early signaling transductions such as PDGF beta‐receptor, extracellular signal‐regulated kinase (ERK) 1/2, Akt, p38, and JNK phosphorylation. These results suggest that hesperetin inhibits PDGF‐BB‐induced rat aortic VSMCs proliferation via G0/G1 arrest in association with modulation of the expression or activation of cell‐cycle regulatory proteins, which may contribute to the beneficial effect of grapefruits and oranges on cardiovascular system. J. Cell. Biochem. 104: 1–14, 2008.

The inhibitory effect and mechanism of luteolin 7-glucoside on rat aortic vascular smooth muscle cell proliferation.

The abnormal proliferation of aortic vascular smooth muscle cells (VSMCs) plays a central role in the pathogenesis of atherosclerosis and restenosis after angioplasty and possibly also in the development of hypertension. The present study was designed to examine the inhibitory effects and the mechanism of luteolin 7-glucoside (L7G) on the platelet-derived growth factor (PDGF)-BB-induced proliferation of VSMCs. L7G significantly inhibited the PDGF-BB-induced proliferation and the DNA synthesis of the VSMCs in a concentration-dependent manner. Preincubation of the VSMCs with L7G significantly inhibited the PDGF-BB- induced extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and the phospholipase C (PLC)-λ1 activation. However, L7G had almost no affect on the phosphorylation of PDGF-β receptor tyrosine kinase, which was induced by PDGF-BB. These results suggest that L7G inhibits the PDGF-BB-induced proliferation of VSMCsvia the blocking of PLC-γ1, Akt, and ERK1/2 phosphorylation.

Antithrombotic and antiplatelet activities of fenofibrate, a lipid-lowering drug

Fenofibrate, a lipid-lowering drug, inhibits hydroxyl-methylglutaryl coenzyme A (HMG-CoA)-reductase activity, thus reducing cholesterol synthesis and increasing the clearance of circulating LDL-cholesterol via the high affinity receptor system. In addition, fenofibrate has beneficial effects such as the inhibition of tissue factor expression, antithrombotic effect and anti-inflammatory effect. The aim of this study was to investigate the effects of fenofibrate on thrombus formation in vivo and platelet activation in vitro and ex vivo. The carotid arteries of male Sprague-Dawley rats were subjected to chemical injury by FeCl(3), and then blood flow was measured with a blood flowmeter. Fenofibrate (200 and 400mg/kg/day for 1 week) delayed the time to occlusion by 61.3% (p<0.05, n=10) and 90.7% (p<0.01, n=10), respectively. Fenofibrate also significantly inhibited ex vivo platelet aggregations induced by collagen (7.5microg/ml) (p<0.01, n=11) and ADP (10microM) (p<0.01, n=11), respectively, but did not affect coagulation times following activated partial thromboplastin and prothrombin activation, indicating the antithrombotic effect was mediated by its inhibition on platelet activation rather than coagulation system. This antiplatelet activity was revealed to be mediated by the suppression of thromboxane A(2) receptor, cytosolic calcium mobilization, and cyclooxygenase (COX)-1 activity. Taken together, we demonstrate that fenofibrate can significantly inhibit artery thrombus formation in vivo, which may be due to antiplatelet activity via the inhibition of thromboxane A(2) receptor, cytosolic calcium mobilization and COX-1 activity, and the beneficial effect of fenofibrate on cardiovascular system may be also due to its modulation of platelet activation.

Antiplatelet and antithrombotic activities of Korean Red Ginseng

The antiplatelet and antithrombotic activities of Korean Red Ginseng (KRG) were examined on rat carotid artery thrombosisin vivo, and platelet aggregationin vitro andex vivo. Administration of KRG to rats not only prevented carotid artery thrombosisin vivo in a dose-dependent manner, but also significantly inhibited ADP- and collagen-induced platelet aggregationex vivo, while failed to prolong coagulation times such as activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating the antithrombotic effect of KRG might be due to its antiplatelet aggregation rather than anticoagulation effect. In line with the above observations, KRG inhibited U46619-, arachidonic acid-, collagen- and thrombin-induced rabbit platelet aggregationin vitro in a concentration-dependent manner, with IC50 values of 620±12, 823 ±22, 722±21 and 650±14 μg/mL, respectively. Accordingly, KRG also inhibited various agonists-induced platelet serotonin secretions as it suppressed platelet aggregation. These results suggest that KRG has a potent antithrombotic effectin vivo, which may be due to antiplatelet rather than anticoagulation activity, and KRG intake may be beneficial to the individuals with high risks of thrombotic and cardiovascular diseases.

Epothilone B Inhibits Neointimal Formation after Rat Carotid Injury through the Regulation of Cell Cycle-Related Proteins

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenetic factor of vascular disorders such as atherosclerosis and restenosis after angioplasty. Epothilone B, a novel potential antitumor compound, has a potent effect on preventing postangioplasty restenosis. Therefore, we established an in vivo rat carotid injury model and examined the potential effects of epothilone B on cardiovascular disease. We found that epothilone B potently prevented neointimal formation and in vivo VSMCs proliferation. In addition, we also showed that epothilone B significantly inhibited 5% fetal bovine serum (FBS)- and 50 ng/ml platelet-derived growth factor (PDGF)-BB-induced proliferation and cell cycle progression in rat aortic VSMCs. Furthermore, FBS and PDGF-BB induced the activations of extracellular signal-regulated kinases 1 and 2, Akt, phospholipase C γ 1, and PDGF-receptor β chain tyrosine kinase were not changed by epothilone B. However, epothilone B treatment caused a significant decrease in the level of cyclin-dependent protein kinase (CDK) 2, whereas it caused no change in the levels of cyclin E and down-regulated the phosphorylation of retinoblastoma, which plays a critical role in cell cycle regulation. Furthermore, levels of p27, an inhibitor of cyclin E/CDK2 complex, were significantly increased in VSMCs treated with epothilone B, indicating that this might be a major molecular mechanism for the inhibitory effects of epothilone B on the proliferation and cell cycle of VSMCs. These findings suggest that epothilone B can inhibit neointimal formation via the cell cycle arrest by the regulation of the cell cycle-related proteins in VSMCs.

YSK2821, a newly synthesized indoledione derivative, inhibits cell proliferation and cell cycle progression via the cell cycle-related proteins by regulating phosphatidylinositol-3 kinase cascade in vascular smooth muscle cells

Indoledione derivatives have pronounced biological effects, i.e., cytotoxic activities against cancer cell lines and antifungal and antibacterial activities. The present study was designed to investigate the effects of YSK2821, a newly synthesized indoledione derivative, on platelet-derived growth factor (PDGF-BB)-induced vascular smooth muscle cell (VSMC) proliferation, as well as the molecular mechanisms of the anti-proliferative effects of YSK2821 in VSMCs. We found that YSK2821 caused the accumulation of cells in the G1 phase of the cell cycle and inhibited [3H]-thymidine incorporation. We demonstrated that YSK2821 remarkably decreased Akt kinase phosphorylation as the mechanism by which YSK2821 suppressed cell signal transduction events in VSMC proliferation. Furthermore, in terms of the effects of YSK2821 on cell cycle-related proteins, YSK2821 enhanced the expression of the cyclin-dependent protein kinase (CDK) inhibitor p27 and down-regulated CDK2 and cyclin E expression, but did not affect CDK4 and cyclin D1 expression. YSK2821 also inhibited the phosphorylation of Rb, a key regulator in the cell cycle. These results indicate that YSK2821, a newly synthesized indoledione derivative, may inhibit VSMC proliferation via a phosphatidylinositol (PI)-3 kinase-dependent pathway, and thus shed light on a novel role for YSK2821 as a potential preventive regulator of cardiovascular disease.

JM91, a newly synthesized indoledione derivative, inhibits rat aortic vascular smooth muscle cells proliferation and cell cycle progression through inhibition of ERK1/2 and Akt activations

The increased potential for growth of vascular smooth muscle cells (VSMCs) is a key abnormality in the development of atherosclerosis and postangioplasty restenosis. Platelet-derived growth factor (PDGF)-BB is a potent mitogen for VSMCs that plays an important role in the intimal accumulation of VSMCs. This study examined the effect of JM91, a newly synthesized indoledione derivative, on the proliferation of PDGF-BB-stimulated rat aortic VSMCs. The antiproliferative effect of JM91 on rat aortic VSMCs was examined by cell counting and [(3)H]thymidine incorporation assay. The pre-incubation of JM91 (0.5-3.0 microM) significantly inhibited the proliferation and DNA synthesis of 25 ng/mL PDGF-BB-stimulated rat aortic VSMCs in a concentration-dependent manner. JM91 inhibited the PDGF-BB-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt kinase, while had no effect on PLCgamma1 and PDGF-Rbeta activation. In addition, treatment with JM91 (0.5-3.0 microM) induced cell-cycle arrest in the G(1) phase, which was associated with the down-regulation of cyclins and CDKs. These findings suggest that the inhibitory effects of JM91 against proliferation, DNA synthesis and cell cycle progression of PDGF-BB-stimulated rat aortic VSMCs are mediated by the suppression of the ERK1/2 and PI3K/Akt signaling pathways. Furthermore, JM91 may be a potential antiproliferative agent for the treatment of atherosclerosis and angioplasty restenosis.