[Show abstract][Hide abstract] ABSTRACT: WNT signaling plays multiple roles in skeletal myogenesis during gestation and postnatal stages. The R-spondin (RSPO) family of secreted proteins and their cognate receptors, members of leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, have emerged as new regulatory components of the WNT signaling pathway. We previously showed that RSPO2 promoted myogenic differentiation via activation of WNT/β-catenin signaling in mouse myoblast C2C12 cells in vitro. However, the molecular mechanism by which RSPO2 regulates myogenic differentiation is unknown. Herein, we show that depletion of the LGR4 receptor severely disrupted myogenic differentiation and significantly diminished the response to RSPO2 in C2C12 cells, showing a requirement of LGR4 in RSPO signaling during myogenic differentiation. We identify the TGFβ antagonist follistatin (Fst) as a key mediator of RSPO-LGR4 signaling in myogenic differentiation. We further demonstrate that Fst is a direct target of the WNT/β-catenin pathway. Activation and inactivation of β-catenin induced and inhibited Fst expression, respectively, in both C2C12 cells and mouse embryos. Specific TCF1/LEF1 binding sites within the promoter and intron 1 region of the Fst gene were required for RSPO2 and WNT/β-catenin-induced Fst expression. This study uncovers a molecular crosstalk between WNT/β-catenin and TGFβ signaling pivotal in myogenic differentiation.
[Show abstract][Hide abstract] ABSTRACT: Recent developments in molecular programming of mesodiencephalic dopaminergic (mdDA) neurons have led to the identification of many transcription factors playing a role in mdDA specification. LIM homeodomain transcription factor Lmx1a is essential for chick mdDA development, and for the efficient differentiation of ES-cells towards a dopaminergic phenotype. In this study, we aimed towards a more detailed understanding of the subtle phenotype in Lmx1a-deficient (dreher) mice, by means of gene expression profiling. Transcriptome analysis was performed, to elucidate the exact molecular programming underlying the neuronal deficits after loss of Lmx1a. Subsequent expression analysis on brain sections, confirmed that Nurr1 is regulated by Lmx1a, and additional downstream targets were identified, like Pou4f1, Pbx1, Pitx2, C130021l20Rik, Calb2 and Rspo2. In line with a specific, rostral-lateral (prosomer 2/3) loss of expression of most of these genes during development, Nurr1 and C130021l20Rik were affected in the SNc of the mature mdDA system. Interestingly, this deficit was marked by the complete loss of the Wnt/b-catenin signaling activator Rspo2 in this domain. Subsequent analysis of Rspo2-/- embryos revealed affected mdDA neurons, partially phenocopying the Lmx1a mutant. To conclude, our study revealed that Lmx1a is essential for a rostral-lateral subset of the mdDA neuronal field, where it might serve a critical function in modulating proliferation and differentiation of mdDA progenitors through the regulation of the Wnt activator Rspo2.
PLoS ONE 09/2013; 8(9):e74049. DOI:10.1371/journal.pone.0074049 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, the R-spondin (RSPO) family of proteins has emerged as important regulators of WNT signaling. Considering the wide spectrum of WNT signaling functions in normal biological processes and disease conditions, there has been a significantly growing interest in understanding the functional roles of RSPOs in multiple biological processes and determining the molecular mechanisms by which RSPOs regulate the WNT signaling pathway. Recent advances in the RSPO research field revealed some of the in vivo functions of RSPOs and provided new information regarding the mechanistic roles of RSPO activity in regulation of WNT signaling. Herein, we review recent progress in RSPO research with an emphasis on signaling mechanisms and biological functions.
The international journal of biochemistry & cell biology 09/2012; 44(12):2278-2287. DOI:10.1016/j.biocel.2012.09.006 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Outgrowth and fusion of the lateral and medial nasal processes and of the maxillary process of the first branchial arch are integral to lip and primary palate development. Wnt9b mutations are associated with cleft lip and cleft palate in mice; however, the cause of these defects remains unknown. Here, we report that Wnt9b(-/-) mice show significantly retarded outgrowth of the nasal and maxillary processes due to reduced proliferation of mesenchymal cells, which subsequently results in a failure of physical contact between the facial processes that leads to cleft lip and cleft palate. These cellular defects in Wnt9b(-/-) mice are mainly caused by reduced FGF family gene expression and FGF signaling activity resulting from compromised canonical WNT/β-catenin signaling. Our study has identified a previously unknown regulatory link between WNT9B and FGF signaling during lip and upper jaw development.
Development 03/2012; 139(10):1821-30. DOI:10.1242/dev.075796 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: R-spondins are a recently characterized family of secreted proteins that activate Wnt/β-catenin signaling. Herein, we determine R-spondin2 (Rspo2) function in craniofacial development in mice. Mice lacking a functional Rspo2 gene exhibit craniofacial abnormalities such as mandibular hypoplasia, maxillary and mandibular skeletal deformation, and cleft palate. We found that loss of the mouse Rspo2 gene significantly disrupted Wnt/β-catenin signaling and gene expression within the first branchial arch (BA1). Rspo2, which is normally expressed in BA1 mesenchymal cells, regulates gene expression through a unique ectoderm-mesenchyme interaction loop. The Rspo2 protein, potentially in combination with ectoderm-derived Wnt ligands, up-regulates Msx1 and Msx2 expression within mesenchymal cells. In contrast, Rspo2 regulates expression of the Dlx5, Dlx6, and Hand2 genes in mesenchymal cells via inducing expression of their upstream activator, Endothelin1 (Edn1), within ectodermal cells. Loss of Rspo2 also causes increased cell apoptosis, especially within the aboral (or caudal) domain of the BA1, resulting in hypoplasia of the BA1. Severely reduced expression of Fgf8, a survival factor for mesenchymal cells, in the ectoderm of Rspo2(-/-) embryos is likely responsible for increased cell apoptosis. Additionally, we found that the cleft palate in Rspo2(-/-) mice is not associated with defects intrinsic to the palatal shelves. A possible cause of cleft palate is a delay of proper palatal shelf elevation that may result from the small mandible and a failure of lowering the tongue. Thus, our study identifies Rspo2 as a mesenchyme-derived factor that plays critical roles in regulating BA1 patterning and morphogenesis through ectodermal-mesenchymal interaction and a novel genetic factor for cleft palate.
[Show abstract][Hide abstract] ABSTRACT: R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this
study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH)
class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression
was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells.
In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation.
Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed
MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts
its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive
regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Diet is one of the most important factors that influence the risks for cardiovascular diseases. Genistein, an isoflavone found in soy, may benefit the cardiovascular system. Here, we investigated the effect of genistein on platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic smooth muscle cells (RASMCs). Genistein significantly inhibited 25 ng/ml PDGF-BB-induced RASMC proliferation and [3H]-thymidine incorporation into DNA at 10, 20, and 40 microM. In accordance with these findings, genistein blocked the PDGF-BB-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Western blot analysis showed that genistein not only inhibited phosphorylation of retinoblastoma protein (pRb) and expression of cyclin E, cyclin-dependent kinase (CDK) 2, and proliferating cell nuclear antigen (PCNA) protein, but also inhibited downregulation of cyclin-dependent kinase inhibitor (CKI) p27kip1. However, genistein did not affect p21cip1, CDK4, and cyclin D1 expression or early signal transduction through PDGF beta-receptor, extracellular signal-regulated kinases 1/2 (ERK1/2), Akt, and phospholipase C (PLC) gamma1 phosphorylation. These results suggest that genistein inhibits PDGF-BB-induced RASMC proliferation via G0/G1 arrest in association with induction of p27kip1, which may contribute to the beneficial effects of genistein on the cardiovascular system.
[Show abstract][Hide abstract] ABSTRACT: 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.
European Journal of Pharmacology 06/2008; 586(1-3):74-81. DOI:10.1016/j.ejphar.2008.02.076 · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 [(3)H]-thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 microM. In accordance with these findings, hesperetin revealed blocking of the PDGF-BB-inducible progression through G(0)/G(1) 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) p27(kip1), while did not affect CKI p21(cip1), p16(INK4), 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 G(0)/G(1) 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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 microM, respectively. In line with the inhibition of collagen-induced platelet aggregation, EGCG revealed blocking of the collagen-mediated phospholipase (PL) Cgamma2 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 PLCgamma2, 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.
[Show abstract][Hide abstract] ABSTRACT: NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC(50) values of 0.71 +/- 0.2, 0.82 +/- 0.3, and 0.45 +/- 0.1 microM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A(2) receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA(2) formation, but had no effect on the PGD(2) production, indicating an inhibitory effect on TXA(2) synthase, which was supported by a TXA(2) synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA(2) formation converted from PGH(2). On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA(2) synthase activity and TXA(2) receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12.
[Show abstract][Hide abstract] ABSTRACT: Epothilone D (Epo-D) is a paclitaxel-like microtubule-stabilizing agent that was isolated from the myxobacterium Sorangium cellulosum. Although Epo-D can inhibit proliferation in multiple tumor cell lines, the effect of Epo-D on neointimal hyperplasia after angioplasty has not been reported. The aim of the present study was to investigate the effects of Epo-D on neointimal hyperplasia using an in vivo rat carotid artery injury model. We demonstrated that local Epo-D treatment significantly reduced neointimal hyperplasia after in vivo rat carotid artery injury, and Epo-D potently inhibited DNA synthesis, cell cycle progression and cell proliferation after FBS- and PDGF-BB-stimulation; PDGF-BB has been identified as the most potent growth factor for stimulating the proliferation of activated rat aortic smooth muscle cells (RASMCs). To clarify the specific effects of Epo-D on cell cycle machinery, we examined its effects on cyclin-dependent kinase (CDK)2, CDK4, cyclin E, p27, and retinoblastoma (Rb) proteins as cell cycle-related proteins in cellular lysates from PDGF-BB-stimulated RASMCs. Epo-D treatment significantly decreased the level of CDK2 protein, but did not change the levels of CDK4 and cyclin E proteins. Furthermore, Epo-D inhibited the phosphorylation of Rb, a key regulator of the G1 to S phase transition in the cell cycle. These findings suggest that Epo-D may regulate the cell cycle G1-checkpoint proteins as its major molecular mechanism for inhibiting neointimal hyperplasia after in vivo rat carotid artery injury.
[Show abstract][Hide abstract] ABSTRACT: In previous studies of the root bark of Cudrania tricuspidata, various isoprenylated xanthones and flavonoids were isolated, some of which have anticancer, hepatoprotective, and antiperoxidative activities. Cytokines and growth factors are involved in the regulation of vascular smooth muscle cells (VSMCs) in atherosclerotic plaques. To assess whether cudraflavanone A isolated from the root bark of C. tricuspidata may be useful in the prevention of atherosclerosis or restenosis after angioplasty, we investigated the ability of cudraflavanone A to inhibit VSMCs growth under 25 ng/mL platelet-derived growth factor BB (PDGF-BB)-stimulated conditions. Cudraflavanone A (0.1-1 microM) significantly inhibited PDGF-BB-induced cell numbers in a concentration-dependent manner. The antigrowth effects of cudraflavanone A on VSMCs were also examined in [3H]-thymidine incorporation and cell cycle assays. Consistent with the inhibitory effect on cell number, PDGF-BB-stimulated [3H]-thymidine incorporation and cell cycle progression in VSMCs was also concentration-dependently reduced by cudraflavanone A. Furthermore, PDGF-BB markedly activated PDGF-beta receptor (PDGF-Rbeta) tyrosine kinase activity, leading to activation of intracellular signals required for VSMC growth. However, PDGF-BB-induced this kinase activity was not affected by cudraflavanone A. PDGF-BB also increased the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), Akt, and phospholipase C gamma (PLCgamma)1, which are important signaling molecules in cell growth. Cudraflavanone A (0.1-1 microM) suppressed PDGF-BB-stimulated Akt activation, which is involved in cell survival, but had no effect on the activation of ERK1/2 and PLCgamma1. Selective modification of Akt activation by cudraflavanone A in VSMCs may suppress intimal thickening after angioplasty and plaque formation in atherosclerosis. These results suggest that cudraflavanone A from C. tricuspidata inhibits PDGF-BB-induced rat aortic VSMC growth via an Akt-dependent pathway.
[Show abstract][Hide abstract] ABSTRACT: Diet can be one of the most important factors that influence risks for atherothrombotic diseases. Hesperetin included in grapefruits and oranges is one candidate that may benefit the cardiovascular system. Here, we investigated antiplatelet activity of hesperetin in vitro. In addition, possible antiplatelet mechanism was also investigated. Hesperetin concentration-dependently inhibited washed rabbit platelet aggregation induced by collagen and arachidonic acid, with IC50 of 20.5+/-3.5 and 69.2+/-5.1 microM, respectively, while has little effect on thrombin- or U46619-, a thromboxane (TX) A2 mimic, mediated platelet aggregation, suggesting that hesperetin may selectively inhibit collagen- and arachidonic acid-mediated signal transduction. In accordance with these findings, hesperetin revealed blocking of the collagen-mediated phospholipase (PL) C-gamma2 phosphorylation, and caused concentration-dependent decreases of cytosolic calcium mobilization, arachidonic acid liberation and serotonin secretion. In addition, hesperetin inhibited arachidonic acid-mediated platelet aggregation by interfering with cyclooxygenase-1 activity as established by the measurement of arachidonic acid-mediated TXA2 and prostaglandin D2 formations as well as cyclooxygenase-1 and TXA2 synthase activity assays. Taken together, the present results provide a cellular mechanism for the antiplatelet activity of hesperetin through inhibition of PLC-gamma2 phosphorylation and cyclooxygenase-1 activity, which may contribute to the beneficial effects of grapefruits and oranges on cardiovascular system.
[Show abstract][Hide abstract] ABSTRACT: 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 gamma 1, and PDGF-receptor beta 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.
Journal of Pharmacology and Experimental Therapeutics 06/2007; 321(2):648-55. DOI:10.1124/jpet.106.117622 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyperproliferation of platelet-derived growth factor (PDGF)-BB-induced vascular smooth muscle cell (VSMC) is a hallmark of atherosclerosis and related vascular disorders. In the previous study, we reported that KTJ740 [2-chloro-3-(4-(ethylcarboxy)-phenyl)-amino-1,4-naphthoquinone], a newly synthesized vitamin K derivative, has potent antithrombotic effects in mice and antiplatelet activity in vitro and ex vivo. In the present study, we have tested that KTJ740 could inhibit PDGF-BB-stimulated VSMC proliferation. We have examined the potential inhibitory effect of this compound on rat aortic smooth muscle cells (RASMCs). Our results show that this compound significantly inhibits PDGF-BB-stimulated RASMC number and DNA synthesis in a concentration-dependent manner. Furthermore, we have examined its effect on cell cycle progression by flow cytometry. KTJ740 treatment resulted in a significant arrest in cell cycle progression of RASMCs induced by PDGF-BB, and this effect was achieved by suppressing activation of PDGF-beta receptor (PDGF-Rbeta) tyrosine kinase pathway. These results suggest that a possibility of KTJ740 can be a potential agent to control vascular disorders and its antiproliferative mechanism may be mediated through PDGF-Rbeta tyrosine kinase-dependent signaling pathway.
[Show abstract][Hide abstract] ABSTRACT: Platelet derived growth factor (PDGF)-BB is one of the most potent vascular smooth muscle cell (VSMC) proliferative factors, and abnormal VSMC proliferation by PDGF-BB plays an important role in the development and progression of cardiovascular problems, including restenosis after coronary angioplasty and atherosclerosis. Previous phytochemical studies on the stems or root barks of Cudrania tricuspidata (Moraceae) resulted in the isolation of various isoprenylated xanthones and flavonoids, some of which have anti-cancer, hepatoprotective, anti-inflammatory and anti-oxidant activities. In the present study, we investigated the antiproliferative effect of cudratricusxanthone A isolated from the root bark of C. tricuspidata and its underlying mechanism in VSMCs. Antiproliferative effects of cudratricusxanthone A on VSMCs were examined by direct cell counting and [3H]-thymidine incorporation assays. Cudratricusxanthone A inhibited [3H]-thymidine incorporations into DNA in VSMCs that occurred in response to treatment with 50 ng/ml PDGF-BB. PDGF-BB-stimulated DNA synthesis was significantly reduced to 86.1, 80.2, 64.2 and 25.1% at concentrations of 0.1, 1, 2 and 3 microM, respectively. Moreover, pre-treatment with cudratricusxanthone A (0.1-3 microM) suppressed this PDGF-BB-stimulated cell number in a concentration-dependent manner. The inhibition percentages were 11.1, 22.7, 51.3 and 81.5% at the concentrations of 0.1, 1, 2 and 3 microM, respectively. We also investigated the mechanism of antiproliferative effects by cudratricusxanthone A in PDGF-BB-stimulated VSMCs. In Western blot analysis, 50 ng/ml PDGF-BB-stimulated phospholipase C (PLC)gamma1, Ras, and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylations were inhibited by cudratricusxanthone A (0.1-3 microM). Consisted with these findings, cudratricusxanthone A inhibited PDGF-receptor beta chain (PDGF-Rbeta) phosphorylation induced by PDGF-BB in a concentration-dependent manner. These findings suggest that the inhibitory effects of cudratricusxanthone A on DNA synthesis and proliferation by PDGF-BB-stimulated VSMCs are mediated by the suppressions of the PDGF-Rbeta and its downstream signaling pathways. Our observation may explain in part mechanistic basis for the prevention of cardiovascular diseases, such as atherosclerosis and restenosis after coronary angioplasty by cudratricusxanthone A.