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AIM: To investigate effect of naringenin on ADP-induced platelet aggregation and its possible mechanism.METHODS: The levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured in the platelets with ADP stimulation using ELISA in the presence or absence of different concentrations of naringenin.The effect of naringenin at different concentrations on the change of phosphodiesterase (PDE) activity was measured by high efficiency liquid chromatography.The effects of naringenin at different concentrations on phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at positions Ser157 and Ser239 in washed platelets with ADP stimulation were analyzed by Western blot.The phosphorylation of VASP at Ser239 was also analyzed in the presence of protein kinase A (PKA), protein kinase G (PKG), or protein kinase C (PKC) inhibitors before incubation with naringenin.The platelet aggregation was measured in the presence of PKA or PKG inhibitors before incubation with naringenin.RESULTS: Naringenin elevated cGMP levels significantly but not cAMP levels in the platelets with ADP stimulation in a dose-dependent manner.Naringenin inhibited PDE activity.Naringenin increased the phosphorylation of VASP at Ser239 in a dose-dependent manner in the platelets with ADP stimulation but only modest changes in the phosphorylation at position Ser157.The phosphorylation level of VASP at Ser239 position was inhibited when the platelets were treated with PKA inhibitor before incubation with naringenin.Incubation of platelets with neither PKG nor PKC inhibitors before treatment with naringenin affect the phosphorylation of VASP at Ser239.Pretreatment with PKA inhibitor but not PKG inhibitor significantly reversed the antiplatelet aggregation by naringenin in ADP-stimulated platelets.CONCLUSION: Naringenin may inhibit platelet activation through the elevation of cGMP-and PKA-mediated VASP phosphorylation.
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In the past few decades, accumulated experimental and clinical evidences suggest that cyclic guanosine monophos-phate (cGMP) plays an important role in atheroselerosis. Vasodilator-stimulated phosphoprotein (VASP), a major downstream compo-nent of the cGMP signaling cascade, is an actin-binding protein which regulates cell adhesion, morphology change, cell motility and cell proliferation. It has shown that VASP is associated with many factors influencing atherosclerosis, and it plays an important role in the genesis and development of atherosclerosis. The relationships between the occurrence of atherosclerosis and risk factors for athero-sclerosis such as hypertension, hyperglycaemia and hyperlipidemia are reviewed in this article.
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Objective Resistin,also known as an adipose tissue-specific secretory factor,is involved in the development and progression of artherosclerosis by inducing dysfunction of endothelial cells,but its action mechanism has been rarely studied.The purpose of this study was to investigate the effect of resistin on the function of human coronary artery endothelial cells (HCAECs) and the underlying mechanisms.Methods We constructed the lentiviral vasodilator-stimulated phosphoprotein (VASP) shRNA interfering sequence (the LV-siVASP group) and a negative control vector (the LV-sicntr group),transfected HCAECs with VASP siRNA or control siRNA,and determined the VASP mRNA and protein expressions by quantitative RT-PCR and Western blot,respectively.We treated the HCAECs with resistin at the concentrations of 0,10,50,100,and 200 ng/mL followed by-detection of its effects on the proliferation and migration of the cells by MTT and Transwell chamber assay,respectively.We made comparisons between the LV-siVASP and LV-sicntr groups in the proliferation and migration of the HCAECs treated with 100 ng/mL resistin,the expression of vascular endothelial growth factor receptor-2 (VEGFR2) by immunofluorescent staining,and the activity of RhoA by pull-down assays.Results Compared with the LV-sicntr group,the LV-siVASP group showed significantly inhibited expressions of RNA (100％ vs [68.1±0.8]％,P＜0.05) and the VASP protein (100％ vs [59.3± 1.7] ％,P＜0.05) Treatment with resistin at 50-200 ng/mL markedly increased the proliferation of the HCAECs at 48 hours (P＜0.05) and induced a dose-dependent promotion of their migration at 24 hours (P＜0.05).No significant difference was observed in the expression VEGFR2 between the two groups,while the activity if RhoA was remarkably reduced in the LV-siVASP group ([41.3±3.1] ％) as compared with the LV-sicntr group (P＜0.05).Conclusion Resistin promotes the proliferation and migration of HCAECs,and the influence of VASP ablation on the action of resistin is associated with the decreased activity of RhoA.
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Obesity has quickly become a worldwide pandemic, causing major adverse health outcomes such as dyslipidemia, type 2 diabetes mellitus, cardiovascular disease and cancers. Obesity-induced insulin resistance is the key for developing these metabolic disorders, and investigation to understand the molecular mechanisms involved has been vibrant for the past few decades. Of these, low-grade chronic inflammation is suggested as a critical concept in the development of obesity-induced insulin resistance, and the anti-inflammatory effect of nitric oxide (NO) signaling has been reported to be linked to improvement of insulin resistance in multiple organs involved in glucose metabolism. Recently, a body of evidence suggested that vasodilatory-stimulated phosphoprotein (VASP), a downstream mediator of NO signaling plays a crucial role in the anti-inflammatory effect and improvement of peripheral insulin resistance. These preclinical studies suggest that NO/VASP signaling could be an ideal therapeutic target in the treatment of obesity-related metabolic dysfunction. In this review, we introduce studies that investigated the protective role of NO/VASP signaling against obesity-related inflammation and insulin resistance in various tissues.
Subject(s)
Adipose Tissue , Cardiovascular Diseases , Diabetes Mellitus, Type 2 , Dyslipidemias , Endothelium, Vascular , Glucose , Inflammation , Insulin Resistance , Insulin , Liver , Macrophages , Metabolism , Nitric Oxide , Obesity , PandemicsABSTRACT
Objective To assess the consistencies of VerifyNow system, thrombelastography (TEG), vasodilator-stimulated phosphoprotein (VASP), and PL-11 platelet analyzer in detecting the antiplatelet function of clopidogrel, and to discuss the clinical application values. Methods Totally 98 consecutive inpatients with ST-segment elevation myocardial infarction (STEMI), non-(NSTEMI), or coronary artery in-stent restenosis (SR) were included in this study. The patients were given a loading dose of 600-mg clopidogrel for 6 hours, 300-mg clopidogrel for at 24 hours, or chronic clopidogrel therapy (75 mg daily for ≥ 7 days) before the procedure. And then the antiplatelet effects were evaluated by VerifyNow, TEG, VASP and PL-11 platelet analyzer simultaneously, with the results of VerifyNow taken as the gold standard and P2Y12 reaction unit (PRU) ≥ 208 taken as high on-clopidogel treatment platelet reactivity (HTPR). Correlation analysis was done for the four methods, and area under ROC curve (AUC) was used to evaluate the value of each method for HTPR. Results The platelet inhibition rate detected by VerifyNow was positively correlated with that by TEG (r = 0. 234, P < 0. 05); by contrast, it was negatively correlated with the platelet reactivity index (PRI) measured by VASP, the maximum platelet aggregation rate (MAR) by PL-11 and the maximum adenosine diphosphate (MA-ADP) by TEG (r = –0. 299, P<0. 01; r = –0. 330, P< 0.05; r = –0. 237, P<0. 05). The PRU values was negatively correlated with the platelet inhibition rate detected by VerifyNow (r = –0.815, P < 0. 01). The area under ROC curve of PL-11 platelet analyzer was the highest (0. 644). Conclusion TEG, VASP, and PL-11 platelet function testing systems all have consistency with the “gold standard” VerifyNow in some extent, with PL-11 platelet analyzer showing the highest sensitivity and specificity.
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<p><b>Objective</b>To investigate the relationship of the expression of vasodilator-stimulated phosphoprotein (VASP) with the metastasis and prognosis of prostate cancer.</p><p><b>METHODS</b>Prostate cancer PC3 cells were infected with VASP shRNA and control shRNA lentiviruses, respectively. The invasive ability of the PC3 cells was determined by transwell migration assay, the expression of VASP in the prostate cancer tissue from 56 patients was detected by immunohistochemistry, and the survival rate of the patients was analyzed according to the VASP expression levels and follow-up data after radical prostatectomy.</p><p><b>RESULTS</b>VASP shRNA lentivirus significantly inhibited the expression of VASP and decreased the invasive ability of the PC3 cells as compared with the results obtained in the scramble shRNA and blank control groups (P<0.05). The survival analysis of the 56 prostate cancer patients showed that the time of biochemical recurrence was markedly shorter in the VASP positive and strongly positive groups than in the VASP-negative cases (P<0.05), but with no statistically significant difference between the former two groups (P>0.05).</p><p><b>CONCLUSIONS</b>VASP is involved in the regulation of the invasive ability of prostate cancer PC3 cells, and the differences in the VASP expression are related to the prognosis of prostate cancer.</p>
Subject(s)
Humans , Male , Cell Adhesion Molecules , Metabolism , Cell Line, Tumor , Immunohistochemistry , Lentivirus , Microfilament Proteins , Metabolism , Neoplasm Metastasis , Phosphoproteins , Metabolism , Prognosis , Prostatectomy , Prostatic Neoplasms , Pathology , General Surgery , RNA, Small Interfering , Survival RateABSTRACT
Objective To use the flow cytometry to detect the platelet vasodilator-stimulated phosphoprotein(VASP)phospho-rylation level and to evaluate the clopidogrel curative effect after PCI.Methods 17 cases in the control group without any drug in-tervention and 26 cases of acute coronary syndrome(ACS)after PCI operation with clopidogrel were selected.Platelet VASP phos-phorylation levels at being selecting and on 7 d after anti-platelet therapy were detected by the flow cytometry and the platelet reac-tivity index (PRI)was calculated.Results The PRI after anti-platelet therapy in the ACS group was decreased significantly,the difference between before treatment and after treatment had statistical significance (P <0.05 ).Conclusion The platelet VASP phosphorylation level detected by the flow cytometry can specifically evaluate the effect of clopidogrel.
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Objective To evaluate the changes of platelet activity before and after anti-platelet treatment in pa?tients with coronary heart disease, and their responsiveness to clopidogrel through detecting the phosphorylation levels of va?sodilator stimulated phosphoprotein (VASP). Methods Twenty-eight cases of healthy people were selected as control group . Patients with chronic stable angina pectoris (CSA, n=95) were randomly divided into A (48 cases) group and B (47 cases) group,and were given clopidogrel 75 mg/d or 150 mg/d respectively;Patients with non ST segment elevation acute coronary syndrome (NST-ACS, n=67) were all given 300 mg loading dose of clopidogrel at the first time, then randomly divid?ed into C (33 cases) group and D (34 cases) group, and given clopidogrel 75 mg/d and 150 mg/d respectively. Blood were tak?en to examine the phosphorylation levels of serum VASP by ELISA before taking clopidogrel, at time point of loading dose and the fifth day of clopidogrel administration . Results Before treatment, phosphorylation levels of serum VASP were low?er in A, B, C, D groups than those in the normal control group(P0.05).②In group C and group D, phosphorylation levels of serum VASP were significantly increased at loading dose and the fifth day of clopidogrel administration than those before treatment (P 0.05). Conclusion The phosphorylation level of serum VASP was lower in patients with coronary heart disease than that in normal control group. Clopidogrel can improve the phosphorylation level of serum VASP in NST-ACS patients .
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Objective To investigate the mechanism of cyclic adenosine monophosphate / protein kinase A signal transduction pathway in severe acute pancreatitis(SAP)-associated lung injury.Methods Seventy-two male healthy SD rats were completely randomized into three groups:sham operation (SO) group(n =8),SAP group and SAP plus H89 (cAMP inhibitor) group,then the latter two groups were divided into four sub-groups with eight rats in each sub-group according to the sampling time of 3,6,12 and 24 h,and the total numbers of groups were nine.The content change of TNF-α and IL-1β in serum,protein levels of cAMP-dependent protein kinase catalytic subunit (PKA C) and phosphorylated vasodilator-stimulated phosphoprotein(p-VASP) and the expression of VSAP mRNA in lung tissue were detected by enzyme-linked immunosorbent assay (ELISA),immunohistochemistry and quantitative real time PCR,respectively.Pathological changes of the pancreas and lung tissues were also observed.Results Compared with the SO group,the serum levels of TNF-α and IL-1β in the SAP group were obviously increased at different time points(P ＜0.05).Pathological changes of the pancreas and lung tissues were aggravated significantly.The protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were increased significantly (P ＜0.05)which peaked at 12 h in the SAP group [TNF-α was (266.07 ± 17.14) pg/mL,IL-1β(169.17 ±25.92) pg/mL,PKA C(210.69 ±6.32) × 103,p-VASP (56.62 ±0.57) × 103,VASP mRNA(2.06 ±0.21)],which had positive correlation with the serum level of TNF-α and IL-1β.Compared with the SAP group,pathological changes of the pancreas and lung tissues were alleviated significantly,the protein levels of PKA C,p-VASP and the expression of VSAP mRNA in lung tissue were decreased significantly in the SAP plus H89 group at different time points(P ＜ 0.05).Conclusion The cyclic adenosine monophosphate / protein kinase A signal transduction pathway is found to participate in the pathological process of SAP-associated lung injury through the up-regulations of TNF-α,IL-1 β and phospho-VASP.
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AIM: To investigate the effect of vasodilator-stimulated phosphoprotein (VASP) phosphorylation on the cell migration induced by platelet-derived growth factor BB (PDGF-BB), and to identify which phosphorylation site was more important among the three phosphorylation sites, namely Ser157,Ser239 and Thr278. METHODS: Two phosphorylation mutants of VASP, pcDNA3.1(+)/VASP-S157A and pcDNA3.1(+)/VASP-S239A, were constructed and respectively transfected into the cultured ECV304 cells by means of liposome. The stable expression cells were screened by using antibiotic G418. Protein expression of VASP was measured by Western blotting. The ECV304 cell migration was evaluated using Transwell chamber. RESULTS: Compared to control group, the cell migration was significantly inhibited in ECV304 transfected with VASP-S157A and VASP-S239A (P<0.05), although slight differences existed between VASP-S157A and VASP-S239A transfected cells (P>0.05). CONCLUSION: VASP mutation on the phosphorylation sites of Ser157 and Ser239 inhibits cell migration, and the phosphorylation sites of Ser157 and Ser239 both greatly affect the function of VASP.
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Objective To observe the effects of vasedilator stimulated phosphoprotein (VASP) of small intestine mucous membrane on the intestinal barrier dysfunction during hemorrhagic shock (HS) in rats. Methods Forty Wistar rats were divided into normal group, HS 1 hour, HS 2 hours and HS 4 hours groups, as well as HS 2 hours + cyclic adenosine monophosphate (cAMP) treatment group. The activity of sermn diamine oxidase (DAO), content of hematoplasma D-lactic acid of each group were determined, and their relationship with the expression of VASP was analyzed. Results The expression of VASP was increased, serum DAO activity and hematoplasma D-lactic acid content were decreased by cAMP in rats at 2 hours after HS. There were differences upon the levels of VASP expression, DAO activity and hematoplasma D-lactic acid content between HS 2 hours group and HS 2 hours + cAMP treatment group ( t = 18.62, 9.28, 2.83, P < 0.05 ). The serum DAO activity increased while VASP expression decreased significantly after HS, which showed an obvious negative correlation between the two indexes (r=-0.95, P<0.05). Conclusions The decrease of VASP contributes to the intestinal barrier dysfunction after HS in rats, while the intestinal barrier dysfunction can be alleviated by cAMP.
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Objective To observe the relationship between zonula occludens protein subunit 1 (ZO-1) and the regulatory effect of vasodilator stimulated phosphoprotein (VASP) on the intestinal barrier function in rats of hemorrhagic shock (HS). Methods The intestinal barrier function was reflected by the hematoplasma D-lactic acid content,and the mucous membrane of small intestine from HS rats at different time after shock (1,2 and 4 h) were adopted to determine the expressions of VASP,phospho-VASP and ZO-1. cAMP,the agonist of VASP phosphorylation,was employed to observe the change of the above mentioned molecules and the intestinal barrier function following HS. Results (1) VASP expression of small intestine mucous membrane was decreased significantly after HS (P