Head to Head Comparison of Two Point-of-care Platelet Function Tests Used for Assessment of On-clopidogrel Platelet Reactivity in Chinese Acute Myocardial Infarction Patients Undergoing Percutaneous Coronary Intervention / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Platelet function tests are widely used in clinical practice to guide personalized antiplatelet therapy. In China, the thromboelastography (TEG) test has been well accepted in clinics, whereas VerifyNow, mainly used for scientific research, has not been used in routine clinical practice. The aim of the current study was to compare these two point-of-care platelet function tests and to analyze the consistency between the two tests for evaluating on-clopidogrel platelet reactivity in Chinese acute myocardial infarction patients undergoing percutaneous coronary intervention (PCI).</p><p><b>METHODS</b>A total of 184 patients admitted to Fuwai Hospital between August 2014 and May 2015 were enrolled in the study. On-clopidogrel platelet reactivity was assessed 3 days after PCI by TEG and VerifyNow using adenosine diphosphate as an agonist. Based on the previous reports, an inhibition of platelet aggregation (IPA) <30% for TEG or a P2Y12 reaction unit (PRU) >230 for VerifyNow was defined as high on-clopidogrel platelet reactivity (HPR). An IPA >70% or a PRU <178 was defined as low on-clopidogrel platelet reactivity (LPR). Correlation and agreement between the two methods were analyzed using the Spearman correlation coefficient (r) and kappa value (κ), respectively.</p><p><b>RESULTS</b>Our results showed that VerifyNow and TEG had a moderate but significant correlation in evaluating platelet reactivity (r = -0.511). A significant although poor agreement (κ = 0.225) in identifying HPR and a significantly moderate agreement in identifying LPR (κ = 0.412) were observed between TEG and VerifyNow. By using TEG as the reference for comparison, the cutoff values of VerifyNow for the Chinese patients in this study were identified as PRU >205 for HPR and PRU <169 for LPR.</p><p><b>CONCLUSIONS</b>By comparing VerifyNow to TEG which has been widely used in clinics, VerifyNow could be an attractive alternative to TEG for monitoring on-clopidogrel platelet reactivity in Chinese patients.</p>

Inhibitory effect of iron on in vitro proliferation of smooth muscle cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Iron is a biocorrodible metal that might be used in bioabsorbable stents. This study investigated the effects at the cellular and protein levels of soluble divalent iron (ferrous gluconate) and soluble trivalent iron (ferric chloride) on the proliferation of human aortic smooth muscle cell (HASMC) in vitro.</p><p><b>METHODS</b>The water-soluble tetrazolium (WST-1) test was used to evaluate the effect of iron on proliferation of HASMC and Western blotting was used to measure the levels of signaling proteins involved in proliferative and apoptosis pathways.</p><p><b>RESULTS</b>HASMC proliferation was inhibited in a concentration dependent manner after treatment with soluble divalent and trivalent iron at concentrations of 100-500 µmol/L. Western blotting analysis showed that the proliferating cell nuclear antigen (PCNA) expression following treatment with soluble divalent iron and trivalent iron at 100, 300 and 500 µmol/L was reduced compared to the control. The PCNA expression decreased with increasing iron concentration and to a greater extent with the trivalent iron than with the divalent iron treatment group. The p53 expression was markedly increased in a concentration dependent manner in both iron treatment groups.</p><p><b>CONCLUSION</b>The soluble divalent iron and, to a greater degree trivalent iron, inhibited HASMC proliferation in a dosedependent manner, which may be attributed to reduction of PCNA expression and increase of p53 expression.</p>

Protein kinase A-mediated cardioprotection of Tongxinluo relates to the inhibition of myocardial inflammation, apoptosis, and edema in reperfused swine hearts / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Our previous studies have demonstrated that Tongxinluo (TXL), a traditional Chinese medicine, can protect hearts against no-reflow and reperfusion injury in a protein kinase A (PKA)-dependent manner. The present study was to investigate whether the PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis.</p><p><b>METHODS</b>In a 90-minute ischemia and 3-hour reperfusion model, minipigs were randomly assigned to sham, control, TXL (0.05 g/kg, gavaged one hour prior to ischemia), and TXL + H-89 (a PKA inhibitor, intravenously and continuously infused at 1.0 µg/kg per minute) groups. Myocardial no-reflow, necrosis, edema, and apoptosis were determined by pathological and histological studies. Myocardial activity of PKA and myeloperoxidase was measured by colorimetric method. The expression of PKA, phosphorylated cAMP response element-binding protein (p-CREB) (Ser(133)), tumor necrosis factor α (TNF-α), P-selectin, apoptotic proteins, and aquaporins was detected by Western blotting analysis.</p><p><b>RESULTS</b>TXL decreased the no-reflow area by 37.4% and reduced the infarct size by 27.0% (P < 0.05). TXL pretreatment increased the PKA activity and the expression of Ser(133) p-CREB in the reflow and no-reflow myocardium (P < 0.05). TXL inhibited the ischemia-reperfusion-induced elevation of myeloperoxidase activities and the expression of TNF-α and P-selectin, reduced myocardial edema in the left ventricle and the reflow and no-reflow areas and the expression of aquaporin-4, -8, and -9, and decreased myocytes apoptosis by regulation of apoptotic protein expression in the reflow and no-reflow myocardium. However, addition of the PKA inhibitor H-89 counteracted these beneficial effects of TXL.</p><p><b>CONCLUSION</b>PKA-mediated cardioprotection of TXL against no-reflow and reperfusion injury relates to the inhibition of myocardial inflammation, edema, and apoptosis in the reflow and no-reflow myocardium.</p>

Ischemic preconditioning attenuates myocardial no-reflow and reperfusion injury after revascularization of acute myocardial infarction by reducing myocardial edema via the protein kinase A pathway / 中华心血管病杂志

<p><b>OBJECTIVE</b>Myocardial edema plays an important role in the development of myocardial no-reflow and reperfusion injury after the revascularization of acute myocardial infarction (AMI). The present study investigated whether the effect of ischemic preconditioning (IPC) against myocardial no-reflow and reperfusion injury was related to the reduction of myocardial edema through the protein kinase A (PKA) pathway.</p><p><b>METHODS</b>Twenty-four minipigs were randomized into sham, AMI, IPC, and IPC + H-89 (PKA inhibitor, 1.0 µg · kg(-1) · min(-1)) groups. The area of no-reflow (ANR), area of necrosis (AN), and water content in left ventricle and ischemic-myocardium and non-ischemic area were determined by pathological studies. Microvascular permeability was determined by FITC-labeled dextran staining. Cardiomyocyte cross-sectional area (CSA) and mitochondria cross-sectional area (MSA) were evaluated by histological analysis. Myocardial expression of aquaporins (AQPs) was detected by Western blot.</p><p><b>RESULTS</b>Compared with the MI group, the sizes of no-reflow and infarct were reduced by 31.9% and 46.6% in the IPC group (all P < 0.01), water content was decreased by 5.7% and 4.6% in the reflow and no-reflow myocardium of the IPC group (all P < 0.05), microvascular permeability and cardiomyocytes swelling in the reflow area were inhibited by 29.8% and 21.3% in the IPC group (all P < 0.01), mitochondrial water accumulation in the reflow and no-reflow areas of the IPC group were suppressed by 45.5% and 34.8% respectively (all P < 0.01), and the expression of aquaporin-4, -8, and -9 in the reflow and no-reflow myocardium were blocked in the IPC group. However, these beneficial effects of IPC were partially abolished in the IPC + H-89 group.</p><p><b>CONCLUSIONS</b>The cardioprotective effects of IPC against no-reflow and reperfusion injury is partly related to the reduction of myocardial edema by inhibition of microvascular permeability and aquaporins up-regulation via PKA pathway.</p>

Impact of cytochrome P450 2C19 polymorphisms on outcome of cardiovascular events in clopidogrel-treated Chinese patients after percutaneous coronary intervention / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the impact of cytochrome P450 (CYP) 2C19 681G > A polymorphism on long-term prognosis of clopidogrel-treated Chinese patients after percutaneous coronary intervention (PCI).</p><p><b>METHODS</b>Between January 1, 2009 and August 31,2009, 267 patients with coronary heart disease who received PCI and treated with clopidogrel for 12 months were enrolled. CYP2C19 * 2 was detected by MALDI-TOF MS and patients were grouped into CYP2C19 * 1/ * 1 (n = 130) and CYP2C19 * 2 carriers group (n = 137). Follow-up was 12 months. The primary endpoint was angina recurrence, urgent coronary revascularization, acute myocardial infarction, stent thrombosis, death and the combined end points.</p><p><b>RESULTS</b>Baseline data were similar between two groups (P > 0.05). Urgent coronary revascularization and the combined end points occurred more frequently in CYP2C19 * 2 carriers than in CYP2C19 * 1/* 1 patients (7.3% vs. 1.5% and 8.0% vs. 2.3% respectively, all P < 0.05). But incidence of angina recurrence, acute myocardial infarction, stent thrombosis and death was similar between two groups (all P > 0.05). Hazard risk of 1 year cumulative survival of CYP2C19 * 2 carriers group was significantly higher than CYP2C19 * 1/ * 1 group after PCI ( HR = 3.59, 95% CI: 1.02 - 12.87, P < 0.05).</p><p><b>CONCLUSION</b>CYP2C19 681G > A polymorphism is a determinant of prognosis in coronary heart disease patients receiving chronic clopidogrel treatment after PCI.</p>

Atorvastatin protects swine bone marrow mesenchymal stem cells from apoptosis through AMPK but not PI3K/Akt pathway / 中华心血管病杂志

<p><b>OBJECTIVE</b>The effect of mesenchymal stem cells (MSCs) transplantation is poor because of the harsh environment post infarction. Our previous studies have proven that Statins could enhance the implanted bone marrow MSCs survival, but the exact mechanism remained to be clarified. We hypothesized that atorvastatin (Ator) could protect MSCs from hypoxia and serum-free (H/SF) induced apoptosis and investigated the potential mechanisms.</p><p><b>METHODS</b>Chinese mini-swine's bone marrow derived MSCs were cultured in vitro and exposed to hypoxia and H/SF, Ator of various concentrations (0.001 - 10 µmol/L), AMPK inhibitor-compound C (CC), PI3K inhibitor-LY294002 (LY), Ator + CC and Ator + LY. Cell apoptosis was assessed using Annexin V/Prospidine Iodine kit by flow cytometry. Phosphorylation of AMPK, Akt, endothelial nitric oxide synthase (eNOS) level and phosphorylation were tested with Western blot. Real Time-PCR was performed to analyze the gene expression of AMPK, Akt and eNOS.</p><p><b>RESULTS</b>MSCs apoptosis in Ator (0.01 - 10 µmol/L) treated H/SF groups was significantly reduced compared with H/SF group (1.94% - 6.10% vs. 10.94%, P < 0.01 or 0.05). Apoptosis was higher in Ator + CC group than in 1 µmol/L Ator group (4.94% ± 0.98% vs. 2.59% ± 0.84%, P < 0.01) and similar between Ator + LY and 1 µmol/L Ator group (2.02% ± 0.45% vs. 2.59% ± 0.84%, P > 0.05). The gene expressions of AMPK, Akt and eNOS were significantly upregulated in atorvastatin treated groups. Meanwhile, phosphorylation of AMPK and eNOS increased in MSCs treated with atorvastatin (P < 0.01 or 0.05). Phosphorylation of eNOS significantly correlated with AMPK phosphorylation (r = 0.599, P = 0.004), but not with Akt phosphorylation (P = 0.263).</p><p><b>CONCLUSIONS</b>Atorvastatin can protect MSCs from H/SF induced apoptosis through AMPK pathway, which resulting in activation of eNOS.</p>

Role of vascular endothelial growth factor, interleukin-2 and tumor necrosis factor-alpha in diabetic retinopathy / 中华实验眼科杂志

BackgroundDiabetic retinopathy (DR) is a progressive vision-threatening complication of diabetes mellitus,but its pathogenic mechanism is still unclear. Recent studies showed that it may be associated with the inflammation response of retinal capillary. Cytokines can cause induction of proinflammatory and adhesion molecules and thereby increase monocyte endothelial cell adhesion, which is now accepted as the early key event in the development of DR. ObjectiveThe present study was to determine the relationship between the stages of DR and the levels of serum vascular endothelial growth factor (VEGF) , interleukin-2 ( IL-2 ), tumor necrosis factor-alpha (TNF-α) in diabetic patients. Methods This was a pilot case-controlled study. Ninety patients with type 2 diabetes mellitus were included in this clinical trial and 30 healthy individuals were enrolled as controls. The patients were grouped into the non-diabetic retinopathy(NDR) group,background DR group and proliferative DR(PDR) group according to the results from ophthalmoscopic examination and fundus fluorescein angiography(FFA) ,with 30 patients for each group. The levels of serum VEGF,IL-2,TNF-α were assayed by ELISA and compared among the 4 groups.Written informed consent was obtained from each subject before received any related medical examination to this study. ResultsThe mean serum VEGF levels were(217.35±27. 87)ng/L,(298.31±49.26)ng/L,and(341.23±40. 18)ng/L, respectively, and mean serum IL-2 levels were( 12. 12± 1. 57 )ng/L, (16.43 ±2. 26 )ng/L, and (21.36±0. 86) ng/L,respectively and mean serum TNF-α levels were( 11.63±0. 94) ng/L, ( 17. 52±0. 65) ng/L,and(22. 01±0. 87 ) ng/L respectively in the patients with NDR ,background DR and PDR, showing significant differences from healthy controls with( 193.46±37. 39 ) ng/L for serum VEGF, ( 8. 99 ±0. 57 ) ng/L for serum IL-2 and ( 7.31 ±0. 52 ) ng/L for serum TNF-α ( F =126. 38, P＜0. 0 1 ;F =120. 37, P＜0. 01 ;F =99. 84, P＜0. 01 ). The levels of serum VEGF, IL-2, and TNF-α in the patients with the NDR,background DR and PDR were increased significantly. The level of serum VEGF showed the positively significant correlation with serum IL-2 level and TNF-α level ( r =0. 749, P ＜ 0.01 ; r =0. 631,P＜0. 01 ). The serum levels of VEGF, IL-2 and TNF-α showed a significantly positive correlation with the prolongation and severity of DR(r=0. 791 ,P＜0. 01 ;r=0. 665 ,P＜0. 01 ;r=0. 632,P＜0. 01 ). ConclusionsVEGF, IL-2 and TNF-α play active roles in the generation and development of diabetic retinopathy, and the level of serum VEGF is closely associated with the levels of serum IL-2 and TNF-α. during the development of DR.

Pretreatment with Tongxinluo protects porcine myocardium from ischaemia/reperfusion injury through a nitric oxide related mechanism / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The traditional Chinese medicine Tongxinluo can protect myocardium against ischaemia/reperfusion injury, but the mechanism of its action is not well documented. We examined the involvement of nitric oxide in the protective role of Tongxinluo.</p><p><b>METHODS</b>Miniswine were randomized to four groups of seven: sham, control, Tongxinluo and Tongxinluo coadministration with a nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA, 10 mg/kg i.v.). Three hours after administration of Tongxinluo, the animals were anaesthetised and the left anterior descending coronary artery ligated and maintained in situ for 90 minutes followed by 3 hours of reperfusion before death. Area of no reflow and necrosis and risk region were determined pathologically by planimetry. The degree of neutrophil accumulation in myocardium was obtained by measuring myeloperoxidase activity and histological analysis. Myocardial endothelial nitric oxide synthase activity and vascular endothelial cadherin content were measured by colorimetric method and immunoblotting analysis respectively.</p><p><b>RESULTS</b>Tongxinluo significantly increased the local blood flow and limited the infarct and size of no reflow. Tongxinluo also attenuated myeloperoxidase activity and neutrophil accumulation in histological sections and maintained the level of vascular endothelial cadherin and endothelial nitric oxide synthase activity in the reflow region when compared with control group. The protection of Tongxinluo was counteracted by coadministration with L-NNA.</p><p><b>CONCLUSIONS</b>Tongxinluo may limit myocardial ischaemia and protect the heart against reperfusion injury. Tongxinluo regulates synthesis of nitric oxide by altering activity of endothelial nitric oxide synthase.</p>

Gelatin microspheres containing vascular endothelial growth factor enhances the efficacy of bone marrow mesenchymal stem cells transplantation in a swine model of myocardial infarction / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the efficacy of transplantation of mesenchymal stem cells (MSC) with gelatin microspheres containing vascular endothelial growth factor in ischemic regions in infracted swine hearts.</p><p><b>METHODS</b>Twelve Chinese mini swines with infarction were randomized to receive autogenetic MSC injection to the peri-infarction area of left ventricular wall (MSC group, n = 6) or MSC transplantation with gelatin hydrogel microspheres incorporating vascular endothelial growth factor (VEGF-MSC group, n = 6). Three weeks later, left ventricular function was assessed by magnetic resonance imaging (MRI). The contrast of the MSC hypointense lesion was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region. Myocardial capillary density, the number of DAPI positive MSC and the apoptotic MSC were also determined.</p><p><b>RESULTS</b>The diameter of the microspheres averaged (104.0 +/- 22.6) microm. At 24 hours after transplantation, MSC were identified by MRI as large intramyocardial signal voids at injection sites which persisted up to 3 weeks. There was no significant difference in the contrast of the lesions and in the size of the lesions at 24 hours between two groups. At 3 weeks after injection, the size of the lesions and the contrast of the lesion were decreased (P < 0.05) in both groups. The capillary density of the injection site was significantly more in the MSC-VEGF microsphere group than that in MSC group [(15.2 +/- 5.4)/HPF vs. (10.2 +/- 5.0)/HPF, t = 2.43, P < 0.05], and there were more dense DAPI labeled MSC per high power fields in injection sites of MSC-VEGF microsphere group than that in MSC group [(354 +/- 83)/HPF vs. (278 +/- 97)/HPF, t = 3.14, P < 0.05]. Moreover, the apoptosis rate of MSCs of MSCs-VEGF microsphere group was less than that of MSC group [(6.4 +/- 4.1)% vs. (11.9 +/- 4.8)%, t = 2.97, P < 0.05].</p><p><b>CONCLUSIONS</b>MSC transplantation with gelatin hydrogel microspheres incorporating VEGF enhanced the efficacy of MSC in this swine model of myocardial infarction. MRI tracking of MSC is feasible and represents a preferred method for studying the engraftment of MSCs in infracted tissue.</p>

Killing effect of coexpressing cytosine deaminase and thymidine kinase on rat vascular smooth muscle cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, such as atherosclerosis and restenosis after balloon angioplasty. Herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and E. coli cytosine deaminase (CD)/5-fluorocytosine (5-Fc) suicide gene systems have been successfully employed in cardiovascular gene therapy, respectively. We reasoned that coexpression of both HSV-TK with CD suicide genes would lead to increased cell killing. To test this imagine, the adenoviral vectors expressing TK and/or CD genes were developed and tested on vascular smooth muscle cells.</p><p><b>METHODS</b>Adenoviral vectors, including Ad-EF1alpha-CD-cytomegalovirus (CMV)-TK coexpressing both CD and TK double suicide genes, Ad-EF1alpha-CD and Ad-CMV-TK expressing CD and TK respectively, and control vector Ad-CMV-LacZ, were constructed and prepared with homologous recombination in RecA + E. coli cells. Integration and expression of CD and/or TK gene were identified by PCR and Western blot. Primary cultured VSMCs were infected at a multiplicity of infection (MOI) of 20 with exposure to their matching prodrugs 5-Fc and GCV. Cell mortality was measured by methyl thiazolyl tetrazolium (MTT) assays. Flow cytometry analysis was used to detect cell death. Apoptotic cells were analyzed using Hoechst 33342 fluorescence dye as a DNA probe. Genomic DNA cleavage of apoptotic VSMCs was tested by agarose gel electrophoresis.</p><p><b>RESULTS</b>Recombinant adenovirus expressing CD and/or TK suicide genes were successfully constructed. Both single and double suicide genes could be integrated into adenoviral genome and expressed. Cytotoxic effects of Ad-EF1alpha-CD-CMV-TK double suicide genes combined with 5-Fc and GCV were higher than those of Ad-CMV-TK and Ad-EF1alpha-CD single gene groups. The rate of cell survival was only (9 +/- 3)% in the Ad-EF1alpha-CD-CMV-TK group, but (37 +/- 3)% in the Ad-CMV-TK and (46 +/- 4)% in the Ad-EF1alpha-CD groups (P < 0.05). Flow cytometry analysis indicated that the killing mechanisms of the groups were different. Necrosis and apoptosis were involved in the mechanism of the double gene group. Based on the DNA stainability with Hoechst 33342, the apoptotic rates of VSMCs in the Ad-EF1alpha-CD-CMV-TK [(11.0 +/- 2.1)%] and Ad-CMV-TK [(12.0 +/- 2.2)%] groups were higher than those in Ad-CMV-LacZ [(1.2 +/- 0.11)%] and Ad-EF1alpha-CD [(5.0 +/- 1.8)%] groups (P < 0.05, respectively). DNA smear could be observed in both Ad-CMV-TK and Ad-EF1alpha-CD-CMV-TK groups after administration of prodrugs.</p><p><b>CONCLUSIONS</b>The killing effect on rat VSMCs mediated by adenoviral CD/TK double suicide genes is superior to that of single suicide gene. The killing mechanism of recombinant adenovirus coexpressing CD/TK double suicide genes is mainly through cytotoxic effect and apoptosis.</p>

Effect of antisense thrombin receptor and p21 double gene co-expression system on the proliferation and apoptosis in human aortic smooth muscle cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To focus on the study of the effect on proliferation and apoptosis of human aortic smooth muscle cells (ASMC) by adeno-associated virus (AAV) vector carrying antisense thrombin receptor (ATR) and p21 double gene co-expression system.</p><p><b>METHODS</b>Cultured human AMSC was infected with recombinant AAV containing ATR, p21 single gene and AP double gene respectively. The integration and expression of genes were confirmed by semi-quantitative RT-PCR. The anti-proliferation effect was determined by MTT assay. Cell cycle and apoptotic cell counts were measured through Flow Cytometry. The rate of apoptotic cells was examined with acridine orange/ethidium bromide(AO/EB) stain.</p><p><b>RESULTS</b>RT-PCR indicated that the exogenous genes had been integrated into ASMC. The rates of cell survival were decreased by 16.67%, 21.60%, and 29.4% and the cell counts of G0/G1 phase were (61.8 +/- 2.9)%, (82.5 +/- 4.0)%, (80.4 +/- 6.1)% in ATR, p21 and AP group respectively after rAAV infected 4 days. The level and area of apoptotic peak were greater in AP double gene than ATR and p21 single gene. Cell stain indicated that apoptotic cells were (7.2 +/- 3.3)%, (10.7 +/- 5.6)%, and (18.3 +/- 2.7)% in each transgene group compared with (1.5 +/- 0.8)% in control group.</p><p><b>CONCLUSION</b>AP double gene co-expression system has powerful effect for inhibiting proliferation and inducing apoptosis ASMC than ATR and p21 single gene and that is a superior way for gene therapy to restenosis.</p>