| BackgroundsA total of1single nucleotide polymorphisms (SNPS) of platelet endothelial cell adhesion molecule-1(PECAM-1) have been identified, three of which have been testified to be associated with human diseases, namely Leul25Val, Asn563Ser and ly670Arg. Genetic mutations participate in abnormal clotting mechanism, and a study suggested more than30%of genetic mutations of individuals were involved in the development process of deep vein thrombosis. The correlation of genetic polymorphism with deep vein thrombosis has been studied as a focus. More and more gene polymorphism was involved in the coagulation mechanism and correlated with the formation of venous thrombosis. PECAM-1gene has been reported to be associated thrombosis severity in DVT model of mice. In order to investigate the correlation between PECAM-1gene and patients with DVT, we studied PECAM-1gene polymorphism and vein thrombosis susceptibility in Part Ⅰ.The mechanism of PEC AM-1participating in the anti-thrombotic effect of gas signal molecule-H2S in Part Ⅱ. This topic on the basis of clinical cases, the use of the special advantage of animal experiments, We mean to provide valuable clues for the diagnosis and treatment of DVT based on clinical cases and reliable research method. And our research results have not been reported before.Previous studies have reported the gene polymorphism of PECAM-1Leul25Val correlated with atherosclerosis diseases, such as coronary heart disease (CHD), myocardial infarction (MI), etc. However, none consensus have been reached. We performed a meta-analysis in Part Ⅲ in order to clarify the correlation of PEC AM-1gene polymorphism and vascular diseases from the point of view of evidence-based medicine (EBM).Part1Platelet endothelial cell adhesion molecule-1Leu125Val gene polymorphism is associated with deep vein thrombosisDeep vein thrombosis (DVT) and pulmonary embolism (PE) are considered to be two different stages of venous thromboembolism (VTE). Deep vein thrombosis is a common disease with an incidence of1%o per year. DVT in lower extremity progresses to post-thrombotic syndrome (PTS) if timely and effective treatment is not administrated, which seriously threaten the patient’s quality of life. DVT is thought to be induced by a joint various factors, including genetic, environmental and behavioral aspects. Previous studies have testified that the action of adhesion of platelet to endothelial cell plays a crucial role during initiating and promoting venous thrombosis. Rapid aggregation and adhesion of platelet on the injured intimal can help repairing intimal. However, the relationship and possible mechanism between cell adhesion molecules and DVT remain unclear. And the role of cell adhesion molecules, in particular platelet endothelial cell adhesion molecule-1in DVT has become a focus.Platelet endothelial cell adhesion molecule-1(PECAM-1), aka CD31, is a cell surface glycoprotein with a molecular weight of130-kDa, which is also known as a member of cellular adhesive molecular immunoglobulin superfamily. PECAM-1mediates adhesion of endothelial cells to extracellular matrix and white blood cells to endothelial cells. It also participates in cell signal transduction, vascular inflammation and white blood cells migration across endothelial cells. PECAM-1promotes the activities of platelet aggregation, activation and release by the receptor ligand reaction with other adhesion molecules, which facilitates the development of primary or secondary thrombosis. Previous studies reveal that PEC AM-1gene knockout mice presented more thrombus and longer thrombolysis process. And high level of serum PEC AM-1significantly delayed thrombolysis.Purpose and methodWe mean to detect the distribution of Leu125Val, Asn563Ser, Gly670Arg by PCR and the level of serum PEC AM-1by Elisa in patient and control groups. The colored Doppler examination was also performed. The relationship between serum PECAM-1level, PECAM-1gene polymorphism, especially Leul25Val polymorphism loci and the occurrence and development of DVT were clarified based on the above studies. Our research meant to explore the prevention and treatment of DVT and would provide new genetic markers of early screening for individuals at high risk of DVT.ResultsGene frequency and allele frequency of PECAM-1Leu125Val, Asn563Ser, and Gly670Arg were detected in DVT group and control group, respectively. Frequency of genotype Leu/Leu, Leu/Val, Val/Val of Leu125Val in DVT group were12.2%,47.8%and12.2%, respectively, while frequency of allele Leu and Val were36.1%and36.1%respectively. In control group, frequency of genotype Leu/Leu, Leu/Val, Val/Val were24.0%,24.0%,22.1%, while frequency of allele Leu and Val were51.0%,49.0%. The polymorphism of Leu125Val genotype frequency and allele frequency were significantly different (P<0.01). Leu125Val genotypes and alleles were obviously higher in DVT group than in control group (X2=10.25, P<0.01; X2=9.85, P<0.01). The correlation of Val/Val genotype to increased risk of DVT was much more obvious than that of Leu/Leu genotype (OR=3.57;95%CI,2.47-5.16, P=0.008). However, genotype frequency and allele frequency of Asn563Ser and Gly670Arg polymorphism loci between DVT group and control group were not significant (P>0.05). The result suggested PECAM-1polymorphism loci, especially Val/Val of Leul25Val genotype, correlated to the risk of DVT significantly.Elisa revealed that sPECAM-1was significantly higher in DVT group than in control group (83.4±23.5ng/ml vs60.4±19.4ng/ml, P<0.01). In DVT group, sPECAM-1was also higher in Leu/Val and Val/Val genotype than in Leu/Leu genotype (P<0.01). sPECAM-1level was higher in Val/Val genotypes than in Leu/Val genotypes (P<0.05).Doppler ultrasound compression test was performed to evaluate thrombosis load in all patients early after diagnosis and treatment and28days after treatment, respectively. Score of thrombus baseline was significantly higher in Leu125Val Val/Val genotype than in Leu/Leu and Leu/Val genotypes. Thrombus score (baseline score minus thrombus score at28days after treatment) was significantly lower in Leu/Val and Val/Val genotypes than in Leu/Leu genotype. The result suggested Val alleles contributed to delayed thrombolysis in DVT patients.ConclusionLeu/Leu, Leu/Val and Val/Val genotype of PEC AM-1Leu125Val correlated to the risk of DVT significantly (P<0.05). Compared with allele125Leu, correlation of allele125Val to increased risk of DVT was much more obvious (P<0.01). Serum sPECAM-1level was higher in DVT group than in control group, and Leu/Val and Val/Val genotypes also presented with increased thrombus load and delayed thrombolysis. Therefore, Leu125Val, known as polymorphism loci of PECAM-1gene, may play a key role during the occurrence and development of DVT, possibly by regulating serum PECAM-1. Part2Experimental Research of the Mechanism of PECAM-1Participating in the Antithrombotic Effect of Hydrogen SulfideDeep vein thrombosis (DVT) is initiated by the inflammatory cell aggregation and adhesion to vascular endothelial cell, which activate platelets and induce thrombosis. The adhesion of platelet to endothelial cell mediated by the interaction between platelets and monocytes, neutrophils is a key step during the pathology progress of venous thrombosis. These interactions are regulated by a variety of cellular adhesive moleculars.PECAM-1exhibits strong inhibition function of platelet aggregation and antithrombotic effect in vivo. Previous experiments showed that PECAM-1gene knockout mice presented much more severe arteriovenous thrombosis and delayed thrombolysis. Inflammation induces thrombosis by promoting high level of coagulation state of blood and up-regulated vascular permeability. PECAM-1exhibits antithrombotic effect based on its anti-inflammatory effects in endothelial cells.Hydrogen sulfide (H2S) is a kind of gas signal molecule, which participates in the regulation of various physiological and pharmacological reactions. Hydrogen sulfide can prevent thrombosis by inhibiting vascular inflammation and platelet aggregation. Previous research has shown that certain drugs containing H2S presented clear antithrombotic effect.In this study, we meant to explore whether PECAM-1participates in the inhibition of platelet aggregation and thrombosis by hydrogen sulphide and the significance of PECAM-1during this process by using small interfering RNA (siRNA). Nitric oxide synthase inhibitor (sinistral nitro arginine methyl ester, L NAME) was also used to investigate whether these effect depended on the nitric oxide signaling pathways. The regulation effect of H2S on PECAM-1was also studied.Methods1.Platelet aggregation function testTo observe whether NaHS is nitric oxide-dependent by performing platelet aggregation function test using multiplate impedance aggregometry.2. Test of adhesion of fibrinogen by flow cytometryNaHS-treated blood and FITC-labeled fibrinogen antibody were incubated for20minutes. Data was acquired by flow parameters by FACSCalibur from5000cells and analyzed by CellQuest software (Version3.3; BD Biosciences). 3.Establishment of DVT model in miceA total of60C57BL/6mice were randomly divided into3groups, including control group (injected by saline), NaHS group (injected by10mmol/kg body weight), NaHS+L-NAME group (injected by100mg/kg body weight). Thrombosis was induced by clamping of femoral vein for3seconds on each of3location using mosquito clamps. Femoral venous thrombus formation, severity and thrombolysis were observed after48hours. Vein thrombosis was weighed, and femoral vein and its branch was also collected.4.Western blottingThe expression of eNOS,iNOS and PECAM-1in vascular endothelium and blood were detected by western blotting. The protein level was quantified by Image J software.5. small interfering RNA (siRNA)A specific siRNA for PECAM-1gene was designed and transfected by liposome in HUVECs cells. After48hours of transfection, PECAM-1protein was detected by western blotting to observe inhibition effect of siRNA.6. Platelet adhesion activity testHUVECs were used to study the platelet adhesion activity treated by hydrogen sulfide. HUVECs were inoculated on the cover glass and stained by crystal violet staining method. The number of platelet HUVECs cells adhered by platelet and the proportion of the total HUVECs cells were anslyzed under optical microscope.Results1.Hydrogen sulfide inhibited platelet functionNaHS inhibited platelet aggregation induced by arachidonic acid, thrombin receptor activation peptide, ADP and collagen in a dose-dependent manner, respectively. L-NAME can significantly weaken inhibition of NaHS on platelet aggregation induced by ADP. NaHS decreased adhesion of fibrinogen stimulated by ADP in a dose-dependent manner.2. Hydrogen sulphide exhibited antithrombotic activity Compared with DVT mice in control group, vein thrombosis proceeded much more slowly in NaHS group and presented smaller thrombus at48hours after clamping. The inhibition of thrombosis of NaHS was attenuated by L-NAME. Compared with control group, a higher level of eNOS and iNOS were expressed in NaHS group. The expression of eNOS and iNOS protein were significantly reduced by L-NAME compared with both NaHS group and control group.3. Hydrogen sulfide up-regulated PEC AM-1expression in platelets and endothelial cellsNaHS up-regulated PECAM-1protein expression of platelet significantly in a dose-dependent manner. Compared with control group, a higher level of PECAM-1protein expression was presented in NaHS group. PECAM-1protein expression was reduced significantly by L-NAME, but remained higher than that in control group. NaHS inhibited platelet Akt phosphorylation (Ser473) in a dose-dependent manner.4. PECAM-1participated antithrombotic effects of hydrogen sulfidePECAM-1expression in HUVECs was inhibited by application of small interfering RNA (siRNA). Transfected HUVECs was co-cultured with platelet, ADP activated platelets, and ADP activated plus NaHS treated platelets for1hour, respectively. ADP significantly increased the adhesion activity between HUVECs and platelet. siRNA-PECAM-1significantly increased the adhesion activity between HUVECs and platelet and attenuated platelet inhibition function of hydrogen sulfide.ConclusionHydrogen sulfide exhibited inhibition of platelet aggregation in vitro and inhibition of thrombosis in vivo, possibly by up-regulation of eNOS, iNOS and PECAM-1protein expression. PECAM-1protein participated in the antithrombotic function of platelet inhibition of hydrogen sulfide, and was a downstream treatment targets. H2S-NO-PECAM-1signaling pathway was involved in the development of thrombosis and might clarify the mechanism of platelet inhibition and antithrombotic effect mechanism of hydrogen sulfide. Part3Association of Leu125Val polymorphisms in the PECAM-1gene with the risk of atherosclerosis disease(CHD):a meta-analysisInflammation plays a critical role in the initiation and progression of atherosclerosis diseases. Platelet activation, aggregation and thrombus formation are regarded as key steps in the pathogenesis of atherosclerosis diseases. Previous studies indicated that platelet endothelial cell adhesion molecule-1(PECAM-1) is constitutively expressed on the surface of circulating monocytes, platelets, neutrophils, specific classes of T cells, and endothelial cells. PECAM-1has been a focus because several publications suggested that genetic polymorphism loci of PECAM-1were associated with several diseases. The association of genetic polymorphism (Leu125Val, C373G, rs668) with atherosclerosis diseases, particularly coronary atherosclerosis cardiac heart disease (CHD) has been a focus of clinical research.Several published literatures investigated the relation between a polymorphism (Leu125Val) in platelet endothelial cell adhesion molecule-1(PECAM-1) gene and risk of atherosclerosis diseases and did not reach the same conclusion. To shed light on these inconclusive findings, we performed a meta-analysis of studies relating the PECAM-1genetic polymorphism (Leu125Val, C373G) to the risk of atherosclerosis diseases.In this study, we explored all published literatures about the correlation of Leu125Val of PECAM-1with atherosclerosis disease, including case-control study and cohort study. A meta-analysis was performed based on a strict standard of literature screening and quality evaluation. We mean to explore the correlation of PECAM-1gene polymorphism Leul25Val and the risk of atherosclerotic disease and provide new clues for further research on vascular diseases.ObjectiveWe mean to explore the evidence of the correlation of PECAM-1gene polymorphism Leu125Val and the risk of atherosclerotic disease, particularly CHD, by a combined quantitative evaluation.MethodsWe identified literatures by searching PubMed, EMBASE, Chinese National Knowledge Infrastructure databases (CNKI) and Wanfang database in China. Data from eligible studies were extracted for meta-analysis. The risk of atherosclerotic disease associated with PEC AM-1genetic polymorphism (Leu125Val) was estimated by pooled odds ratios (ORs) and95%confidence intervals (95%CIs). The software Review Manager (Version5.2) was used for meta-analysis. Publication bias was tested by funnel plot.ResultsA total of102articles were retrieved, including66Chinese literatures and36English literatures, among of which2articles focused on carotid atherosclerosis cerebral infarction. There was no arteriosclerosis occlusion of lower extremities. Finally,15studies comprising3696cases and3940controls fulfilled the inclusion criteria.All the15independent studies focused on the correlation of Leul25Val (125c/G) polymorphism with susceptibility of. Forest plot revealed the meta-analysis result of PEC AM-1gene polymorphism with CHD, including a systematic review, patient and control group, the weight, the OR and95%confidence interval.Heterogeneity test of dominant heredity [(LL+LV) vs VV] revealed Chi2=100.53, P<0.001, Ⅰ2=86%, while heterogeneity test of recessive heredity [(W+LV) vs LL] revealed Chi2=34.99, P=0.001, Ⅰ2=60%. Heterogeneity test of allele genes revealed Chi2=68.55, P<0.001, Ⅰ2=80%. Analysis of random effects model revealed OR=1.15,0.84-1.56(95%CI), P=0.38of (LL+LV) vs VV; OR=0.96,0.79-1.17(95%CI), P=0.69of (VV+LV) vs LL; OR=1.08,0.92-1.27(95%CI), P=0.80 of V vs L. Our results did not show that Leu125Val polymorphism in PECAM-1gene was associated with the risk of atherosclerosis disease (CHD).ConclusionMeta-analysis revealed that Leu125Val polymorphism in PECAM-1gene is not a susceptibility marker of atherosclerosis disease(CHD). |
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