[The effect of VEGF antisense oligonucleotides combined with low molecular weight heparin on the growth and metastasis of mice Lewis lung cancer].

OBJECTIVE: To investigate the inhibitory effect of vascular endothelial growth factor (VEGF) antisense oligonucleotides or/and dalteparin sodium (fragmin) on tumor growth and metastasis of mice Lewis lung cancer. METHODS: 40 mice with Lewis lung cancer were randomizedly divided into five groups: control group, VEGF antisense oligonucleotides (ASODN) group, VEGF mismatch sense oligonucleotides (MSODN) group, low molecular weight heparin (LMWH) group, and combined group. Sodium chloride, VEGF-ASODN, VEGF -MSODN, fragmin, and VEGF-ASODN plus fragmin were given respectively (once every two days, 15 times altogether). The volume and weight of subcutaneous tumors were measured, and the rates of lung metastasis were detected by HE staining. The microvessel density (MVD) in tumor mass were measured by immunohistochemistry staining. VEGF protein level in tumor tissue were detected by Western blot assay. RESULTS: After treatment, the tumor growth inhibitory rates were 47.34%, 27.31% and 59.03%, and the rates of lung metastasis were 37.5%, 37.5% and 25% in ASODN, LMWH, and the combined group, respectively. Tumor MVD and VEGF protein expression of the above three groups were lower than those of the control group. There was a significant difference in regard to the tumor growth inhibitory rates and MVD between the above three treated groups and the control group as well as the MSODN group (P < 0.05). CONCLUSIONS: VEGF-ASODN and fragmin may down-regulate VEGF gene expression and inhibit angiogenesis, Combined use of fragmin can enhance anti- tumor effect of VEGF-ASODN.

[Relationship between polymorphisms of plasminogen activator inhibitor-1 promoter gene and pulmonary thromboembolism in Chinese Han population].

OBJECTIVE: To determine the prevalence of polymorphisms in the plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G polymorphisms in Chinese Han population and to investigate whether they are associated with pulmonary thromboembolism (PTE). METHODS: Samples of peripheral venous blood were collected from 101 patients with PTE diagnosed by high probability of lung ventilation/perfusion scan and/or multi-slice CT pulmonary angiography (CTPA) as well as medical history and clinical manifestations, 67 males and 34 females, aged 48 +/- 15, and 101 age and sex-matched healthy controls from the same geographic area as controls. The genome DNA was extracted from the whole blood using potassium iodide-phenol-chloroform method. Polymerase chain reaction (PCR), denaturing high performance liquid chromatography (DHPLC), and sequence analysis were used to screen the single nucleotide polymorphisms and the genotype distribution of -675 4G/5G located in the promoter region of the PAI-1 gene. RESULTS: The frequencies of the allele 4G of PAI-1 gene in the controls were 0.495, significantly lower than in the PTE patients (0.733, chi(2) = 24.060, P < 0.01). The frequencies of the allele 5G of PAI-1 gene in the controls were 0.505, significantly higher than that in the PET patients. The genotype frequency of 4G4G of the PET patients was 57.4%, significantly higher than that of the controls (30.7%, P = 0.000). The genotype frequencies of 4G5G and 5G5G of the PET patients were 31.7% and 10.9% respectively, not significantly different from those of the controls (37.6 and 31.7% respectively). The presence of 4G allele of PAI-1 gene was found to be a greater risk factor for PTE. In comparison with the controls, the OR of 4G4G + 4G5G, 4G4G, and 4G5G in the PET patients were 3.794 (1.786 - 8.060), 5.443 (2.416 - 12.260), and 2.450 (1.067 - 5.623) respectively with the P values of 0.001, 0.000, and 0.035 respectively. CONCLUSION: The 4G/5G and 4G/4G genotypes are associated with the pathogenesis of PET.T.

[The role of polymorphonuclear cells in lung ischemia-reperfusion injury in a canine model of pulmonary thromboembolism].

OBJECTIVE: To explore the effects of polymorphonuclear cells (PMN) on lung ischemia-reperfusion (I/R) injury in a canine model of pulmonary thromboembolism. METHODS: Fifteen dogs were divided into three groups: a sham group (n = 5), an ischemia group (n = 5) and a reperfusion group (n = 5). PMN in the whole blood were isolated with density gradient centrifugation. Apoptosis rates of the PMN was measured through flow cytometer after the cells were labeled by Annexin V-FITC before embolectomy and at 2, 4, 6 h after the operation. The myeloperoxidase (MPO) concentrations in lung homogenates were measured by ELISA. Alveolar PMN in the reperfusion lobar were observed by optical microscopy. The lung ultrastructure was studied by transmission electron microscopy. RESULTS: Alveolar PMN infiltration and the concentrations of MPO in the reperfusion group were significantly higher than those in the ischemia group (PMN (31 +/- 11) vs (8 +/- 4)/ten high power fields, MPO (11.7 +/- 1.6) vs (9.1 +/- 0.5) microg/L, P < 0.05). In the reperfusion group, abundant inflammatory cell infiltration was observed, predominantly with PMN in the alveoli. Apoptosis rates of the blood PMN at 6 h after reperfusion were much lower than before reperfusion (3.0 +/- 2.5)% vs (7.4 +/- 4.5)%, P < 0.05). At 4 and 6 h after operation, the PMN apoptosis rates in the reperfusion group were significantly lower than the ischemia group (4 h: (4.8 +/- 2.6)% vs (9.3 +/- 2.0)%, 6 h: (3.0 +/- 2.5)% vs (8.0 +/- 1.6)%, P < 0.05). PMN attaching firmly to the alveolar septum were observed by electron microscope. CONCLUSION: PMN with enhanced activities and decreased apoptosis rate, are involved in the cellular mechanisms of the lung I/R injury in this model of pulmonary thromboembolism.

[Effects of different durations of thromboembolism on blood gases, hemodynamics, pulmonary arteriography and thrombo-pathology in a canine model with selective embolization of pulmonary lobar arteries].

OBJECTIVE: To investigate the effects of different durations of thromboembolism on blood gases, hemodynamic parameters, pulmonary arteriography and thrombo-pathology in an animal model mimicking chronic pulmonary thromboembolism (PTE). METHODS: Sixteen dogs were embolized with five thrombi developed by autologous blood into the left lower pulmonary artery (n = 15) and the right lower pulmonary artery (n = 1, used to confirm the available method of selective embolization). The 15 dogs were divided into three groups: sham group (n = 5), one-week group (n = 5) and two-week group (n = 5) according to the different durations of embolization. Swan-Ganz catheter was used to guide a plastic duct, through which the thrombi were injected selectively into the left or right lower pulmonary artery by X-ray fluoroscopy. Local pulmonary arteriography of lower pulmonary arteries was taken. Blood pressure (BP), and blood gases were measured. Central venous pressure (CVP), mean pulmonary arterial pressure (MPAP), pulmonary arteriole wedge pressure (PAWP), and cardiac output (CO) were recorded, and pulmonary vascular resistance (PVR) was calculated. Each dog underwent muscular injection with tranexamic acid for one or two weeks to prevent thrombolysis. The lower lung lobe was dissected to confirm the thromboembolism after one or two weeks. The lung sections were stained with phosphotungstic acid hematoxylin (PTAH) to observe thromboemboli with optical microscopy. RESULTS: In the PTE group, PaO(2)/FiO(2), MPAP and PVR changed significantly as compared to baseline values (P < 0.05) after one hour of embolization, with MPAP increasing from (15 +/- 3) mm Hg to (21 +/- 4) mm Hg, PVR increasing from (178 +/- 114) mm Hg.s/L to (404 +/- 260) mm Hg.s/L, and PaO(2)/FiO(2) decreasing from (508 +/- 58) mm Hg to (395 +/- 100) mm Hg; these parameters returned to the baseline values one or two weeks later. After embolization, pulmonary arteriography demonstrated lower lobar artery cut-off perfusion defects. One week later, pulmonary arteriography demonstrated irregularities and stiffness of the arterial wall, enlarged proximal part of lower pulmonary artery and cut-off perfusion defects. Poor filling at embolus site was evident after embolization for two weeks. In the 1-week PTE group, organized tissue covered with the blue-purple fibrin nest was observed in the thrombus with PTAH stain. In the two-week group, the well organized thrombi were partially recanalized and surrounded and invaded by hyperplastic tissues from pulmonary artery wall. CONCLUSIONS: A canine model mimicking chronic PTE can be established by the use of fibrinolytic inhibitor tranexamic acid. Different manifestations on pulmonary arteriography and varied degree of organization of thrombi are evident at different times after embolization.

Relationship between polymorphisms of genes encoding microsomal epoxide hydrolase and glutathione S-transferase P1 and chronic obstructive pulmonary disease.

BACKGROUND: Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD). However, only 10% - 20% of chronic heavy cigarette smokers develop symptomatic disease. COPD is most likely the result of complex interactions between environmental and genetic factors. Genetic susceptibility to COPD might depend on the variations in enzyme activities that detoxify cigarette smoke products, such as microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST). In this study, we investigated the relationship between polymorphisms in the genes encoding mEH and glutathione S-transferase P1 (GSTP1) and COPD in a Chinese population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find mEH polymorphism in exon 3 (Tyr113-->His), exon 4 (His139-->Arg) and GSTP1 polymorphism in exon 5 (Ile105-->Val) in 100 COPD patients and 100 age- and sex-matched healthy controls. RESULTS: The proportion of mEH exon 3 heterozygotes was significantly higher in patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarette years was 2.96 (95% CI 1.24 - 7.09). There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and the controls. When COPD patients were non-smokers, the OR of very slow activity genotype versus other genotypes was more than 1.00; and when COPD patients were smokers (current smokers and ex-smokers), the OR was less than 1.00. There was no significant difference in GSTP1 polymorphism adjusted by age, sex, BMI and smoking between COPD patients and the controls. CONCLUSIONS: mEH exon 3 heterozygotes might be associated with susceptibility to COPD in China. The interaction might exist between mEH genotype and smoke. The gene polymorphism for GSTP1 might not be associated with susceptibility to COPD in the Chinese population.

[Expression of matrix metalloproteinases in lung tissue and levels of some cytokines in bronchoalveolar lavage fluid in the obstructive emphysema rat models].

OBJECTIVE: To observe the changes of matrix metalloproteinase-2 (MMP-2), MMP-9 and interleukin-10 (IL-10), tumor necrosis factor-alpha (TNFalpha) in lung tissue of the obstructive emphysema rat models and to evaluate the relationship between these changes and emphysema formation. METHODS: The rat emphysema models were established by exposure to cigarette smoking. Pulmonary function tests were performed to evaluate the forced expiratory volume in 0.3 second (FEV(0.3)), FEV(0.3)/forced vital capacity (FVC) and functional residual capacity (FRC). The morphological indices of emphysema were measured by computer image analyzer. The protein expression and enzymatic activity of MMPs in lung tissue were observed. The contents of IL-10 and TNFalpha in bronchoalveolar lavage fluid (BALF) were measured. RESULTS: Pulmonary function test showed that in model group the FEV(0.3)/FVC was decreased, whereas the FRC was increased significantly than those in control group (P < 0.01, respectively). There was a significant decrease in the relative content of elastin in lung tissue in model group than that in control group. The expression and enzymatic activity of MMP-2 and MMP-9 in lung tissue, the counts of total leukocytes and neutrophils and the level of TNFalpha in BALF were significantly elevated, but the level of IL-10 in BALF was significantly reduced in model group compared with those in control group (P < 0.01, respectively). There were statistically significant positive correlations between the enzymatic activity of MMP-2, MMP-9 and the total leukocyte counts (P < 0.01, respectively), and significant negative correlations between the enzymatic activity of MMP-2, MMP-9 and the content of elastin (P < 0.05, respectively). CONCLUSIONS: MMP-2, MMP-9, IL-10 and TNFalpha may play an important role in the formation of obstructive emphysema in rat models caused by passive smoking.

[Association between polymorphisms in the microsomal epoxide hydrolase (mEH) gene and chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the association between polymorphisms in the microsomal epoxide hydrolase (mEH) gene and susceptibility to chronic obstructive pulmonary disease (COPD) in a Chinese population. METHODS: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed to genotype mEH polymorphisms in exon3 (Tyr113-->His) and exon4 (His139-->Arg) in 100 COPD patients and 100 age and sex matched healthy controls. RESULTS: (1) The proportion of mEH heterozygotes in exon3 was significantly higher in the patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarettes years was 2.96 (95% CI 1.24 - 7.09). (2) There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and controls. (3) When COPD patients were nonsmokers, the OR of very slow activity genotype versus other genotypes was more than 1.00, and when COPD patients were smokers (Current smokers and ex-smokers), the OR was less than 1.00. CONCLUSIONS: (1) mEH heterozygotes in exon3 might be associated with the susceptibility to COPD in China. (2) The interaction might be existed between mEH genotype and smoke.

[A canine model of acute pulmonary thromboembolism induced by autologous radioactive blood clots].

OBJECTIVE: To establish a canine model of pulmonary thromboembolism (PTE) for evaluating the effects of thrombolytic therapy. METHODS: The preparations of radioactive blood clots in vitro were made from fresh whole blood (from 6 donors) mixed with (99m)Tc-SC. After eluting the clots with saline solution, the stability and evenness of (99m)Tc-SC in the clots were determined. Then a canine model of PTE induced by these clots was established and the rates of spontaneous lysis were measured by the regions of interest (ROI) technique and the in vitro method. RESULTS: (99m)Tc-SC was stable in the radioactive blood clots after elution, and the radioactivities in the thrombi were well-distributed. The rates of thrombolysis were (6.2 +/- 4.0)% as measured by ROI and (6.0 +/- 2.7)% by the in vitro method. CONCLUSIONS: (99m)Tc-SC is stable and well-distributed in blood clots. A canine model of PTE can be induced by autologous radioactive blood clots.

[Experimental study of the thrombolytic effects in a canine model of pulmonary thromboembolism induced by autologous radioactive blood clots].

OBJECTIVE: To compare the thrombolytic effects of the two dosing regimes with urokinase (UK) in a canine model of pulmonary thromboembolism induced by radioactive blood clots. METHODS: Seventeen dogs were randomly assigned into three groups: the control group, the UK(2h) group (UK infused over 2 hours) and the UK(12h) group (UK given over 12 hours). The thrombolytic differences was investigated among the three groups. Thrombolysis was assessed by continuously counting over both lung fields with single photon emission computed tomography (SPECT) and calculated by regions of interest (ROI) technology and by counting radioactivity in the lung in vitro. The extent of thrombolysis was calculated as the difference between the radioactivity originally incorporated in the clot (decay-corrected) and the radioactivity in the lung in vitro. RESULTS: In three groups, the lysis rates measured by ROI technology were (6.2 +/- 4.0)%, (39.5 +/- 13.9)%, and (16.9 +/- 8.9)% respectively, and (6.0 +/- 2.7)%, (42.8 +/- 12.4)%, and (17.7 +/- 9.3)% by the method in vivo. The thrombolytic ratio of the UK(2h) group was significantly higher than that of the other two groups (P < 0.01), and there was no marked difference between the control group and the UK(12h) group. There was a thrombolytic peak in the UK(2h) group at the first four hours after infusion of agent. CONCLUSIONS: For the fresh thrombi, the UK(2h) regime is superior to the UK(12h) due to its higher thrombolytic ratio and prompt thrombolytic property. The model and methods are highly reliable.

[Association between polymorphisms in the gene coding for glutathione S-transferase P1 and chronic obstructive pulmonary disease].

OBJECTIVE: To investigate the association between polymorphisms in the gene coding for glutathione S-transferase P1 (GSTP1) and susceptibility to chronic obstructive pulmonary disease (COPD) in a Chinese population. METHODS: This was a pilot study of the molecular epidemiology in patients with COPD. The research design was a case-control study. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed to genotype GSTP1 polymorphisms in exon 5 in 100 COPD patients and 100 age and sex matched healthy controls. Logistic regression analysis method was used to calculate the odds ratio (OR) and the 95% confidence interval (CI). RESULT: There was no significant difference in GSTP1 polymorphisms adjusted by age, sex, body mass index and smoking between COPD patients and controls. CONCLUSION: The gene polymorphism for GSTP1 was not associated with susceptibility to COPD in the Chinese population.