Endothelin-1 And Prostacyclin Are Involved In The Pathogenesis Of Chronic Obstructive Pulmunary Disease

Chapter 1 Evaluation of Endothelin-1 in patients with stable chronic obstuctive pulmonary diseaseObjectiveThe purpose of this study was to examine the levels of endothelin-1 (ET-1) in the bronchoalveolar lavage fluid(BALF),induced sputum and plasma in patients with stable chronic obstructive pulmonary disease (COPD) and to elucidate whether ET-1 is implicated in the pathological process of COPD.MethodsThirteen patients with COPD,14 healthy smokers and 14 normal controls were enrolled in the study.BALF and induced sputum were collected.Cells in BALF and sputum suspension were counted and classified with Wright's stain.The ET-1 levels were measured by radioimmunoassay in all of samples.Results1.The differences of FEV1%pre,FEV1/FVC%and PEF between healthy smokers and normal controls weren't significant(p＞0.05). FEV1%pre,FEV1/FVC%and PEF in COPD patients were significantly lower than those in healthy smokers and normal controls(p＜0.01).2.The total BALF and sputum cells in healthy smokers and patients with COPD were significantly higher than those in control group(p＜0.01).In addition,the numbers of alvelor macrophages(AM) and neukocytes(PMN) in both healthy smokers and patients with COPD were significantly higher than those in control group(p＜0.01).3.There wasn't significant difference in the ET-1 levels in BALF of three groups(p＞0.05).The sputum ET-1 levels in healthy smokers and patients with COPD were significantly higher than those in control group (p＜0.05).The ET-1 levels in plasma in COPD patient were significantly higher than those in healthy smokers and control group(p＜0.05).4.The numbers of AM were inversely correlated with FEV1%pre and FEV₁%FVC(r=-0.480,p＜0.01;r=-0.545,p＜0.01) while the numbers of PMN were inversely correlated with FEV1%pre and FEV₁%FVC(r=-0.677,p＜0.01;r=-0.773,p＜0.01) in patients with stable COPD.ET-1 levels in sputum and plasma were inversely correlated with FEV1%pre in stable COPD patients(r=-0.723,p＜0.01;r=-0.801, p＜0.01).In addition,the increase in sputum ET-1 levels was correlated with the increase of the AM numbers and plasma ET-1 levels(r=0.543,p＜0.01;r=0.864,p＜0.01).Conclusion1.There are chronic inflammatory process in airway of stable COPD and both PMN and AM contribute to the process.2.ET-1 may have a role in contributing to airway inflammation and airway remodeling in the pathological process of COPD.Chapter 2 The expression of prostacyclin is decreased in patients with stable chronic obstuctive pulmonary diseaseObjectiveTo investigate the expression of prostacyclin(PGI₂) synthase (PGI₂S) and 6-keto-PGF 1αthat is a production of endogenous PGI₂ by non-enzymatic hydration in the supematants of lung homogenates in patients with stable COPD.MethodsThe lung tissues were obtained from 12 patients with stable COPD and 10 controls.The expression of PGI₂S protein was assessed by immunohistochemistry using paraffin-embedded sections.Concentration of 6-keto-PGF1αwas measured by ELISA in the supernatants of lung homogenates.Sections were scored by a pathologist in blinded fashion for PGI₂S staining and expressed as a ratio in small/medium blood vessels and airway epithelia.Results1.FEV1%pre and FEV1/FVC%in COPD patients were significantly lower than those in controls(p＜0.01). 2.The differences in PGI₂S staining were statistically significant in pulmonary small/medium-sized vessels and airway epithelia(p＜0.05) between COPD patients and controls.3.The concentration of 6-keto-PGF1αin the supernatants of lung homogenates in COPD group was significantly lower than that of the control group[(2.6±0.4) vs(16.2±2.8) ng/ml,p＜0.05].ConclusionsThe expression of PGI₂ is decreased in COPD patients and PGI₂ may be involved in the pathogenesis of COPDChapter3 Treatment with Endothelin-1 receptor antagonists prevents pulmonary emphysema in ratsObjectiveEndothelin(ET) is involved with physiologic functions related to respiratory system and seems to implicate the pathogenesis of pulmonary emphysema.We hypothesized that ET-1 plays an important role in the pathogenesis of emphysema and,thus the ET receptor antagonists may have protective role in the development of emphysema.MethodsSprague-Dawley rats(n=24) were divided into four groups:(1) control group,(2) cigarette smoke extract(CSE) group,(3) CSE+ BQ-123 group(a selective ETA receptor antagonist),and(4) CSE+ bosentan group(a mixed ETA/ETB receptor antagonist).CSE was injected intraperitoneally once a week for three weeks and BQ-123 and bosentan were administered daily for the same duration.Apoptosis index, caspase-3 activity,MMP-2 and MMP-9 activities and TNF-αand IL-1βconcentrations were measured in the lung tissues.Antioxidant activity were measured in the serums.Results1.We firstly confirmed the emphysematous destruction in the lungs of experimental rats induced by the intraperitoneal injection of CSE within 3 weeks.The MLI and DI were significantly increased in the CSE group(108.7±6.8μm and 62.2±7.0%,respectively) compared with control group(69.8±6.6μm,p＜0.01 and 13.9±2.7%,p＜0.01, respectively).However,the MLI and DI were significantly decreased in the BQ-123(89.0±7.4μm and 41.5%±4.5%,respectively) and bosentan groups(81.9±6.1μm and 44.0%μ8.5%,respectively) compared with CSE rats(p＜0.01,respectively).2.The TUNEL-positive cells were markedly distributed in the peribronchioles,intra-alveoli,and septal areas of the emphysematous lungs in CSE rats comparing with the lungs of control rats.The AI was significantly higher in CSE group(20.3±0.9%) than control group(2.9±0.9%,p＜0.01).And the AI was significantly reduced in BQ-123 group(9.9±2.3%,p＜0.01) and bosentan group(8.8±0.6%,p＜0.01) compared with that in CSE group.3.The caspase-3 positive cells were markedly distributed in the emphysematous lungs of CSE group comparing with the stained cells in the lungs of control rats.These positive cells were apparently reduced in the BQ-123 and bosentan groups compared with the stained cells in CSE group.Comparing with the control group,expression of caspase-3 was prominently enhanced in CSE groups,but almost no changes in both the BQ-123 and bosentan groups.The relative density of the caspase-3 toβ-actin by the densitometry analysis showed that the protein level of the cleaved caspase-3 was increased in the CSE rats(218.2±64.7%) compared with that in control rats(98.2±34.3%,p＜0.05).Expectedly, both BQ-123 and bosentan treatments markedly inhabited the increases of the cleaved caspase-3 protein levels in rats injected with CSE (BQ-123:90.4±32.9%,Bosentan:84.9±30.8%,p＜0.05 in comparison to CSE).4.Rats injected with CSE showed increased MMP-2 and MMP-9 activities in their lung tissue homogenates(MMP-2:172.1±43.5%; MMP-9:168.9±38.6%) compared with the activities in the control rats (MMP-2:105.5±27.1%,p＜0.05;MMP-9:94.5±27.1%,p＜0.05). Expectedly,MMP-2 and MMP-9 activities were reduced significantly in both BQ-123(MMP-2:109.6±32.8%,p＜0.05;MMP-9:99.9±50.0%, p＜0.05) and bosentan groups(MMP-2:95.1±14.1%,p＜0.01; MMP-9:68.4±19.9%,p＜0.01) compared with the activities in the CSE group.5.The levels of TNF-αand IL-1βwere significantly increased in the CSE group in comparison to those in controls(p＜0.05, respectively).BQ-123 and bosentan significantly prevented the increases of the levels of TNF-αand IL-1βin lungs of rats with injection of CES (p＜0.05).In addition,the levels of TNF-αand IL-1βin the lungs were positively correlated with the AI in the lungs of rats(r=0.693,p＜0.01; r=0.555,p＜0.01,respectively).6.The serum antioxidant activity was significantly decreased in the CSE group(1653.4±504.7μmol/L) in comparison to that of controls (2438.9±235.6μmol/L,p＜0.01).The BQ-123 and bosentan significantly prevented the decrease of the serum antioxidant activity in the rats injected with CSE(BQ123:2551.1±144.8μmol/L,p＜0.01; Bosentan:2597.0±409.8μmol/L,p＜0.01).ConclusionET-1 receptor antagonists protect against the development of emphysema probably by decelerating apoptosis,inhibiting proteolytic enzyme activity,reducing inflammatory cytokine levels and improving antioxidant activity.ET-1 antagonists may represent a new therapeutic option in the treatment of emphysema in humans.Chapter 4 A novel protective effect of a prostacyclin analogue in the development of cigarette smoke extract-induced emphysemaObjectiveThe aim of this study was to determine whether the administration of PGI₂ analogue,beraprost(BPS) would attenuate the development of CSE-induced emphysema in a rat model and,further,to elucidate the molecular mechanisms involved with its effect.Methods Sprague-Dawley rats(n=24) were randomly divided into 4 groups:normal group,CSE group,low dose BPS group(BPS-1) and high dose BPS group(BPS-2).The CSE-PBS solution was injected intraperitoneally with 1 ml once a week.BPS was administered daily via a gastric gavage for 21 days.BPS was started at the day of the first CSE injection.TUNEL was performed to observe the DNA damaged cells and the expression of caspase-3 was determined by immunohistochemistry and Western Blotting.MMP-2 and MMP-9 activities were investigated by gelatin zymography and TNF-αand IL-1βconcentrations were measured by ELISA.We also analyze the serum antioxidant activity by bioantioxidant power(BAP) test.Results1.We confirmed that intraperitoneal injection of CSE in rats caused emphysematous destruction of the lung within 3 weeks.The MLI and DI were significantly increased in the CSE group compared with the values in the control group(MLI:108.7±6.8μm vs.69.8±6.6μm,p＜0.01 and DI:62.2±7.0%vs.13.9±2.7%,p＜0.01,respectively).The MLI and DI were significantly reduced in the lungs in the BPS-1 group(MLI: 83.9±10.5μm,p＜0.01;DI:49.3±7.5%,p＜0.01) and BPS-2 group (MLI:77.0±7.1μm,p＜0.01;DI:47.1±6.9%,p＜0.01) compared with those in the CSE group.2.The concentration of 6-keto-PGF1αwas significantly reduced in the lung tissues of the rats with CSE-induced emphysema(40.6±29.3μg/mg) compared with those of control rats(173.2±49.3μg/mg,p＜0.01).BPS treatment markedly increased the level of 6-keto-PGF1αcompared with that in CSE injected rats(BPS-1:134.4±52.1μg/mg,p＜0.05;BPS-2:432.9±71.5μg/mg,p＜0.01).It is curious that the 6-keto-PGF1αlevel in high-dose BPS group was three-folds higher than that in groups of control and low-dose BPS rats(p＜0.01).3.The TUNEL-positive(TUNEL⁺) cells were frequently localized in the peribronchiolar,intra-alveolar,and septal structures in the CSE-injected rats.The DNA damaged cells were very rare in the normal rats(AI:2.9±0.9%),while the TUNEL⁺ cells in CSE group were much more frequent(AI:20.3±0.9%,p＜0.01).BPS greatly reduced the number of TUNEL⁺ cells in the lungs of CSE treated emphysematous rats(AI:8.9±2.5%in BPS-1 group and 7.8±1.3%in BPS-2 group,p＜0.01 vs.controls for each).4.Rats injected with CSE showed a large number of caspase-3⁺ alveolar septal cells compared with the cells in the control and BPS treated rats.The protein levels of the cleaved form of caspase-3 were increased in the rats with CSE-induced emphysema(270.1±34.7%) compared with those of control rats(146.3±20.4%,p＜0.05).BPS treatment markedly reduced the protein level of cleaved caspase-3 compared with that in CSE injected rats(BPS-1:162.7±53.3%,p＜0.05;BPS-2:154.0±32.8%,p＜0.05).5.Rats injected with CSE showed increased MMP-2 and MMP-9 activities in their lung tissue homogenates(MMP-2:215.2±33.5%,p＜0.01;MMP-9:190.5±60.0%,p＜0.05) compared with the activity in the control rats(MMP-2:98.2±38.4%,MMP-9:84.1±29%).MMP-2 and MMP-9 activities were reduced in the two BPS treatment groups, but statistical significance occurred in MMP-2 activity of both low- and high-dose BPS treatment rats(BPS-1:148.1±45.3%,p＜0.05;BPS-2: 116.8＜53.3%,p＜0.01,respectively) while in MMP-9 activity in only high dose BPS treatment rats(97.3±40.6%,p＜0.05).6.The levels of TNF-αand IL-1βwere significantly increased in the CSE group compared with those in controls(p＜0.01).And high-dose BPS was significantly reduced the levels of TNF-αand IL-1βin the lungs of CSE treated emphysematous rats(p＜0.01).The levels of TNF-αand IL-1βwere positively correlated with the AI in the lungs of all rats including the control rats and emphysema rats induced by CSE injection(r=0.657,p＜0.01 and r=0.530,p＜0.01,respectively).7.The serum antioxidant activity was significantly decreased in the CSE treated rats(1653.4±504.7μmol/L,p＜0.05) compared with the control rats(2438.9±235.6μmol/L).BPS treatment significantly prevented the reduction of antioxidant activity in rats injected with CSE (BPS-1:2420±434.4μmol/L,p＜0.05,BPS-2:2344.8±664.6μmol/L, p＜0.05,in comparison to CSE group). 8.The cAMP levels were 2.31±1.04μg/mg in the lung tissues of BPS-1 group and 3.83±2.83μg/mg in BPS-2 group,in contrast to the undetectable cAMP levels in both the control rats and CSE-injected rats (less than 1.05μg/mg).ConclusionBPS protects against the development of CSE-induced emphysema by attenuating apoptosis,inhibiting proteolytic enzyme activity, reducing inflammatory cytokine levels and augmenting antioxidant activity.BPS may potentially represent a new therapeutic option in the prevention of emphysema in humans in prospect.