Study On The Pathological Characteristics Of COPD Related With Biomass Fuels Exposure And Possible Involvement Of Vegf In Its Development

Chronic obstructive pulmonary disease(COPD)is a serious threat to human health.At present,it is the fourth killer in the world.It is expected to rise to the third killer in 2020.Biomass fuels smoke(BMF)exposure is an important risk factor for COPD.The BMF smoke exposure population in China is very large,and the task of prevention and control of COPD caused by BFM(BMF-COPD)is particularly arduous.The analysis of the pathological characteristics of BMF-COPD and the possible mechanisms underlying its specific phenotype are of great significance in guiding clinical diagnosis and treatment.In this study,the self-developed software integration system was used to analyze the lung CT image data of BMF-COPD patients,and HE staining and apoptosis detection were used to observe the lung pathological changes of COPD rat model induced by BMF smoke exposure.At the same time,thirteen cytokines in peripheral blood of BMF-COPD patients which are closely related to the pathogenesis of COPD,were measured,and their correlation with pulmonary imaging and pathological changes of lung tissue were analyzed.Based on the finding that vascular endothelial growth factor(VEGF)is significantly related to the changes of pulmonary vessels and peripheral alveolar structure,the changes of CT images of the lung in the patients who have used VEGF antagonist were analyzed retrospectively to explore its possible role and significance in the pathological changes of BMF-COPD.Part I Lung CT characteristic of BMF-COPD patients and analysis of serum cytokinesObjective:CT images of lungs and serum cytokines in patients with BMF-COPD were analyzed,and their imaging characteristics and cytokine changes were studied.To study the pathological characteristics of BMF-COPD patients by analyzing the lung CT.Methods:1 Analysis of CT characteristics of lungs in patients with BMF-COPD(1)Subjects:8 people with BMF-COPD,8 healthy people with BMF exposure were recruited from the COPD community screening database,and 8 healthy people without smoking and BMF exposure were recruited from physical examination population of the first people's Hospital of Guangzhou city.The baseline characteristic data were collected.Informed consents were signed.(2)Development of lung CT analysis integrated system:Based on the method of Matsuoka,a lung CT image analysis system that could detect small blood vessels in the lung was developed.(3)Airway thickness,emphysema and the proposition of pulmonary small vessels in lung CT were analyzed in all the groups.(4)The characteristics of CT image of BMF-COPD patients was analyzed by reviewing the literature and comparing the CT image characteristics of COPD patients who smoke and have not been exposed to BMF smoke.Methods:2 Determination of serum cytokines in patients with BMF-COPD(1)Subjects:221 subjects were recruited from community screen COPD-data bank of four cities including Guangzhou and Shaoguan,including 59 cases of BMF-COPD in COPD stable stage,162 healthy subjects,which included 36 healthy ones without smoking and BMF exposure,32 healthy ones with BMF exposure,68 healthy ones with cigarette smoke exposure and 26 healthy persons with smoking and BMF exposure.(2)Collect baseline information,detect lung function,collect serum samples,etc.were collected.All subjects signed informed consent.(3)The serum concentrations of 13 cytokines were detected by Luminex system.(4)Statistical analysis:Correlation analysis of cytokine and BMF exposure was analyzed by bivariate correlation analysis.The comparison of cytokine levels of in different subgroups of COPD were studied by single factor analysis of variance.BMF exposure threshold was determined by Python programming:Each exposure was set as cut-off.The data above and below the cut-off were divided into two groups,and the correlation analysis of cytokines and exposure was performed on both groups.When cytokine was significantly correlated with the exposure.The exposure at the cut-off point was defined as the exposure threshold.Results:1 CT imaging results of lungs(1)There were no significant differences in baseline data(age,gender,BMI)among the three groups of subjects.Of the 8 patients with BMF-COPD,one patients was in GOLD stage I and seven patients were in GOLD stage II.(2)Distribution of emphysema:there were only a few areas of LAA-950 in healthy groups,and a larger area of LAA-950 in BMF-control groups(P>0.05),and a significant larger area of LAA-950 in BMF-COPD patients(P<0.05).The distribution of emphysema in all the groups was as follows:The upper lung/lower lung ratio of emphysema was 0.72 in the healthy group,0.87 in the BMF-control group,and 0.88in the BMF-COPD group.However,there was no significant difference in the overall distribution of different parts in all the groups(P>0.05)(3)The proportion of small vessels:the proportion of total small vessels and the proportion of small vessels in middle lung and lower lung in the BMF healthy group and BMF-COPD group were significantly lower than that in healthy control group,P all<0.05.The proportion of small vessels in patients with BMF-COPD group was lower than that in BMF healthy group,but there was no significant difference(P>0.05).(4)Airway remodeling sign:among the BMF-COPD group,the BMF-control group and the healthy control group,there was no significant difference of the airway wall thickness(WA%)and the inner diameter of the right upper apical segment(P>0.05).The small airway wall area percentage(WA%)in BMF-COPD was 82.2�3.58%,which was thicker than that in the BMF-control group(76.61�3.20%),P<0.05.There was no significant difference of the small airway wall thickness between BMF-control group(76.61�3.20%)and the healthy group(77.33�2.45%),P>0.05.(5)The proportion of LAA-950 was negatively correlated with the proportion of pulmonary small vessels in the healthy group and the BMF-COPD group(r=-0.67,P=0.03).However,there was no correlation between the proportion of pulmonary small vessels and airway thickness(r=-0.12,P=0.54).(6)In the literature,CT imaging of the lungs of smoking-COPD patients is mainly emphysema of the upper lobe,especially in patients with GOLD stage I and GOLD stage II.The small blood vessels in the upper lobe are reduced,and small airway remodeling are found in smoking healthy people.After comparative analysis,the emphysema of BMF-COPD was evenly distributed in the lungs,and the reduction of small blood vessels was more pronounced in the lower and middle lungs.No small airway remodeling was observed in the BMF-control group.2 The results of serum cytokine detection(1)There was no significant difference in the age and BMI among all the groups.In the BMF-COPD group,according to GOLD classification,there were 39 patients in GOLD stage I,16 patients in GOLD stage II,4 patients in GOLD stage III,and no patients in GOLD stage IV.According to the COPD subgroup classification,there were 37 patients in GOLD group A,18 patients in GOLD group B,3 patients in GOLD group C,and 1 patient in GOLD group D.(2)For eleven inflammatory cytokines,including IL-1?,IL-4,IL-5,IL-6,IL-13,IL-17,Eotaxin,IFN-?,MIP-1?,MIP-1?,IP-10,there was no significant difference among different subgroups of COPD,P>0.05.For the correlation between serum cytokine concentration and BMF exposure,only IL-4 and IFN-?had weak negative correlation,P<0.05.The growth factor PDGF-BB involved in airway remodeling and vascular remodeling was not related to BMF exposure,and there was no significant difference of PDGF-BB serum concentration among different subgroups of COPD,P>0.05.No positive threshold BMF exposure was found in PDGF-BB-related study.(3)The serum concentration of VEGF,which is related to airway remodeling,emphysema,angiogenesis and vascular endothelial cell apoptosis,was not only related to the BMF exposure,but also to the severity of COPD:After BMF exposure?40 years*hours,serum VEGF concentration was significantly negatively correlated with BMF exposure(P=0.01,r=-0.33).For COPD patients with mild symptoms(CAT score?10),VEGF was negatively correlated with BMF exposure(P=0.046,r=-0.33).For COPD patients with severe symptoms(CAT score?10)and higher risk(COPD group D),the VEGF level is 49.28�34.77 pg/ml,which is significantly lower than patients in COPD group B(121.6�54.03 pg/ml),P=0.04.In the healthy group,the serum VEGF is 82.15�52.87 pg/ml in BMF healthy group,which was significantly lower than that in the smoking healthy group(133.1�86.94pg/ml),P<0.05.Brief summary:1.Compared with the CT imaging results of COPD caused by smoking,those exposed to BMF have their own characteristics.No airway remodeling was found in the BMF-control group,but there were extensive reductions in small blood vessels.BMF-COPD is characterized by evenly distributed emphysema and a significant decrease in the proportion of small and middle pulmonary vessels.The proportion of small blood vessels was significantly inversely related to the proportion of emphysema.2.Serum VEGF expression was correlated with BMF exposure and negatively correlated with COPD severity.Part II Correlation between lung lesions and vascular endothelial growth factor in BMF-RCOPD rat modelsObjective:To analyze the pathological characteristics in lung tissue of BMF-RCOPD rat models and its relationship with VEGF,so as to explore the possible role of VEGF in the development of BMF-COPD.Methods:1 Ten SD rats of 6-8 weeks of age were randomly divided into BMF smoke exposure group(BMF group)and control group.By fir firing method,,rats in BMF-RCOPD group were exposed in BMF smoke for 6 months:twice a day,2 hours each time.Rats in control group were kept in the clean air for 6 months.2 HE staining were used to evaluate the pathological changes of lung:the thickness of airway wall,the parameters of emphysema,which including MLI,MAN and MAA.Apoptosis of vascular endothelial cells was detected by vascular endothelial cells labeling plus fluorescence TUNEL staining.3 VEGF expression in rat lung tissue were detected by Western blot and immunohistochemistry.4.Bivariate correlation analysis was performed between VEGF expression in rat lung tissue and lung pathological changes.Results:1 In HE staining sections,COPD-like changes were observed after 6 months of BMF exposure,which were manifested as cilia shedding of airway epithelial cells,infiltration of inflammatory cells,thickening of airway wall,enlargement of alveolus cavity(an emphysema-like change).2 After 6 months of BMF exposure,the thickness of airway wall in the BMF group was 37.44(26.40-57.68)um,which was significantly thicker than the control group(20.42(17.40-24.95)um)(P<0.05),for the indexes of emphysema,MLI,MAA increased,MAN decreased in BMF group,P all<0.05.The specific values were as follows:MLI length in the peripheral and central regions of BMF group was 103.9�15.36um and 89.92�14.74um,respectively,both of which were longer than that in the peripheral(64.91�4.51um)and central region(68.70�6.17um)of control group,P<0.05,MAN in the peripheral and central regions of the BMF group were 100.00�30.38/nm~2 and 102.6�25.75/nm~2,respectively,both of which were significantly lower than that in the peripheral(200.60�53.39/nm2)and the central region.(214.40�86.13 cells/nm2)of control group,P all<0.05;MAA in the peripheral region of the BMF group was 10497�3818 um~2,which was higher than that in the peripheral(4986�1967 um~2)and central area(4680�2553um~2),P<0.05;MAA in the central lung region of the BMF group was 9466�2339um~2,with no significant difference that in the peripheral region(4986�1967 um2)and the central region(4680�2553um2)of control group,both P>0.05.3 The apoptosis number of pulmonary vascular endothelial cells was 316(273-416)in BMF group,which is significant higher than in control group 27(12.7-103.6),P=0.00.The number of normal vascular endothelial cells in BMF group is 122(73-227),which is significant lower than that in control group(547(450-984),P=0.02.4 VEGF expression in lung tissue of rats:The expression of VEGF in lung tissue of rats in BMF group was significantly lower than that in control group,in Western blot detection(0.43�0.07vs 0.68�0.06,P=0.04).In immunohistochemistry detection,VEGF was strongly expressed in the bronchial epithelial cells and smooth muscle cells,while the expression in the alveolar epithelial cells was weaker than that in the bronchial epithelium in control group.After 6 months of BMF exposure,the relative values of VEGF in the central and peripheral regions of the lungs in the BMF group were 6050(5101-8581)and 2454(2156-3248),respectively.The relative values of VEGF in the central and peripheral regions of the lungs in the control group were10150(9722-11235)and 9800(6071-13950),respectively.The expression of VEGF in both the central and peripheral areas of the lung was significantly lower than that in the control group(both P<0.05).In the BMF-RCOPD group,the VEGF expression in the peripheral region was significantly lower than in the central region,P<0.05.There was no significant difference of VEGF expression in the bronchus between BMF group(6.80(5.76-37.6))and control group(21.51(16.16-41.51)),P>0.05.5 VEGF expression in lung parenchyma was negatively correlated with the proportion of emphysema,P<0.05.VEGF expression in lung parenchyma was significantly negatively correlated with MLI(r=-0.63,P=0.00),significantly positively correlated with MAN(r=0.70,P=0.00),and significantly negatively correlated with MAA(r=-0.63,P=0.00).There was no correlation between the expression of VEGF in the bronchus and the thickness of bronchial wall(P=0.21,r=-0.42).6 The expression of VEGF in lung tissue was negatively correlated with the number of endothelial cell apoptosis(P=0.02,r=-0.73).Brief summary:After 6 months of BMF exposure,the rats showed COPD manifestations:increased airway wall thickness,emphysema-like changes,decreased number of pulmonary vascular endothelial cells and increased apoptosis of pulmonary vascular endothelial cells.The expression of VEGF in the lung parenchyma of the BMF-RCOPD group was significantly reduced,and the expression of VEG in lung tissue was inversely correlated with the severity of emphysema and the number of pulmonary vascular endothelial cells apoptosis.There was no significant change in the bronchial VEGF expression between control group and BMF group,and VEGF expression in the bronchus did not correlate with the thickness of the bronchial wall.Part III The effect of VEGF monoclonal antibody on airway wall thickness and emphysema in patients with lung cancer and colorectal cancerObjective:The lung CT features of lung cancer and colon patients treated with VEGF monoclonal antibody were analyzed retrospectively,and the effects of VEGF monoclonal antibody on emphysema and airway remodeling were analyzed.Methods:1.Retrospective study design:Patients with lung cancer and colorectal cancer over 40 years old treated in in Guangzhou No.1 People's Hospital from 2017 to2018 were recruited.Those who were treated with bevacizumab were included in VEGF monoclonal antibody group,and those treated patients with no bevacizumab and similar chemotherapy regime were included in control group.Exclusion criteria:use of other targeted inhibitors,incomplete data,co-morbidities and other conditions,which affected lung CT assessment.Healthy people and BMF-COPD patients recruited in Part I study were taken as normal control group and BMF-COPD control group.2 CT analysis:the HRCT of the two groups before and after treatment and after drug withdrawal were analyzed,and the changes of emphysema,airway wall thickness and the proposition of small blood vessels were compared.Diffuse emphysema is defined as centrilobular emphysema with or without paralobular emphysema or bullae.localized emphysema is defined as pure paralobular emphysema or bullae.Results:1 In VEGF monoclonal antibody group,18 cases were recruited,including 4cases with diffuse emphysema,2 cases with localized emphysema and 12 cases without emphysema.In the control group,69 patients were recruited,including 25 patients with diffuse emphysema,14 patients with localized emphysema and 44 patients without emphysema.There was no significant difference between the two groups in lung cancer/colorectal cancer ratio,gender,age,emphysema/non emphysema ratio(P>0.05).The diffuse emphysema patients in VEGF monoclonal group had the similar COPD manifestations as BMF-COPD group:with no significant difference in bronchi wall thickness and proportion of emphysema and small airway wall thickness(P all>0.05).2.For patients with localized emphysema,there was no significant difference in the change of emphysema between VEGF monoclonal antibody group and control group(P>0.05).For patients with diffuse emphysema,the change of small airway wall thickness had no significant difference between VEGF monoclonal antibody group and control group(P>0.05),and the change of emphysema percent is significantly higher in VEGF monoclonal antibody group than control group(P<0.05):The fusion and enlargement of paralobular emphysema or bullae were more obvious.3.After bevacizumab treatment,3 patients without emphysema developed emphysema.After discontinuation of treatment,emphysema could basically return to the level of pre-treatment.5 The increase of LAA-950%by 3%after bevacizumab treatment was set as cut-off.The VEGF monoclonal antibody group was divided into emphysema increase group and non-response group.There was no significant difference in the course of bevacizumab treatment between the groups(P=0.08).Brief summary:The decrease of VEGF is related to the occurrence of emphysema and has little relation with airway remodeling.After VEGF monoclonal antibody intervention,emphysema in patients with localized emphysema did not change significantly.In patients with diffuse emphysema,airway remodeling did not improve significantly,but emphysema worsened;emphysema occurred in some patients with non-emphysema,and it can be recovered after stopping treatment.Conclusion1 CT signs of BMF-RCOPD are characterized by extensive small blood vessel changes and evenly distributed emphysema.2.BMF smoke exposure can reduce the expression of VEGF in the lungs of rats and increase the apoptosis of vascular endothelial cells.The expression of VEGF is negatively correlated with the proportion of emphysema and the number of vascular endothelial cells.3.VEGF monoclonal antibody has little improvement on airway remodeling and can aggravate emphysema.