Analysis Of Interrelationship Of Circulating Matrix Metalloproteinase-9, TNF-α During Bone Metabolism Abnormality In COPD Patients With Osteoporosis

Background and purpose:Chronic obstructive pulmonary disease (COPD) is a common chronic respiratory disease characterized by airflow limitation, It has a high and increasing prevalence and mortality. By2020, COPD may become the third most common fatal disease in the world. Osteoporosis, a condition characterized by low bone mass and bone microarchitectural deterioration which predisposes to fragility fracture, is one of the most important systemic complications of COPD. The incidence of osteoporosis among COPD patients is significantly higher than in the general population. Clinical sequelae of serious osteoporosis, including compression fractures and kyphosis, can further reduce vital capacity, thereby further compromising respiratory status in patients with COPD. A correlation between reduced lung function and bone mineral density (BMD) is suggested by some studies, however, the mechanisms between COPD and osteoporosis are not yet fully clear.Emphysema is a kind of chronic pulmonary disease characterized by abnormal and permanent airspace enlargement beyond the terminal bronchioles accompanied by destruction of the alveolar walls. The most widely accepted theory of the pathogenesis is a proteinase-antiproteinase imbalance. Recent studies have shown that the balance between matrix metalloproteinases (MMPs) and their inhibitors(TIMPs) plays an important role in the breakdown and remodelling of ECM in the alveolar walls in COPD and asthma patients. MMPs, a group of calcium-activated zinc ion endopeptidases widely found in a variety of connective tissues, play an essential role in the degradation of the extracellular matrix and basement membrane enzyme. They are differentially expressed during different stages of COPD development. The TIMPs are endogenous natural inhibitors of MMPs. The airway MMP-9/TIMP-1ratio is considered a biomarker of airway tissue destruction and of the dynamic equilibrium of repair.In addition to lung disease, MMPs have been suggested to have a important role in bone remodelling and turnover. MMP-9is produced by osteoclasts and osteoclast precursor cells,the monocytes also. It is essential for recruitment of preosteoclasts into bone and their local migration into the diaphysis. In contrast, TIMP-1prevents bone resorption.Experimental studies indicate that TNF-α is a multifaceted cytokine and a well-recognized systemic inflammatory factor related to the occurrence and development of a variety of the systemic complications of COPD. TNF-a is also an important regulator of bone metabolism and remodelling. It maybe stimulate osteoclast differentiation in a synergistic manner. TNF-α,IL-1β,\IL-6are all well-established inflammatory cytokines involved in the systemic inflammation of COPD. There is an association between distinct serum inflammatory mediators and quantitative CT measurements of emphysema. Simultaneously, IL-1β, IL-6and TNF-a have been shown to be important modulators of bone metabolism and remodeling by stimulating osteoclast differentiation in a synergistic fashion. These cytokines may not only regulate osteoclastogenesis via their effects on stromal cells, but also act directly on osteoclasts and their precursors. In addition, they also interact with the osteoporosis-related OPG/RANK/RANKL protein system.The OPG/RANK/RANKL system is an important regulator of bone metabolism, and participates in the development of post-menopausal osteoporosis. This was a major breakthrough in the field of osteoporosis and bone metabolism and opened a new perspective on bone metabolic balance and osteoclast biology. RANKL and OPG are the promoting and inhibitory factors of osteoclast differentiation, respectively. They maintain a certain ratio within the body. Imbalances in this ratio can disrupt bone metabolism, leading to reduced bone mass/osteoporosis among other bone diseases. Whether MMP-9/TIMP-1, TNF-a,the OPG/RANK/RANKL pathway is involved in the pathogenesis of osteoporosis in COPD has not been studied.Osteoporosis is a metabolic condition characterized by decreased bone mass and strength due to increased bone turnover, which compromises bone architecture and results in increased fracture risk. Bone is a metabolically active tissue that undergoes continuous remodelling by two counter acting processes, namely bone formation and bone resorption. These processes rely on the activity of osteoclasts, osteoblasts and osteocytes. Acceleration of bone turnover, accompanied with a disruption of the coupling between these cellular activities,plays an established role in the pathogenesis of metabolic bone diseases, such as osteoporosis. In the past few decades, Many research has been working on biochemical markers which may reflect the rate of bone turnover. A large number of studies have shown that serum or urine biochemical markers are related to bone loss and fractures, which is important to identify high-risk patients. The biochemical markers of bone turnover and their applications in clinical trials can test the efficacy of drugs and how they supply the measurement of bone mass. Targeted use of biochemical markers can be further optimized to identify high-risk patients, the process of drug development and clinical monitoring of the efficacy of osteoporosis treatment. Biochemical markers are bound to be used widely because of their advantages of non-invasive, easy repeatability and timeliness.In this study, the correlations among serum MMP-9and its cognate inhibitor TIMP-1, TNF-a, and the OPG/RANK/RANKL system,bone biochemical markers were assessed, and their associations with impairment of lung function and bone mineral density in patients with COPD was evaluated.This study include two parts:Part I Analysis of interrelationship of circulating matrix metalloproteinase-9, TNF-a, and OPG/RANK/RANKL system in COPD patients with osteoporosisObjective:To study the relationship between matrix metalloproteinase-9(MMP-9), its cognate inhibitor TIMP-1, inflammatory cytokine TNF-a, and OPG/RANK/RANKL system in COPD patients with osteoporosis.Methods:1. Patients and sample collection:Ninety male patients diagnosed with clinically stable COPD were enrolled from Sep2010to May2012. Female patients were excluded from the study to avoid the influence of postmenopausal osteoporosis. COPD was diagnosed according to the criteria issued by the Global Initiative for Chronic Obstructive Lung Disease (GOLD). These ninety patients were divided into three groups based on BMD:those with COPD and normal BMD (T-score>-1.0), those with COPD with osteopenia (T score-1.0to-2.4), and those with COPD and osteoporosis (T-score≤-2.5). Each group contained30patients. All patients were more than40years old and all were current or former smokers. The exclusion criteria were as follows:(1)serious cardiovascular and cerebrovasculardiseases, liver or renal function failure,consciousness disorder,(2) history of respiratory diseases other than COPD, such as asthma, bronchiectasis,(3) history of bone disease, such as fractures,osteomalacia,(4) rheumatic diseases,such as rheumatoid arthritis,(5) metabolic, or inflammatory disease,such as hyperthyrosis, hyperparathyroidism,(6) inhaled, oral, or intravenous corticosteroid treatment within the last3months,(7) medication which may influence bone metabolism within the previous one year, including those being treated for osteoporosis,(8) occurrence of malignancy within the previous five years. All subjects provided informed consent before joining the study.On the recruiting day, all patients completed pulmonary function test and the life quality assessment using the COPD Assessment Test (CAT) and the measurement of BMD. The BMD of lumbar spine (L1-L4) and bilateral femoral neck was measured using dual X-ray absorptiometry(DXA). BMD is here expressed in g/cm2and a T-score, which was used for diagnosis according to the World Health Organization guidelines as follows:normal BMD:T score greater than-1at both sites (lumbar spine and FN); osteopenia:T score less than or equal to-1but greater than-2.5at either site; osteoporosis:T score less than or equal to-2.5at either site. Ninety patients were divided into three groups of30patients each based on BMD: those with COPD and normal BMD, those with COPD with osteopenia, and those with COPD and osteoporosis.2. The measurement of cytokines(Enzyme-linked immunosorbent assay,ELISA):Fasting blood samples were collected by venipuncture the next morning. These were centrifuged at1000×g for5min at room temperature to produce serum samples, which were stored at-80℃until analysis.High-sensitivity MMP-9(pro-MMP-9), TIMP-1, and TNF-α levels were determined using sandwich ELISA kits. OPG/RANK/RANKL concentrations were measured using ELISA kits too.3. Statistics:Data are expressed as the mean±SD and analyzed using the Statistical Package for the Social Sciences, SPSS13.0(SPSS Inc, Chicago, IL, U.S.). Data from the three experimental groups were analyzed with normally distributed test(shapiro-wilk test),one-way ANOVA with a post-hoc Tukey’s test, and Pearson’s correlation analysis. The relationships among parameters were evaluated using multiple linear stepwise regression analysis. A p-value<0.05was considered statistically significant.Results:The age, BMI, smoking index, FEV1/FVC, FEV1%Pre, and CAT scores of the COPD patients were compared between all groups of patients. COPD patients with osteoporosis had significantly lower BMI and higher CAT scores (all P<0.01) than COPD patients with normal BMD and those with low BMD. There was no significant difference in age, smoking index, FEV1/FVC, or FEV1%Pre(P>0.05). As expected, based on the study design, the lumbar spine and femoral neck BMD was significantly lower in the groups of COPD with low BMD (P<0.05, P<0.01) and COPD with osteoporosis (all P<0.01) than in COPD with normal BMD.Pearson analysis revealed positively correlations between BMI and the lumbar spine BMD (r=0.278, P<0.01) and femoral neck BMD (r=0.273, P<0.01); negative correlations between CAT scores and the lumbar spine BMD (r=-0.331, P<0.01) and femoral neck BMD (r=-0.486, P<0.01). The serum level of MMP-9, MMP-9/TIMP-1ratio, the serum level of TNF-a increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.05or P<0.01). However, no difference in the serum level of TIMP-1was detected among the three groups(P>0.05). The serum levels of OPG, RANKL, the RANKL/OPG ratio increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.01). However, no difference existed in the level of RANK among these three groups(P>0.05).Pearson analysis revealed negative correlations between MMP-9serum levels and the lumbar spine BMD (r=-0.432, P<0.01) and femoral neck BMD (r=-0.697, P<0.01) and FEV1%Pre (r=-0.226, P<0.05); between RANKL serum levels and the lumbar spine BMD (r=-0.434, P<0.01) and femoral neck BMD (r=-0.538, P<0.01) and FEV1%Pre (r=-0.325, P<0.01); and between the RANKL/OPG ratio and the lumbar spine BMD (r=-0.345, P<0.01) and femoral neck BMD (r=-0.420, P<0.01) and FEV1%Pre (r=-0.459, P<0.05). Other cytokines, such as TIMP-1, TNF-a, OPG, and RANK, did not correlate with the lumbar spine or femoral neck BMD or FEV1%Pre (P>0.05). FEV1%Pre was positively correlated with the lumbar spine and femoral neck BMD (r=0.338, P<0.01, r=0.409, P<0.01).MMP-9was positively correlated with TNF-a (r-0.370, P<0.001) and OPG(r=0.306, P<0.01) and RANKL/OPG ratio (r=0.318, P<0.01).Conclusions:Osteoporosis is one of the most important systemic complications of COPD. Negative correlations between the serum levels of MMP-9,RANKL, the RANKL/OPG ratio and the lumbar spine BMD, femoral neck BMD,FEV1%Pre. Other cytokines, such as TIMP-1, TNF-α, OPG, and RANK, did not correlate with the lumbar spine BMD or femoral neck BMD or FEV1%Pre. MMP-9, TNF-a, and the OPG/RANK/RANKL system may be closely interrelated and may play interactive roles in pathogenesis of osteoporosis in COPD. Part Ⅱ Analysis of interrelationship of circulating matrix metalloproteinase-9, TNF-a, bone biochemical markers and bone mineral density in COPD patientsObjective:To study the relationship between matrix metalloproteinase-9(MMP-9), its cognate inhibitor TIMP-1, inflammatory cytokine TNF-a, bone biochemical markers and bone mineral density(BMD) in COPD patients.Methods:Ninety male patients diagnosed with clinically stable COPD were enrolled, who were divided into three groups based on bone mineral density(BMD):those with COPD and normal BMD, those with COPD with low but non-osteoporotic BMD, and those with COPD and osteoporosis. Each group contained30patients.All these patients completed pulmonary function,dual x-ray absorptiometry(DXA) measurements and COPD assessment test (CAT).All these patients were measured of MMP-9, TIMP-1, TNF-a, and serum bone alkaline phosphatase(sBAP)、serum osteocalcin(sOC)、bone cross-linked C-telopeptides of type Icollagen(sCTX) by ELISA.Results:COPD patients with osteoporosis had significantly lower BMI and higher CAT scores (all P<0.01), than COPD patients with normal or low BMD. There was no significant difference in age, smoking index, FEV1/FVC, or FEV1%Pre (P>0.05). As expected, based on the study design, the lumbar spine and femoral neck BMD was significantly lower in the groups of COPD with low BMD (P<0.05, P<0.01) and COPD with osteoporosis (all P<0.01) than in COPD with normal BMD. The serum level of MMP-9, the MMP-9/TIMP-1ratio, the serum level of TNF-a increased successively from COPD patients with normal BMD to those with low BMD to those with osteoporosis(P<0.05or P<0.01).However, no significant difference in the serum level of TIMP-1was detected among the three groups(P>0.05).The serum levels of BAP、OC were all significantly lower in COPD patients with osteoporosis and those with low BMD than in those with normal BMD (all P <0.01). However, no significant difference in levels of sBAP、sOC was detected between COPD with osteoporosis and in those with low BMD (P>0.05). The level of sCTX was significantly higher in the groups of COPD with osteoporosis than those with low BMD and those with normal BMD(all P<0.01). However, no difference in the level of sCTX was detected between COPD with low BMD and those with normal BMD (P>0.05).Pearson analysis revealed negative correlations between MMP-9serum levels and the lumbar spine BMD (r=-0.432, P<0.01) and femoral neck BMD (r=-0.697, P<0.01) and FEV1%Pre (r=-0.226, P<0.05);The BMD of lumbar spine and femoral neck、the lung function FEV1%Pre were positively correlated with sBAP level (r=0.418, P<0.01,and r=0.702, P<0.01,and r=0.295, P<0.01, respectively), sOC level (r=0.338, P<0.01,and r=0.574, P<0.01, and r=0.278, P<0.01, respectively), and negative correlated with sCTX level (r=-0.418, P<0.01,and r=-0.602, P<0.01,and r=-0.243, P<0.05, respectively). However, they had no significant correlation with TIMP-1, TNF-a. The ratio of MMP-9/TIMP-1was negative correlated with the levels of sBAP, sOC(r=-0.525, P<0.01; r=-0.460, P<0.01) and positively correlated with sCTX (r=0.635, P<0.01). The level of TIMP-1was positively correlated with sCTX (r=0.236, P<0.05), TNF-a was negative correlated with sBAP(r=-0.350, P<0.01) and positively correlated with sCTX (r=0.370, P<0.01). But no difference existed between the level of TNF-a with sOC (P>0.05).Conclusions:These results suggest that circulating MMP-9,TNF-a and the bone biochemical markers maybe closely interrelated in bone turnover state and may play important roles in pathogenesis of osteoporosis in COPD. Bone biochemical markers suggest increased bone turnover in COPD.