Chronic Obstructive Pulmonary Diseased Human Lung Fibroblasts Secrete Increased Matrix Metalloproteases -12 and -8

INTRODUCTION: According to the American Lung Association, 16.5 million people have Chronic Obstructive Pulmonary Disease (COPD), the third leading cause of death in the United States. COPD is a condition in which the airways of the lung are inflamed, and the alveoli thicken leading to decreased oxygen exchange and the ability to breathe. The thickening of the airway is in part due to abnormal remodeling of the extracellular matrix. Dysregulation of Matrix Metalloprotease (MMP) activity leads to the breakdown of collagen type I, a necessary component of the extracellular matrix in the lung tissue. In this study, we measure MMP-8 and MMP-12 production by Human Lung Fibroblasts (HLFB) reared in serum-free media (normal (NHLFB) and COPD Diseased (DHLFB)). METHODS: Commercially available early passage HLFB established from lung tissue, obtained during a biopsy, were cultured and passaged in our laboratory through passage 4. Both normal (NHLFB) and diseased (DHLFB) cells were viable when cultured at subconfluence for 24 hours in a serum-free medium. We collected conditioned media (secreted fraction) and cell-associated matrix (incorporated fraction) from the cultures following 24 hours in serum-free medium or treatment with 5ug Extracellular Matrix Protection Factor-2 (ECPF-2). ECPF-2 is a novel inhibitor of MMP-8 and -12 activity. Utilizing commercially available ELISA kits, we measured intact collagen type I (Chondrex), degraded collagen type I (CUSABIO), MMP-8 (Quantikine, Abcam), MMP-12 (Abcam), and MMP-9 (Abcam). RESULTS: There were detectable levels of MMP-12 (ng/culture) and MMP-8 (pg/culture) in both the secreted (conditioned media) and incorporated (extracellular matrix) fractions. Overall, DHLFB produced 3-fold more secreted MMP-12 [150ng/culture versus 50ng/culture] and 14-fold less incorporated MMP-12 [20ng/culture versus 300ng/culture] than the NHLFB. In the case of MMP-8, DHLFB produced 2-fold less secreted MMP-8 [30pg/culture versus 80pg/culture], while the incorporated fractions of MMP-8 were relatively similar in both normal and diseased cultures. In line with these findings, the diseased lung fibroblasts secreted approximately 10-fold higher levels of degraded collagen type I, confirming the diseased nature of the cells. DISCUSSION: Our findings suggest that MMP-8 has a role in the increased degradation of the extracellular matrix in COPD since collagen type I is a preferred substrate for MMP-8. But the role of MMP-12 in the disease process may be more indirect and involve elastolysis that could disrupt the interactions with collagen type I and facilitate collagen degradation by allowing access to the fibril by MMP-8. Future studies will measure elastin turnover in these cultures to get a clearer picture of the dysregulation of the extracellular matrix associated with COPD