Proteomic Analysis of Altered Extracellular Matrix Turnover in Bleomycin-induced Pulmonary Fibrosis

Fibrotic disease is characterized by the pathological accumulation of extracellular matrix (ECM) proteins. Surprisingly, very little is known about the synthesis and degradation rates of the many proteins and proteoglycans that constitute healthy or pathological extracellular matrix. A comprehensive understanding of altered ECM protein synthesis and degradation during the onset and progression of fibrotic disease would be immensely valuable. We have developed a dynamic proteomics platform that quantifies the fractional synthesis rates of large numbers of proteins via stable isotope labeling and LC/MS-based mass isotopomer analysis. Here, we present the first broad analysis of ECM protein kinetics during the onset of experimental pulmonary fibrosis. Mice were labeled with heavy water for up to 21 days following the induction of lung fibrosis with bleomycin. Lung tissue was subjected to sequential protein extraction to fractionate cellular, guanidine-soluble ECM proteins and residual insoluble ECM proteins. Fractional synthesis rates were calculated for 34 ECM proteins or protein subunits, including collagens, proteoglycans, and microfibrillar proteins. Overall, fractional synthesis rates of guanidine-soluble ECM proteins were faster than those of insoluble ECM proteins, suggesting that the insoluble fraction reflected older, more mature matrix components. This was confirmed through the quantitation of pyridinoline cross-links in each protein fraction. In fibrotic lung tissue, there was a significant increase in the fractional synthesis of unique sets of matrix proteins during early (pre-1 week) and late (post-1 week) fibrotic response. Furthermore, we isolated fast turnover subpopulations of several ECM proteins (e.g. type I collagen) based on guanidine solubility, allowing for accelerated detection of increased synthesis of typically slow-turnover protein populations. This establishes the presence of multiple kinetic pools of pulmonary collagen in vivo with altered turnover rates during evolving fibrosis. These data demonstrate the utility of dynamic proteomics in analyzing changes in ECM protein turnover associated with the onset and progression of fibrotic disease.The extracellular matrix (ECM)¹ comprises an intricate network of cell-secreted collagens, proteoglycans, and glycoproteins providing structural and mechanical support to every tissue. The dynamic interplay between cells and ECM also directs cell proliferation, migration, differentiation, and apoptosis associated with normal tissue development, homeostasis, and repair (1, 2). Tissue repair following acute injury is typically characterized by the recruitment of inflammatory cells, enzymatic degradation of ECM immediately adjacent to the damaged tissue site, and subsequent infiltration of fibroblasts depositing new ECM. However, in the case of chronic tissue injury and inflammation, abnormal signaling pathways can stimulate uncontrolled ECM protein deposition, ultimately resulting in fibrosis and organ failure (3–6). In fact, fibrotic diseases including idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis, and cardiovascular disease have been estimated to account for over 45% of deaths in the developed world (1).Despite the wide prevalence of fibrotic diseases, there is currently a paucity of anti-fibrotic drug treatments and diagnostic tests (7, 8). Median survival rates for idiopathic pulmonary fibrosis, for example, range from only two to five years following diagnosis (9, 10). Failure in the development of successful anti-fibrotic treatments can in part be attributed to a poor understanding of the active and dynamic role played by the ECM during various stages of fibrotic disease. ECM components influence myofibroblast differentiation not only through their modulation of fibrogenic growth factor activity (e.g. TGF-β), but also through mechanotransductive pathways whereby cells interpret altered ECM mechanical properties (3, 5, 11–13). The search for novel target pathways in the development of anti-fibrotic therapies would benefit from a better understanding of dynamic ECM synthesis and degradation associated with the various stages of fibrotic disease.The combination of stable isotope labeling and proteomic analysis provides a new approach for interrogating dynamic changes in ECM protein synthesis associated with fibrotic disease. We have developed a platform termed “dynamic proteomics,” whereby protein synthesis rates from tissue samples are measured following the administration of stable isotope tracers (e.g.²H, ¹⁵N) (14). Label incorporation into newly synthesized proteins is assessed via LC/MS analysis of mass isotopomer distributions in peptides derived from parent proteins through enzymatic degradation, providing a means to quantify the fractional synthesis rate (FSR) of individual proteins over the labeling period. Unlike traditional static proteomic techniques, this strategy provides valuable information regarding which proteins are actively synthesized or degraded during any specific stage of the disease process. Moreover, as measurements of label incorporation do not fluctuate based on the amount or yield of protein isolated (14–16), dynamic proteomic strategies also offer additional robustness relative to traditional quantitative proteomic techniques.The detection of ECM components in highly cellular tissues such as liver and lung poses an additional stumbling block in the proteomic analysis of fibrotic ECM. The identification of less abundant matrix components is limited by the overwhelming number of cellular proteins present in standard homogenized tissue samples. Standard global protein fractionation techniques (e.g. gel electrophoresis) are inefficient at enriching targeted subsets of proteins. Tissue decellularization techniques commonly utilized in regenerative medicine offer a novel approach toward the enrichment of ECM proteins prior to proteomic analysis (17). Tissue samples are incubated under mechanical agitation in the presence of weak detergents that solubilize cell membranes, releasing cellular protein components into solution while keeping the surrounding structural ECM intact. This technique has recently been applied in the compositional proteomic analysis of cardiovascular, lung, and colon tissues, leading to the identification of ECM-related proteins previously not associated with those tissues (11, 18–20).We present here the first study to combine dynamic proteomics with tissue decellularization in order to analyze altered ECM protein synthesis associated with pulmonary fibrosis. Bleomycin and sham-dosed mice were labeled for up to three weeks with heavy water (²H₂O), and lung tissue was subsequently collected and fractionated into cellular and extracellular components. Further fractionation of ECM based on guanidine solubility resulted in the identification of protein fractions with kinetically distinct characteristics composed of a variety of collagens, basement membrane proteoglycans, and microfibrillar proteins. Label incorporation into ECM proteins in sham-dosed control lungs was generally faster in the guanidine-soluble fraction, suggesting that the insoluble pool reflected more stable, slower-turnover matrix components. In bleomycin-dosed lungs, however, there was a significant increase in the synthesis of both guanidine-soluble and insoluble ECM proteins. These labeling and fractionation methods should be easily adaptable to a variety of animal and human tissue types and could provide a new approach toward actively monitoring the dynamic changes in ECM synthesis and composition associated with fibrotic disease.