The Use of RNA Interference to Mitigate Pulmonary Fibrosis in Response to Asbestos Exposure

Kinsey, Sarah, Bachelor of Arts, May 2015 Human Biology and Psychology The Use of RNA Interference to Mitigate Pulmonary Fibrosis in Response to Asbestos Exposure Faculty Mentor: Dr. Elizabeth A. Putnam The adverse health effects of exposure to asbestos are widely known and have been well documented. When a person is diagnosed with asbestosis, a chronic lung disease caused by inhaling asbestos fibers, few treatment options exist, none of which halt or reverse the progression of the disease. The rapidly growing field of gene therapy offers new avenues for potential treatments worthy of investigation. The detrimental effects of asbestos exposure are due to the physiological response of the lungs to asbestos fibers in the form of fibrosis, a result of excess extracellular collagen deposition. A protein called SPARC (Secreted Protein Acidic and Rich in Cysteine) has been identified in previous studies as being a matricellular protein involved in the fibrotic response following asbestos exposure. Using the principle of RNA interference, we aimed to knock down the expression of SPARC in our studies, hypothesizing that a reduction in SPARC expression would yield a reduction in fibrosis. In our in vitro experiments, we prepared several viral constructs containing a SPARC-specific short hairpin RNA (shRNA), and identified the most effective construct using primary mouse lung fibroblasts. The most effective construct was used to infect asbestos-exposed and control mice. The presence of fibrosis was measured in three distinct ways: 1. Histologically to visually observe the presence of fibrosis, 2. Using RT-PCR to measure the presence of SPARC mRNA, 3. Using a Western Blot to measure the presence of collagen. We expected to observe that the mice treated with the SPARC shRNA containing virus experienced less fibrosis, and had less SPARC mRNA and collagen present in their lungs than control mice. Unfortunately, none of the mice in our study developed fibrosis after asbestos exposure, as had been previously demonstrated. We intend to reevaluate our instillation method, among other procedures, before proceeding with future studies. Mitigating Pulmonary Fibrosis Post Asbestos Exposure – Page 3 Introduction Asbestos is a broad term used to describe a group of silicate minerals in the form of thin fibers (1). These minerals are naturally occurring and were mined extensively in the early 1900’s for their substantial commercial utility due their high tensile strength and heat resistant properties (1) (2). Asbestosis can be defined as the interstitial fibrosis caused by the inhalation and deposition of asbestos fibers in the lungs (1). The most common symptom of this disease is dyspnea, though a nonproductive cough and chest pain are also typical (1). The molecular mechanisms underlying asbestosis are complex and have yet to be fully elucidated. To date, we understand that asbestos triggers the accumulation of alveolar macrophages and an inflammatory response (3). Subsequently, broad pulmonary involvement is observed, indicated by the loss of alveolar epithelial cells, fibroblast proliferation and collagen deposition (See Fig. 1) (3). Due to the long, thin shape of the asbestos fibers, the alveolar macrophages are unable to ingest them. This frustrated phagocytosis results in the release of transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), among other growth factors and cytokines; these encourage the deposition of collagen in the walls of the alveoli and elsewhere, resulting in fibrosis (3). In addition to the release of macrophage products, recent studies demonstrate that alveolar epithelial cell apoptosis is sufficient to induce pulmonary fibrosis (3). One protein that appears to be a promising target in the fibrosis pathway is SPARC (Secreted Protein Acidic and Rich in Cysteine), also known as osteonectin or BM-40. This 43kD matricellular protein is known to interact with cell-surface receptors and the extracellular matrix (ECM) without contributing to ECM structure and is activated during tissue renewal, remodeling and repair as well as embryonic development (4). In her doctoral thesis of 2009 entitled, The Influence of SPARC on Collagen Deposition in Asbestos-Induced Pulmonary Fibrosis, Smartt summarizes the current understanding of this protein as follows, “Since its discovery, SPARC has been shown to function as a modulator of growth factor activity (5) (6) (7) (8), a counteradhesive protein (7) (9), a modulator of cell proliferation (10) (6) and migration (11) (12) a regulator of the extracellular matrix (13) (14), and a cell cycle inhibitor (10)” (4). Additionally, SPARC has been shown to stimulate the TGF-β signaling system (8). A 2010 study used RNA interference to knock down the expression of SPARC in a murine model with bleomycin-induced fibrosis and demonstrated that inhibition of SPARC Mitigating Pulmonary Fibrosis Post Asbestos Exposure – Page 4 significantly reduced pulmonary and epithelial fibrosis and collagen expression (15). These results were promising, but would they hold true in an asbestos-induced fibrosis model? However, a study published in 2011 indicated that the effect of SPARC might not be as clear as was previously thought. Those results established that SPARC-inhibited mice demonstrate decreased collagen deposition compared to controls at low doses of bleomycin, but increased collagen deposition at high doses (16). A 2013 study showed that, in addition to SPARC stimulating TGF-β as was demonstrated in 2003, SPARC expression is also upregulated by TGFβ (17). The study also found that SPARC expression was necessary for the production of hydrogen peroxide (H2O2) in fibroblasts, which contributes to epithelial damage (17). Clearly, these findings collectively demonstrate that SPARC plays a role in the development of fibrosis. The aim of this study was to determine the effect of SPARC inhibition on collagen deposition and pulmonary fibrosis in a murine model after asbestos-induced fibrosis. Crocidolite asbestos was used for these studies to allow for comparison with other data in the field. (3) Figure 1: A Hypothetical Model of Asbestos-Induced Pulmonary Fibrosis (3)