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A2211 - Nuclear Heparan Sulfate Upregulates Pro-Fibrotic Gene Expression And.is Abundant in Pulmonary Fibroblasts from IPF Patients
Author Block: W. B. LaRiviere, S. M. Haeger, S. Liao, A. R. Lara, E. P. Schmidt, Y. Yang; Pulmonary and Critical Care, University of Colorado Denver, Aurora, CO, United States.
RATIONALE: Heparan sulfate (HS) is a linear polysaccharide found ubiquitously in mammalian tissues, often anchored to cell-surface proteoglycans. Lung injury induces activation of heparanase, a glucuronidase that degrades HS from cell surfaces, releasing HS fragments. These fragments may be internalized into the nucleus, where they are associated with alterations in histone acetyltransferase activity and cell cycle progression. The presence and impact of nuclear HS translocation in chronic, progressive lung injury is unexplored. METHODS. We performed immunofluorescence to determine the expression of heparanase and localization of HS fragments within lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) and murine models of IPF (bleomycin). We used cell fractionation and Western blotting to determine nuclear translocation of HS in mouse lung fibroblasts (MLFs) after enzymatic release of cell-surface HS. To define the pathophysiological consequences of nuclear HS translation, we performed HS nucleofection on MLFs and measured changes in the expression of pro-fibrotic genes via qPCR. All experiments were performed with > 5 biological replicates per group. RESULTS: In human patients and mouse models of IPF, both heparanase expression and nuclear HS localization were increased. Enzymatic degradation of cell-surface HS from MLFs induced TGF-β (Tgfb1) and collagen type I (Col1a1) expression. 24 hours after MLF nucleofection with FITC-labeled HS, we observed a mean 2-fold increase in α smooth muscle actin (Acta2) (p=0.0021) expression, 5-fold increase in Tgfb1 expression (p=0.0054), and an 8-fold increase in Col1a1 expression (p=0.0005) vs empty lipofectamine control. While induction of profibrotic genes required nuclear translocation, treatment of MLF with HS without lipofectamine was sufficient to induce Cola1a1 and Acta2 expression (p=0.012 and p=0.0003, respectively). CONCLUSIONS: Nuclear transfection of HS into primary mouse lung fibroblasts produces a detectable change in pro-fibrotic gene expression, including Tgfb1, Acta2, and Col1a1. Unexpectedly, treatment with FITC-HS alone also exerted an influence on α smooth muscle actin expression, but not on Tgfb1expression and Col1a1, in a manner independent of detectable nuclear import. Taken together, these results suggest that nuclear import of heparan sulfate is capable of upregulating profibrotic gene expression and may play a role in the pathogenesis of IPF.