Lung Fibroblast to Myofibroblast Transition is Cell Density Dependent Mary T. Doolin1, Kimberly M. Stroka, Ph.D.1 1Fischell Department of Bioengineering, University of Maryland, College Park, MD Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung characterized by the differentiation of resident fibroblasts into contractile myofibroblasts that deposit excessive extracellular matrix (ECM). There are no effective treatments for IPF, and the median survival after diagnosis is approximately 3 years. Concomitant with increased ECM deposition and crosslinking, there is increased substrate stiffness and confinement experienced by cells. It has been shown that fibroblasts cultured on stiff substrates are more likely to undergo fibroblast to myofibroblast transition (FMT) due to increased actomyosin contractility. However, the effect of confining forces from the ECM or neighboring cells on FMT is unknown. Methods: Normal human lung fibroblasts (HLFs) were harvested, then seeded at various densities onto tissue culture polystyrene or into collagen I coated polydimethylsiloxane (PDMS) micropillar arrays of various spacing (50, 20, 10, or 5 μm). HLFs were allowed to attach overnight, then serum starved before treatment with TGF-β1 to induce FMT. Cells were treated with TGF-β1 under serum-free conditions for 1-5 days, then processed for immunofluorescence staining or western blot. FMT was evaluated by α-smooth muscle actin (αSMA) staining localized to f-actin stress fibers, in the case of immunofluorescence staining, or increased αSMA protein expression, in the case of western blot.

Figure 1 HLFs cultured for 24 or 72 hours with 2, 5, or 10 ng/mL TGF-β1 or control and stained for f-actin (green), α- Figure 2 α-SMA protein expression in HLFs treated SMA (red), and nucleus (blue). Scale bar is 100 μm. with TGF-β1 or control within PDMS micropillars. Results: HLFs seeded at 5,000 cells/cm2 displayed more αSMA-rich stress fibers with increased TGF-β1 treatment time and with increased TGF-β1 concentration (Figure 1). Therefore, we seeded 5,000 cells/cm2 within micropillar arrays and treated with 10 ng/mL TGF- β1 for 72 hours. Interestingly, there was no difference in αSMA protein expression in TGF-β1 treated groups relative to control, except on tissue culture polystyrene (TCPS, Figure 2). We observed that cells grew to a higher degree of confluence on collagen I coated 2D TCPS than on any collagen I coated PDMS surface. Therefore, we next investigated if cell density may affect FMT. HLFs seeded at medium to high densities (5,000 or 50,000 cells/cm2) displayed more αSMA-rich stress fibers when incubated with 10 ng/mL TGF-β1 relative to control (Figure 3). However, when HLFs were seeded at a low density (500 cells/cm2), αSMA remained diffuse in both the treated and control group. Conclusions: FMT appears to depend on the concentration and duration of TGF- β1 treatment, however only HLFs near confluence transition into mature myofibroblasts. More studies will elucidate if this is due to cell-cell Figure 3 HLFs seeded at various densities and treated adhesions, cell spreading, secreted factors, or with TGF-β1 or control for 5 days. α-SMA (red), f-actin another phenomenon. (green), and nucleus (blue). Scale bar is 50 μm.