Assistant Professor University of Michigan-Dearborn Wixom, Michigan, United States
Introduction: : Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease that is characterized by stiff scar formation in the lung tissue. Rather than properly repairing and recovering from repetitive damage to the alveolar epithelium, individuals with IPF undergo a dysregulated tissue remodeling process. An excess production of pro-fibrotic mediators, such as TGF-β1, causes pulmonary fibroblasts to migrate, proliferate, and activate into highly contractile myofibroblasts. These myofibroblasts then secrete excessive amounts of extracellular matrix (ECM), contributing to the progressive scar formation. Additionally, fibroblasts are highly mechanically active, and the stiffening of the tissue from excess ECM creates a positive feedback loop and causes further fibroblast activation. IPF is considered to be male dominated, as males are more susceptible to IPF and tend to have a poorer prognosis, though sex as a biological variable in IPF is not well understood. Thus, understanding the sex-based differences in pulmonary fibroblast functional phenotypes can give us insights on disease progression and treatment.
Materials and
Methods: : Primary pulmonary fibroblast cell lines were isolated from porcine male and female lung tissues. Macroscale contraction studies were done on cells encapsulated in 3D bovine collagen hydrogels at varying cell concentrations. The diameter of collagen hydrogels with a concentration of 0.25 million cells per milliliter were measured daily over 4 days. Additionally, the diameter of collagen gels with a concentration of 2 million cells per milliliter was measured every 20 minutes over an 8 hour period. Contraction studies were similarly done for collagen hydrogels stimulated with TGF-β1 at 2 ng/mL. Gel sizes were compared between groups each day and each hour for the low and high concentrations respectively. A one-phase decay model was used to compare the decay constant and plateau values for the 4 day contraction profile, while a simple linear regression was used to compare the slopes of the 8 hour profile. High cellular concentration gels utilized in the contraction studies and cells grown on 2D tissue culture plates were collected after 24 hours of culture for mRNA isolation and gene expression analysis. Gene expression was assessed using qPCR and fold change was quantified using the 2^(-ddCT) method utilizing GAPDH as the housekeeping gene and 2D male samples as the control. All experiments were run with 3 biological replicates and 3-6 technical replicates. Independent t tests were used to directly compare normally distributed groups, while Mann-Whitney tests were used to compare nonparametric groups.
Results, Conclusions, and Discussions:: Low cell concentration gels were cultured over a 4 day period. Male gel diameters were found to be significantly smaller than females from day 1 to 4 (Fig. 1A). Females, however, showed a significantly greater response to TGF-β1 on days 2-4 (Fig. 1C). Additionally, significant differences were observed between plateau values of males and females, and TGF-β1 stimulus significantly decreased the plateau values for both males and females (Fig. 1F). Because there is a greater contraction observed in the first 24 hours, cell concentration was increased to study the contraction in this period. Male gel diameters were found to be significantly smaller than females at multiple timepoints after 2 hours of culture, as well as when stimulated with TGF-β1 (Fig. 2A-2B). TGF-β1 stimulus caused a significantly more negative slope in both males and females, with stimulated males exhibiting a faster decline than females (Fig. 2D). Gene expression analysis showed a significant difference between males and female alpha smooth muscle actin expression when treated with TGF-β1 in both 2D and 3D culture (Fig. 3A). In 3D culture, TGF-β1 expression was significantly greater in females with and without prior TGF-β1 stimulation (Fig. 3C). Collagen 3 expression was found to be significantly different between males and females in both 2D and 3D culture (Fig. 3F).
Overall, male cells were found to be more contractile under baseline conditions compared to females. While male cells initially contract faster when subjected to TGF-β1, later, females exhibit a greater TGF-β1 response relative to their baseline contraction. Interestingly, females exhibit an up-regulation of TGF-β1 expression under both baseline conditions and TGF-β1 stimulus. Future directions include quantifying contraction on the cellular level, as well as confirming qPCR results with protein secretion analyses. Other fibroblast behaviors will also be studied to characterize their phenotype, such as morphological analyses and proliferation. These findings aim to improve our understanding of how sex differences influence disease progression through their effects on pulmonary fibroblast behaviors.
