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A2089 - Adventitial Fibroblast Phenotype Is Dependent on Matrix and Interstitial Fluid Flow: Implications for Pulmonary Hypertension
Author Block: M. Yeager1, K. L. Colvin2; 1Pediatrics Critical Care; Bioengineering, University of Colorado at Denver, Aurora, CO, United States, 2Pediatrics Critical Care; Bioengineering, University of Colorado Denver, Aurora, CO, United States.
Rationale: Pulmonary artery hypertension (PAH) is hypothesized to be a disorder involving hyper-proliferative pulmonary vascular cells. In vivo, the pulmonary artery adventitial and peri-adventitial spaces undergo fibrotic remodeling. In vitro, adventitial fibroblasts from individuals with PAH are reported to be highly proliferative vs. controls. There are few studies available using three-dimensional culture techniques that incorporate both interstitial fluid flow and culture on biologically relevant substrates. We assessed the impact of matrix stiffness and architecture as well as interstitial fluid flow on the phenotype of adventitial fibroblasts isolated from rats with PAH vs. controls.
Methods: We purchased human pulmonary artery fibroblasts and established primary cultures of adventitial fibroblasts from rats and assessed their purity using cell sorting and immunofluorescence. We cultured adventitial fibroblasts in the absence of flow, in low and high rates of interstitial fluid flow, and in five hydrogels representing a range of stiffness and architecture. In each condition, we measured cell proliferation and migration by time-lapse fluorescent microscopy combined with cell tracking software. Cytokine and chemokine release was measured by enzyme-linked immunosorbent assay. Fibroblast gene expression was measured by quantitative reverse-transcriptase polymerase chain reaction and immunoblotting.
Results: We found that fibroblasts isolated from control rats vs. PAH rats were in fact mixed cultures of endothelium, smooth muscle, inflammatory cells, and neurons. Both sorted and unsorted cultures of human pulmonary artery fibroblasts and those from rats with and without PAH all exhibited highly variable rates of proliferation on plastic substrates. However, all adventitial fibroblast proliferation rates were much lower relative to HeLa, HEK293, HCT116, or LNCaP cancer cells. Furthermore, adventitial fibroblasts adopted a range of proliferation rates and phenotypic appearances depending on substrate stiffness and architecture, preferring softer, collagen containing hydrogels with round to oval void space architecture. Fibroblasts from all rats demonstrated increased directional migration with increasing fluid flow rates. Increased expression of tenascin-c and focal adhesion kinase correlated to higher migration rate.
Conclusions:
Neither control nor PAH adventitial fibroblasts are highly proliferative, a finding that is consistent with the low rates of adventitial fibroblast proliferation observed in end-stage PAH in both humans and animal models. Flow-tuned three-dimensional culture on biologically relevant substrates provides the opportunity to study adventitial fibroblasts in environments that more closely match normal and fibrotic lung matrix architecture and material properties. Our observations provide a clearer understanding of how adventitial fibroblasts impact the pathobiology of PH, which may lead to novel therapies.