Featured Article: TGF-β1 dominates extracellular matrix rigidity forinducing differentiation of human cardiac fibroblasts tomyofibroblasts

摘要

Cardiac fibroblasts and their activated derivatives, myofibroblasts, play a critical role in wound healing after myocardial injury and often contribute to long-term pathological outcomes, such as excessive fibrosis. Thus, defining the microenvironmental factors that regulate the phenotype of cardiac fibroblasts and myofibroblasts could lead to new therapeutic strategies. Both chemical and biomechanical cues have previously been shown to induce myofibroblast differentiation in many organs and species. For example, transforming growth factor beta 1, a cytokine secreted by neutrophils, and rigid extracellular matrix environments have both been shown to promote differentiation. However, the relative contributions of transforming growth factor beta 1 and extracellular matrix rigidity, two hallmark cues in many pathological myocardial microenvironments, to the phenotype of human cardiac fibroblasts are unclear. We hypothesized that transforming growth factor beta 1 and rigid extracellular matrix environments would potentially have a synergistic effect on the differentiation of human cardiac fibroblasts to myofibroblasts. To test this, we seeded primary human adult cardiac fibroblasts onto coverslips coated with polydimethylsiloxane of various elastic moduli, introduced transforming growthfactor beta 1, and longitudinally quantified cell phenotype by measuringexpression of α-smooth muscle actin, the most robust indicator ofmyofibroblasts. Our data indicate that, although extracellular matrix rigidityinfluenced differentiation after one day of transforming growth factor beta 1treatment, ultimately transforming growth factor beta 1 superseded extracellularmatrix rigidity as the primary regulator of myofibroblast differentiation. Wealso measured expression of POSTN, FAP, andFSP1, proposed secondary indicators offibroblast/myofibroblast phenotypes. Although these genes partially trended withα-smooth muscle actin expression, they were relatively inconsistent. Finally, wedemonstrated that activated myofibroblasts incompletely revert to a fibroblastphenotype after they are re-plated onto new surfaces without transforming growthfactor beta 1, suggesting differentiation is partially reversible. Our resultsprovide new insights into how microenvironmental cues affect human cardiacfibroblast differentiation in the context of myocardial pathology, which isimportant for identifying effective therapeutic targets and dictating supportingcell phenotypes for engineered human cardiac disease models.Impact statementHeart disease is the leading cause of death worldwide. Many forms of heartdisease are associated with fibrosis, which increases extracellular matrix(ECM) rigidity and compromises cardiac output. Fibrotic tissue issynthesized primarily by myofibroblasts differentiated from fibroblasts.Thus, defining the cues that regulate myofibroblast differentiation isimportant for understanding the mechanisms of fibrosis. However, previousstudies have focused on non-human cardiac fibroblasts and have not testedcombinations of chemical and mechanical cues. We tested the effects ofTGF-β1, a cytokine secreted by immune cells after injury, and ECM rigidityon the differentiation of human cardiac fibroblasts to myofibroblasts. Ourresults indicate that differentiation is initially influenced by ECMrigidity, but is ultimately superseded by TGF-β1. This suggests thattargeting TGF-β signaling pathways in cardiac fibroblasts may havetherapeutic potential for attenuating fibrosis, even in rigidmicroenvironments. Additionally, our approach can be leveraged to engineermore precise multi-cellular human cardiac tissue models.