Amy D. Bradshaw

The prevalence of fibrotic disease and its contribution to organ failure have wide-ranging consequences in terms of both morbidity and mortality and are particularly relevant in chronic conditions that afflict aging populations. The paucity of treatment options for those with fibrosis-dependent complications illustrates the challenge and underlying complexity of controlling and reducing extracellular matrix (ECM) content once fibrosis has been established. Legitimately, a major focus of research in fibrosis has centered on transcriptional regulation of fibrillar collagen, particularly collagen I, and factors that induce the expression of genes encoding the fibrillar collagens. However, knowledge that other facets of extracellular matrix biology, in addition to fibrillar collagen content, also make significant contributions to fibrosis is appreciated with emerging significance. Herein, a summary of some recent advances in uncovering critical fibroblast activation states, ECM organization, and composition of fibrotic ECM, including basement membrane components, is discussed. In addition, evidence in support of distinct fibroblast phenotypes in fibrotic tissues, which once established, limit regression of fibrosis despite alleviation of the initiating pathology, is given. As the capacity to reduce established fibrosis has the potential for profound translational significance across organs, more research into each of these important processes is merited.

Background: Myocardial fibrosis is prevalent in cardiomyopathies that result in heart failure with reduced ejection fraction. Heart failure with reduced ejection fraction treated with a left ventricular assist device (LVAD) yields hemodynamic unloading and may provide partial cardiomyocyte recovery, but contemporary studies reveal no consistent reductions in fibrosis. This study tested the hypothesis that, despite normalization of hemodynamic overload by LVAD, fibrosis and fibroblast activation persist resulting in sustained increases in myocardial stiffness. Methods and Results: Tissues from subjects with heart failure with reduced ejection fraction undergoing LVAD implantation (pre‐LVAD), from transplanted hearts with LVAD (post‐LVAD) or without cardiac pathology (control) were collected. Quantification of myocardial stiffness and collagen content revealed significant increases in pre‐LVAD versus control that remained elevated in post‐LVAD. Myocardial fibroblast populations increased in pre‐ and post‐LVAD hearts versus control. Control, pre‐LVAD, and post‐LVAD fibroblasts were isolated and plated on substrates with mechanical stiffnesses reflective of normal (≈2 kPa) or fibrotic (≈8 kPa) myocardium. Quantification of collagen I and α‐smooth muscle actin production demonstrated that control fibroblasts were responsive to substrate stiffness, whereas pre‐ and post‐LVAD fibroblasts were unresponsive and exhibited no significant differences on either substrate. Bulk‐RNA sequence analysis revealed changes in gene expression in pre‐LVAD versus control fibroblasts including mechano‐sensitive pathways that appear to be uncoupled, resulting in increased expression of genes implicated in proliferation, whereas mechano‐sensing genes were decreased. Conclusions: These data support that sustained cardiac hemodynamic overload leads to a phenotypic conversion in fibroblasts in which the capacity to detect changes in mechanical input is muted, thus contributing to retention of collagen content and stiffness in both pre‐ and post‐LVAD hearts.

Epithelial adherens junctions (AJs) are cell-cell adhesion complexes that are influenced by tissue mechanics, such as those emanating from the extracellular matrix (ECM). Here, we introduce a mechanism whereby epithelial AJs can also regulate the ECM. We show that the AJ component PLEKHA7 regulates levels and activity of the key ECM remodeling components MMP1 and LOX in well-differentiated colon epithelial cells, through the miR-24 and miR-30c miRNAs. PLEKHA7 depletion in epithelial cells results in LOX-dependent ECM remodeling in culture and in the colonic mucosal lamina propria in mice. Furthermore, PLEKHA7-depleted cells exhibit increased migration and invasion rates that are MMP1- and LOX-dependent, and form colonies in 3D cultures that are larger in size and acquire aberrant morphologies in stiffer matrices. These results reveal an AJ-mediated mechanism, through which epithelial cells drive ECM remodeling to modulate their behavior, including acquisition of phenotypes that are hallmarks of conditions such as fibrosis and tumorigenesis. Teaser Epithelial cells instruct ECM remodeling to modulate their behavior, as a result of adherens junction and miRNA disruption.

Our novel findings highlight the dynamic changes in myocardial macrophage populations that occur in response to PO and after alleviation of PO. Our data demonstrated, for the first time, a potential benefit of macrophages in contributing to collagen degradation and the partial regression of interstitial fibrosis following normalization of hemodynamic load.

Human induced pluripotent stem cell derived cardiac fibroblasts (hiPSC-CFs) play a critical role in modeling human cardiovascular diseases in vitro. However, current culture substrates used for hiPSC-CF differentiation and expansion, such as Matrigel and tissue culture plastic (TCPs), are tissue mismatched and may provide pathogenic cues. Here, we report that hiPSC-CFs differentiated on Matrigel and expanded on tissue culture plastic (M-TCP-iCFs) exhibit transcriptomic hallmarks of activated fibroblasts limiting their translational potential. To alleviate pathogenic activation of hiPSC-CFs, we utilized decellularized extracellular matrix derived from porcine heart extracellular matrix (HEM) to provide a biomimetic substrate for improving hiPSC-CF phenotypes. We show that hiPSC-CFs differentiated and expanded on HEM (HEM-iCFs) exhibited reduced expression of hallmark activated fibroblast markers versus M-TCP-iCFs while retaining their cardiac fibroblast phenotype. HEM-iCFs also maintained a reduction in expression of hallmark genes associated with pathogenic fibroblasts when seeded onto TCPs. Further, HEM-iCFs more homogenously integrated into an hiPSC-derived cardiac organoid model, resulting in improved cardiomyocyte sarcomere development. In conclusion, HEM provides an improved substrate for the differentiation and propagation of hiPSC-CFs for disease modeling.