Tschumperlin, Daniel J.

Extracellular matrix stiffness controls DNA damage response.

Abstract Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.

Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40–60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-β-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.

Objective This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). Design All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. Results The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A , the gene that encodes the Na + channel Na V 1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na + current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. Conclusions A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na + channel Na V 1.5. Within this set, a novel Na V 1.5 regulator, let-7f, resulted in decreased Na V 1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na V 1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Abstract Introduction Impaired plasminogen activation (PA) is causally related to the development of lung fibrosis. Prior studies demonstrate that enhanced PA in the lung limits the severity of scarring following injury and in vitro studies indicate that PA promotes matrix degradation and fibroblast apoptosis. These findings led us to hypothesize that increased PA in an in vivo model would enhance the resolution of established lung fibrosis in conjunction with increased myofibroblast apoptosis. Methods Transgenic C57BL/6 mice with doxycycline inducible lung-specific urokinase plasminogen activator (uPA) expression or littermate controls were treated (day 0) with bleomycin or saline. Doxycycline was initiated on days 1, 9, 14, or 21. Lung fibrosis, stiffness, apoptosis, epithelial barrier integrity, and inflammation were assessed. Results Protection from fibrosis with uPA upregulation from day 1 through day 28 was associated with reduced parenchymal stiffness as determined by atomic force microscopy. Initiation of uPA expression beginning in the late inflammatory or the early fibrotic phase reduced stiffness and fibrosis at day 28. Induction of uPA activity in mice with established fibrosis decreased lung collagen and lung stiffness while increasing myofibroblast apoptosis. Upregulation of uPA did not alter lung inflammation but was associated with improved epithelial cell homeostasis. Conclusion Restoring intrapulmonary PA activity diminishes lung fibrogenesis and enhances the resolution of established lung fibrosis. This PA-mediated resolution is associated with increased myofibroblast apoptosis and improved epithelial cell homeostasis. These studies support the potential capacity of the lung to resolve existing scar in murine models.

Pharmacologic stimulation of a G protein–coupled receptor preferentially expressed on fibroblasts reverses experimental lung and liver fibrosis.

ABSTRACT Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFβ-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFβ-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFβ-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene PPARGC1A (PGC1α) in response to TGFβ stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFβ-mediated fibroblast activation via targeted gene repression.

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is thought to present both molecular and mechanical barriers to neuronal regeneration. The scar consists of a meshwork of reactive glia and deposited, cross-linked, extracellular matrix (ECM) that has long been assumed to present a mechanically “stiff” blockade. However, remarkably little quantitative information is available about the rheological properties of chronically injured spinal tissue. In this study we utilize atomic force microscopy microindentation to provide quantitative evidence of chronic mechanical stiffening after SCI. Using the results of this tissue characterization, we assessed the sensitivity of both mouse and human astrocytes in vitro and determined that they are exquisitely mechanosensitive within the relevant range of substrate stiffness observed in the injured/uninjured spinal cord. We then utilized a novel immune modifying nanoparticle (IMP) treatment as a tool to reveal fibrotic scarring as one of the key drivers of mechanical stiffening after SCI in vivo . We also demonstrate that glial scar-forming astrocytes form a highly aligned, anisotropic network of glial fibers after SCI, and that IMP treatment mitigates this pathological alignment. Taken together, our results identify chronic mechanical stiffening as a critically important aspect of the complex lesion milieu after SCI that must be considered when assessing and developing potential clinical interventions for SCI.

Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex, respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator–activated receptor γ coactivator 1 α gene ( PPARGC1A, encoding PGC1α). Both TGF-β and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of the CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGF-β– and matrix stiffness–promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.