Jones, Dakota L.

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.

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.

Abstract Reciprocal epithelial–mesenchymal interactions are pivotal in lung development, homeostasis, injury, and repair. Organoids have been used to investigate such interactions, but with a major focus on epithelial responses to mesenchyme and less attention to epithelial effects on mesenchyme. In the present study, we used nascent organoids composed of human and mouse lung epithelial and mesenchymal cells to demonstrate that healthy lung epithelium dramatically represses transcriptional, contractile, and matrix synthetic functions of lung fibroblasts. Repression of fibroblast activation requires signaling via the bone morphogenetic protein (BMP) pathway. BMP signaling is diminished after epithelial injury in vitro and in vivo, and exogenous BMP4 restores fibroblast repression in injured organoids. In contrast, inhibition of BMP signaling in healthy organoids is sufficient to derepress fibroblast matrix synthetic function. Our results reveal potent repression of fibroblast activation by healthy lung epithelium and a novel mechanism by which epithelial loss or injury is intrinsically coupled to mesenchymal activation via loss of repressive BMP signaling.

AbstractAdipose-derived mesenchymal stem cells (AMSCs) offer potential as a therapeutic option for clinical applications in musculoskeletal regenerative medicine because of their immunomodulatory functions and capacity for trilineage differentiation. In preparation for a phase I clinical trial using AMSCs to treat patients with osteoarthritis, we carried out preclinical studies to assess the safety of human AMSCs within the intra-articular joint space. Culture-expanded human AMSCs grown in human platelet-lysate were delivered via intra-articular injections into normal healthy rabbit knees and knees at risk for the development of osteoarthritis after bilateral medial anterior hemimeniscectomy. Treatment outcomes and safety were evaluated by assessing the general health, function, and behavior of the animals. Joint tissues were analyzed by x-ray, magnetic resonance imaging, and histopathology. Intra-articular AMSC therapy was well tolerated in this study. We did not observe adverse systemic reactions, nor did we find evidence of damage to intra-articular joint tissues. Thus, the data generated in this study show a favorable safety profile for AMSCs within the joint space in support of a phase I clinical trial evaluating the clinical utility of AMSCs to treat osteoarthritis.

ABSTRACTPreservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA‐seq) and real‐time reverse transcription quantitative polymerase chain reaction (RT‐qPCR) to assess genome‐wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of “early response genes” that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of “early response genes” and reduced levels of ECM‐related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1950–1959, 2016.

ABSTRACTLike any foreign object, orthopaedic implants are susceptible to infection when introduced into the human body. Without additional preventative measures, the absolute number of annual prosthetic joint infections will continue to rise, and may exceed the capacity of health care systems in the near future. Bacteria are difficult to eradicate from synovial joints due to their exceptionally diverse taxonomy, complex mechanistic attachment capabilities, and tendency to evolve antibiotic resistance. When a primary orthopaedic implant fails from prosthetic joint infection, surgeons are generally challenged by limited options for intervention. In this review, we highlight the etiology and taxonomic groupings of bacteria known to cause prosthetic joint infections, and examine their key mechanisms of attachment. We propose that antimicrobial strategies should focus on the most harmful bacteria taxa within the context of occurrence, taxonomic diversity, adhesion mechanisms, and implant design. Patient‐specific identification of organisms that cause prosthetic joint infections will permit assessment of their biological vulnerabilities. The latter can be targeted using a range of antimicrobial techniques that exploit different colonization mechanisms including implant surface attachment, biofilm formation, and/or hematogenous recruitment. We anticipate that customized strategies for each patient, joint, and prosthetic component will be most effective at reducing prosthetic joint infections, including those caused by antibiotic‐resistant and polymicrobial bacteria. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:177–186, 2016.