Changes in Nascent Chromatin Structure Regulate Activation of the Pro-fibrotic Transcriptome and Myofibroblast Emergence in Organ Fibrosis

Cell reprogramming to a myofibroblast responsible for the pathological accumulation of extracellular matrix is fundamental to the onset of fibrosis. Here, we explored how condensed chromatin structure marked by H3K72me3 becomes modified to allow for activation of repressed genes to drive emergence of myofibroblasts. In the early stages of myofibroblast precursor cell differentiation, we discovered that H3K27me3 demethylase enzymes UTX/KDM6B creates a delay in the accumulation of H3K27me3 on nascent DNA revealing a period of decondensed chromatin structure. This period of decondensed nascent chromatin structure allows for binding of pro-fibrotic transcription factor, Myocardin-related transcription factor A (MRTF-A) to nascent DNA. Inhibition of UTX/KDM6B enzymatic activity condenses chromatin structure, prevents MRTF-A binding, blocks activation of the pro-fibrotic transcriptome, and results in an inhibition of fibrosis in lens and lung fibrosis models. Our work reveals UTX/KDM6B as central coordinators of fibrosis, highlighting the potential to target its demethylase activity to prevent organ fibrosis

Partial bladder outlet obstruction alters Ca2+ sensitivity of force, but not of MLC phosphorylation, in bladder smooth muscle.

Partial bladder outlet obstruction in the rabbit produces changes in bladder function similar to those seen clinically in patients with obstructive uropathies. Whole organ function is significantly altered, as are the smooth muscle cells inside the bladder wall. This study was designed to determine whether outlet obstruction alters smooth muscle function at the level of contractile filaments. Rabbit bladders were partially obstructed for 2 wk. Triton X-100 was used to provide a detergent-skinned bladder smooth muscle preparation that would allow control of the intracellular environment while the ability to shorten and develop force is maintained. Ca2+-force and Ca2+-myosin light chain (MLC) phosphorylation relations and maximal velocity of shortening were determined. The Ca2+ sensitivity of force was significantly lower in tissues from animals subjected to outlet obstruction compared with tissues from control animals. In contrast, no difference was noted in the Ca2+ sensitivity of MLC phosphorylation. Maximal levels of stress and MLC phosphorylation were similar in both animal groups. Maximal velocity of shortening was significantly slower in tissues from outlet-obstructed animals at all Ca2+ concentrations compared with tissues from control animals. Ultrastructurally, detergent skinning had little effect on structural integrity. Moreover, tissues from obstructed animals showed an increase in the number of sarcolemmal attachment plaque structures. We suggest that partial bladder outlet obstruction produces deleterious (e.g., decrease in Ca2+ sensitivity of force) and compensatory (e.g., increase in membrane attachment plaques) changes in bladder smooth muscle cells

Trametinib prevents mesothelial-mesenchymal transition and ameliorates abdominal adhesion formation

Background: Intra-abdominal adhesions are a major cause of morbidity after abdominal or gynecologic surgery. However, knowledge about the pathogenic mechanism(s) is limited, and there are no effective treatments. Here, we investigated a mouse model of bowel adhesion formation and the effect(s) of an Federal Drug Administration-approved drug (trametinib) in preventing adhesion formation. Materials and methods: C57BL/6 mice were used to develop a consistent model of intra-abdominal adhesion formation by gentle cecal abrasion with mortality rates of <10%. Adhesion formation was analyzed histologically and immunochemically to characterize the expression of pro-fibrotic marker proteins seen in pathologic scaring and included alpha smooth muscle actin (alpha SMA) and fibronectin EDA (FNEDA) which arises from alternative splicing of the fibronectin messenger RNA resulting in different protein isoforms. Trichrome staining assessed collagen deposition. Quantitative polymerase chain reaction analysis of RNA isolated from adhesions by laser capture microscopy was carried out to assess pro-fibrotic gene expression. To block adhesion formation, trametinib was administered via a subcutaneous osmotic pump. Results: Adhesions were seen as early as post-operative day 1 with extensive adhesions being formed and vascularized by day 5. The expression of the FNEDA isoform occurred first with subsequent expression of alpha SMA and collagen. The drug trametinib was chosen for in vivo studies because it effectively blocked the mesothelial to mesenchymal transition of rat mesothelium. Trametinib, at the highest dose used (3 mg/kg/d), prevented adhesion formation while at lower doses, adhesions were usually limited, as evidenced by the presence of FNEDA isoform but not alpha SMA. Conclusions: Cecal abrasion in mice is a reliable model to study abdominal adhesions, which can be ameliorated using the MEK1/2 inhibitor trametinib. While blocking adhesion formation at the cell and molecular levels, trametinib, at the therapeutic doses utilized, did not impair the wound healing at the laparotomy site.

Changes in nascent chromatin structure regulate activation of the pro-fibrotic transcriptome and myofibroblast emergence in organ fibrosis

Summary: Cell reprogramming to a myofibroblast responsible for the pathological accumulation of extracellular matrix is fundamental to the onset of fibrosis. Here, we explored how condensed chromatin structure marked by H3K72me3 becomes modified to allow for activation of repressed genes to drive emergence of myofibroblasts. In the early stages of myofibroblast precursor cell differentiation, we discovered that H3K27me3 demethylase enzymes UTX/KDM6B creates a delay in the accumulation of H3K27me3 on nascent DNA revealing a period of decondensed chromatin structure. This period of decondensed nascent chromatin structure allows for binding of pro-fibrotic transcription factor, Myocardin-related transcription factor A (MRTF-A) to nascent DNA. Inhibition of UTX/KDM6B enzymatic activity condenses chromatin structure, prevents MRTF-A binding, blocks activation of the pro-fibrotic transcriptome, and results in an inhibition of fibrosis in lens and lung fibrosis models. Our work reveals UTX/KDM6B as central coordinators of fibrosis, highlighting the potential to target its demethylase activity to prevent organ fibrosis