Investigation of mesalazine as an antifibrotic drug following myocardial infarction in male mice.

OBJECTIVES: Myocardial infarction (MI) initiates a complex reparative response during which damaged cardiac muscle is replaced by connective tissue. While the initial repair is essential for survival, excessive fibrosis post-MI is a primary contributor to progressive cardiac dysfunction, and ultimately heart failure. Currently, there are no approved drugs for the prevention or the reversal of cardiac fibrosis. Therefore, we tested the therapeutic potential of repurposed mesalazine as a post-MI therapy, as distinct antifibrotic effects have recently been demonstrated. METHODS: At 8 weeks of age, MI was induced in male C57BL/6J mice by LAD ligation. Mesalazine was administered orally at a dose of 100 µg/g body weight in drinking water. Fluid intake, weight development, and cardiac function were monitored for 28 days post intervention. Fibrosis parameters were assessed histologically and via qPCR. RESULTS: Compared to controls, mesalazine treatment offered no survival benefit. However, no adverse effects on heart and kidney function and weight development were observed, either. While total cardiac fibrosis remained largely unaffected by mesalazine treatment, we found a distinct reduction of perivascular fibrosis alongside reduced cardiac collagen expression. CONCLUSIONS: Our findings warrant further studies on mesalazine as a potential add-on therapy post-MI, as perivascular fibrosis development was successfully prevented.

Systemic mesalazine treatment prevents spontaneous skin fibrosis in PLK2-deficient mice.

Skin fibrosis is a complex biological remodeling process occurring in disease like systemic sclerosis, morphea, or eosinophilic fasciitis. Since the knowledge about the underlying pathomechanisms is still incomplete, there is currently no therapy, which prevents or reverses skin fibrosis sufficiently. The present study investigates the role of polo-like kinase 2 (PLK2) and the pro-fibrotic cytokine osteopontin (OPN) in the pathogenesis of cutaneous fibrosis and demonstrates the antifibrotic effects of systemic mesalazine treatment in vivo. Isolated primary dermal fibroblasts of PLK2 wild-type (WT) and knockout (KO) mice were characterized in vitro. Skin thickness and histoarchitecture were studied in paraffin-embedded skin sections. The effects of mesalazine treatment were examined in isolated fibroblasts and PLK2 KO mice, which were fed 100 µg/g mesalazine for 6 months via the drinking water. Compared to WT, PLK2 KO fibroblasts displayed higher spontaneous myofibroblast differentiation, reduced proliferation rates, and overexpression of the fibrotic cytokine OPN. In vitro, 72 h of treatment with 10 mmol/L mesalazine induced phenotype conversion in PLK2 KO fibroblasts and attenuated OPN expression by inhibiting ERK1/2. In vivo, dermal myofibroblast differentiation, collagen accumulation, and skin thickening were prevented by mesalazine in PLK2 KO. Plasma creatinine levels indicated good tolerability of systemic long-term mesalazine treatment. The current study reveals a spontaneous fibrotic skin phenotype and ERK1/2-dependent OPN overexpression in PLK2 KO mice. We provide experimental evidence for the antifibrotic effectiveness of systemic mesalazine treatment to prevent fibrosis of the skin, suggesting further investigation in experimental and clinical settings.

Genetic Deletion of Polo-Like Kinase 2 Induces a Pro-Fibrotic Pulmonary Phenotype.

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.

Transferability of miRNA-technology to bioprocessing: Influence of cultivation mode and media.

The biopharmaceutical industry strives for improvement of their production processes. In recent years, miRNAs have been shown to positively impact the production capacity of recombinant CHO cells, especially with regard to difficult to express proteins. Effective and reliable gene regulation of process relevant target genes by miRNAs is a prerequisite for integrating them into the toolbox of industrial cell engineering strategies. However, most studies rely on transient transfection of miRNA mimics; there is low standardization in evaluation of miRNA function and little knowledge on transferability of effects found during transient expression to stable expression during industry relevant fed-batch cultivation. In order to provide more insight into this topic, we used the pcDNA6.2 vector for stable miRNA overexpression during batch and fed-batch cultivation in CHO DG44 cells, optimized the vector, and compared the miRNA levels and effects with those achieved by transfection of miRNA mimics. We found that miR-1 downregulated TWF1 mRNA in different recombinant CHO DG44 clones in a dose-dependent manner during transient batch cultivation. Cells stably overexpressing miR-1 also showed a TWF1 mRNA downregulation when cultivated in batch mode using in-house medium 1. However, when the cells stably overexpressing miR-1 were cultivated in fed-batch mode using in-house medium 2. Consequently, a change of cultivation mode and medium seems to have an impact on target gene regulation by miRNA. Taken together, our findings highlight the importance to standardize miRNA evaluations and test miRNAs in the final application environment.

Repurposing mesalazine against cardiac fibrosis in vitro.

Cardiovascular diseases are exacerbated and driven by cardiac fibrosis. TGFß induces fibroblast activation and differentiation into myofibroblasts that secrete excessive extracellular matrix proteins leading to stiffening of the heart, concomitant cardiac dysfunction, and arrhythmias. However, effective pharmacotherapy for preventing or reversing cardiac fibrosis is presently unavailable. Therefore, drug repurposing could be a cost- and time-saving approach to discover antifibrotic interventions. The aim of this study was to investigate the antifibrotic potential of mesalazine in a cardiac fibroblast stress model. TGFß was used to induce a profibrotic phenotype in a human cardiac fibroblast cell line. After induction, cells were treated with mesalazine or solvent control. Fibroblast proliferation, key fibrosis protein expression, extracellular collagen deposition, and mechanical properties were subsequently determined. In response to TGFß treatment, fibroblasts underwent a profound phenoconversion towards myofibroblasts, determined by the expression of fibrillary αSMA. Mesalazine reduced differentiation nearly by half and diminished fibroblast proliferation by a third. Additionally, TGFß led to increased cell stiffness and adhesion, which were reversed by mesalazine treatment. Collagen 1 expression and deposition-key drivers of fibrosis-were significantly increased upon TGFß stimulation and reduced to control levels by mesalazine. SMAD2/3 and ERK1/2 phosphorylation, along with reduced nuclear NFκB translocation, were identified as potential modes of action. The current study provides experimental pre-clinical evidence for antifibrotic effects of mesalazine in an in vitro model of cardiac fibrosis. Furthermore, it sheds light on possible mechanisms of action and suggests further investigation in experimental and clinical settings.