GPCR-induced YAP activation sensitizes fibroblasts to profibrotic activity of TGFß1.

Tissue fibrosis is a pathological condition characterized by uncontrolled fibroblast activation that ultimately leads to organ failure. The TGFß1 pathway, one of the major players in establishment of the disease phenotype, is dependent on the transcriptional co-activators YAP/TAZ. We were interested whether fibroblasts can be sensitized to TGFß1 by activation of the GPCR/YAP/TAZ axis and whether this mechanism explains the profibrotic properties of diverse GPCR ligands. We found that LPA, S1P and thrombin cooperate in human dermal fibroblasts with TGFß1 to induce extracellular matrix synthesis, myofibroblast marker expression and cytokine secretion. Whole genome expression profiling identified a YAP/TAZ signature behind the synergistic profibrotic effects of LPA and TGFß1. LPA, S1P and thrombin stimulation led to activation of the Rho-YAP axis, an increase of nuclear YAP-Smad2 complexes and enhanced expression of profibrotic YAP/Smad2-target genes. More generally, dermal, cardiac and lung fibroblast responses to TGFß1 could be enhanced by increasing YAP nuclear levels (with GPCR ligands LPA, S1P, thrombin or Rho activator) and inhibited by decreasing nuclear YAP (with Rho inhibitor, forskolin, latrunculin B or 2-deoxy-glucose). Thus, we present here a conceptually interesting finding that fibroblast responses to TGFß1 can be predicted based on the nuclear levels of YAP and modulated by stimuli/treatments that change YAP nuclear levels. Our study contributes to better understanding of fibrosis as a complex interplay of signalling pathways and proposes YAP/TAZ as promising targets in the treatment of fibrosis.

A novel genomic signature with translational significance for human idiopathic pulmonary fibrosis.

The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat-human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-ß1-treated primary human lung fibroblasts and transforming growth factor-ß1-treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model-human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease.

In vitro and in vivo antibacterial evaluation of cadazolid, a new antibiotic for treatment of Clostridium difficile infections.

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 µg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.

Identification of cathepsin L as a potential sex-specific biomarker for renal damage.

The renin-angiotensin system is a well-known regulator of blood pressure and plays an important role in the pathogenesis of cardiovascular disease and renal damage. Genetic factors, including single nucleotide polymorphisms and sex, are increasingly recognized as potential risk factors for the development of cardiovascular disease. Double transgenic rats (dTGRs), harboring human renin and angiotensinogen genes, were used in this study to investigate potential sex differences influencing renal function and renal gene expression. dTGR males and females had comparable increases in blood pressure, whereas body weight, albuminuria/proteinuria, and urine flow rate were higher in males. At 8 weeks of age, renal plasma flow and glomerular filtration rate were proportionally lower in males, and renal vascular resistance tended to be higher. Males developed more severe tubulointerstitial and vascular lesions. By the end of week 8, 40%of the males but none of the females had died. Genome expression studies were performed with RNA from kidneys of 7-week-old male and female dTGRs and control rats to further investigate the sex-related differences on a molecular level. Forty-five genes showed sex-dependent expression patterns in dTGRs that were significantly different compared to controls. Cathepsin L, one of the genes differentially expressed between the sexes, was also shown to be strongly associated with the degree of renal injury. In dTGRs, urinary cathepsin L at week 7 was higher in males (nanograms per 24 hours: male, 512±163; female, 132±70). These results reveal a potential new biomarker for the personalized diagnosis and management of chronic kidney disease.