Protease‐activated receptor (PAR)‐2 is required for PAR‐1 signalling in pulmonary fibrosis

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease‐activated receptor (PAR)‐1 and PAR‐2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin‐induced lung fibrosis is diminished in both PAR‐1 and PAR‐2 deficient mice. We thus have been suggested that combined inactivation of PAR‐1 and PAR‐2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR‐1 and PAR‐2 agonists in the absence or presence of specific PAR‐1 or PAR‐2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild‐type and PAR‐2 deficient mice with or without a specific PAR‐1 antagonist (P1pal‐12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR‐1 and/or PAR‐2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR‐1 and PAR‐2 did not show any additive effects on these pro‐fibrotic responses. Strikingly, PAR‐2 deficiency as well as pharmacological PAR‐1 inhibition reduced bleomycin‐induced pulmonary fibrosis to a similar extent. PAR‐1 inhibition in PAR‐2 deficient mice did not further diminish bleomycin‐induced pulmonary fibrosis. Finally, we show that the PAR‐1‐dependent pro‐fibrotic responses are inhibited by the PAR‐2 specific antagonist. Targeting PAR‐1 and PAR‐2 simultaneously is not superior to targeting either receptor alone in bleomycin‐induced pulmonary fibrosis. We postulate that the pro‐fibrotic effects of PAR‐1 require the presence of PAR‐2.     

Age and sex differences in kidney microRNA expression during the life span of F344 rats

Background

Growing evidence suggests that epigenetic mechanisms of gene regulation may play a role in susceptibilities to specific toxicities and adverse drug reactions. MiRNAs in particular have been shown to be important regulators in cancer and other diseases and show promise as predictive biomarkers for diagnosis and prognosis. In this study, we characterized the global kidney miRNA expression profile in untreated male and female F344 rats throughout the life span. These findings were correlated with sex-specific susceptibilities to adverse renal events, such as male-biased renal fibrosis and inflammation in old age.

Methods

Kidney miRNA expression was examined in F344 rats at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age in both sexes using Agilent miRNA microarrays. Differential expression was determined using filtering criteria of ≥1.5 fold change and ANOVA or pairwise t-test (FDR <5%) to determine significant age and sex effects, respectively. Pathway analysis software was used to investigate the possible roles of these target genes in age- and sex-specific differences.

Results

Three hundred eleven miRNAs were found to be expressed in at least one age and sex. Filtering criteria revealed 174 differentially expressed miRNAs in the kidney; 173 and 34 miRNAs exhibiting age and sex effects, respectively. Principal component analysis revealed age effects predominated over sex effects, with 2-week miRNA expression being much different from other ages. No significant sexually dimorphic miRNA expression was observed from 5 to 8 weeks, while the most differential expression (13 miRNAs) was observed at 21 weeks. Potential target genes of these differentially expressed miRNAs were identified.

Conclusions

The expression of 56% of detected renal miRNAs was found to vary significantly with age and/or sex during the life span of F344 rats. Pathway analysis suggested that 2-week-expressed miRNAs may be related to organ and cellular development and proliferation pathways. Male-biased miRNA expression at older ages correlated with male-biased renal fibrosis and mononuclear cell infiltration. These miRNAs showed high representation in renal inflammation and nephritis pathways, and included miR-214, miR-130b, miR-150, miR-223, miR-142-5p, miR-185, and miR-296*. Analysis of kidney miRNA expression throughout the rat life span will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13293-014-0019-1) contains supplementary material, which is available to authorized users.     

Crosstalk of cardiomyocytes and fibroblasts in co-cultures

Electromechanical function of cardiac muscle depends critically on the crosstalk of myocytes with non-myocytes. Upon cardiac fibrosis, fibroblasts translocate into infarcted necrotic tissue and alter their communication capabilities. In the present in vitro study, we determined a multiple parameter space relevant for fibrotic cardiac tissue development comprising the following essential processes: (i) adhesion to substrates with varying elasticity, (ii) dynamics of contractile function, and (iii) electromechanical connectivity. By combining electric cell-substrate impedance sensing (ECIS) with conventional optical microscopy, we could measure the impact of fibroblast–cardiomyocyte ratio on the aforementioned parameters in a non-invasive fashion. Adhesion to electrodes was quantified via spreading rates derived from impedance changes, period analysis allowed us to measure contraction dynamics and modulations of the barrier resistance served as a measure of connectivity. In summary, we claim that: (i) a preferred window for substrate elasticity around 7 kPa for low fibroblast content exists, which is shifted to stiffer substrates with increasing fibroblast fractions. (ii) Beat frequency decreases nonlinearly with increasing fraction of fibroblasts, while (iii) the intercellular resistance increases with a maximal functional connectivity at 75% fibroblasts. For the first time, cardiac cell–cell junction density-dependent connectivity in co-cultures of cardiomyocytes and fibroblasts was quantified using ECIS.     

METTL3/N6‐methyladenosine/ miR‐21‐5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF‐κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR‐21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF‐kB signalling pathway. However, whether miR‐21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6‐methyladenosine (m⁶A) modification‐dependent primary microRNA (pri‐microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR‐21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK‐2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR‐21‐5p mimic or miR‐21‐5p inhibitor. We found that the levels of miR‐21‐5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF‐kB pathway was activated by miR‐21‐5p, confirming that miR‐21‐5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR‐21‐5p maturation. Our research is the first to demonstrate the role of the METTL3‐m⁶A‐miR‐21‐5p‐SPRY1/ERK/NF‐kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

S-nitrosothiols increases cystic fibrosis transmembrane regulator expression and maturation in the cell surface

S-nitrosothiols (SNOs) are endogenous signaling molecules with a broad spectrum of beneficial airway effects. SNOs are normally present in the airway, but levels tend to be low in cystic fibrosis (CF) patients. We and others have demonstrated that S-nitrosoglutathione (GSNO) increases the expression, maturation, and function of wild-type and mutant F508del cystic fibrosis transmembrane conductance regulator (CFTR) in human bronchial airway epithelial (HBAE) cells. We hypothesized that membrane permeable SNOs, such as S-nitrosoglutathione diethyl ester (GNODE) and S-nitroso-N-acetyl cysteine (SNOAC) may be more efficient in increasing the maturation of CFTR. HBAE cells expressing F508del CFTR were exposed to GNODE and SNOAC. The effects of these SNOs on the expression and maturation of F508del CFTR were determined by cell surface biotinylation and Western blot analysis. We also found for the first time that GNODE and SNOAC were effective at increasing CFTR maturation at the cell surface. Furthermore, we found that cells maintained at low temperature increased cell surface stability of F508del CFTR whereas the combination of low temperature and SNO treatment significantly extended the half-life of CFTR. Finally, we showed that SNO decreased the internalization rate of F508del CFTR in HBAE cells. We anticipate identifying the novel mechanisms, optimal SNOs, and lowest effective doses which could benefit cystic fibrosis patients.     

Structural insights into PDZ-mediated interaction of NHERF2 and LPA2, a cellular event implicated in CFTR channel regulation

The formation of CFTR–NHERF2–LPA₂ macromolecular complex in airway epithelia regulates CFTR channel function and plays an important role in compartmentalized cAMP signaling. We previously have shown that disruption of the PDZ-mediated NHERF2–LPA₂ interaction abolishes the LPA inhibitory effect and augments CFTR Cl⁻ channel activity in vitro and in vivo. Here we report the first crystal structure of the NHERF2 PDZ1 domain in complex with the C-terminal LPA₂ sequence. The structure reveals that the PDZ1–LPA₂ binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four LPA₂ residues contributing to specific interactions. Comparison of the PDZ1–LPA₂ structure to the structure of PDZ1 in complex with a different peptide provides insights into the diverse nature of PDZ1 substrate recognition and suggests that the conformational flexibility in the ligand binding pocket is involved in determining the broad substrate specificity of PDZ1. In addition, the structure reveals a small surface pocket adjacent to the ligand-binding site, which may have therapeutic implications. This study provides an understanding of the structural basis for the PDZ-mediated NHERF2–LPA₂ interaction that could prove valuable in selective drug design against CFTR-related human diseases.     

Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.     

Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.