Fibrosis is a significant global health problem associated with many inflammatory and degenerative diseases affecting multiple organs, individually or simultaneously. Fibrosis develops when extracellular matrix (ECM) remodeling becomes excessive or uncontrolled and is associated with nearly all forms of heart disease. Cardiac fibroblasts and myofibroblasts are the main effectors of ECM deposition and scar formation. The heart is a complex multicellular organ, where the various resident cell types communicate between themselves and with cells of the blood and immune systems. Exosomes, which are small extracellular vesicles, (EVs), contribute to cell-to-cell communication and their pathophysiological relevance and therapeutic potential is emerging. Here, we will critically review the role of endogenous exosomes as possible fibrosis mediators and discuss the possibility of using stem cell-derived and/or engineered exosomes as anti-fibrotic agents.
Cardiac fibrosis occurs in a wide range of cardiac diseases and is characterised by the transdifferentiation of cardiac fibroblasts (FB) into myofibroblasts (MFB). Myofibroblasts produce large quantities of extracellular matrix proteins, resulting in myocardial scar. The antifibrotic effect of the bile acid ursodeoxycholic acid (UDCA) is established in cases of liver fibrosis but not the adult myocardium.Our hypothesis is: UDCA is antifibrotic in the adult heart, mediated by the membrane bile acid receptor Takeda G protein-coupled receptor 5 (TGR5).We constructed a predictive network of fibrosis using RNA-seq datasets. We found that UDCA and it’s analogue INT-777, both reduced MFB markers in rat and human FBs and living myocardial slices (LMS). Utilising a knock-out mouse model, we show that the antifibrotic effect of UDCA is mediated by TGR5. Finally, we performed RNA-seq upon UDCA-treated human FB and integrated with our network of fibrosis, establishing the mechanism of TGR5 agonists.
Background Circular RNAs (circRNAs) are an emerging class of noncoding RNAs stemming from the splicing and circularization of pre-mRNAs exons. CircRNAs can regulate transcription and splicing, sequester microRNAs acting as “sponge” and inducing the respective targets, and bind to RNA binding proteins. Recently, they have been found deregulated in dilated cardiomyopathies (DCM), one of the cardiovascular diseases with the worst rate of morbidity and mortality, and whose molecular mechanisms are only partially known. Purpose Therein, we will evaluate in ischemic DCM patients the modulation of 17 circRNAs, 14 out of them obtained from literature data on DCM ischemic or not, while the other 3 were circRNAs not characterized in the heart previously. The study aims to identify circRNAs candidates for further functional characterization in DCM. In addition, as differential expression (DE) analysis is not easily performed for circRNAs in RNA-seq datasets, the validated circRNAs will be used to set up the most specific and sensitive bioinformatics pipeline for circRNA-DE analysis. Methods We designed divergent and convergent specific primers for 17 circRNAs and their host gene, respectively, and their amplification efficiency was measured by RT-qPCR. Transcripts expression was measured in left ventricle biopsies of 12 patients affected by non end-stage ischemic HF and of 12 matched controls. Results We identified cPVT1, cANKRD17, cBPTF as DE, and validated the modulation of 5 out of the 14 DCM-related circRNAs (cHIPK3, cALPK2, cPCMTD1, cNEBL, cSLC8A1), while cPDRM5, cTTN1 showed opposite modulation, which may be due to the specific disease condition. All of them were modulated differently from the respective host gene. CircRNA/miRNA interactions were predicted using Starbase 3.0. Next, mRNAs-targets of the identified miRNAs were predicted by mirDIP 4.1 and intersected with gene expression datasets of the same patients, previously obtained by microarray analysis. We found that cBPTF and cANKRD17 might sponge 12 and 2 miRNAs, respectively. Enrichment analysis of the relevant targets identified several important pathways implicated in DCM, such as MAPK, FoxO, EGFR, VEGF and Insulin/IGF pathways. In addition, deep RNA-Seq analysis that is currently ongoing and the validated circRNAs will be used to optimize the bioinformatics pipeline for circRNA DE analysis. Conclusions We identified a subset of circRNAs deregulated in ischemic HF potentially implicated in HF pathogenesis.
Background Cardiac fibrosis is associated with inflammation and extracellular matrix (ECM) accumulation. A pro-fibrotic cytokine, IL11 induces cardiac fibroblasts conversion to myofibroblasts expressing α-smooth muscle actin (α-SMA) and ECM. MicroRNAs (miRNAs) are a class of small non-coding RNAs which participate in regulation of gene expression; Although mainly intracellular, miRNAs can be released into the blood stream where they can be readily detected. Purpose To screen miRNAs upregulated following IL11 triggered conversion of rat cardiac fibroblasts into myofibroblasts. To validate these miRNAs as potential diagnostic biomarkers of cardiac fibrosis by testing their level in blood plasma and septum of aortic valve stenosis (AVS) patients. Methods and results With a bioinformatical approach (Figure 1), we predicted miRNAs which can target proteins involved in TGFβ and IL-11 pathways of fibrosis progression. Of a vast number of miRNAs, we identified 7 strong candidates. After qPCR validation, we found miRNA-27b-5p and miRNA-497 to be significantly upregulated in rat cardiac fibroblasts treated by IL11 (5 ng/ul) but not TGFβ1 (100 ng/ul), values are 2–ΔΔCt: (3±1.5) and (5.2±2.2) (p-value <0.05) for miRNA-27b-5p and miRNA-497 respectively. Next, we overexpressed these two miRNAs separately in rat cardiac fibroblasts. With immunostaining we observed a (18.3±6.8)% increase in the percentage of α-SMA positive cells for miR-27b-5p and a (38.0±8.3)% increase for miR-497. Moreover, we detected with qPCR a significant up-regulation of α-SMA expression (−ΔΔCt = 3.4±0.9 for miR-27b-5p; −ΔΔCt = 8.2±0.7 for miR-497) in cells overexpressing miRNA27b-5p and miRNA497. Furthermore, we found that levels of both miRNA-27b-5p and miRNA-497 were significantly higher in blood plasma (p=0.0002, p=0.04) of AVS patients compared to age and sex matched control group of healthy donors (Figure 2) and heart septum (p=0.0004, p=0.04) of AVS patients compared to septum of healthy donors that could not be used for transplantation. In addition, quantification of Sirius red staining and immunohistochemistry for Col1a1 displayed significant ECM accumulation in AVS patients (p=0.04). Conclusions We found miRNA-497 and miRNA-27b-5p to be pro-fibrotic in rat fibroblasts. Importantly, we found both miRNAs to be up-regulated in the peripheral blood of AVS patients. FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): Roman Tikhomirov PhD studentship is supported by a fellowship from the University of Verona, ItalyEEU-Cardiac RNA cost action CA17129
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