Exosomal miRNAs as Potential Biomarkers to Monitor Phosphodiesterase 5 Inhibitor Induced Anti-Fibrotic Effects on CCl4 Treated Rats

Abstract

MicroRNAs (miRNAs) are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of non-alcoholic fatty liver disease. Here, we investigated the phosphodiesterase 5 (PDE5) inhibitor induced effects on hepatic and plasma exosomal miRNA expression in CCl4-treated rats. In the present study, hepatic miRNA profiling was conducted using the Nanostring nCounter technology and mRNA profiling using RNA sequencing from PDE5 treated rats in the model of CCl4-induced liver fibrosis. To evaluate if the PDE5 inhibitor affected differentially expressed miRNAs in the liver can be detected in plasma exosomes, qRT-PCR specific assays were used. In livers from CCl4-treated rats, the expression of 22 miRNAs was significantly increased (>1.5-fold, adj. p < 0.05), whereas the expression of 16 miRNAs was significantly decreased (>1.5-fold, adj. p < 0.05). The majority of the deregulated miRNA species are implicated in fibrotic and inflammatory processes. The PDE5 inhibitor suppressed the induction of pro-fibrotic miRNAs, such as miR-99b miR-100 and miR-199a-5p, and restored levels of anti-fibrotic miR-122 and miR-192 in the liver. In plasma exosomes, we observed elevated levels of miR-99b, miR-100 and miR-142-3p after treatment with the PDE5-inhibitor compared to CCl4/Vehicle-treated. Our study demonstrated for the first time that during the development of hepatic fibrosis in the preclinical model of CCl4-induced liver fibrosis, defined aspects of miRNA regulated liver pathogenesis are influenced by PDE5 treatment. In conclusion, miRNA profiling of plasma exosomes might be used as a biomarker for NASH progression and monitoring of treatment effects.

Extracellular Vesicles in Organ Fibrosis: Mechanisms, Therapies, and Diagnostics

“…Serum exosomal miR-103-3p levels allowed discrimination between HBV patients with no, mild or severe hepatic fibrosis, consistent with the role of exosomal miR-103 in macrophage-mediated activation of HSC (see above) [ 270 ]. Plasma EVs from CCl 4 -treated rats contained upregulated mir-122, -99b, and -192 and downregulated miR-100 and these were variably differentially expressed when the rats were treated with anti-fibrotic phosphodiesterase 5 inhibitor supporting their potential as biomarkers during fibrosis therapy [ 304 ]. Finally, in Schistosoma infection, levels of serum exosomal miR-92a-3p, 146a-5p and 532-5p discriminated patients with grades I-III hepatic fibrosis from those with no hepatic fibrosis, while exosomal miR-146a-5p also showed promise for discriminating grades 0–I (mild) hepatic fibrosis from grades II–III (severe) hepatic fibrosis [ 305 ].…”

Exosomal microRNAs as diagnostic and therapeutic biomarkers in non-malignant liver diseases

“…Serum exosomal miR-146a-5p has the potential to serve as a biomarker for grading liver fibrosis due to schistosomiasis (Cai et al 2020). Furthermore, exosomal microRNA biomarkers that enable us to predict the effects of prognostic anti-fibrotic treatments, such as the phosphodiesterase 5 inhibitor were identified in vivo; these included exosomal miR-99b, miR-100, and miR-142-3p (Broermann et al 2020). Their clinical implications should be further evaluated.…”

Discovery of Potent Phosphodiesterase-9 Inhibitors for the Treatment of Hepatic Fibrosis

“…All reagents were purchased from commercial suppliers and used directly without further purification. 1 H NMR and 13 C NMR spectra were recorded at room temperature on a Bruker AVANCE III 400 instrument or a Bruker Ascend TM 500 instrument with tetramethylsilane (TMS) as an internal standard. The following abbreviations are used in the spectra: s (singlet), d (doublet), dd (two doublets), t (triplet), q (quartet), and m (multiplet).…”

“…Procedures used to perform the hERG inhibition, human CYP450 inhibition, acute toxicity, molecular docking and molecular dynamic simulations, 1 H NMR, 13 C NMR, HRMS, and purity of the tested compounds (PDF) Molecular formula strings and some data (CSV)…”