Anti-inflammatory, Anti-fibrotic and pro-cardiomyogenic effects of genetically engineered extracellular vesicles enriched in miR-1 and miR-199a on human cardiac fibroblasts

Rationale Emerging evidence indicates that stem cell (SC)- derived extracellular vesicles (EVs) carrying bioactive miRNAs are able to repair damaged or infarcted myocardium and ameliorate adverse remodeling. Fibroblasts represent a major cell population responsible for scar formation in the damaged heart. However, the effects of EVs on cardiac fibroblast (CFs) biology and function has not been investigated. Objective To analyze the biological impact of stem cell-derived EVs (SC-EVs) enriched in miR-1 and miR-199a on CFs and to elucidate the underlying molecular mechanisms. Methods and Results Genetically engineered human induced pluripotent stem cells (hiPS) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) expressing miR-1 or miR-199a were used to produce miR-EVs. Cells and EVs were thoughtfully analyzed for miRNA expression using RT-qPCR method. Both hiPS-miRs-EVs and UC-MSC-miRs-EVs effectively transferred miRNAs to recipient CFs, however, hiPS-miRs-EVs triggered cardiomyogenic gene expression in CFs more efficiently than UC-MSC-miRs-EVs. Importantly, hiPS-miR-1-EVs exhibited cytoprotective effects on CFs by reducing apoptosis, decreasing levels of pro-inflammatory cytokines (CCL2, IL-$1\beta$, IL-8) and downregulating the expression of a pro-fibrotic gene – $\alpha$-smooth muscle actin ($\alpha$-SMA). Notably, we identified a novel role of miR-199a-3p delivered by hiPS-EVs to CFs, in triggering the expression of cardiomyogenic genes (NKX2.5, TNTC, MEF2C) and ion channels involved in cardiomyocyte contractility (HCN2, SCN5A, KCNJ2, KCND3). By targeting SERPINE2, miR-199a-3p may reduce pro-fibrotic properties of CFs, whereas miR-199a-5p targeted BCAM and TSPAN6, which may be implicated in downregulation of inflammation. Conclusions hiPS-EVs carrying miR-1 and miR-199a attenuate apoptosis and pro-fibrotic and pro-inflammatory activities of CFs, and increase cardiomyogenic gene expression. These finding serve as rationale for targeting fibroblasts with novel EV-based miRNA therapies to improve heart repair after myocardial injury

The impact of noradrenergic neurotoxin DSP-4 and noradrenaline transporter knockout (NET-KO) on the activity of liver cytochrome P450 3A (CYP3A) in male and female mice

Background Our earlier studies have shown that the brain noradrenergic system regulates cytochrome P450 (CYP) in rat liver via neuroendocrine mechanism. In the present work, a comparative study on the effect of intraperitoneal administration of the noradrenergic neurotoxin DSP-4 and the knockout of noradrenaline transporter (NET-KO) on the CYP3A in the liver of male and female mice was performed. Methods The experiments were conducted on C57BL/6J WT and $NET^{–/–}$ male/female mice. DSP-4 was injected intraperitoneally as a single dose (50 mg/kg ip.) to WT mice. The activity of CYP3A was measured as the rate of 6β-hydroxylation of testosterone in liver microsomes. The CYP3A protein level was estimated by Western blotting. Results DSP-4 evoked a selective decrease in the noradrenaline level in the brain of male and female mice. At the same time, DSP-4 reduced the CYP3A activity in males, but not in females. The level of CYP3A protein was not changed. The NET knockout did not affect the CYP3A activity/protein in both sexes. Conclusions The results with DSP-4 treated mice showed sex-dependent differences in the regulation of liver CYP3A by the brain noradrenergic system (with only males being responsive), and revealed that the NET knockout did not affect CYP3A in both sexes. Further studies into the hypothalamic–pituitary–gonadal hormones in DSP-4 treated mice may explain sex-specific differences in CYP3A regulation, whereas investigation of monoaminergic receptor sensitivity in the hypothalamic/pituitary areas of $NET^{–/–}$ mice will allow for understanding a lack of changes in the CYP3A activity in the NET-KO animals