MicroRNAs regulate the maladaptation of endothelial cells (ECs) to naturally occurring disturbed blood flow at arterial bifurcations resulting in arterial inflammation and atherosclerosis in response to hyperlipidemic stress. Here, we show that reduced endothelial expression of the RNAse Dicer, which generates almost all mature miRNAs, decreases monocyte adhesion, endothelial C–X–C motif chemokine 1 (CXCL1) expression, atherosclerosis and the lesional macrophage content in apolipoprotein E knockout mice (Apoe−/−) after exposure to a high-fat diet. Endothelial Dicer deficiency reduces the expression of unstable miRNAs, such as miR-103, and promotes Krüppel-like factor 4 (KLF4)-dependent gene expression in murine atherosclerotic arteries. MiR-103 mediated suppression of KLF4 increases monocyte adhesion to ECs by enhancing nuclear factor-κB-dependent CXCL1 expression. Inhibiting the interaction between miR-103 and KLF4 reduces atherosclerosis, lesional macrophage accumulation and endothelial CXCL1 expression. Overall, our study suggests that Dicer promotes endothelial maladaptation and atherosclerosis in part by miR-103-mediated suppression of KLF4.

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

Dicer plays an atheroprotective role by coordinately regulating the inflammatory response and lipid metabolism in macrophages through enhancing fatty acid-fueled mitochondrial respiration, suggesting that promoting Dicer/miR-10a-dependent metabolic reprogramming in macrophages has potential therapeutic implications to prevent atherosclerosis.

The consumption of wine and spirits, traditionally aged in oak barrels, exposes humans to roburin ingestion. These molecules belong to a class of ellagitannins (ETs), and their only known source is oak wood. Very little is currently known about roburin bioavailability and biological activity. We reported for the first time human absorption of roburins from a French oak wood (Quercus robur) water extract (Robuvit) by measuring the increase of total phenols (from 0.63 ± 0.06 to 1.26 ± 0.18 μg GAE equiv/mL plasma) and the appearance of roburin metabolites (three different glucoronidate urolithins and ellagic acid), in plasma, after 5 days of supplementation. Robuvit supplementation induced also the increase of plasma antioxidant capacity from 1.8 ± 0.05 to 1.9 ± 0.01 nmol Trolox equiv/mL plasma. Moreover, utilizing a combined ex vivo cell culture approach, we assessed the effect of Q. robur metabolites (present in human serum after supplementation) on gene expression modulation, utilizing an Affymetrix array matrix, in endothelial, neuronal, and keratinocyte cell lines. The functional analysis reveals that Robuvit metabolites affect ribosome, cell cycle, and spliceosome pathways.

Blood flow at arterial bifurcations and curvatures is naturally disturbed. Endothelial cells (ECs) fail to adapt to disturbed flow, which transcriptionally direct ECs toward a maladapted phenotype, characterized by chronic regeneration of injured ECs. MicroRNAs (miRNAs) can regulate EC maladaptation through targeting of protein-coding RNAs. However, long noncoding RNAs (lncRNAs), known epigenetic regulators of biological processes, can also be miRNA targets, but their contribution on EC maladaptation is unclear. Here we show that hyperlipidemia- and oxLDL-induced upregulation of miR-103 inhibits EC proliferation and promotes endothelial DNA damage through targeting of novel lncWDR59. MiR-103 impedes lncWDR59 interaction with Notch1-inhibitor Numb, therefore affecting Notch1-induced EC proliferation. Moreover, miR-103 increases the susceptibility of proliferating ECs to oxLDL-induced mitotic aberrations, characterized by an increased micronucleic formation and DNA damage accumulation, by affecting Notch1-related β-catenin co-activation. Collectively, these data indicate that miR-103 programs ECs toward a maladapted phenotype through targeting of lncWDR59, which may promote atherosclerosis.

Hyperglycemia contributes to dysregulate endothelial function associated with diabetes, leading to initiation and propagation of vascular complications and dysfunction. Caffeic acid (CA), a dietary hydroxycinnamic acid abundant in coffee, has been reported to exert antidiabetic effects in rat models. Herein, we investigated the molecular effects of physiological concentrations of CA (10 nM) against endothelial dysfunction induced by high glucose (HG) in human endothelial cells (HUVECs). HUVECs were exposed to HG 25 mM, to mimic diabetic condition, in presence of CA. Intracellular redox status (reduced glutathione, superoxide dismutase (SOD) and total antioxidant activity levels), and NF-κB pathway were examined. We also evaluated the involvement of NF-E2-related factor 2 (Nrf2)/electrophile responsive element (EpRE) pathway. Our data show that CA inhibits HG-induced nuclear translocation of NF-κB and the downstream expression of endothelial adhesion molecule 1 and restores antioxidant levels by upregulating Nrf2/EpRE pathway. Our data suggest that CA can suppress several aspects of HG-induced endothelial dysfunction through the modulation of intracellular redox status controlled by the transcription factor Nrf2. These findings highlight that low physiological concentration of CA achievable specifically upon food consumption are able to prevent endothelial dysfunction associated with inflammation and oxidative stress induced by high concentration of glucose. © 2016 BioFactors, 43(1):54-62, 2017.

The respiratory system is one of the most affected targets of SARS-CoV-2. Various therapies have been utilized to counter viral-induced inflammatory complications, with diverse success rates. Pending the distribution of an effective vaccine to the whole population and the achievement of “herd immunity”, the discovery of novel specific therapies is to be considered a very important objective. Here, we report a computational study demonstrating the existence of target motifs in the SARS-CoV-2 genome suitable for specific binding with endogenous human micro and long non-coding RNAs (miRNAs and lncRNAs, respectively), which can, therefore, be considered a conceptual background for the development of miRNA-based drugs against COVID-19. The SARS-CoV-2 genome contains three motifs in the 5′UTR leader sequence recognized by selective nucleotides within the seed sequence of specific human miRNAs. The seed of 57 microRNAs contained a “GGG” motif that promoted leader sequence-recognition, primarily through offset-6mer sites able to promote microRNAs noncanonical binding to viral RNA. Similarly, lncRNA H19 binds to the 5′UTR of the viral genome and, more specifically, to the transcript of the viral gene Spike, which has a pivotal role in viral infection. Notably, some of the non-coding RNAs identified in our study as candidates for inhibiting SARS-CoV-2 gene expression have already been proposed against diverse viral infections, pulmonary arterial hypertension, and related diseases.

In order to study the effects of vitamin C supplementation on gene expression and compare its action between physiological and inflammatory conditions, a pilot study was set up utilizing microarray and qPCR technologies. Five healthy volunteers were supplemented with 1 g vitamin C (Redoxon®) per day for five consecutive days. Peripheral blood mononuclear cells (PBMNC) were isolated before and just after the last supplementation, and RNA was isolated for the Affymetrix gene 1.0 ST chip analysis. PBMNC were also, ex vivo, treated with LPS, and gene expression was quantified by means of a “Human NFkB Signaling” qPCR array. Only a very moderate effect on the baseline gene expression modulation was associated with vitamin C supplementation. However, in spite of the limited number of subjects analyzed, vitamin C supplementation resulted in a markedly different modulation of gene expression upon the inflammatory stimulus, specifically at the level of the MyD88-dependent pathway and of the anti-inflammatory cytokine IL-10 synthesis. This study suggests that vitamin C supplementation in healthy subjects, not selected according to a specific genetic profile, consuming an adequate amount of vitamin C, and having a satisfactory vitamin C plasma concentration at the baseline, does not result in a significant modification of gene expression profile. Under this satisfactory micronutrient status, supplementation of vitamin C is “buffered” within a homeostatic physiological equilibrium. Differently, following a second “hit” constituted of an inflammatory stimulus such as LPS, able to trigger a critical burst to the normal physiological state, the higher availability of ascorbic acid emerges, and results in a significant modulation of cell response.