MicroRNAs and obesity-induced endothelial dysfunction: key paradigms in molecular therapy

Abstract

The endothelium plays a pivotal role in maintaining vascular health. Obesity is a global epidemic that has seen dramatic increases in both adult and pediatric populations. Obesity perturbs the integrity of normal endothelium, leading to endothelial dysfunction which predisposes the patient to cardiovascular diseases. MicroRNAs (miRNAs) are short, single-stranded, non-coding RNA molecules that play important roles in a variety of cellular processes such as differentiation, proliferation, apoptosis, and stress response; their alteration contributes to the development of many pathologies including obesity. Mediators of obesity-induced endothelial dysfunction include altered endothelial nitric oxide synthase (eNOS), Sirtuin 1 (SIRT1), oxidative stress, autophagy machinery and endoplasmic reticulum (ER) stress. All of these factors have been shown to be either directly or indirectly caused by gene regulatory mechanisms of miRNAs. In this review, we aim to provide a comprehensive description of the therapeutic potential of miRNAs to treat obesity-induced endothelial dysfunction. This may lead to the identification of new targets for interventions that may prevent or delay the development of obesity-related cardiovascular disease.

Keywords: Cardiovascular diseases; Endothelial dysfunction; MicroRNAs; Obesity.

Fig. 1

Summarized scheme of miRNAs biogenesis and mechanism of action: In the nucleus, Pol II and Pol III RNA polymerases transcribe the coding sequences of miRNAs. Drosha binds to DGCR8 cofactor to catalyze the formation of pre-miRNA. Pre-miRNA is then translocated by the exportins system from the nucleus to the cytoplasm, where it is then cleaved by the Dicer-TRBP complex to form a 22nt-dsRNA. Within the cytoplasm, the 22nt-dsRNA interacts with AGO proteins to form the RISC complex, while the passenger strand is degraded. The 22nt-RNA guide chain constitutes a mature miRNA that guides the RISC complex towards the 3-UTR regions of the mRNA targets. This interaction either represses their translation or induces their degradation. Pol: polymerase; Drosha: ribonuclease III double-stranded RNA-specific endoribonuclease; DGCR8: DiGeorge syndrome chromosomal region 8; Dicer: helicase with RNase motif; TRBP: TAR RNA binding protein; AGO: Argonaute protein; RISC: RNA-induced silencing complex; UTR: untranslated region

Fig. 2

Obesity induces endothelial dysfunction. Obesity induces a decrease in NO bioavailability by: reducing eNOS activation and/or expression; negatively regulating SIRT1; and inducing cellular stresses including oxidative stress, ER stress and autophagy disruption. All of these effects lead to endothelial dysfunction. NO: Nitric Oxide; eNOS: endothelial Nitric Oxide Synthase; SIRT1: Sirtuin 1; ER: endoplasmic reticulum

Fig. 3

Obesity, eNOS, miRNAs and endothelial function. Ca²⁺ activates eNOS which then converts l-Arginine to l-Citrulline, producing NO as a byproduct. NO, synthetized through eNOS phosphorylation in the endothelium, will diffuse to the smooth muscle cells and increase the levels of cGMP, which then induce vasodilatation. During obesity, higher levels of miR-24, miR-155, miR-15b, miR-16, miR-221/222 and miR-765 are expressed and directly inhibit eNOS translation, thereby causing endothelial dysfunction. Other miRNAs have been shown to affect eNOS through indirect signaling pathways such as AKT (miR-21, miR-26, miR-221/222, and miR-486) and Caveolin1 (miR-132 and miR-124). eNOS: endothelial Nitric Oxide Synthase; NO: Nitric Oxide; cGMP: cyclic guanosine monophosphate; Cav1: Caveolin 1; AKT: Protein kinase B

Fig. 4

Obesity, SIRT1, miRNAs and endothelial function. SIRT1 directly deacetylates eNOS, PGC-1 α, p53, and FoxO1 leading to enhanced NO bioavailability and mitochondrial biogenesis as well as, decreased senescence, angiogenesis, apoptosis, and oxidative stress. All of these effects allow for the maintenance of endothelial homeostasis. Levels of miRNAs that directly target SIRT1 and thereby modulate endothelial function can be either increased (miR-204, miR-34a, miR-132, miR-217 and miR-200c) or decreased (miR-149) in obesity. SIRT1: Sirtuin 1; eNOS: endothelial Nitric Oxide Synthase; NO: Nitric Oxide; ROS: Reactive oxygen species; SOD2: Superoxide dismutase; P53: Tumor protein; FoxO: Forkhead box; PGC-1 α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha

Fig. 5

Obesity, oxidative stress, miRNAs and endothelial function. Superoxide anions (O₂^·) are mainly produced by NOX2, NOX4 and mitochondria through electron transport chain complex I and III. Under physiological conditions, O₂^· are highly regulated by antioxidant mechanism through SOD that detoxifies O₂^· to hydrogen peroxide (H₂O₂), which is then converted to water (H₂O) by catalase or glutathione peroxidase (GSHPx) and peroxiredoxins (PRDXs). Obesity interferes with the normal workings of this system. By decreasing miR-448-3p and miR-19b, it increases NOX2 levels. At the same time, it decreases miR-25 while increasing miR-146a, which can cause up or downregulation of NOX4 levels respectively (NOX4 has dual effect). Additionally, obesity increases the levels of miR-210 and miR-29, both of which affect the mitochondrial respiratory chain. Moreover, obesity modulates the levels of miRNAs that regulate the antioxidant system (e.g. miR-21 and miR-17, both of which target SOD, with miR-17 additionally targeting GSHPx; and miR-23b and miR-200c, both of which target PRDXs). Altogether, the modulation of miRNA levels during obesity leads to excessive production of O₂^· and consequently endothelial dysfunction. SOD: Superoxide dismutase; GSHPx: glutathione peroxidase; PRDXs: peroxiredoxins; NOX2: catalytic, membrane-bound subunit of NADPH oxidase; NOX4: NADPH oxidase homolog

Fig. 6

Obesity, autophagy flux, miRNAs and endothelial function. Autophagy flux consists of a series of steps including initiation, elongation, fusion of the autophagosome with the lysosome and degradation. Autophagy induction is controlled by mTOR and AMPK, which tightly regulate the ULK complex: ULK1, ATG13 and FIP200. By modulating miRNA levels, obesity regulates autophagy induction. During obesity, increased levels of miR-155, miR-199a-5b and miR-101 are expressed and all directly target mTOR. miR-148b and miR-451, also increased during obesity, target AMPK. miR-199a, miR-28a, miR-106b and miR-17-5p target ULK1, while miR-20a and miR-20b target FIP200. Beclin1, which also plays an important role in autophagy initiation, is targeted by miR-506-3p, miR-216a, miR-129-5p and miR-376b. These four miRNAs are known to be increased during obesity. Autophagosome elongation involves ATG5, ATG7, ATG 12, ATG-16L1, LC3 and p62. ATG 5 can be suppressed by miR-181a and miR-374a; ATG7 by miR-210, miR-188-3p and miR-137; ATG 12 by miR-30d and miR-630 and ATG 16-L1 by miR-20a and miR-96. All these miRNAs are increased during obesity. LC3-II is post-transcriptionally controlled by miR-204, which is known to be induced during obesity. P62 is controlled by miR-17, miR-20, miR-93 and miR-106, which are all also increased during obesity. The fusion and degradation step is controlled by UVRAG, LAMP-1/2, RAB7, VAMP8 and STX17. All of these membrane fusion factors are tightly regulated by miRNAs. UVRAG is suppressed by miR-374, miR-630, miR-125, and miR-351; Lamp1 is inhibited by miR-23a and miR-320a, while LAMP2 is inhibited by miR-487-5p; Rab7, VAMP8 and STX17 are regulated by miR-30c, miR-96, and miR-124, respectively. All of these miRNAS are induced by obesity. In summary, the modulation that takes place in miRNA levels during obesity leads to autophagy flux disruption-induced endothelial dysfunction. ULK1: unc-51-like kinase 1; FIP200: focal adhesion kinase family interacting protein of 200 kDa; mTOR: mammalian target of rapamycin; ATG: autophagy-related protein; LC3: Microtubule-associated protein 1A/1B-light chain 3; p62: sequestrome 1; UVRAG: UV resistance-associated gene; VAMP8: vesicle-associated membrane protein; STX17: Syntaxin; LAMP: Lysosome-associated membrane proteins; Rab7: Ras-related protein

Fig. 7

Obesity, ER stress, miRNAs and endothelial function. Under ER stress, BIP will activate the three UPR sensor pathways that are initiated by PERK, ATF6, and IRE1. MicroRNAs involved in the UPR pathway and evidenced linkage to obesity and endothelial dysfunction are listed in this diagram. miR-30d, miR-181a, and miR-199a-5p have all been shown to directly target BIP. MiR-204, miR-23a, miR-27a and miR-24 directly target and inhibit PERK signaling, which then causes ER-stress-induced cell death. ATF4, located downstream of PERK, is directly inhibited by miR-214 and miR-663. CHOP, which is regulated by ATF4, is suppressed by miR-211. IRE1α and its downstream signaling XBP1 make up the second arm of the UPR; the former is targeted by miR-1291, miR-23a, miR-27a and miR-24, while the latter is targeted by miR-214. Finally, in the third arm of UPR, miR-221, miR-145 and miR-494 directly target ATF6. ER: endoplasmic reticulum; ATF6: Activating transcription factor 6; IRE1α inositol requiring enzyme 1 alpha; and PERK protein kinase-like ER kinase; BIP: Binding Immunoglobulin Protein, also known as GRP78; UPR: unfolded protein response, ATF 4: Activating transcription factor 4; CHOP: CCAAT-enhancer-binding protein homologous protein; XBP1: X box binding protein 1; EI2 α: Eukaryotic Initiation Factor 2 alpha