RNA N6-methyladenosine modulates endothelial atherogenic responses to disturbed flow in mice

Atherosclerosis preferentially occurs in atheroprone vasculature where human umbilical vein endothelial cells (HUVECs) are exposed to disturbed flow. Disturbed flow is associated with vascular inflammation and focal distribution. Recent studies have revealed the involvement of epigenetic regulation in atherosclerosis progression. N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA, but its function in endothelial atherogenic progression remains unclear. Here, we show that m6A mediates the EGFR signaling pathway during EC activation to regulate the atherosclerotic process. Oscillatory stress (OS) reduced the expression of METTL3, the primary m6A methyltransferase. Through m6A sequencing and functional studies, we determined that m6A mediates the mRNA decay of the vascular pathophysiology gene EGFR which leads to EC dysfunction. m6A modification of the EGFR 3'UTR accelerated its mRNA degradation. Double mutation of the EGFR 3'UTR abolished METTL3-induced luciferase activity. Adenovirus-mediated METTL3 overexpression significantly reduced EGFR activation and endothelial dysfunction in the presence of OS. Furthermore, TSP-1, an EGFR ligand, was specifically expressed in atheroprone regions without being affected by METTL3. Inhibition of the TSP-1/EGFR axis by using shRNA and AG1478 significantly ameliorated atherogenesis. Overall, our study revealed that METTL3 alleviates endothelial atherogenic progression through m6A-dependent stabilization of EGFR mRNA, highlighting the important role of RNA transcriptomics in atherosclerosis regulation.

Shear Stress Alterations Activate BMP4/pSMAD5 Signaling and Induce Endothelial Mesenchymal Transition in Varicose Veins

Chronic venous diseases, including varicose veins, are characterized by hemodynamic disturbances due to valve defects, venous insufficiency, and orthostatism. Veins are physiologically low shear stress systems, and how altered hemodynamics drives focal endothelial dysfunction and causes venous remodeling is unknown. Here we demonstrate the occurrence of endothelial to mesenchymal transition (EndMT) in human varicose veins. Moreover, the BMP4-pSMAD5 pathway was robustly upregulated in varicose veins. In vitro flow-based assays using human vein, endothelial cells cultured in microfluidic chambers show that even minimal disturbances in shear stress as may occur in early stages of venous insufficiency induce BMP4-pSMAD5-based phenotype switching. Furthermore, low shear stress at uniform laminar pattern does not induce EndMT in venous endothelial cells. Targeting the BMP4-pSMAD5 pathway with small molecule inhibitor LDN193189 reduced SNAI1/2 expression in venous endothelial cells exposed to disturbed flow. TGFβ inhibitor SB505124 was less efficient in inhibiting EndMT in venous endothelial cells exposed to disturbed flow. We conclude that disturbed shear stress, even in the absence of any oscillatory flow, induces EndMT in varicose veins via activation of BMP4/pSMAD5-SNAI1/2 signaling. The present findings serve as a rationale for the possible use of small molecular mechanotherapeutics in the management of varicose veins.

Vitexin inhibits APEX1 to counteract the flow-induced endothelial inflammation

Vascular endothelial cells are exposed to shear stresses with disturbed vs. laminar flow patterns, which lead to proinflammatory vs. antiinflammatory phenotypes, respectively. Effective treatment against endothelial inflammation and the consequent atherogenesis requires the identification of new therapeutic molecules and the development of drugs targeting these molecules. Using Connectivity Map, we have identified vitexin, a natural flavonoid, as a compound that evokes the gene-expression changes caused by pulsatile shear, which mimics laminar flow with a clear direction, vs. oscillatory shear (OS), which mimics disturbed flow without a clear direction. Treatment with vitexin suppressed the endothelial inflammation induced by OS or tumor necrosis factor-α. Administration of vitexin to mice subjected to carotid partial ligation blocked the disturbed flow-induced endothelial inflammation and neointimal formation. In hyperlipidemic mice, treatment with vitexin ameliorated atherosclerosis. Using SuperPred, we predicted that apurinic/apyrimidinic endonuclease1 (APEX1) may directly interact with vitexin, and we experimentally verified their physical interactions. OS induced APEX1 nuclear translocation, which was inhibited by vitexin. OS promoted the binding of acetyltransferase p300 to APEX1, leading to its acetylation and nuclear translocation. Functionally, knocking down APEX1 with siRNA reversed the OS-induced proinflammatory phenotype, suggesting that APEX1 promotes inflammation by orchestrating the NF-κB pathway. Animal experiments with the partial ligation model indicated that overexpression of APEX1 negated the action of vitexin against endothelial inflammation, and that endothelial-specific deletion of APEX1 ameliorated atherogenesis. We thus propose targeting APEX1 with vitexin as a potential therapeutic strategy to alleviate atherosclerosis.

Endothelial Yin Yang 1 Phosphorylation at S118 Induces Atherosclerosis Under Flow

Rationale: Disturbed flow occurring in arterial branches and curvatures induces vascular endothelial cell (EC) dysfunction and atherosclerosis. We postulated that disturbed flow plays important roles in modulating phosphoprotein expression profiles to regulate endothelial functions and atherogenesis. Objective: The goal of this study is to discover novel site-specific phosphorylation alterations induced by disturbed flow in ECs to contribute to atherosclerosis. Methods and Results: Quantitative phosphoproteomics analysis of ECs exposed to disturbed flow with low and oscillatory shear stress (OS, 0.5plusminus4 dynes/cm 2 ) vs. pulsatile flow with high shear stress (PS, 124plusminus dynes/cm 2 ) revealed that OS induces serine (S)118 phosphorylation of Yin Yang 1 (phospho-YY1 S118 ) in ECs. Elevated phospho-YY1 S118 level in ECs was further confirmed to be present in the disturbed flow regions in experimental animals and human atherosclerotic arteries. This disturbed flow-induced EC phospho-YY1 S118 is mediated by casein kinase 2α (CK2α) through its direct interaction with YY1. Yeast two-hybrid library screening and in situ proximity ligation assays demonstrate that phospho-YY1 S118 directly binds zinc finger with KRAB and SCAN domains 4 (ZKSCAN4) to induce promoter activity and gene expression of human double minute 2 (HDM2), which consequently induces EC proliferation through down-regulations of p53 and p21 CIP1 . Administration of apolipoprotein E-deficient (ApoE -/- ) mice with CK2-specific inhibitor tetrabromocinnamic acid or atorvastatin inhibits atherosclerosis formation through down-regulations of EC phospho-YY1 S118 and HDM2. Generation of novel transgenic mice bearing EC-specific overexpression of S118-non-phosphorylatable mutant of YY1 in ApoE -/- mice confirms the critical role of phospho-YY1 S118 in promoting atherosclerosis through EC HDM2. Conclusions: Our findings provide new insights into the mechanisms by which disturbed flow induces endothelial phospho-YY1 S118 to promote atherosclerosis, thus indicating phospho-YY1 S118 as a potential molecular target for atherosclerosis treatment.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

A Computational Fluid Dynamics Protocol for The Hemodynamic Analysis of a Microfluidic Model of Thrombosis

Thrombosis is both attributed to biochemical agonists and mechanical stresses applied to platelets. Whilst the effect of biochemical agonists has been extensively studied, the mechanosensitive factors remain poorly defined. Stenotic microfluidic channels mimic the narrowing vessels, providing the real-time observation of platelets under disturbed flow. Though the experimental analysis of platelets in disturbed flow confirms the mechanosensitive behavior of platelets, it cannot explicate detailed thresholds for platelet activation. Computational Fluid Dynamics (CFD) could be utilized alongside experimental analysis to characterize thresholds for platelet behavior under imposed shear stress. CFD simulations, however, are prone to uncertainties and errors which should be minimized to obtain compelling results. Hereby, we have presented a CFD protocol for researchers in the field of microfluidic and hemodynamic studies.

Punicalagin Attenuates Disturbed Flow-Induced Vascular Dysfunction by Inhibiting Force-Specific Activation of Smad1/5

BackgroundPathophysiological vascular remodeling in response to disturbed flow with low and oscillatory shear stress (OSS) plays important roles in atherosclerosis progression. Pomegranate extraction (PE) was reported having anti-atherogenic effects. However, whether it can exert a beneficial effect against disturbed flow-induced pathophysiological vascular remodeling to inhibit atherosclerosis remains unclear. The present study aims at investigating the anti-atherogenic effects of pomegranate peel polyphenols (PPP) extraction and its purified compound punicalagin (PU), as well as their protective effects on disturbed flow-induced vascular dysfunction and their underlying molecular mechanisms.MethodsThe anti-atherogenic effects of PPP/PU were examined on low-density lipoprotein receptor knockout mice fed with a high fat diet. The vaso-protective effects of PPP/PU were examined in rat aortas using myograph assay. A combination of in vivo experiments on rats and in vitro flow system with human endothelial cells (ECs) was used to investigate the pharmacological actions of PPP/PU on EC dysfunction induced by disturbed flow. In addition, the effects of PPP/PU on vascular smooth muscle cell (VSMC) dysfunction were also examined.ResultsPU is the effective component in PPP against atherosclerosis. PPP/PU evoked endothelium-dependent relaxation in rat aortas. PPP/PU inhibited the activation of Smad1/5 in the EC layers at post-stenotic regions of rat aortas exposed to disturbed flow with OSS. PPP/PU suppressed OSS-induced expression of cell cycle regulatory and pro-inflammatory genes in ECs. Moreover, PPP/PU inhibited inflammation-induced VSMC dysfunction.ConclusionPPP/PU protect against OSS-induced vascular remodeling through inhibiting force-specific activation of Smad1/5 in ECs and this mechanism contributes to their anti-atherogenic effects.

In Vivo Function of Flow-Responsive Cis -DNA Elements of the Endothelial Nitric Oxide Synthase Gene: A Role for Chromatin-Based Mechanisms

Background: Endothelial nitric oxide synthase (eNOS) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress (SS). Disturbed flow with lower average SS decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains two distinct flow-responsive cis -DNA elements in the promoter, the Shear Stress Response Element (SSRE) and the Krüppel-Like Factor (KLF) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis -DNA elements remains unknown. Methods: Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro . DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Results: Wildtype mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). Surprisingly, the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis -element necessary for eNOS transcription in vivo . The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wildtype mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo . Conclusions: We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic SS in vivo . Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.