Activation of Nrf2 inhibits atherosclerosis in ApoE-/- mice through suppressing endothelial cell inflammation and lipid peroxidation.

BACKGROUND: Nuclear erythroid 2-related factor 2 (Nrf2), a transcription factor, is critically involved in the regulation of oxidative stress and inflammation. However, the role of endothelial Nrf2 in atherogenesis has yet to be defined. In addition, how endothelial Nrf2 is activated and whether Nrf2 can be targeted for the prevention and treatment of atherosclerosis is not explored. METHODS: RNA-sequencing and single-cell RNA sequencing analysis of mouse atherosclerotic aortas were used to identify the differentially expressed genes. In vivo endothelial cell (EC)-specific activation of Nrf2 was achieved by injecting adeno-associated viruses into ApoE-/- mice, while EC-specific knockdown of Nrf2 was generated in Cdh5CreCas9floxed-stopApoE-/- mice. RESULTS: Endothelial inflammation appeared as early as on day 3 after feeding of a high cholesterol diet (HCD) in ApoE-/- mice, as reflected by mRNA levels, immunostaining and global mRNA profiling, while the immunosignal of the end-product of lipid peroxidation (LPO), 4-hydroxynonenal (4-HNE), started to increase on day 10. TNF-α, 4-HNE, and erastin (LPO inducer), activated Nrf2 signaling in human ECs by increasing the mRNA and protein expression of Nrf2 target genes. Knockdown of endothelial Nrf2 resulted in augmented endothelial inflammation and LPO, and accelerated atherosclerosis in Cdh5CreCas9floxed-stopApoE-/- mice. By contrast, both EC-specific and pharmacological activation of Nrf2 inhibited endothelial inflammation, LPO, and atherogenesis. CONCLUSIONS: Upon HCD feeding in ApoE-/- mice, endothelial inflammation is an earliest event, followed by the appearance of LPO. EC-specific activation of Nrf2 inhibits atherosclerosis while EC-specific knockdown of Nrf2 results in the opposite effect. Pharmacological activators of endothelial Nrf2 may represent a novel therapeutic strategy for the treatment of atherosclerosis.

Induction of KLF2 by Exercise Activates eNOS to Improve Vasodilatation in Diabetic Mice.

Diabetic endothelial dysfunction associated with diminished endothelial nitric oxide (NO) synthase (eNOS) activity accelerates the development of atherosclerosis and cardiomyopathy. However, the approaches to restore eNOS activity and endothelial function in diabetes remain limited. The current study shows that enhanced expression of Krüppel-like factor 2 (KLF2), a shear stress-inducible transcription factor, effectively improves endothelial function through increasing NO bioavailability. KLF2 expression is suppressed in diabetic mouse aortic endothelium. Running exercise and simvastatin treatment induce endothelial KLF2 expression in db/db mice. Adenovirus-mediated endothelium-specific KLF2 overexpression enhances both endothelium-dependent relaxation and flow-mediated dilatation, while it attenuates oxidative stress in diabetic mouse arteries. KLF2 overexpression increases the phosphorylation of eNOS at serine 1177 and eNOS dimerization. RNA-sequencing analysis reveals that KLF2 transcriptionally upregulates genes that are enriched in the cyclic guanosine monophosphate-protein kinase G-signaling pathway, cAMP-signaling pathway, and insulin-signaling pathway, all of which are the upstream regulators of eNOS activity. Activation of the phosphoinositide 3-kinase-Akt pathway and Hsp90 contributes to KLF2-induced increase of eNOS activity. The present results suggest that approaches inducing KLF2 activation, such as physical exercise, are effective to restore eNOS activity against diabetic endothelial dysfunction. ARTICLE HIGHLIGHTS: Exercise and statins restore the endothelial expression of Krüppel-like factor 2 (KLF2), which is diminished in diabetic db/db mice. Endothelium-specific overexpression of KLF2 improves endothelium-dependent relaxation and flow-mediated dilation through increasing nitric oxide bioavailability. KLF2 promotes endothelial nitric oxide synthase (eNOS) coupling and phosphorylation in addition to its known role in eNOS transcription. KLF2 upregulates the expression of several panels of genes that regulate eNOS activity.

Molecular mechanisms and therapeutic perspectives of peroxisome proliferator-activated receptor α agonists in cardiovascular health and disease.

The prevalence of cardiovascular disease (CVD) has been rising due to sedentary lifestyles and unhealthy dietary patterns. Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor regulating multiple biological processes, such as lipid metabolism and inflammatory response critical to cardiovascular homeostasis. Healthy endothelial cells (ECs) lining the lumen of blood vessels maintains vascular homeostasis, where endothelial dysfunction associated with increased oxidative stress and inflammation triggers the pathogenesis of CVD. PPARα activation decreases endothelial inflammation and senescence, contributing to improved vascular function and reduced risk of atherosclerosis. Phenotypic switch and inflammation of vascular smooth muscle cells (VSMCs) exacerbate vascular dysfunction and atherogenesis, in which PPARα activation improves VSMC homeostasis. Different immune cells participate in the progression of vascular inflammation and atherosclerosis. PPARα in immune cells plays a critical role in immunological events, such as monocyte/macrophage adhesion and infiltration, macrophage polarization, dendritic cell (DC) embedment, T cell activation, and B cell differentiation. Cardiomyocyte dysfunction, a major risk factor for heart failure, can also be alleviated by PPARα activation through maintaining cardiac mitochondrial stability and inhibiting cardiac lipid accumulation, oxidative stress, inflammation, and fibrosis. This review discusses the current understanding and future perspectives on the role of PPARα in the regulation of the cardiovascular system as well as the clinical application of PPARα ligands.

Sema3G activates YAP and promotes VSMCs proliferation and migration via Nrp2/PlexinA1.

BACKGROUND: Diabetes exacerbates neointima formation after vascular procedures, manifested by accelerated proliferation and migration of vascular smooth muscle cells (VSMCs). Semaphorin 3G (Sema3G), secreted mainly from endothelial cells (ECs), regulates various cellular functions and vascular pathologies. However, the function and potential mechanism of ECs-derived Sema3G in VSMCs under diabetic condition remain unclear. OBJECTIVE: To investigate the role and the mechanism of ECs-derived Sema3G in the regulation of VSMCs proliferation and migration. RESULTS: ECs-derived Sema3G promoted human aortic SMCs (HASMCs) cell cycle progression and proliferation. Sema3G upregulated the expression of MMP2 and MMP9, which might explain the increased HASMCs migration by Sema3G. Inhibition of Nrp2/PlexinA1 mitigated the effect of Sema3G on promoting HASMCs proliferation and migration. Mechanistically, Sema3G inhibited LATS1 and activated YAP via Nrp2/PlexinA1. Verteporfin, an FDA-approved YAP pathway inhibitor, counteracted Sema3G-induced cyclin E and cyclin D1 expression. Besides, Sema3G expression was upregulated in ECs of diabetic mouse aortas. Serum Sema3G level was increased in type 2 diabetic patients and mice. Moreover, compared to chow diet-fed mice, high-fat diet (HFD)-fed obese mice showed thicker neointima and higher Sema3G expression in vasculature after femoral injury. CONCLUSIONS: Our results indicated that ECs-derived Sema3G under diabetic condition activated YAP and promoted HASMCs proliferation and migration via Nrp2/PlexinA1. Thus, inhibition of Sema3G may hold therapeutic potential against diabetes-associated intimal hyperplasia.

SOX4 is a novel phenotypic regulator of endothelial cells in atherosclerosis revealed by single-cell analysis.

INTRODUCTION: Atherosclerotic complications represent the leading cause of cardiovascular mortality globally. Dysfunction of endothelial cells (ECs) often initiates the pathological events in atherosclerosis. OBJECTIVES: In this study, we sought to investigate the transcriptional profile of atherosclerotic aortae, identify novel regulator in dysfunctional ECs and hence provide mechanistic insights into atherosclerotic progression. METHODS: We applied single-cell RNA sequencing (scRNA-seq) on aortic cells from Western diet-fed apolipoprotein E-deficient (ApoE-/-) mice to explore the transcriptional landscape and heterogeneity of dysfunctional ECs. In vivo validation of SOX4 upregulation in ECs were performed in atherosclerotic tissues, including mouse aortic tissues, human coronary arteries, and human renal arteries. Single-cell analysis on human aortic aneurysmal tissue was also performed. Downstream vascular abnormalities induced by EC-specific SOX4 overexpression, and upstream modulators of SOX4 were revealed by biochemical assays, immunostaining, and wire myography. Effects of shear stress on endothelial SOX4 expression was investigated by in vitro hemodynamic study. RESULTS: Among the compendium of aortic cells, mesenchymal markers in ECs were significantly enriched. Two EC subsets were subsequently distinguished, as the 'endothelial-like' and 'mesenchymal-like' subsets. Conventional assays consistently identified SOX4 as a novel atherosclerotic marker in mouse and different human arteries, additional to a cancer marker. EC-specific SOX4 overexpression promoted atherogenesis and endothelial-to-mesenchymal transition (EndoMT). Importantly, hyperlipidemia-associated cytokines and oscillatory blood flow upregulated, whereas the anti-diabetic drug metformin pharmacologically suppressed SOX4 level in ECs. CONCLUSION: Our study unravels SOX4 as a novel phenotypic regulator during endothelial dysfunction, which exacerbates atherogenesis. Our study also pinpoints hyperlipidemia-associated cytokines and oscillatory blood flow as endogenous SOX4 inducers, providing more therapeutic insights against atherosclerotic diseases.

Endothelial UCP2 Is a Mechanosensitive Suppressor of Atherosclerosis.

BACKGROUND: Inflamed endothelial cells (ECs) trigger atherogenesis, especially at arterial regions experiencing disturbed blood flow. UCP2 (Uncoupling protein 2), a key mitochondrial antioxidant protein, improves endothelium-dependent relaxation in obese mice. However, whether UCP2 can be regulated by shear flow is unknown, and the role of endothelial UCP2 in regulating inflammation and atherosclerosis remains unclear. This study aims to investigate the mechanoregulation of UCP2 expression in ECs and the effect of UCP2 on endothelial inflammation and atherogenesis. METHODS: In vitro shear stress simulation system was used to investigate the regulation of UCP2 expression by shear flow. EC-specific Ucp2 knockout mice were used to investigate the role of UCP2 in flow-associated atherosclerosis. RESULTS: Shear stress experiments showed that KLF2 (Krüppel-like factor 2) mediates fluid shear stress-dependent regulation of UCP2 expression in human aortic and human umbilical vein ECs. Unidirectional shear stress, statins, and resveratrol upregulate whereas oscillatory shear stress and proinflammatory stimuli inhibit UCP2 expression through altered KLF2 expression. KLF2 directly binds to UCP2 promoter to upregulate its transcription in human umbilical vein ECs. UCP2 knockdown induced expression of genes involved in proinflammatory and profibrotic signaling, resulting in a proatherogenic endothelial phenotype. EC-specific Ucp2 deletion promotes atherogenesis and collagen production. Additionally, we found endothelial Ucp2 deficiency aggravates whereas adeno-associated virus-mediated EC-Ucp2 overexpression inhibits carotid atherosclerotic plaque formation in disturbed flow-enhanced atherosclerosis mouse model. RNA-sequencing analysis revealed FoxO1 (forkhead box protein O1) as the major proinflammatory transcriptional regulator activated by UCP2 knockdown, and FoxO1 inhibition reduced vascular inflammation and disturbed flow-enhanced atherosclerosis. We showed further that UCP2 level is critical for phosphorylation of AMPK (AMP-activated protein kinase), which is required for UCP2-induced inhibition of FoxO1. CONCLUSIONS: Altogether, our studies uncover that UCP2 is novel mechanosensitive gene under the control of fluid shear stress and KLF2 in ECs. UCP2 expression is critical for endothelial proinflammatory response and atherogenesis. Therapeutic strategies enhancing UCP2 level may have therapeutic potential against atherosclerosis.

Activation of AMPK/miR-181b Axis Alleviates Endothelial Dysfunction and Vascular Inflammation in Diabetic Mice.

Hyperglycemia in diabetes mellitus impairs endothelial function and disrupts microRNA (miRNA) profiles in vasculature, increasing the risk of diabetes-associated complications, including coronary artery disease, diabetic retinopathy, and diabetic nephropathy. miR-181b was previously reported to be an anti-inflammatory mediator in vasculature against atherosclerosis. The current study aimed to investigate whether miR-181b ameliorates diabetes-associated endothelial dysfunction, and to identify potential molecular mechanisms and upstream inducer of miR-181b. We found that miR-181b level was decreased in renal arteries of diabetic patients and in advanced glycation end products (AGEs)-treated renal arteries of non-diabetic patients. Transfection of miR-181b mimics improved endothelium-dependent vasodilation in aortas of high fat diet (HFD)/streptozotocin (STZ)-induced diabetic mice, accompanied by suppression of superoxide overproduction and vascular inflammation markers. AMPK activator-induced AMPK activation upregulated miR-181b level in human umbilical vein endothelial cells (HUVECs). Chronic exercise, potentially through increased blood flow, activated AMPK/miR-181b axis in aortas of diabetic mice. Exposure to laminar shear stress upregulated miR-181b expression in HUVECs. Overall, our findings highlight a critical role of AMPK/miR-181b axis and extend the benefits of chronic exercise in counteracting diabetes-associated endothelial dysfunction.

Branched-chain amino acid supplementation impairs insulin sensitivity and promotes lipogenesis during exercise in diet-induced obese mice.

OBJECTIVE: Branched-chain amino acids (BCAAs) are popular dietary supplements for exercise. However, increased BCAA levels positively correlate with obesity and diabetes. The metabolic impact of BCAA supplementation on insulin sensitivity during exercise is less understood. METHODS: Male C57BL/6 mice were fed for 12 weeks with a high-fat diet, normal chow diet, or BCAA-restricted high-fat diet. They were subjected to running exercise with or without BCAA treatment for another 12 weeks. RESULTS: Exercise reduced body weight, improved insulin sensitivity, lowered BCAAs in plasma, and inhibited the upregulation of BCAAs and metabolites caused by BCAA supplementation in the subcutaneous white adipose tissue (sWAT) of obese mice. BCAA supplementation reversed insulin sensitivity ameliorated by exercise. The phosphorylation of protein kinase B (Ser473 and Ser474) was decreased by BCAAs in the sWAT of obese mice. However, BCAA supplementation had no such effects in lean mice. BCAAs also increased the expression of fatty acid synthase and other lipogenesis genes in the sWAT of exercised obese mice. BCAA restriction had no effect on body weight and insulin sensitivity in obese mice. CONCLUSIONS: BCAA supplementation impaired the beneficial effect of exercise on glycolipid metabolism in obese but not lean mice. Caution should be taken regarding the use of BCAAs for individuals with obesity who exercise.

Restoration of Autophagic Flux Improves Endothelial Function in Diabetes Through Lowering Mitochondrial ROS-Mediated eNOS Monomerization.

Endothelial nitric oxide synthase (eNOS) monomerization and uncoupling play crucial roles in mediating vascular dysfunction in diabetes, although the underlying mechanisms are still incompletely understood. Increasing evidence indicates that autophagic dysregulation is involved in the pathogenesis of diabetic endothelial dysfunction; however, whether autophagy regulates eNOS activity through controlling eNOS monomerization or dimerization remains elusive. In this study, autophagic flux was impaired in the endothelium of diabetic db/db mice and in human endothelial cells exposed to advanced glycation end products or oxidized low-density lipoprotein. Inhibition of autophagic flux by chloroquine or bafilomycin A1 were sufficient to induce eNOS monomerization and lower nitric oxide bioavailability by increasing mitochondrial reactive oxygen species (mtROS). Restoration of autophagic flux by overexpressing transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, decreased endothelial cell oxidative stress, increased eNOS dimerization, and improved endothelium-dependent relaxations (EDRs) in db/db mouse aortas. Inhibition of mammalian target of rapamycin kinase (mTOR) increased TFEB nuclear localization, reduced mtROS accumulation, facilitated eNOS dimerization, and enhanced EDR in db/db mice. Moreover, calorie restriction also increased TFEB expression, improved autophagic flux, and restored EDR in the aortas of db/db mice. Taken together, the findings of this study reveal that mtROS-induced eNOS monomerization is closely associated with the impaired TFEB-autophagic flux axis leading to endothelial dysfunction in diabetic mice.

KLF2 Mediates the Suppressive Effect of Laminar Flow on Vascular Calcification by Inhibiting Endothelial BMP/SMAD1/5 Signaling.

[Figure: see text].

A high methionine and low folate diet alters glucose homeostasis and gut microbiome.

Hyperhomocysteinemia (HHcy) is considered as a risk factor for several complications, including cardiovascular and neurological disorders. A high methionine low folate (HMLF) diet chronically causes HHcy by accumulating homocysteine in the systemic circulation. Elevated Hcy level is also associated with the incidence of diabetes mellitus. However, very few studies focus on the impact of HMLF diet on glucose homeostasis, and that on gut microbiome profile. HHcy was induced by feeding C57BL/6 mice a HMLF diet for 8 weeks. The HMLF diet feeding resulted in a progressive body weight loss, and development of slight glucose intolerance and insulin resistance in HHcy mice. Notably, the HMLF diet alters the gut microbiome profile and increases the relative abundance of porphyromonadaceae family of bacteria in HHcy mice. These findings provide new insights into the roles of dysregulated glucose homeostasis and gut flora in the pathogenesis of HHcy-related complications.

A GLP-1 analog lowers ER stress and enhances protein folding to ameliorate homocysteine-induced endothelial dysfunction.

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular diseases and increases mortality in type 2 diabetic patients. HHcy induces endoplasmic reticulum (ER) stress and oxidative stress to impair endothelial function. The glucagon-like peptide 1 (GLP-1) analog exendin-4 attenuates endothelial ER stress, but the detailed vasoprotective mechanism remains elusive. The present study investigated the beneficial effects of exendin-4 against HHcy-induced endothelial dysfunction. Exendin-4 pretreatment reversed homocysteine-induced impairment of endothelium-dependent relaxations in C57BL/6 mouse aortae ex vivo. Four weeks subcutaneous injection of exendin-4 restored the impaired endothelial function in both aortae and mesenteric arteries isolated from mice with diet-induced HHcy. Exendin-4 treatment lowered superoxide anion accumulation in the mouse aortae both ex vivo and in vivo. Exendin-4 decreased the expression of ER stress markers (e.g., ATF4, spliced XBP1, and phosphorylated eIF2α) in human umbilical vein endothelial cells (HUVECs), and this change was reversed by cotreatment with compound C (CC) (AMPK inhibitor). Exendin-4 induced phosphorylation of AMPK and endothelial nitric oxide synthase in HUVECs and arteries. Exendin-4 increased the expression of endoplasmic reticulum oxidoreductase (ERO1α), an important ER chaperone in endothelial cells, and this effect was mediated by AMPK activation. Experiments using siRNA-mediated knockdown or adenoviral overexpression revealed that ERO1α mediated the inhibitory effects of exendin-4 on ER stress and superoxide anion production, thus ameliorating HHcy-induced endothelial dysfunction. The present results demonstrate that exendin-4 reduces HHcy-induced ER stress and improves endothelial function through AMPK-dependent ERO1α upregulation in endothelial cells and arteries. AMPK activation promotes the protein folding machinery in endothelial cells to suppress ER stress.

Pharmacological basis and new insights of resveratrol action in the cardiovascular system.

Resveratrol (trans-3,4',5-trihydroxystilbene) belongs to the family of natural phytoalexins. Resveratrol first came to our attention in 1992, following reports of the cardioprotective effects of red wine. Thereafter, resveratrol was shown to exert antioxidant, anti-inflammatory, anti-proliferative, and angio-regulatory effects against atherosclerosis, ischaemia, and cardiomyopathy. This article critically reviews the current findings on the molecular basis of resveratrol-mediated cardiovascular benefits, summarizing the broad effects of resveratrol on longevity regulation, energy metabolism, stress resistance, exercise mimetics, circadian clock, and microbiota composition. In addition, this article also provides an update, both preclinically and clinically, on resveratrol-induced cardiovascular protection and discusses the adverse and inconsistent effects of resveratrol reported in both preclinical and clinical studies. Although resveratrol has been claimed as a master anti-aging agent against several age-associated diseases, further detailed mechanistic investigation is still required to thoroughly unravel the therapeutic value of resveratrol against cardiovascular diseases at different stages of disease development. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.

Endothelial TFEB (Transcription Factor EB) Restrains IKK (IκB Kinase)-p65 Pathway to Attenuate Vascular Inflammation in Diabetic db/db Mice.

Objective- TFEB (transcription factor EB) was recently reported to be induced by atheroprotective laminar flow and play an anti-atherosclerotic role by inhibiting inflammation in endothelial cells (ECs). This study aims to investigate whether TFEB regulates endothelial inflammation in diabetic db/db mice and the molecular mechanisms involved. Approach and Results- Endothelial denudation shows that TFEB is mainly expressed in ECs in mouse aortas. Western blotting shows TFEB total protein level decreases whereas the p-TFEB S142 (phosphorylated form of TFEB) increases in db/db mouse aortas, suggesting a decreased TFEB activity. Adenoviral TFEB overexpression reduces endothelial inflammation as evidenced by decreased expression of vascular inflammatory markers in db/db mouse aortas, and reduced expression of a wide range of adhesion molecules and chemokines in human umbilical vein ECs. Monocyte attachment assay shows TFEB suppresses monocyte adhesion to human umbilical vein ECs. RNA sequencing of TFEB-overexpressed human umbilical vein ECs suggested TFEB inhibits NF-κB (nuclear factor-kappa B) signaling. Indeed, luciferase assay shows TFEB suppresses NF-κB transcriptional activity. Mechanistically, TFEB suppresses IKK (IκB kinase) activity to protect IκB-α from degradation, leading to reduced p65 nuclear translocation. Inhibition of IKK by PS-1145 abolished TFEB silencing-induced inflammation in human umbilical vein ECs. Lastly, we identified KLF2 (Krüppel-like factor 2) upregulates TFEB expression and promoter activity. Laminar flow experiment showed that KLF2 is required for TFEB induction by laminar flow and TFEB is an anti-inflammatory effector downstream of laminar flow-KLF2 signaling in ECs. Conclusions- These findings suggest that TFEB exerts anti-inflammatory effects in diabetic mice and such function in ECs is achieved by inhibiting IKK activity and increasing IκBα level to suppress NF-κB activity. KLF2 mediates TFEB upregulation in response to laminar flow.

Inhibition of miR-92a Suppresses Oxidative Stress and Improves Endothelial Function by Upregulating Heme Oxygenase-1 in db/db Mice.

AIMS: Inhibition of microRNA-92a (miR-92a) is reported to suppress endothelial inflammation and delay atherogenesis. We hypothesize that miR-92a inhibition protects endothelial function through suppressing oxidative stress in diabetic db/db mice. RESULTS: In this study, we found elevated expression of miR-92a in aortic endothelium from db/db mice and in renal arteries from diabetic subjects. Endothelial cells (ECs) exposed to advanced glycation end products (AGEs) and oxidized low-density lipoprotein express higher level of miR-92a. Overexpression of miR-92a impairs endothelium-dependent relaxations (EDRs) in C57BL/6 mouse aortas. Overexpression of miR-92a suppresses expression of heme oxygenase-1 (HO-1), a critical cytoprotective enzyme, whereas inhibition of miR-92a increases HO-1 expression in human umbilical vein ECs (HUVECs) and db/db mouse aortas. Importantly, miR-92a inhibition by Ad-anti-miR-92a improved EDRs and reduced reactive oxygen species (ROS) production in db/db mouse aortas. HO-1 inhibition by SnMP or HO-1 knockdown by shHO-1 reversed the suppressive effect of miR-92a inhibition on ROS production induced by AGE treatment in C57BL/6 mouse aortas. In addition, SnMP reversed miR-92a inhibition-induced improvement of EDRs in AGE-treated C57BL/6 mouse aortas and in db/db mouse aortas. INNOVATION: Expression of miR-92a is increased in diabetic aortic endothelium and inhibition of miR-92a exerts vasoprotective effect in diabetic mice through HO-1 upregulation in ECs. CONCLUSION: MiR-92a expression is elevated in diabetic ECs. MiR-92a overexpression impairs endothelial function and suppresses HO-1 expression in ECs. Inhibition of miR-92a attenuates oxidative stress and improves endothelial function through enhancing HO-1 expression and activity in db/db mouse aortas. Antioxid. Redox Signal. 28, 358-370.

MicroRNAs Regulating Reactive Oxygen Species in Cardiovascular Diseases.

SIGNIFICANCE: Oxidative stress caused by overproduction of reactive oxygen species (ROS) in cells is one of the most important contributors to the pathogenesis of cardiovascular and metabolic diseases such as hypertension and atherosclerosis. Excessive accumulation of ROS impairs, while limiting oxidative stress protects cardiovascular and metabolic function through various cellular mechanisms. Recent Advances: MicroRNAs (miRNAs) are novel regulators of oxidative stress in cardiovascular cells that modulate the expression of redox-related genes. This article summarizes recent advances in our understanding of how miRNAs target major ROS generators, antioxidant signaling pathways, and effectors in cells of the cardiovascular system. CRITICAL ISSUES: The role of miRNAs in regulating ROS in cardiovascular cells is complicated because miRNAs can target multiple redox-related genes, act on redox regulatory pathways indirectly, and display context-dependent pro- or antioxidant effects. The complex regulatory network of ROS and the plethora of targets make it difficult to pin point the role of miRNAs and develop them as therapeutics. Therefore, these properties should be considered when designing strategies for therapeutic or diagnostic development. FUTURE DIRECTIONS: Future studies can gain a better understanding of redox-related miRNAs by investigating their own regulatory mechanisms and the dual role of ROS in the cardiovascular systems. The combination of improved study design and technical advancements will reveal newer pathophysiological importance of redox-related miRNAs.

Integrin-YAP/TAZ-JNK cascade mediates atheroprotective effect of unidirectional shear flow.

The Yorkie homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1), effectors of the Hippo pathway, have been identified as mediators for mechanical stimuli. However, the role of YAP/TAZ in haemodynamics-induced mechanotransduction and pathogenesis of atherosclerosis remains unclear. Here we show that endothelial YAP/TAZ activity is regulated by different patterns of blood flow, and YAP/TAZ inhibition suppresses inflammation and retards atherogenesis. Atheroprone-disturbed flow increases whereas atheroprotective unidirectional shear stress inhibits YAP/TAZ activity. Unidirectional shear stress activates integrin and promotes integrin-Gα13 interaction, leading to RhoA inhibition and YAP phosphorylation and suppression. YAP/TAZ inhibition suppresses JNK signalling and downregulates pro-inflammatory genes expression, thereby reducing monocyte attachment and infiltration. In vivo endothelial-specific YAP overexpression exacerbates, while CRISPR/Cas9-mediated Yap knockdown in endothelium retards, plaque formation in ApoE-/- mice. We also show several existing anti-atherosclerotic agents such as statins inhibit YAP/TAZ transactivation. On the other hand, simvastatin fails to suppress constitutively active YAP/TAZ-induced pro-inflammatory gene expression in endothelial cells, indicating that YAP/TAZ inhibition could contribute to the anti-inflammatory effect of simvastatin. Furthermore, activation of integrin by oral administration of MnCl2 reduces plaque formation. Taken together, our results indicate that integrin-Gα13-RhoA-YAP pathway holds promise as a novel drug target against atherosclerosis.

Inhibition of miR-200c Restores Endothelial Function in Diabetic Mice Through Suppression of COX-2.

Endothelial dysfunction plays a crucial role in the development of diabetic vasculopathy. Our initial quantitative PCR results showed an increased miR-200c expression in arteries from diabetic mice and patients with diabetes. However, whether miR-200c is involved in diabetic endothelial dysfunction is unknown. Overexpression of miR-200c impaired endothelium-dependent relaxations (EDRs) in nondiabetic mouse aortas, whereas suppression of miR-200c by anti-miR-200c enhanced EDRs in diabetic db/db mice. miR-200c suppressed ZEB1 expression, and ZEB1 overexpression ameliorated endothelial dysfunction induced by miR-200c or associated with diabetes. More importantly, overexpression of anti-miR-200c or ZEB1 in vivo attenuated miR-200c expression and improved EDRs in db/db mice. Mechanistic study with the use of COX-2(-/-) mice revealed that COX-2 mediated miR-200c-induced endothelial dysfunction and that miR-200c upregulated COX-2 expression in endothelial cells through suppression of ZEB1 and increased production of prostaglandin E2, which also reduced EDR. This study demonstrates for the first time to our knowledge that miR-200c is a new mediator of diabetic endothelial dysfunction and inhibition of miR-200c rescues EDRs in diabetic mice. These new findings suggest the potential usefulness of miR-200c as the target for drug intervention against diabetic vascular complications.

Bone Morphogenic Protein 4-Smad-Induced Upregulation of Platelet-Derived Growth Factor AA Impairs Endothelial Function.

OBJECTIVE: Bone morphogenic protein 4 (BMP4) is an important mediator of endothelial dysfunction in cardio-metabolic diseases, whereas platelet-derived growth factors (PDGFs) are major angiogenic and proinflammatory mediator, although the functional link between these 2 factors is unknown. The present study investigated whether PDGF mediates BMP4-induced endothelial dysfunction in diabetes mellitus. APPROACH AND RESULTS: We generated Ad-Bmp4 to overexpress Bmp4 and Ad-Pdgfa-shRNA to knockdown Pdgfa in mice through tail intravenous injection. SMAD4-shRNA lentivirus, SMAD1-shRNA, and SMAD5 shRNA adenovirus were used for knockdown in human and mouse endothelial cells. We found that PDGF-AA impaired endothelium-dependent vasodilation in aortas and mesenteric resistance arteries. BMP4 upregulated PDGF-AA in human and mouse endothelial cells, which was abolished by BMP4 antagonist noggin or knockdown of SMAD1/5 or SMAD4. BMP4-impared relaxation in mouse aorta was also ameliorated by PDGF-AA neutralizing antibody. Tail injection of Ad-Pdgfa-shRNA ameliorates endothelial dysfunction induced by Bmp4 overexpression (Ad-Bmp4) in vivo. Serum PDGF-AA was elevated in both diabetic patients and diabetic db/db mice compared with nondiabetic controls. Pdgfa-shRNA or Bmp4-shRNA adenovirus reduced serum PDGF-AA concentration in db/db mice. PDGF-AA neutralizing antibody or tail injection with Pdgfa-shRNA adenovirus improved endothelial function in aortas and mesenteric resistance arteries from db/db mice. The effect of PDGF-AA on endothelial function in mouse aorta was also inhibited by Ad-Pdgfra-shRNA to inhibit PDGFRα. CONCLUSIONS: The present study provides novel evidences to show that PDGF-AA impairs endothelium-dependent vasodilation and PDGF-AA mediates BMP4-induced adverse effect on endothelial cell function through SMAD1/5- and SMAD4-dependent mechanisms. Inhibition of PGDF-AA ameliorates vascular dysfunction in diabetic mice.

Regulators and effectors of bone morphogenetic protein signalling in the cardiovascular system.

Bone morphogenetic proteins (BMPs) play key roles in the regulation of cell proliferation, differentiation and apoptosis in various tissues and organs, including the cardiovascular system. BMPs signal through both Smad-dependent and -independent cascades to exert a wide spectrum of biological activities. Cardiovascular disorders such as abnormal angiogenesis, atherosclerosis, pulmonary hypertension and cardiac hypertrophy have been linked to aberrant BMP signalling. To correct the dysregulated BMP signalling in cardiovascular pathogenesis, it is essential to get a better understanding of how the regulators and effectors of BMP signalling control cardiovascular function and how the dysregulated BMP signalling contributes to cardiovascular dysfunction. We hence highlight several key regulators of BMP signalling such as extracellular regulators of ligands, mechanical forces, microRNAs and small molecule drugs as well as typical BMP effectors like direct downstream target genes, mitogen-activated protein kinases, reactive oxygen species and microRNAs. The insights into these molecular processes will help target both the regulators and important effectors to reverse BMP-associated cardiovascular pathogenesis.