The PTM profiling of CTCF reveals the regulation of 3D chromatin structure by O-GlcNAcylation.

CCCTC-binding factor (CTCF), a ubiquitously expressed and highly conserved protein, is known to play a critical role in chromatin structure. Post-translational modifications (PTMs) diversify the functions of protein to regulate numerous cellular processes. However, the effects of PTMs on the genome-wide binding of CTCF and the organization of three-dimensional (3D) chromatin structure have not been fully understood. In this study, we uncovered the PTM profiling of CTCF and demonstrated that CTCF can be O-GlcNAcylated and arginine methylated. Functionally, we demonstrated that O-GlcNAcylation inhibits CTCF binding to chromatin. Meanwhile, deficiency of CTCF O-GlcNAcylation results in the disruption of loop domains and the alteration of chromatin loops associated with cellular development. Furthermore, the deficiency of CTCF O-GlcNAcylation increases the expression of developmental genes and negatively regulates maintenance and establishment of stem cell pluripotency. In conclusion, these results provide key insights into the role of PTMs for the 3D chromatin structure.

High-density lipoprotein regulates angiogenesis by affecting autophagy via miRNA-181a-5p.

We previously demonstrated that normal high-density lipoprotein (nHDL) can promote angiogenesis, whereas HDL from patients with coronary artery disease (dHDL) is dysfunctional and impairs angiogenesis. Autophagy plays a critical role in angiogenesis, and HDL regulates autophagy. However, it is unclear whether nHDL and dHDL regulate angiogenesis by affecting autophagy. Endothelial cells (ECs) were treated with nHDL and dHDL with or without an autophagy inhibitor. Autophagy, endothelial nitric oxide synthase (eNOS) expression, miRNA expression, nitric oxide (NO) production, superoxide anion (O2•-) generation, EC migration, and tube formation were evaluated. nHDL suppressed the expression of miR-181a-5p, which promotes autophagy and the expression of eNOS, resulting in NO production and the inhibition of O2•- generation, and ultimately increasing in EC migration and tube formation. dHDL showed opposite effects compared to nHDL and ultimately inhibited EC migration and tube formation. We found that autophagy-related protein 5 (ATG5) was a direct target of miR-181a-5p. ATG5 silencing or miR-181a-5p mimic inhibited nHDL-induced autophagy, eNOS expression, NO production, EC migration, tube formation, and enhanced O2•- generation, whereas overexpression of ATG5 or miR-181a-5p inhibitor reversed the above effects of dHDL. ATG5 expression and angiogenesis were decreased in the ischemic lower limbs of hypercholesterolemic low-density lipoprotein receptor null (LDLr-/-) mice when compared to C57BL/6 mice. ATG5 overexpression improved angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Taken together, nHDL was able to stimulate autophagy by suppressing miR-181a-5p, subsequently increasing eNOS expression, which generated NO and promoted angiogenesis. In contrast, dHDL inhibited angiogenesis, at least partially, by increasing miR-181a-5p expression, which decreased autophagy and eNOS expression, resulting in a decrease in NO production and an increase in O2•- generation. Our findings reveal a novel mechanism by which HDL affects angiogenesis by regulating autophagy and provide a therapeutic target for dHDL-impaired angiogenesis.

Dual action of macrophage miR-204 confines cyclosporine A-induced atherosclerosis.

BACKGROUND AND PURPOSE: Atherosclerosis induced by cyclosporine A (CsA), an inhibitor of the calcineurin/nuclear factor of activated T cells (NFAT) pathway, is a major concern after organ transplantation. However, the atherosclerotic mechanisms of CsA remain obscure. We previously demonstrated that calcineurin/NFAT signalling inhibition contributes to atherogenesis via suppressing microRNA-204 (miR-204) transcription. We therefore hypothesised that miR-204 is involved in the development of CsA-induced atherosclerosis. EXPERIMENTAL APPROACH: ApoE-/- mice with macrophage-miR-204 overexpression were generated to determine the effects of miR-204 on CsA-induced atherosclerosis. Luciferase reporter assays and chromatin immunoprecipitation sequencing were performed to explore the targets mediating miR-204 effects. KEY RESULTS: CsA alone did not significantly affect atherosclerotic lesions or serum lipid levels. However, it exacerbated high-fat diet-induced atherosclerosis and hyperlipidemia in C57BL/6J and ApoE-/- mice, respectively. miR-204 levels decreased in circulating monocytes and plaque lesions during CsA-induced atherosclerosis. The upregulation of miR-204 in macrophages inhibited CsA-induced atherosclerotic plaque formation but did not affect serum lipid levels. miR-204 limited the CsA-induced foam cell formation by reducing the expression of the scavenger receptors SR-BII and CD36. SR-BII was post-transcriptionally regulated by mature miR-204-5p via 3'-UTR targeting. Additionally, nuclear-localised miR-204-3p prevented the CsA-induced binding of Ago2 to the CD36 promoter, suppressing CD36 transcription. SR-BII or CD36 expression restoration dampened the beneficial effects of miR-204 on CsA-induced atherosclerosis. CONCLUSION AND IMPLICATIONS: Macrophage miR-204 ameliorates CsA-induced atherosclerosis, suggesting that miR-204 may be a potential target for the prevention and treatment of CsA-related atherosclerotic side effects.

High-density lipoprotein regulates angiogenesis by long non-coding RNA HDRACA.

Normal high-density lipoprotein (nHDL) can induce angiogenesis in healthy individuals. However, HDL from patients with coronary artery disease undergoes various modifications, becomes dysfunctional (dHDL), and loses its ability to promote angiogenesis. Here, we identified a long non-coding RNA, HDRACA, that is involved in the regulation of angiogenesis by HDL. In this study, we showed that nHDL downregulates the expression of HDRACA in endothelial cells by activating WW domain-containing E3 ubiquitin protein ligase 2, which catalyzes the ubiquitination and subsequent degradation of its transcription factor, Kruppel-like factor 5, via sphingosine 1-phosphate (S1P) receptor 1. In contrast, dHDL with lower levels of S1P than nHDL were much less effective in decreasing the expression of HDRACA. HDRACA was able to bind to Ras-interacting protein 1 (RAIN) to hinder the interaction between RAIN and vigilin, which led to an increase in the binding between the vigilin protein and proliferating cell nuclear antigen (PCNA) mRNA, resulting in a decrease in the expression of PCNA and inhibition of angiogenesis. The expression of human HDRACA in a hindlimb ischemia mouse model inhibited the recovery of angiogenesis. Taken together, these findings suggest that HDRACA is involved in the HDL regulation of angiogenesis, which nHDL inhibits the expression of HDRACA to induce angiogenesis, and that dHDL is much less effective in inhibiting HDRACA expression, which provides an explanation for the decreased ability of dHDL to stimulate angiogenesis.

Bestrophin3 Deficiency in Vascular Smooth Muscle Cells Activates MEKK2/3-MAPK Signaling to Trigger Spontaneous Aortic Dissection.

BACKGROUND: Aortic dissection (AD) is a fatal cardiovascular disorder without effective medications due to unclear pathogenic mechanisms. Bestrophin3 (Best3), the predominant isoform of bestrophin family in vessels, has emerged as critical for vascular pathological processes. However, the contribution of Best3 to vascular diseases remains elusive. METHODS: Smooth muscle cell-specific and endothelial cell-specific Best3 knockout mice (Best3SMKO and Best3ECKO, respectively) were engineered to investigate the role of Best3 in vascular pathophysiology. Functional studies, single-cell RNA sequencing, proteomics analysis, and coimmunoprecipitation coupled with mass spectrometry were performed to evaluate the function of Best3 in vessels. RESULTS: Best3 expression in aortas of human AD samples and mouse AD models was decreased. Best3SMKO but not Best3ECKO mice spontaneously developed AD with age, and the incidence reached 48% at 72 weeks of age. Reanalysis of single-cell transcriptome data revealed that reduction of fibromyocytes, a fibroblast-like smooth muscle cell cluster, was a typical feature of human ascending AD and aneurysm. Consistently, Best3 deficiency in smooth muscle cells decreased the number of fibromyocytes. Mechanistically, Best3 interacted with both MEKK2 and MEKK3, and this interaction inhibited phosphorylation of MEKK2 at serine153 and MEKK3 at serine61. Best3 deficiency induced phosphorylation-dependent inhibition of ubiquitination and protein turnover of MEKK2/3, thereby activating the downstream mitogen-activated protein kinase signaling cascade. Furthermore, restoration of Best3 or inhibition of MEKK2/3 prevented AD progression in angiotensin II-infused Best3SMKO and ApoE-/- mice. CONCLUSIONS: These findings unveil a critical role of Best3 in regulating smooth muscle cell phenotypic switch and aortic structural integrity through controlling MEKK2/3 degradation. Best3-MEKK2/3 signaling represents a novel therapeutic target for AD.

Ponatinib modulates the metabolic profile of obese mice by inhibiting adipose tissue macrophage inflammation.

Obesity-induced metabolic syndrome is a rapidly growing conundrum, reaching epidemic proportions globally. Chronic inflammation in obese adipose tissue plays a key role in metabolic syndrome with a series of local and systemic effects such as inflammatory cell infiltration and inflammatory cytokine secretion. Adipose tissue macrophages (ATM), as one of the main regulators in this process, are particularly crucial for pharmacological studies on obesity-related metabolic syndrome. Ponatinib, a multi-targeted tyrosine kinase inhibitor originally used to treat leukemia, has recently been found to improve dyslipidemia and atherosclerosis, suggesting that it may have profound effect on metabolic syndrome, although the mechanisms underlying have not yet been revealed. Here we discovered that ponatinib significantly improved insulin sensitivity in leptin deficient obese mice. In addition to that, ponatinib treatment remarkably ameliorated high fat diet-induced hyperlipidemia and inhibited ectopic lipid deposition in the liver. Interestingly, although ponatinib did not reduce but increase the weight of white adipose tissue (WAT), it remarkably suppressed the inflammatory response in WAT and preserved its function. Mechanistically, we showed that ponatinib had no direct effect on hepatocyte or adipocyte but attenuated free fatty acid (FFA) induced macrophage transformation from pro-inflammatory to anti-inflammatory phenotype. Moreover, adipocytes co-cultured with FFA-treated macrophages exhibited insulin resistance, while pre-treat these macrophages with ponatinib can ameliorate this process. These results suggested that the beneficial effects of ponatinib on metabolic disorders are achieved by inhibiting the inflammatory phenotypic transformation of ATMs, thereby maintaining the physiological function of adipose tissue under excessive obesity. The data here not only revealed the novel therapeutic function of ponatinib, but also provided a theoretical basis for the application of multi-target tyrosine kinase inhibitors in metabolic diseases.

Endophilin A2 protects against renal fibrosis by targeting TGF-ß/Smad signaling.

Renal fibrosis underlies all forms of end-stage kidney disease. Endophilin A2 (EndoA2) plays a role in nephrotic syndrome; however, its effect on renal fibrosis remains unknown. Here, we demonstrate that EndoA2 protects against kidney interstitial fibrosis via the transforming growth factor-ß (TGF-ß)/Smad signaling pathway. Mouse kidneys with fibrosis or kidney biopsy specimens from patients with fibrotic nephropathy had lower levels of EndoA2 protein expression than that in kidneys without fibrosis. In vivo overexpression of EndoA2 with the endophilin A2 transgene (EndoA2Tg ) notably prevented renal fibrosis, decreased the protein expression of profibrotic molecules, suppressed tubular injury, and reduced apoptotic tubular cells in the obstructed kidney cortex of mice with unilateral ureteral obstruction (UUO). In vivo and in vitro overexpression of EndoA2 markedly inhibited UUO- or TGF-ß1-induced phosphorylation of Smad2/3 and tubular epithelial cells dedifferentiation. Furthermore, EndoA2 was co-immunoprecipitated with the type II TGF-ß receptor (TßRII), thus inhibiting the binding of the type I TGF-ß receptor (TßRI) to TßRII. These findings indicate that EndoA2 mitigates renal fibrosis, at least partially, via modulating the TGF-ß/Smad signaling. Targeting EndoA2 may be a new potential therapeutic strategy for treatment of renal fibrosis.

Moderate heart rate reduction promotes cardiac regeneration through stimulation of the metabolic pattern switch.

As a biological pump, the heart needs to consume a substantial amount of energy to maintain sustained beating. Myocardial energy metabolism was recently reported to be related to the loss of proliferative capacity in cardiomyocytes (CMs). However, the intrinsic relationship between beating rate and proliferation in CMs and whether energy metabolism can regulate this relationship remains unclear. In this study, we find that moderate heart rate reduction (HRR) induces CM proliferation under physiological conditions and promotes cardiac regenerative repair after myocardial injury. Mechanistically, moderate HRR induces G1/S transition and increases the expression of glycolytic enzymes in CMs. Furthermore, moderate HRR induces a metabolic pattern switch, activating glucose metabolism and increasing the relative proportion of ATP production by the glycolytic pathway for biosynthesis of substrates needed for proliferative CMs. These results highlight the potential therapeutic role of HRR in not only acute myocardial protection but also long-term CM restoration.

Macrophage NFATc3 prevents foam cell formation and atherosclerosis: evidence and mechanisms.

AIMS: Our previous study demonstrated that Ca2+ influx through the Orai1 store-operated Ca2+ channel in macrophages contributes to foam cell formation and atherosclerosis via the calcineurin-ASK1 pathway, not the classical calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Moreover, up-regulation of NFATc3 in macrophages inhibits foam cell formation, suggesting that macrophage NFATc3 is a negative regulator of atherogenesis. Hence, this study investigated the precise role of macrophage NFATc3 in atherogenesis. METHODS AND RESULTS: Macrophage-specific NFATc3 knockout mice were generated to determine the effect of NFATc3 on atherosclerosis in a mouse model of adeno-associated virus-mutant PCSK9-induced atherosclerosis. NFATc3 expression was decreased in macrophages within human and mouse atherosclerotic lesions. Moreover, NFATc3 levels in peripheral blood mononuclear cells from atherosclerotic patients were negatively associated with plaque instability. Furthermore, macrophage-specific ablation of NFATc3 in mice led to the atherosclerotic plaque formation, whereas macrophage-specific NFATc3 transgenic mice exhibited the opposite phenotype. NFATc3 deficiency in macrophages promoted foam cell formation by potentiating SR-A- and CD36-meditated lipid uptake. NFATc3 directly targeted and transcriptionally up-regulated miR-204 levels. Mature miR-204-5p suppressed SR-A expression via canonical regulation. Unexpectedly, miR-204-3p localized in the nucleus and inhibited CD36 transcription. Restoration of miR-204 abolished the proatherogenic phenotype observed in the macrophage-specific NFATc3 knockout mice, and blockade of miR-204 function reversed the beneficial effects of NFATc3 in macrophages. CONCLUSION: Macrophage NFATc3 up-regulates miR-204 to reduce SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis, indicating that the NFATc3/miR-204 axis may be a potential therapeutic target against atherosclerosis.

MiR-302a Limits Vascular Inflammation by Suppressing Nuclear Factor-κ B Pathway in Endothelial Cells.

The inflammatory response of endothelial cells accelerates various vascular diseases. MicroRNAs (miRNAs) participate in diverse cellular processes during inflammation. In the present study, we found that miR-302a is an effective suppressor of vascular inflammation in endothelial cells. It was revealed that miR-302a exhibited a lower level in a lipopolysaccharide (LPS)-induced mouse model and in patients with vascular inflammatory disease. Genetic haploinsufficiency of miR-302 aggravated the LPS-induced vascular inflammatory response in mice, and overexpression of miR-302a attenuated vascular inflammation in mice. Furthermore, overexpression of miR-302a inhibited the synthesis and secretion of adhesion factors in endothelial cells, and suppressed the adhesion of monocytes to endothelium. In the study of molecular mechanism, we found that miR-302a relieved vascular inflammation mainly by regulating the nuclear factor kappa-B (NF-κB) pathway in endothelial cells. The results showed that interleukin-1 receptor-associated kinase4 (IRAK4) and zinc finger protein 91 (ZFP91) were the binding targets of miR-302a. MiR-302a prevented the nuclear translocation of NF-κB by inhibiting phosphorylation of IκB kinase complex ß (IKKß) and inhibitors of κBα (IκBα) via targeting IRAK4. In addition, miR-302a downregulated the expression of NF-κB by directly binding with ZFP91. These findings indicate that miR-302a negatively regulates inflammatory responses in the endothelium via the NF-κB pathway and it may be a novel target for relieving vascular inflammation.

MicroRNA-302a promotes neointimal formation following carotid artery injury in mice by targeting PHLPP2 thus increasing Akt signaling.

The excessive proliferation and migration of smooth muscle cells (SMCs) play an important role in restenosis following percutaneous coronary interventions. MicroRNAs are able to target various genes and involved in the regulation of diverse cellular processes including cell growth and proliferation. In this study we investigated whether and how MicroRNAs regulated vascular SMC proliferation and vascular remodeling following carotid artery injury in mice. We showed that carotid artery injury-induced neointimal formation was remarkably ameliorated in microRNA (miR)-302 heterozygous mice and SMC-specific miR-302 knockout mice. In contrast, delivery of miR-302a adenovirus to the injured carotid artery enhanced neointimal formation. Upregulation of miR-302a enhanced the proliferation and migration of mouse aorta SMC (MASMC) in vitro by promoting cell cycle transition, whereas miR-302a inhibition caused the opposite results. Moreover, miR-302a promoted Akt activation by corporately decreasing Akt expression and increasing Akt phosphorylation in MASMCs. Application of the Akt inhibitor GSK690693 (5 µmol/L) counteracted the functions of miR-302a in promoting MASMC proliferation and migration. We further revealed that miR-302a directly targeted at the 3' untranslated region of PH domain and leucine rich repeat protein phosphatase 2 (PHLPP2) and negatively regulated PHLPP2 expression. Restoration of PHLPP2 abrogated the effects of miR-302a on Akt activation and MASMC motility. Furthermore, knockdown of PHLPP2 largely abolished the inhibition of neointimal formation that was observed in miR-302 heterozygous mice. Our data demonstrate that miR-302a exacerbates SMC proliferation and restenosis through increasing Akt signaling by targeting PHLPP2.

Reduced intracellular chloride concentration impairs angiogenesis by inhibiting oxidative stress-mediated VEGFR2 activation.

Chloride (Cl-) homeostasis is of great significance in cardiovascular system. Serum Cl- level is inversely associated with the mortality of patients with heart failure. Considering the importance of angiogenesis in the progress of heart failure, this study aims to investigate whether and how reduced intracellular Cl- concentration ([Cl-]i) affects angiogenesis. Human umbilical endothelial cells (HUVECs) were treated with normal Cl- medium or low Cl- medium. We showed that reduction of [Cl-]i (from 33.2 to 16.18 mM) inhibited HUVEC proliferation, migration, cytoskeleton reorganization, tube formation, and subsequently suppressed angiogenesis under basal condition, and VEGF stimulation or hypoxia treatment. Moreover, VEGF-induced NADPH-mediated reactive oxygen species (ROS) generation and VEGFR2 axis activation were markedly attenuated in low Cl- medium. We revealed that lowering [Cl-]i inhibited the expression of the membrane-bound catalytic subunits of NADPH, i.e., p22phox and Nox2, and blunted the translocation of cytosolic regulatory subunits p47phox and p67phox, thereby restricting NADPH oxidase complex formation and activation. Furthermore, reduced [Cl-]i enhanced ROS-associated protein tyrosine phosphatase 1B (PTP1B) activity and increased the interaction of VEGFR2 and PTP1B. Pharmacological inhibition of PTP1B reversed the effect of lowering [Cl-]i on VEGFR2 phosphorylation and angiogenesis. In mouse hind limb ischemia model, blockade of Cl- efflux using Cl- channel inhibitors DIDS or DCPIB (10 mg/kg, i.m., every other day for 2 weeks) significantly enhanced blood flow recovery and new capillaries formation. In conclusion, decrease of [Cl-]i suppresses angiogenesis via inhibiting oxidase stress-mediated VEGFR2 signaling activation by preventing NADPH oxidase complex formation and promoting VEGFR2/PTP1B association, suggesting that modulation of [Cl-]i may be a novel therapeutic avenue for the treatment of angiogenic dysfunction-associated diseases.

Hepatocyte TMEM16A Deletion Retards NAFLD Progression by Ameliorating Hepatic Glucose Metabolic Disorder.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease, and the mechanisms underpinning its pathogenesis have not been completely established. Transmembrane member 16A (TMEM16A), a component of the Ca2+-activated chloride channel (CaCC), has recently been implicated in metabolic events. Herein, TMEM16A is shown to be responsible for CaCC activation in hepatocytes and is increased in liver tissues of mice and patients with NAFLD. Hepatocyte-specific ablation of TMEM16A in mice ameliorates high-fat diet-induced obesity, hepatic glucose metabolic disorder, steatosis, insulin resistance, and inflammation. In contrast, hepatocyte-specific TMEM16A transgenic mice exhibit the opposite phenotype. Mechanistically, hepatocyte TMEM16A interacts with vesicle-associated membrane protein 3 (VAMP3) to induce its degradation, suppressing the formation of the VAMP3/syntaxin 4 and VAMP3/synaptosome-associated protein 23 complexes. This leads to the impairment of hepatic glucose transporter 2 (GLUT2) translocation and glucose uptake. Notably, VAMP3 overexpression restrains the functions of hepatocyte TMEM16A in blocking GLUT2 translocation and promoting lipid deposition, insulin resistance, and inflammation. In contrast, VAMP3 knockdown reverses the beneficial effects of TMEM16A downregulation. This study demonstrates a role for TMEM16A in NAFLD and suggests that inhibition of hepatic TMEM16A or disruption of TMEM16A/VAMP3 interaction may provide a new potential therapeutic strategy for NAFLD.

TMEM16A ameliorates vascular remodeling by suppressing autophagy via inhibiting Bcl-2-p62 complex formation.

Rationale: Transmembrane member 16A (TMEM16A) is a component of calcium-activated chloride channels that regulate vascular smooth muscle cell (SMC) proliferation and remodeling. Autophagy, a highly conserved cellular catabolic process in eukaryotes, exerts important physiological functions in vascular SMCs. In the current study, we investigated the relationship between TMEM16A and autophagy during vascular remodeling. Methods: We generated a transgenic mouse that overexpresses TMEM16A specifically in vascular SMCs to verify the role of TMEM16A in vascular remodeling. Techniques employed included immunofluorescence, electron microscopy, co-immunoprecipitation, and Western blotting. Results: Autophagy was activated in aortas from angiotensin II (AngII)-induced hypertensive mice with decreased TMEM16A expression. The numbers of light chain 3B (LC3B)-positive puncta in aortas correlated with the medial cross-sectional aorta areas and TMEM16A expression during hypertension. SMC-specific TMEM16A overexpression markedly inhibited AngII-induced autophagy in mouse aortas. Moreover, in mouse aortic SMCs (MASMCs), AngII-induced autophagosome formation and autophagic flux were blocked by TMEM16A upregulation and were promoted by TMEM16A knockdown. The effect of TMEM16A on autophagy was independent of the mTOR pathway, but was associated with reduced kinase activity of the vacuolar protein sorting 34 (VPS34) enzyme. Overexpression of VPS34 attenuated the effect of TMEM16A overexpression on MASMC proliferation, while the effect of TMEM16A downregulation was abrogated by a VPS34 inhibitor. Further, co-immunoprecipitation assays revealed that TMEM16A interacts with p62. TMEM16A overexpression inhibited AngII-induced p62-Bcl-2 binding and enhanced Bcl-2-Beclin-1 interactions, leading to suppression of Beclin-1/VPS34 complex formation. However, TMEM16A downregulation showed the opposite effects. Conclusion: TMEM16A regulates the four-way interaction between p62, Bcl-2, Beclin-1, and VPS34, and coordinately prevents vascular autophagy and remodeling.

MicroRNA-181a-5p and microRNA-181a-3p cooperatively restrict vascular inflammation and atherosclerosis.

MicroRNAs have emerged as important post-transcriptional regulators of gene expression and are involved in diverse diseases and cellular process. Decreased expression of miR-181a has been observed in the patients with coronary artery disease, but its function and mechanism in atherogenesis is not clear. This study was designed to determine the roles of miR-181a-5p, as well as its passenger strand, miR-181a-3p, in vascular inflammation and atherogenesis. We found that the levels of both miR-181a-5p and miR-181a-3p are decreased in the aorta plaque and plasma of apoE-/- mice in response to hyperlipidemia and in the plasma of patients with coronary artery disease. Rescue of miR-181a-5p and miR-181a-3p significantly retards atherosclerotic plaque formation in apoE-/- mice. MiR-181a-5p and miR-181a-3p have no effect on lipid metabolism but decrease proinflammatory gene expression and the infiltration of macrophage, leukocyte and T cell into the lesions. In addition, gain-of-function and loss-of-function experiments show that miR-181a-5p and miR-181a-3p inhibit adhesion molecule expression in HUVECs and monocytes-endothelial cell interaction. MiR-181a-5p and miR-181a-3p cooperatively receded endothelium inflammation compared with single miRNA strand. Mechanistically, miR-181a-5p and miR-181a-3p prevent endothelial cell activation through blockade of NF-κB signaling pathway by targeting TAB2 and NEMO, respectively. In conclusion, these findings suggest that miR-181a-5p and miR-181a-3p are both antiatherogenic miRNAs. MiR-181a-5p and miR-181a-3p mimetics retard atherosclerosis progression through blocking NF-κB activation and vascular inflammation by targeting TAB2 and NEMO, respectively. Therefore, restoration of miR-181a-5p and miR-181a-3p may represent a novel therapeutic approach to manage atherosclerosis.

Renal inhibition of miR-181a ameliorates 5-fluorouracil-induced mesangial cell apoptosis and nephrotoxicity.

The development of nephrotoxicity largely limits the clinical use of chemotherapy. MiRNAs are able to target various genes and involved in the regulation of diverse cellular processes, including cell apoptosis and death. Our study showed that miR-181a expression was significantly increased after 5-fluorouracil (5-FU) treatment in renal mesangial cells and kidney tissue, which was associated with decreased baculoviral inhibition of apoptosis protein repeat-containing 6 (BIRC6) expression and increased apoptotic rate. Enforced miR-181a expression enhanced 5-FU-induced p53-dependent mitochondrial apoptosis, including declined Bcl-2/Bax ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase-9 and caspase-3 activation. However, inhibition of miR-181a was associated with reduced p53-mediated mitochondrial apoptosis induced by 5-FU. Moreover, miR-181a increased BIRC6 downstream gene p53 protein expression and transcriptional activity by reducing ubiquitin-mediated protein degradation. We found that miR-181a directly targeted 3'-UTR of BIRC6 mRNA and negatively regulated BIRC6 expression. In vivo study, knockdown of miR-181a with adeno-associated virus harboring miR-181a-tough decoy attenuated 5-FU-induced renal cell apoptosis, inflammation and kidney injury. In conclusion, these results demonstrate that miR-181a increases p53 protein expression and transcriptional activity by targeting BIRC6 and promotes 5-FU-induced apoptosis in mesangial cells. Inhibition of miR-181a ameliorates 5-FU-induced nephrotoxicity, suggesting that miR-181a may be a novel therapeutic target for nephrotoxicity treatment during chemotherapy.

Inhibition of Orai1-mediated Ca2+ entry limits endothelial cell inflammation by suppressing calcineurin-NFATc4 signaling pathway.

Orai1-dependent Ca2+ entry plays an essential role in inflammatory response through regulating T cell and macrophage activation and neutrophil infiltration. However, whether Orai1 Ca2+ entry contributes to endothelial activation, one of the early steps of vascular inflammation, remains elusive. In the present study, we observed that knockdown of Orai1 reduced, whereas overexpression of Orai1 potentiated, TNFα-induced expression of adhesion molecules such as ICAM-1 and VCAM-1 in HUVECs, and subsequently blocked adhesion of monocyte to HUVECs. In vivo, Orai1 downregulation attenuated TNFα-induced ICAM-1 and VCAM-1 expression in mouse aorta and the levels of pro-inflammatory cytokines in the serum. In addition, Orai1 knockdown also dramatically decreased the expression of pro-inflammatory cytokines and neutrophil infiltration in the lung after TNFα treatment, and thus protected lung tissue injury. Notably, among all isoforms of nuclear factor of activated T cells (NFATs), TNFα only triggered NFATc4 nuclear accumulation in HUVECs. Knockdown of Orai1 or inhibition of calcineurin prevented TNFα-induced NFATc4 nuclear translocation and reduced ICAM-1 and VCAM-1 expression in HUVECs. Overexpression of NFATc4 further enhanced ICAM-1 and VCAM-1 expression induced by TNFα. Our study demonstrates that Orai1-Ca2+-calcineurin-NFATc4 signaling is an essential inflammatory pathway required for TNFα-induced endothelial cell activation and vascular inflammation. Therefore, Orai1 may be a potential therapeutic target for treatment of inflammatory diseases.

Ca2+/Calmodulin-Dependent Protein Kinase II γ-Dependent Serine727 Phosphorylation Is Required for TMEM16A Ca2+-Activated Cl- Channel Regulation in Cerebrovascular Cells.

BACKGROUND: TMEM16A is a critical component of Ca2+-activated chloride channels (CaCCs) and mediates basilar arterial smooth muscle cell (BASMC) proliferation in hypertensive cerebrovascular remodeling. CaMKII is a negative regulator of CaCC, and four CaMKII isoforms (α, ß, γ and δ) are expressed in vasculature; however, it is unknown which and how CaMKII isoforms affect TMEM16A-associated CaCC and BASMC proliferation.Methods and Results:Patch clamp and small interfering RNA (siRNA) knockdown of different CaMKII isoforms revealed that only CaMKIIγ inhibited native Ca2+-activated chloride currents (ICl.Ca) in BASMCs. The TMEM16A overexpression evoked TMEM16A Cl-current and inhibited angiotensin II (Ang II)-induced proliferation in BASMCs. The co-immunoprecipitation and pull-down assay indicated an interaction between CaMKIIγ and TMEM16A protein. TMEM16A Cl-current was modulated by CaMKIIγ phosphorylation at serine residues in TMEM16A. Serine525 and Serine727 in TMEM16A were mutated to alanine, and only mutation at Ser727 (S727A) reversed the CaMKIIγ inhibition of the TMEM16A Cl-current. Phosphomimetic mutation S727D markedly decreased TMEM16A Cl-current and reversed TMEM16A-mediated suppression of BASMC proliferation, mimicking the inhibitory effects of CaMKIIγ on TMEM16A. A significant increase in CaMKIIγ isoform content was observed in parallel to the decrease of TMEM16A and ICl.Cain basilar artery proliferative remodeling in Ang II-infused mice. CONCLUSIONS: Serine 727 phosphorylation in TMEM16A by CaMKIIγ provides a new mechanism for regulating TMEM16A CaCC activity and Ang II-induced BASMC proliferation.

The oxidized phospholipid POVPC impairs endothelial function and vasodilation via uncoupling endothelial nitric oxide synthase.

Endothelial dysfunction is an early stage of atherosclerosis. We recently have shown that 25-hydroxycholesterol found in atherosclerotic lesions could impair endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase (eNOS). 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), the oxidation product of oxidized low-density lipoprotein, is another proinflammatory lipid and has also been found in atherosclerotic lesions. However, whether POVPC promotes atherosclerosis like 25-hydroxycholesterol remains unclear. The purpose of this study was to explore the effects of POVPC on endothelial function and vasodilation. Human umbilical vein endothelial cells (HUVECs) were incubated with POVPC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation (O2-) were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS), AKT, PKC-ßII and P70S6K as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining. The expression of Bcl-2, Bax, and Cleaved Caspase 3 were detected by immunoblotting. Finally, aortic ring from C57BL6 mice were isolated and treated with POVPC and the endothelium-dependent vasodilation was evaluated. POVPC significantly inhibited HUVECs proliferation, migration, tube formation, decreased NO production but increased O2- generation. POVPC inhibited the phosphorylation of Akt and eNOS at Ser1177, increased activation of PKC-ßII, P70S6K and the phosphorylation of eNOS at Thr495, reduced the association of HSP90 with eNOS. Meanwhile, POVPC induced endothelial cell apoptosis by inhibiting Bcl-2 expression, increasing Bax and cleaved caspase-3 expressions as well as caspase-3 activity and impaired endothelium-dependent vasodilation. These data demonstrated that POVPC impaired endothelial function by uncoupling and inhibiting eNOS as well as by inducing endothelial cell apoptosis. Therefore, POVPC may play an important role in the development of atherosclerosis and may be considered as a potential therapeutic target for atherosclerosis.