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BACKGROUND: AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase that is activated by cellular perturbations associated with ATP depletion or stress. While AMPK modulates the activity of a variety of targets containing a specific phosphorylation consensus sequence, the number of AMPK targets and their influence over cellular processes is currently thought to be limited. RESULTS: We queried the human and the mouse proteomes for proteins containing AMPK phosphorylation consensus sequences. Integration of this database into Gaggle software facilitated the construction of probable AMPK-regulated networks based on known and predicted molecular associations. In vitro kinase assays were conducted for preliminary validation of 12 novel AMPK targets across a variety of cellular functional categories, including transcription, translation, cell migration, protein transport, and energy homeostasis. Following initial validation, pathways that include NAD synthetase 1 (NADSYN1) and protein kinase B (AKT2) were hypothesized and experimentally tested to provide a mechanistic basis for AMPK regulation of cell migration and maintenance of cellular NAD(+) concentrations during catabolic processes. CONCLUSIONS: This study delineates an approach that encompasses both in silico procedures and in vitro experiments to produce testable hypotheses for AMPK regulation of cellular processes.

Background: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models., Methods: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity., Results: Histological and expression profiles of CCM neurovascular lesions ( Slco1c1-iCreERT2;Pdcd10 fl/fl ; Pdcd10 BECKO ) in male and female mice found that sustained mild hypoxia (12% O 2 , 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis., Conclusions: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness., Competing Interests: Disclosures None.

Visualizing spatial assay data in anatomical images is vital for understanding biological processes in cell, tissue, and organ organizations. Technologies requiring this functionality include traditional one-at-a-time assays, and bulk and single-cell omics experiments, including RNA-seq and proteomics. The spatialHeatmap software provides a series of powerful new methods for these needs, and allows users to work with adequately formatted anatomical images from public collections or custom images. It colors the spatial features (e.g. tissues) annotated in the images according to the measured or predicted abundance levels of biomolecules (e.g. mRNAs) using a color key. This core functionality of the package is called a spatial heatmap plot. Single-cell data can be co-visualized in composite plots that combine spatial heatmaps with embedding plots of high-dimensional data. The resulting spatial context information is essential for gaining insights into the tissue-level organization of single-cell data, or vice versa. Additional core functionalities include the automated identification of biomolecules with spatially selective abundance patterns and clusters of biomolecules sharing similar abundance profiles. To appeal to both non-expert and computational users, spatialHeatmap provides a graphical and a command-line interface, respectively. It is distributed as a free, open-source Bioconductor package (https://bioconductor.org/packages/spatialHeatmap) that users can install on personal computers, shared servers, or cloud systems., (© The Author(s) 2024. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

MASH is a prevalent liver disease that can progress to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and ultimately death, but there are no approved therapies. Leukotriene B4 (LTB4) is a potent pro-inflammatory chemoattractant that drives macrophage and neutrophil chemotaxis, and genetic loss or inhibition of its high affinity receptor, leukotriene B4 receptor 1 (BLT1), results in improved insulin sensitivity and decreased hepatic steatosis. To validate the therapeutic efficacy of BLT1 inhibition in an inflammatory and pro-fibrotic mouse model of MASH and fibrosis, mice were challenged with a choline-deficient, L-amino acid defined high fat diet and treated with a BLT1 antagonist at 30 or 90 mg/kg for 8 weeks. Liver function, histology, and gene expression were evaluated at the end of the study. Treatment with the BLT1 antagonist significantly reduced plasma lipids and liver steatosis but had no impact on liver injury biomarkers or histological endpoints such as inflammation, ballooning, or fibrosis compared to control. Artificial intelligence-powered digital pathology analysis revealed a significant reduction in steatosis co-localized fibrosis in livers treated with the BLT1 antagonist. Liver RNA-seq and pathway analyses revealed significant changes in fatty acid, arachidonic acid, and eicosanoid metabolic pathways with BLT1 antagonist treatment, however, these changes were not sufficient to impact inflammation and fibrosis endpoints. Targeting this LTB4-BLT1 axis with a small molecule inhibitor in animal models of chronic liver disease should be considered with caution, and additional studies are warranted to understand the mechanistic nuances of BLT1 inhibition in the context of MASH and liver fibrosis., (Copyright 2023 The Author(s).)

Competing Interests: Disclosures None.

Rationale: Obstructive sleep apnea (OSA)-induced endothelial cell (EC) dysfunction contributes to OSA-related cardiovascular sequelae. The mechanistic basis of endothelial impairment by OSA is unclear. Objectives: The goals of this study were to identify the mechanism of OSA-induced EC dysfunction and explore the potential therapies for OSA-accelerated cardiovascular disease. Methods: The experimental methods include data mining, bioinformatics, EC functional analyses, OSA mouse models, and assessment of OSA human subjects. Measurements and Main Results: Using mined microRNA sequencing data, we found that microRNA 210 (miR-210) conferred the greatest induction by intermittent hypoxia in ECs. Consistently, the serum concentration of miR-210 was higher in individuals with OSA from two independent cohorts. Importantly, miR-210 concentration was positively correlated with the apnea-hypopnea index. RNA sequencing data collected from ECs transfected with miR-210 or treated with OSA serum showed a set of genes commonly altered by miR-210 and OSA serum, which are largely involved in mitochondrion-related pathways. ECs transfected with miR-210 or treated with OSA serum showed reduced [Formula: see text]o 2 rate, mitochondrial membrane potential, and DNA abundance. Mechanistically, intermittent hypoxia-induced SREBP2 (sterol regulatory element-binding protein 2) bound to the promoter region of miR-210, which in turn inhibited the iron-sulfur cluster assembly enzyme and led to mitochondrial dysfunction. Moreover, the SREBP2 inhibitor betulin alleviated intermittent hypoxia-increased systolic blood pressure in the OSA mouse model. Conclusions: These results identify an axis involving SREBP2, miR-210, and mitochondrial dysfunction, representing a new mechanistic link between OSA and EC dysfunction that may have important implications for treating and preventing OSA-related cardiovascular sequelae.

Background: Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 (CCM1 ), CCM2 , and PDCD10 (CCM3 ). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown., Methods: We use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue., Results: Expression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes ( Aldh1l1-EGFP/Rpl10a ) in the presence or absence of CCM lesions ( Slco1c1-iCreERT2;Pdcd10 fl/fl ; Pdcd10 BECKO ) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10 BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b . Pharmacological inhibition of NLRP3 (NOD [nucleotide-binding oligomerization domain]-' LRR [leucine-rich repeat]- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA [carboxyfluorescein-fluorochrome-labeled inhibitors of caspases] caspase-1) in brain endothelial cells from Pdcd10 BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkb fl/fl mice) in chronic Pdcd10 BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects., Conclusions: Our study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects.

Cerebral cavernous malformations (CCMs) are common neurovascular lesions caused by loss-of-function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3), and generally regarded as an endothelial cell-autonomous disease. Here we reported that proliferative astrocytes played a critical role in CCM pathogenesis by serving as a major source of VEGF during CCM lesion formation. An increase in astrocyte VEGF synthesis is driven by endothelial nitric oxide (NO) generated as a consequence of KLF2- and KLF4-dependent elevation of eNOS in CCM endothelium. The increased brain endothelial production of NO stabilized HIF-1α in astrocytes, resulting in increased VEGF production and expression of a "hypoxic" program under normoxic conditions. We showed that the upregulation of cyclooxygenase-2 (COX-2), a direct HIF-1α target gene and a known component of the hypoxic program, contributed to the development of CCM lesions because the administration of a COX-2 inhibitor significantly prevented the progression of CCM lesions. Thus, non-cell-autonomous crosstalk between CCM endothelium and astrocytes propels vascular lesion development, and components of the hypoxic program represent potential therapeutic targets for CCMs.

Vascular endothelial cells (ECs) sense and respond to hemodynamic forces such as pulsatile shear stress (PS) and oscillatory shear stress (OS). Among the metabolic pathways, glycolysis is differentially regulated by atheroprone OS and atheroprotective PS. Studying the molecular mechanisms by which PS suppresses glycolytic flux at the epigenetic, transcriptomic, and kinomic levels, we have demonstrated that glucokinase regulatory protein (GCKR) was markedly induced by PS in vitro and in vivo, although PS down-regulates other glycolysis enzymes such as hexokinase (HK1). Using next-generation sequencing data, we identified the binding of PS-induced Krüppel-like factor 4 (KLF4), which functions as a pioneer transcription factor, binding to the GCKR promoter to change the chromatin structure for transactivation of GCKR. At the posttranslational level, PS-activated AMP-activated protein kinase (AMPK) phosphorylates GCKR at Ser-481, thereby enhancing the interaction between GCKR and HK1 in ECs. In vivo, the level of phosphorylated GCKR Ser-481 and the interaction between GCKR and HK1 were increased in the thoracic aorta of wild-type AMPKα2 +/+ mice in comparison with littermates with EC ablation of AMPKα2 (AMPKα2 -/- ). In addition, the level of GCKR was elevated in the aortas of mice with a high level of voluntary wheel running. The underlying mechanisms for the PS induction of GCKR involve regulation at the epigenetic level by KLF4 and at the posttranslational level by AMPK., Competing Interests: The authors declare no competing interest.

The transmembrane protein heart of glass1 (HEG1) directly binds to and recruits Krev interaction trapped protein 1 (KRIT1) to endothelial junctions to form the HEG1-KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel-like factors 4 and 2 (KLF4 and KLF2), which are implicated in endothelial vascular homeostasis; however, the effect of acute inhibition of the HEG1-KRIT1 interaction remains incompletely understood. Here, we report a high-throughput screening assay and molecular design of a small-molecule HEG1-KRIT1 inhibitor to uncover acute changes in signaling pathways downstream of the HEG1-KRIT1 protein complex disruption. The small-molecule HEG1-KRIT1 inhibitor 2 (HKi2) was demonstrated to be a bona fide inhibitor of the interaction between HEG1 and KRIT1 proteins, by competing orthosterically with HEG1 through covalent reversible interactions with the FERM (4.1, ezrin, radixin, and moesin) domain of KRIT1. The crystal structure of HKi2 bound to KRIT1 FERM revealed that it occupies the same binding pocket on KRIT1 as the HEG1 cytoplasmic tail. In human endothelial cells (ECs), acute inhibition of the HEG1-KRIT1 interaction by HKi2 increased KLF4 and KLF2 mRNA and protein levels, whereas a structurally similar inactive compound failed to do so. In zebrafish, HKi2 induced expression of klf2a in arterial and venous endothelium. Furthermore, genome-wide RNA transcriptome analysis of HKi2-treated ECs under static conditions revealed that, in addition to elevating KLF4 and KLF2 expression, inhibition of the HEG1-KRIT1 interaction mimics many of the transcriptional effects of laminar blood flow. Furthermore, HKi2-treated ECs also triggered Akt signaling in a phosphoinositide 3-kinase (PI3K)-dependent manner, as blocking PI3K activity blunted the Akt phosphorylation induced by HKi2. Finally, using an in vitro colocalization assay, we show that HKi6, an improved derivative of HKi2 with higher affinity for KRIT1, significantly impedes recruitment of KRIT1 to mitochondria-localized HEG1 in CHO cells, indicating a direct inhibition of the HEG1-KRIT1 interaction. Thus, our results demonstrate that early events of the acute inhibition of HEG1-KRIT1 interaction with HKi small-molecule inhibitors lead to: (i) elevated KLF4 and KLF2 gene expression; and (ii) increased Akt phosphorylation. Thus, HKi's provide new pharmacologic tools to study acute inhibition of the HEG1-KRIT1 protein complex and may provide insights to dissect early signaling events that regulate vascular homeostasis., (© 2021 The Authors. FASEB BioAdvances published by the Federation of American Societies for Experimental Biology.)

Objective: Although type 2 diabetes mellitus (T2DM) has been reported as a risk factor for coronavirus disease 2019 (COVID-19), the effect of pharmacologic agents used to treat T2DM, such as metformin, on COVID-19 outcomes remains unclear. Metformin increases the expression of angiotensin converting enzyme 2, a known receptor for severe acute respiratory syndrome coronavirus 2. Data from people with T2DM hospitalized for COVID-19 were used to test the hypothesis that metformin use is associated with improved survival in this population., Methods: Retrospective analyses were performed on de-identified clinical data from a major hospital in Wuhan, China, that included patients with T2DM hospitalized for COVID-19 during the recent epidemic. One hundred and thirty-one patients diagnosed with COVID-19 and T2DM were used in this study. The primary outcome was mortality. Demographic, clinical characteristics, laboratory data, diabetes medications, and respiratory therapy data were also included in the analysis., Results: Of these 131 patients, 37 used metformin with or without other antidiabetes medications. Among the 37 metformin-taking patients, 35 (94.6%) survived and 2 (5.4%) did not survive. The mortality rates in the metformin-taking group versus the non-metformin group were 5.4% (2/37) versus 22.3% (21/94). Using multivariate analysis, metformin was found to be an independent predictor of survival in this cohort (P = .02)., Conclusion: This study reveals a significant association between metformin use and survival in people with T2DM diagnosed with COVID-19. These clinical data are consistent with potential benefits of the use of metformin for COVID-19 patients with T2DM., (© 2020 American Association of Clinical Endocrinologists. Published by Elsevier, Inc. All rights reserved.)

Type 2 diabetes mellitus (T2DM) is a frequent comorbidity of cancer. Hyperinsulinemia secondary to T2DM promotes cancer progression, whereas antidiabetic agents, such as metformin, have anticancer effects. However, the detailed mechanism for insulin and metformin-regulated cancer cell proliferation remains unclear. This study identified a mechanism by which insulin upregulated the expression of c-Myc, sterol regulatory element-binding protein 1 (SREBP1), and acetyl-coenzyme A (CoA) carboxylase 1 (ACC1), which are important regulators of lipogenesis and cell proliferation. Thymine DNA glycosylase (TDG), a DNA demethylase, was transactivated by c-Myc upon insulin treatment, thereby decreasing 5-carboxylcytosine (5caC) abundance in the SREBP1 promoter. On the other hand, metformin-activated AMP-activated protein kinase (AMPK) increased DNA methyltransferase 3A (DNMT3A) activity to increase 5-methylcytosine (5mC) abundance in the TDG promoter. This resulted in decreased TDG expression and enhanced 5caC abundance in the SREBP1 promoter. These findings demonstrate that c-Myc activates, whereas AMPK inhibits, TDG-mediated DNA demethylation of the SREBP1 promoter in insulin-promoted and metformin-suppressed cancer progression, respectively. This study indicates that TDG is an epigenetic-based therapeutic target for cancers associated with T2DM., (© 2020 The Author(s).)

Endothelial dysfunction is critically involved in the pathogenesis of pulmonary arterial hypertension (PAH) and that exogenously administered microRNA may be of therapeutic benefit. Lower levels of miR-483 were found in serum from patients with idiopathic pulmonary arterial hypertension (IPAH), particularly those with more severe disease. RNA-seq and bioinformatics analyses showed that miR-483 targets several PAH-related genes, including transforming growth factor-β (TGF-β), TGF-β receptor 2 (TGFBR2), β-catenin, connective tissue growth factor (CTGF), interleukin-1β (IL-1β), and endothelin-1 (ET-1). Overexpression of miR-483 in ECs inhibited inflammatory and fibrogenic responses, revealed by the decreased expression of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1. In contrast, inhibition of miR-483 increased these genes in ECs. Rats with EC-specific miR-483 overexpression exhibited ameliorated pulmonary hypertension (PH) and reduced right ventricular hypertrophy on challenge with monocrotaline (MCT) or Sugen + hypoxia. A reversal effect was observed in rats that received MCT with inhaled lentivirus overexpressing miR-483. These results indicate that PAH is associated with a reduced level of miR-483 and that miR-483 might reduce experimental PH by inhibition of multiple adverse responses., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Lining the luminal surface of the vasculature, endothelial cells (ECs) are in direct contact with and differentially respond to hemodynamic forces depending on their anatomic location. Pulsatile shear stress (PS) is defined by laminar flow and is predominantly located in straight vascular regions, while disturbed or oscillatory shear stress (OS) is localized to branch points and bifurcations. Such flow patterns have become a central focus of vascular diseases, such as atherosclerosis, because the focal distribution of endothelial dysfunction corresponds to regions exposed to OS, whereas endothelial homeostasis is maintained in regions defined by PS. Deciphering the mechanotransduction events that occur in ECs in response to differential flow patterns has required the innovation of multidisciplinary approaches in both in vitro and in vivo systems. The results from these studies have identified a multitude of shear stress-regulated molecular networks in the endothelium that are implicated in health and disease. This review outlines the significance of scientific findings generated in collaboration with Dr. Shu Chien., (© Author(s).)

Background: Obstructive Sleep Apnea (OSA) is a highly prevalent condition that is associated with several comorbidities including cardiovascular disease (CVD). Recent studies have revealed mixed results as to whether standard OSA therapy reverses CVD in adult patients. Thus, many advocate for earlier recognition of OSA induced CVD, as early as childhood, to prompt treatment antecedent to the onset of irreversible CVD. Here we investigated if the serum level of miR-92a, a known biomarker for CVD, can be used to identify patients with OSA in both children and adults., Methods: Consecutive snoring patients undergoing polysomnography were recruited for determination of circulating miR-92a, in addition to inflammatory and metabolic profiles. We assessed whether circulating miR-92a was associated with OSA severity., Results: Using two separate cohorts of adults (n=57) and children (n=13), we report a significant increase in the serum level of miR-92a in patients with severe OSA (p=0.021) and further demonstrate a significant correlation (Spearman rank correlation 0.308, p=0.010) with serum miR-92a levels and the apnea hypopnea index (AHI), a primary measure of OSA severity. Stepwise regression analysis revealed that serum miR-92a levels were independently associated with AHI (ß=0.332, p=0.003), age (ß=0.394, p=0.002) and LDL cholesterol levels (ß=0.368, p=0.004)., Conclusion: Our study is the first to establish that miR-92a is a useful biomarker for OSA severity in both children and adults. Given the canonical role of miR-92a on endothelial dysfunction, miR-92a may be useful to identify early onset CVD in OSA patients or stratify patient CVD risk to identify those that may benefit from earlier OSA treatment.

Pulsatile shear (PS) and oscillatory shear (OS) elicit distinct mechanotransduction signals that maintain endothelial homeostasis or induce endothelial dysfunction, respectively. A subset of microRNAs (miRs) in vascular endothelial cells (ECs) are differentially regulated by PS and OS, but the regulation of the miR processing and its implications in EC biology by shear stress are poorly understood. From a systematic in silico analysis for RNA binding proteins that regulate miR processing, we found that nucleolin (NCL) is a major regulator of miR processing in response to OS and essential for the maturation of miR-93 and miR-484 that target mRNAs encoding Krüppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS). Additionally, anti-miR-93 and anti-miR-484 restore KLF2 and eNOS expression and NO bioavailability in ECs under OS. Analysis of posttranslational modifications of NCL identified that serine 328 (S328) phosphorylation by AMP-activated protein kinase (AMPK) was a major PS-activated event. AMPK phosphorylation of NCL sequesters it in the nucleus, thereby inhibiting miR-93 and miR-484 processing and their subsequent targeting of KLF2 and eNOS mRNA. Elevated levels of miR-93 and miR-484 were found in sera collected from individuals afflicted with coronary artery disease in two cohorts. These findings provide translational relevance of the AMPK-NCL-miR-93/miR-484 axis in miRNA processing in EC health and coronary artery disease., Competing Interests: The authors declare no conflict of interest.

Activated by AMP-dependent and -independent mechanisms, AMP-activated protein kinase (AMPK) plays a central role in the regulation of cellular bioenergetics and cellular survival. AMPK regulates a diverse set of signaling networks that converge to epigenetically mediate transcriptional events. Reversible histone and DNA modifications, such as acetylation and methylation, result in structural chromatin alterations that influence transcriptional machinery access to genomic regulatory elements. The orchestration of these epigenetic events differentiates physiological from pathophysiological phenotypes. AMPK phosphorylation of histones, DNA methyltransferases and histone post-translational modifiers establish AMPK as a key player in epigenetic regulation. This review focuses on the role of AMPK as a mediator of cellular survival through its regulation of chromatin remodeling and the implications this has for health and disease.

Rationale: Endothelial dysfunction plays an integral role in pulmonary hypertension (PH). AMPK (AMP-activated protein kinase) and ACE2 (angiotensin-converting enzyme 2) are crucial in endothelial homeostasis. The mechanism by which AMPK regulates ACE2 in the pulmonary endothelium and its protective role in PH remain elusive., Objectives: We investigated the role of AMPK phosphorylation of ACE2 Ser680 in ACE2 stability and deciphered the functional consequences of this post-translational modification of ACE2 in endothelial homeostasis and PH., Methods: Bioinformatics prediction, kinase assay, and antibody against phospho-ACE2 Ser680 (p-ACE2 S680) were used to investigate AMPK phosphorylation of ACE2 Ser680 in endothelial cells. Using CRISPR-Cas9 genomic editing, we created gain-of-function ACE2 S680D knock-in and loss-of-function ACE2 knockout (ACE2 -/- ) mouse lines to address the involvement of p-ACE2 S680 and ACE2 in PH. The AMPK-p-ACE2 S680 axis was also validated in lung tissue from humans with idiopathic pulmonary arterial hypertension., Measurements and Main Results: Phosphorylation of ACE2 by AMPK enhanced the stability of ACE2, which increased Ang (angiotensin) 1-7 and endothelial nitric oxide synthase-derived NO bioavailability. ACE2 S680D knock-in mice were resistant to PH as compared with wild-type littermates. In contrast, ACE2-knockout mice exacerbated PH, a similar phenotype found in mice with endothelial cell-specific deletion of AMPKα2. Consistently, the concentrations of phosphorylated AMPK, p-ACE2 S680, and ACE2 were decreased in human lungs with idiopathic pulmonary arterial hypertension., Conclusions: Impaired phosphorylation of ACE2 Ser680 by AMPK in pulmonary endothelium leads to a labile ACE2 and hence is associated with the pathogenesis of PH. Thus, AMPK regulation of the vasoprotective ACE2 is a potential target for PH treatment.

In silico analysis of Big Data is a useful tool to identify putative kinase targets as well as nodes of signaling cascades that are difficult to discover by traditional single molecule experimentation. System approaches that use a multi-tiered investigational methodology have been instrumental in advancing our understanding of cellular mechanisms that result in phenotypic changes. Here, we present a bioinformatics approach to identify AMP-activated protein kinase (AMPK) target proteins on a proteome-wide scale and an in vitro method for preliminary validation of these targets. This approach offers an initial screening for the identification of AMPK targets that can be further validated using mutagenesis and molecular biology techniques.

The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driven, hormetic pathway providing insight for therapeutic approaches against cholesterol associated neuronal disorders.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) acts as a master regulator of cellular energy homeostasis by directly phosphorylating metabolic enzymes and nutrient transporters and by indirectly promoting the transactivation of nuclear genes involved in mitochondrial biogenesis and function. We explored the mechanism of AMPK-mediated induction of gene expression. We identified AMPK consensus phosphorylation sequences in three proteins involved in nucleosome remodeling: DNA methyltransferase 1 (DNMT1), retinoblastoma binding protein 7 (RBBP7), and histone acetyltransferase 1 (HAT1). DNMT1 mediates DNA methylation that limits transcription factor access to promoters and is inhibited by RBBP7. Acetylation of histones by HAT1 creates a more relaxed chromatin-DNA structure that favors transcription. AMPK-mediated phosphorylation resulted in the activation of HAT1 and inhibition of DNMT1. For DNMT1, this inhibition was both a direct effect of phosphorylation and the result of increased interaction with RBBP7. In human umbilical vein cells, pharmacological AMPK activation or pulsatile shear stress triggered nucleosome remodeling and decreased cytosine methylation, leading to increased expression of nuclear genes encoding factors involved in mitochondrial biogenesis and function, such as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), transcription factor A (Tfam), and uncoupling proteins 2 and 3 (UCP2 and UCP3). Similar effects were seen in the aortas of mice given pharmacological AMPK activators, and these effects required AMPK2α. These results enhance our understanding of AMPK-mediated mitochondrial gene expression through nucleosome remodeling., (Copyright © 2017, American Association for the Advancement of Science.)

Objective: Cortactin translocates to the cell periphery in vascular endothelial cells (ECs) on cortical-actin assembly in response to pulsatile shear stress. Because cortactin has putative sites for AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 1 (SIRT1) deacetylation, we examined the hypothesis that AMPK and SIRT1 coregulate cortactin dynamics in response to shear stress., Approach and Results: Analysis of the ability of AMPK to phosphorylate recombinant cortactin and oligopeptides whose sequences matched AMPK consensus sequences in cortactin pointed to Thr-401 as the site of AMPK phosphorylation. Mass spectrometry confirmed Thr-401 as the site of AMPK phosphorylation. Immunoblot analysis with AMPK siRNA and SIRT1 siRNA in human umbilical vein ECs and EC-specific AMPKα2 knockout mice showed that AMPK phosphorylation of cortactin primes SIRT1 deacetylation in response to shear stress. Immunoblot analyses with cortactin siRNA in human umbilical vein ECs, phospho-deficient T401A and phospho-mimetic T401D mutant, or aceto-deficient (9K/R) and aceto-mimetic (9K/Q) showed that cortactin regulates endothelial nitric oxide synthase activity. Confocal imaging and sucrose-density gradient analyses revealed that the phosphorylated/deacetylated cortactin translocates to the EC periphery facilitating endothelial nitric oxide synthase translocation from lipid to nonlipid raft domains. Knockdown of cortactin in vitro or genetic reduction of cortactin expression in vivo in mice substantially decreased the endothelial nitric oxide synthase-derived NO bioavailability. In vivo, atherosclerotic lesions increase in ApoE - /- /cortactin +/- mice, when compared with ApoE -/- /cortactin +/+ littermates., Conclusions: AMPK phosphorylation of cortactin followed by SIRT1 deacetylation modulates the interaction of cortactin and cortical-actin in response to shear stress. Functionally, this AMPK/SIRT1 coregulated cortactin-F-actin dynamics is required for endothelial nitric oxide synthase subcellular translocation/activation and is atheroprotective., (© 2016 American Heart Association, Inc.)

Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction.

Endothelial functions are highly regulated by imposed shear stress in vivo. The characteristics of shear stress determine mechanotransduction events that regulate phenotypic outcomes including redox and inflammatory states. Recent data indicate that microRNAs (miRs) in vascular endothelial cells play an essential role in shear stress-regulated endothelial responses. More specifically, atheroprotective pulsatile flow (PS) induces miRs that inhibit mediators of oxidative stress and inflammation while promoting those involved in maintaining vascular homeostasis. Conversely, oscillatory flow (OS) elicits the opposing networks. This is exemplified by the PS-responsive transcription factor Krüppel-like factor 2 (KLF2), which regulates miR expression but is also regulated by OS-sensitive miRs to ultimately regulate the oxidative and inflammatory state of the endothelium. In this review, we outline important findings demonstrating the multifaceted roles of shear stress-regulated miRs in endothelial redox and inflammatory balance. Furthermore, we discuss the use of algorithms in deciphering signaling networks differentially regulated by PS and OS., (© 2013 Elsevier Inc. All rights reserved.)

Background: The molecular basis for the focal nature of atherosclerotic lesions is poorly understood. Here, we explored whether disturbed flow patterns activate an innate immune response to form the NLRP3 inflammasome scaffold in vascular endothelial cells via sterol regulatory element binding protein 2 (SREBP2)., Methods and Results: Oscillatory flow activates SREBP2 and induces NLRP3 inflammasome in endothelial cells. The underlying mechanisms involve SREBP2 transactivating NADPH oxidase 2 and NLRP3. Consistently, SREBP2, NADPH oxidase 2, and NLRP3 levels were elevated in atheroprone areas of mouse aortas, suggesting that the SREBP2-activated NLRP3 inflammasome causes functionally disturbed endothelium with increased inflammation. Mimicking the effect of atheroprone flow, endothelial cell-specific overexpression of the activated form of SREBP2 synergized with hyperlipidemia to increase atherosclerosis in the atheroresistant areas of mouse aortas., Conclusions: Atheroprone flow induces NLRP3 inflammasome in endothelium through SREBP2 activation. This increased innate immunity in endothelium synergizes with hyperlipidemia to cause topographical distribution of atherosclerotic lesions.

B-cell lymphoma-6 protein (Bcl-6) is a corepressor for inflammatory mediators such as vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 and -3, which function to recruit monocytes to vascular endothelial cells upon inflammation. Poly [ADP ribose] polymerase 1 (PARP-1) is proinflammatory, in part through its binding at the Bcl-6 intron 1 to suppress Bcl-6 expression. We investigated the mechanisms by which PARP-1 dissociates from the Bcl-6 intron 1, ultimately leading to attenuation of endothelial inflammation. Analysis of the PARP-1 primary sequence suggested that phosphorylation of PARP-1 Serine 177 (Ser-177) by AMP-activated protein kinase (AMPK) is responsible for the induction of Bcl-6. Our results show that AMPK activation with treatment of 5-aminoimidazole-4-carboxamide ribonucleotide, metformin, or pulsatile shear stress induces PARP-1 dissociation from the Bcl-6 intron 1, increases Bcl-6 expression, and inhibits expression of inflammatory mediators. Conversely, AMPKα suppression or knockdown produces the opposite effects. The results demonstrate an anti-infamatory pathway linking AMPK, PARP-1, and Bcl-6 in endothelial cells.