Shear stress regulation of miR-93 and miR-484 maturation through nucleolin.

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

MicroRNA-483 amelioration of experimental pulmonary hypertension.

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

Inhibition of the HEG1-KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells

The Kruppel-like Factors 2 and 4 (KLF2/4) are transcription factors and master regulators of endothelial cells (ECs) phenotype and homeostasis. KLF2/4 are important blood-flow-responsive genes within ECs that differentially regulate the expression of factors that confer anti-inflammatory, antithrombotic, and antiproliferative effects in ECs. We found that genetic inactivation of endothelial Krit1 (Krev interaction trapped protein 1) or Heg1 (Heart of glass) led to upregulation of KLF2/4 expression levels. We also observed that vasoprotective proteins, endothelial nitric oxide synthase (eNOS) and thrombomodulin (TM), are upregulated by the increase of KLF2/4 as a result of loss of endothelial KRIT1. Here, we developed a high-throughput screening assay to identify inhibitors of the HEG1-KRIT1 interaction and identified sirtinol (HKi001) as a promising hit inhibitor. The crystal structure of sirtinol bound to the KRIT1 FERM domain confirmed the primary screening results and ultimately led to the identification of a fragment-like inhibitor (HKi002), which occupies the HEG1 pocket producing comparable activity. These findings suggest that these inhibitors block the interaction by competing with the HEG1 for binding to KRIT1 FERM domain. Moreover, our results demonstrate that HKi002 upregulates KLF2/4 gene expression in two types of human ECs. These results reveal an unexpected role of inhibiting the HEG1-KRIT1 interaction and provide a proof-of-concept that pharmacological manipulation of this complex may offer new opportunities to induce expression of KLF2/4 as vasoprotective factors

AMPK: An Epigenetic Landscape Modulator

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

AMPK Mediates Endothelial Function Through the Phosphorylation of Nucleolin and PARP-1

Endothelial cells play an active role in maintaining vascular health. Their response to different flow patterns and circulating molecules initiate signaling cascades that determine endothelial phenotypes and ultimately vascular health or disease. One key molecule that initiates endothelial pro-health cascades is the AMP-activated protein kinase (AMPK). Responding to the onset of cellular stress, AMPK not only maintains the cellular energy status by activating catabolic pathways, while shutting down anabolic ones, it also promotes endothelial differentiation and quiescence while attenuating inflammation. However, the precise signaling events that result in AMPK's beneficial effects remain elusive. Here, we show that both Poly (ADP-ribose) polymerase-1 (PARP-1) and nucleolin (NCL) are phosphorylated by AMPK. PARP-1 is an abundant nuclear protein that once activated, uses NAD+ to "PARylate" itself as well as other protein targets. It serves multiple functions in the cell including DNA repair, epigenetics, mitosis, and transcription by binding to TTGATATAAAT sequences within the genome. The B-cell lymphoma 6 (Bcl-6) intron 1 contains a PARP-1 binding sequence. PARP-1 binds to this sequence it the intron in its inactive state, but upon activation by AMPK phosphorylation, it dissociates allowing transactivation of Bcl-6 protein. Bcl-6 acts as a co-repressor for VCAM-1, MCP-1, and MCP-3, which inhibits the recruitment of macrophages to the endothelium thus attenuating the local inflammatory response. In addition to the phosphorylation of PARP-1, AMPK regulates the transcriptome through the phosphorylation of nucleolin.We also demonstrate that AMPK regulates endothelial health phosphorylation of nucleolin. Nucleolin is a ubiquitous, multifunctional protein that traffics to multiple cellular locations. It facilitates ribosome biogenesis in the nuceolus, transcription in the nucleus, mRNA stability in the cytoplasm, and angiogenesis at the cell surface. Upon phosphorylation of nucleolin by AMPK, nucleolin transiently translocates to the nucleus where it transactivates KLF2 expression. KLF2 is a zinc finger containing shear stress responsive transcription factor that regulates the majority of the transcriptome in response to pulsatile flow. Additionally, KLF2 is involved in the differentiation of endothelial cells and maintains endothelial quiescence. Taken together, AMPK regulates the PARP-1 anti-inflammatory and nucleolin/KLF2 pathways which promote endothelial health

AMPK: An Epigenetic Landscape Modulator

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

spatialHeatmap: visualizing spatial bulk and single-cell assays in anatomical images

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