Single-cell RNA sequencing unveils unique transcriptomic signatures of endothelial cells and role of ENO1 in response to disturbed flow.

Flow patterns exert significant effects on vascular endothelial cells (ECs) to lead to the focal nature of atherosclerosis. Using a step flow chamber to investigate the effects of disturbed shear (DS) and pulsatile shear (PS) on ECs in the same flow channel, we conducted single-cell RNA sequencing analyses to explore the distinct transcriptomic profiles regulated by DS vs. PS. Integrated analysis identified eight cell clusters and demonstrated that DS induces EC transition from atheroprotective to proatherogenic phenotypes. Using an automated cell type annotation algorithm (SingleR), we showed that DS promoted endothelial-to-mesenchymal transition (EndMT) by inducing the transcriptional phenotypes for inflammation, hypoxia responses, transforming growth factor-beta (TGF-ß) signaling, glycolysis, and fatty acid synthesis. Enolase 1 (ENO1), a key gene in glycolysis, was one of the top-ranked genes in the DS-induced EndMT cluster. Pseudotime trajectory analysis revealed that the kinetic expression of ENO1 was significantly associated with EndMT and that ENO1 silencing repressed the DS- and TGF-ß-induced EC inflammation and EndMT. Consistent with these findings, ENO1 was highly expressed in ECs at the inner curvature of the mouse aortic arch (which is exposed to DS) and atherosclerotic lesions, suggesting its proatherogenic role in vivo. In summary, we present a comprehensive single-cell atlas of ECs in response to different flow patterns within the same flow channel. Among the DS-regulated genes, ENO1 plays an important role in DS-induced EC inflammation and EndMT. These results provide insights into how hemodynamic forces regulate vascular endothelium in health and disease.

AMPK Attenuation of ß-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of ß-Arrestin-1-ser330.

BACKGROUND: ß-adrenergic receptor (ß-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of ß-AR remains unclear. METHODS: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates ß-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the ß-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between ß-adrenergic insult and ß-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from ß-arrestin-1-S330A/S330D mutation and ß-adrenergic insult. RESULTS: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to ß-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted ß-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing ß-arrestin-1-S330D (active form) inhibited the ß-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. ß-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the ß-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS: AMPK phosphorylation of ß-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting ß-AR/cAMP/PKA activation. Subsequently, ß-arrestin-1 Ser330 phosphorylation blocks ß-AR-induced cardiac inflammasome activation and remodeling.

BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension.

BACKGROUND: An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-ß (transforming growth factor-ß) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-ß family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-ßR2/R1, and receptor-regulated Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-ß-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH. METHODS: Bioinformatic analyses were used to explore the mechanism by which BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-ß signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension. RESULTS: BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9, which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-ß signaling by downregulating TGF-ß expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3-/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling. CONCLUSIONS: These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-ß signaling in PASMCs. Such rebalance of BMP/TGF-ß pathways is translationally important for PAH alleviation.

Role of inflammasomes in endothelial dysfunction.

The vascular endothelium dynamically responds to environmental cues and plays a pivotal role in maintaining vascular homeostasis by regulating vasomotor tone, blood cell trafficking, permeability and immune responses. However, endothelial dysfunction results in various pathological conditions. Inflammasomes are large intracellular multimeric complexes activated by pathogens or cellular damage. Inflammasomes in vascular endothelial cells (ECs) initiate innate immune responses, which have emerged as significant mediators in endothelial dysfunction, contributing to the pathophysiology of an array of diseases. This review summarizes the mechanisms and ramifications of inflammasomes in ECs and related vascular diseases such as atherosclerosis, abdominal aortic aneurysm, stroke, and lung and kidney diseases. We also discuss potential drugs targeting EC inflammasomes and their applications in treating vascular diseases.

Epigenetic repression of Cend1 by lysine-specific demethylase 1 is essential for murine heart development.

Epigenetic regulation of heart development remains incompletely understood. Here we show that LSD1, a histone demethylase, plays a crucial role in regulating cardiomyocyte proliferation during heart development. Cardiomyocyte-specific deletion of Lsd1 in mice inhibited cardiomyocyte proliferation, causing severe growth defect of embryonic and neonatal heart. In vivo RNA-seq and in vitro functional studies identified Cend1 as a target suppressed by LSD1. Lsd1 loss resulted in elevated Cend1 transcription associated with increased active histone mark H3K4me2 at Cend1 promoter. Cend1 knockdown relieved the cell-cycle arrest and proliferation defect caused by LSD1 inhibition in primary rat cardiomyocytes. Moreover, genetic deletion of Cend1 rescued cardiomyocyte proliferation defect and embryonic lethality in Lsd1 null embryos. Consistently, LSD1 promoted the cell cycle of cardiomyocytes derived from human-induced pluripotent stem cells by repressing CEND1. Together, these findings reveal an epigenetic regulatory mechanism involving the LSD1-CEND1 axis that controls cardiomyocyte proliferation essential for murine heart development.

AURKB/CDC37 complex promotes clear cell renal cell carcinoma progression via phosphorylating MYC and constituting an AURKB/E2F1-positive feedforward loop.

As the second most common malignant tumor in the urinary system, renal cell carcinoma (RCC) is imperative to explore its early diagnostic markers and therapeutic targets. Numerous studies have shown that AURKB promotes tumor development by phosphorylating downstream substrates. However, the functional effects and regulatory mechanisms of AURKB on clear cell renal cell carcinoma (ccRCC) progression remain largely unknown. In the current study, we identified AURKB as a novel key gene in ccRCC progression based on bioinformatics analysis. Meanwhile, we observed that AURKB was highly expressed in ccRCC tissue and cell lines and knockdown AURKB in ccRCC cells inhibit cell proliferation and migration in vitro and in vivo. Identified CDC37 as a kinase molecular chaperone for AURKB, which phenocopy AURKB in ccRCC. AURKB/CDC37 complex mediate the stabilization of MYC protein by directly phosphorylating MYC at S67 and S373 to promote ccRCC development. At the same time, we demonstrated that the AURKB/CDC37 complex activates MYC to transcribe CCND1, enhances Rb phosphorylation, and promotes E2F1 release, which in turn activates AURKB transcription and forms a positive feedforward loop in ccRCC. Collectively, our study identified AURKB as a novel marker of ccRCC, revealed a new mechanism by which the AURKB/CDC37 complex promotes ccRCC by directly phosphorylating MYC to enhance its stability, and first proposed AURKB/E2F1-positive feedforward loop, highlighting AURKB may be a promising therapeutic target for ccRCC.

Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation.

AIMS: Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by a high mortality rate. Pulmonary arterial endothelium cells (PAECs) serve as a primary sensor of various environmental cues, such as shear stress and hypoxia, but PAEC dysfunction may trigger vascular remodelling during the onset of PH. This study aimed to illustrate the role of Sirtuin 7 (SIRT7) in endothelial dysfunction during PH and explore the potential therapeutic strategy for PH. METHODS AND RESULTS: SIRT7 levels were measured in human and murine experimental PH samples. Bioinformatic analysis, immunoprecipitation, and deacetylation assay were used to identify the association between SIRT7 and Krüpple-like factor 4 (KLF4), a key transcription factor essential for endothelial cell (EC) homeostasis. Sugen5416 + hypoxia (SuHx)-induced PH mouse models and cell cultures were used for the study of the therapeutic effect of SIRT7 for PH. SIRT7 level was significantly reduced in lung tissues and PAECs from PH patients and the SuHx-induced PH mouse model as compared with healthy controls. Pulmonary endothelium-specific depletion of Sirt7 increased right ventricular systolic pressure and exacerbated right ventricular hypertrophy in the SuHx-induced PH model. At the molecular level, we identified KLF4 as a downstream target of SIRT7, which deacetylated KLF4 at K228 and inhibited the ubiquitination-proteasome degradation. Thus, the SIRT7/KLF4 axis maintained PAEC homeostasis by regulating proliferation, migration, and tube formation. PAEC dysfunction was reversed by adeno-associated virus type 1 vector-mediated endothelial overexpression of Sirt7 or supplementation with nicotinamide adenine dinucleotide (NAD)+ intermediate nicotinamide riboside which activated Sirt7; both approaches successfully reversed PH phenotypes. CONCLUSION: The SIRT7/KLF4 axis ensures PAEC homeostasis, and pulmonary endothelium-specific SIRT7 targeting might constitute a PH therapeutic strategy.