Smooth muscle Cxcl12 activation is associated with vascular remodeling in flow-induced pulmonary hypertension.

Patients with congenital heart disease (CHD) resulting in significant left-to-right shunting of blood are at risk for the development of pulmonary arterial hypertension (PAH). The underlying mechanism by which pulmonary overcirculation and shear stress lead to vascular remodeling remains unclear. Our study established a new "two-hit" murine model of severe pulmonary hypertension (PH) by combining left pneumonectomy and exposure to hypoxia (LP/Hx). Utilizing transgenic reporter lines, immunofluorescence staining, and advanced microscopy, we conducted cell-lineage tracing experiments for endothelial cells (ECs), smooth muscle cells (SMCs), and pericytes. We identified that SMCs is a primary contributor to distal arteriolar remodeling after LP/Hx. Subsequent qPCR analysis on isolated cells demonstrated that Cxcl12 was upregulated in both ECs and SMCs from LP/Hx animals. Likewise, CXCL12 was overexpressed in the SMC layer of arterioles in patients with acyanotic PAH-CHD. These findings provide novel insights into the contribution of SMCs and Cxcl12 to pulmonary flow-induced vascular remodeling. This newly established murine model of PH will serve as a new tool for research and targeted therapeutics for patients with PAH.

Interplay Between FoxM1 and Dab2 Promotes Endothelial Cell Responses in Diabetic Wound Healing.

Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor (VEGF)-dependent angiogenesis remain uncertain and could reveal new therapeutic targets. In our study, the molecular underpinnings of endothelial dysfunction in diabetes were investigated, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FoxM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell (EC) function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high concentrations glucose treated primary mouse skin ECs, simulating hyperglycemic conditions in diabetes mellitus. In diabetic mice with a genetic EC deficiency of Dab2 angiogenesis was reduced in vivo and in vitro when compared with wild-type mice. Restoration of Dab2 expression by injected mRNA-containing lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. At the same time, FoxM1 was downregulated in skin ECs subjected to high glucose conditions as determined by RNA-sequencing analysis. FoxM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FoxM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. These findings indicate that restoring Dab2 expression through targeted therapies can enhance angiogenesis and wound repair in diabetes. To explore this therapeutic potential, we tested LyP-1-conjugated lipid nanoparticles (LNPs) containing Dab2 or control mRNAs to target ECs and found the former significantly improved wound healing and angiogenesis in diabetic mice. This study provides evidence of the crucial roles of Dab2 and FoxM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.

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.

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.

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.

Multi-Organ System Metabolic Stress and Sex-Divergent Vascular Associations.

Introduction: Women experience excess cardiovascular risk compared to men in the setting of similar metabolic disease burden. This consistent finding could be related to sex differences in the vascular response to various forms of metabolic stress. In this study we examine the association of both systemic and organ-specific metabolic stress with vascular health in women and men. Methods: We conducted an observational study of 4,299 adult participants (52% women, aged 59±13 years) of the National Health and Nutrition Examination Survey (NHANES) 2017-2018 cohort and 110,225 adult outpatients (55% women, aged 64±16 years) of the Cedars-Sinai Medical Center (CSMC) 2019 cohort. We used natural splines to examine the association of systemic and organ-specific measures of metabolic stress including body mass index (BMI), hemoglobin A1c (HbA1c), hepatic FIB-4 score, and CKD-EPI estimated glomerular filtration rate (eGFR) on systolic blood pressure (SBP). Piecewise linear models were generated using normal value thresholds (BMI <25 kg/m 2 , HbA1c <5.7%, FIB-4 <1.3, and eGFR ≥90 ml/min), which approximated observed spline breakpoints. The primary outcome was increase in SBP (relative to a sex-specific physiologic baseline SBP) in association with increase in level of each metabolic measure. Results: Women compared to men demonstrated larger magnitudes and an earlier onset of increase in SBP per increment increase across all metabolic stress measures. The slope of SBP increase per increment of each metabolic measure was greater for women than men particularly for metabolic measures within the normal range, with slope differences of 1.71 mmHg per kg/m2 of BMI, 9.61 mmHg per %HbA1c, 6.45 mmHg per FIB-4 unit, and 0.37 mmHg per ml/min decrement of eGFR in the NHANES cohort (P difference <0.05 for all). Overall results were consistent in the CSMC cohort. Conclusions: Women exhibited greater vascular sensitivity in the setting of multiple types of metabolic stress, particularly in periods representing the transition from metabolic health to disease. These findings underscore the importance of involving early metabolic health interventions as part of efforts to mitigate vascular risks in both women and men.

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.

Early-Onset Hypertension and Sex-Specific Residual Risk for Cardiovascular Disease in Type 2 Diabetes.

OBJECTIVE: To investigate whether the sex disparities in type 2 diabetes-associated cardiovascular disease (CVD) risks may be related to early-onset hypertension that could benefit from intensive blood pressure (BP) control. RESEARCH DESIGN AND METHODS: We analyzed intensive versus standard BP control in relation to incident CVD events in women and men with type 2 diabetes, based on their age of hypertension diagnosis. RESULTS: Among 3,792 adults with type 2 diabetes (49% women), multivariable-adjusted CVD risk was increased per decade earlier age at hypertension diagnosis (hazard ratio 1.11 [1.03-1.21], P = 0.006). Excess risk associated with early-diagnosed hypertension was attenuated in the presence of intensive versus standard antihypertensive therapy in women (P = 0.036) but not men (P = 0.76). CONCLUSIONS: Women with type 2 diabetes and early-onset hypertension may represent a higher-risk subpopulation that not only contributes to the excess in diabetes-related CVD risk for women but may benefit from intensive BP control.

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.

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.

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.

Endothelial Cell Response in Kawasaki Disease and Multisystem Inflammatory Syndrome in Children.

Although Kawasaki disease (KD) and multisystem inflammatory syndrome in children (MIS-C) share some clinical manifestations, their cardiovascular outcomes are different, and this may be reflected at the level of the endothelial cell (EC). We performed RNA-seq on cultured ECs incubated with pre-treatment sera from KD (n = 5), MIS-C (n = 7), and healthy controls (n = 3). We conducted a weighted gene co-expression network analysis (WGCNA) using 935 transcripts differentially expressed between MIS-C and KD using relaxed filtering (unadjusted p < 0.05, >1.1-fold difference). We found seven gene modules in MIS-C, annotated as an increased TNFα/NFκB pathway, decreased EC homeostasis, anti-inflammation and immune response, translation, and glucocorticoid responsive genes and endothelial-mesenchymal transition (EndoMT). To further understand the difference in the EC response between MIS-C and KD, stringent filtering was applied to identify 41 differentially expressed genes (DEGs) between MIS-C and KD (adjusted p < 0.05, >2-fold-difference). Again, in MIS-C, NFκB pathway genes, including nine pro-survival genes, were upregulated. The expression levels were higher in the genes influencing autophagy (UBD, EBI3, and SQSTM1). Other DEGs also supported the finding by WGCNA. Compared to KD, ECs in MIS-C had increased pro-survival transcripts but reduced transcripts related to EndoMT and EC homeostasis. These differences in the EC response may influence the different cardiovascular outcomes in these two diseases.

Epitranscriptional Regulation: From the Perspectives of Cardiovascular Bioengineering.

The central dogma of gene expression involves DNA transcription to RNA and RNA translation into protein. As key intermediaries and modifiers, RNAs undergo various forms of modifications such as methylation, pseudouridylation, deamination, and hydroxylation. These modifications, termed epitranscriptional regulations, lead to functional changes in RNAs. Recent studies have demonstrated crucial roles for RNA modifications in gene translation, DNA damage response, and cell fate regulation. Epitranscriptional modifications play an essential role in development, mechanosensing, atherogenesis, and regeneration in the cardiovascular (CV) system, and their elucidation is critically important to understanding the molecular mechanisms underlying CV physiology and pathophysiology. This review aims at providing biomedical engineers with an overview of the epitranscriptome landscape, related key concepts, recent findings in epitranscriptional regulations, and tools for epitranscriptome analysis. The potential applications of this important field in biomedical engineering research are discussed.

Cyclic stretch promotes vascular homing of endothelial progenitor cells via Acsl1 regulation of mitochondrial fatty acid oxidation.

Endothelial progenitor cells (EPCs) play an important role in vascular repair and re-endothelialization after vessel injury. EPCs in blood vessels are subjected to cyclic stretch (CS) due to the pulsatile pressure, but the role of CS in metabolic reprogramming of EPC, particularly its vascular homing and repair, is largely unknown. In the current study, physiological CS applied to EPCs at a magnitude of 10% and a frequency of 1 Hz significantly promoted their vascular adhesion and endothelial differentiation. CS enhanced mitochondrial elongation and oxidative phosphorylation (OXPHOS), as well as adenosine triphosphate production. Metabolomic study and Ultra-high performance liquid chromatography-mass spectrometry assay revealed that CS significantly decreased the content of long-chain fatty acids (LCFAs) and markedly induced long-chain fatty acyl-CoA synthetase 1 (Acsl1), which in turn facilitated the catabolism of LCFAs in mitochondria via fatty acid ß-oxidation and OXPHOS. In a rat carotid artery injury model, transplantation of EPCs overexpressing Acsl1 enhanced the adhesion and re-endothelialization of EPCs in vivo. MRI and vascular morphology staining showed that Acsl1 overexpression in EPCs improved vascular repair and inhibited vascular stenosis. This study reveals a mechanotransduction mechanism by which physiological CS enhances endothelial repair via EPC patency.

Obstructive Sleep Apnea-induced Endothelial Dysfunction Is Mediated by miR-210.

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]o2 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.

MED1 Regulates BMP/TGF-ß in Endothelium: Implication for Pulmonary Hypertension.

BACKGROUND: Dysregulated BMP (bone morphogenetic protein) or TGF-ß (transforming growth factor beta) signaling pathways are imperative in idiopathic and familial pulmonary arterial hypertension (PAH) as well as experimental pulmonary hypertension (PH) in rodent models. MED1 (mediator complex subunit 1) is a key transcriptional co-activator and KLF4 (Krüppel-like factor 4) is a master transcription factor in endothelium. However, MED1 and KLF4 epigenetic and transcriptional regulations of the BMP/TGF-ß axes in pulmonary endothelium and their dysregulations leading to PAH remain elusive. We investigate the MED1/KLF4 co-regulation of the BMP/TGF-ß axes in endothelium by studying the epigenetic regulation of BMPR2 (BMP receptor type II), ETS-related gene (ERG), and TGFBR2 (TGF-ß receptor 2) and their involvement in the PH. METHODS: High-throughput screening involving data from RNA-seq, MED1 ChIP-seq, H3K27ac ChIP-seq, ATAC-seq, and high-throughput chromosome conformation capture together with in silico computations were used to explore the epigenetic and transcriptional regulation of BMPR2, ERG, and TGFBR2 by MED1 and KLF4. In vitro experiments with cultured pulmonary arterial endothelial cells (ECs) and bulk assays were used to validate results from these in silico analyses. Lung tissue from patients with idiopathic PAH, animals with experimental PH, and mice with endothelial ablation of MED1 (EC-MED1-/-) were used to study the PH-protective effect of MED1. RESULTS: Levels of MED1 were decreased in lung tissue or pulmonary arterial endothelial cells from idiopathic PAH patients and rodent PH models. Mechanistically, MED1 acted synergistically with KLF4 to transactivate BMPR2, ERG, and TGFBR2 via chromatin remodeling and enhancer-promoter interactions. EC-MED1-/- mice showed PH susceptibility. In contrast, MED1 overexpression mitigated the PH phenotype in rodents. CONCLUSIONS: A homeostatic regulation of BMPR2, ERG, and TGFBR2 in ECs by MED1 synergistic with KLF4 is essential for the normal function of the pulmonary endothelium. Dysregulation of MED1 and the resulting impairment of the BMP/TGF-ß signaling is implicated in the disease progression of PAH in humans and PH in rodent models.

Time Domains of Hypoxia Responses and -Omics Insights.

The ability to respond rapidly to changes in oxygen tension is critical for many forms of life. Challenges to oxygen homeostasis, specifically in the contexts of evolutionary biology and biomedicine, provide important insights into mechanisms of hypoxia adaptation and tolerance. Here we synthesize findings across varying time domains of hypoxia in terms of oxygen delivery, ranging from early animal to modern human evolution and examine the potential impacts of environmental and clinical challenges through emerging multi-omics approaches. We discuss how diverse animal species have adapted to hypoxic environments, how humans vary in their responses to hypoxia (i.e., in the context of high-altitude exposure, cardiopulmonary disease, and sleep apnea), and how findings from each of these fields inform the other and lead to promising new directions in basic and clinical hypoxia research.

RNA Sequencing Reveals Beneficial Effects of Atorvastatin on Endothelial Cells in Acute Kawasaki Disease.

Background Damage to the coronary arteries during the acute phase of Kawasaki disease (KD) is linked to inflammatory cell infiltration, myointimal proliferation, and endothelial cell (EC) dysfunction. To understand the response of ECs to KD treatment, we studied the genome-wide transcriptional changes in cultured ECs incubated with KD sera before and after treatment with or without atorvastatin. Methods and Results RNA sequencing of human umbilical vein ECs incubated with pooled sera from patients with acute KD before or after treatment with intravenous immunoglobulin and infliximab revealed differentially expressed genes in interleukin-1, tumor necrosis factor-α, and inflammatory cell recruitment pathways. Subacute sera pooled from patients treated with intravenous immunoglobulin, infliximab, and atorvastatin uniquely induced expression of NOS3, Kruppel like factor (KLF2, and KLF4 (promotes EC homeostasis and angiogenesis) and ZFP36 ring finger protein (ZFP36) and suppressor of cytokine signaling 3 (SOCS3) (suppresses inflammation), and suppressed expression of TGFB2 and DKK1 (induces endothelial-mesenchymal transition) and sphingosine kinase 1 (SPHK1) and C-X-C motif chemokine ligand 8 (CXCL8) (induces inflammation). Conclusions These results suggest that atorvastatin treatment of patients with acute KD may improve EC health, reduce mediators of inflammation produced by ECs, and block KD-induced myofibroblast proliferation.

JAGGED-NOTCH3 signaling in vascular remodeling in pulmonary arterial hypertension.

Within the pulmonary arterial tree, the NOTCH3 pathway is crucial in controlling vascular smooth muscle cell proliferation and maintaining smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension (PAH) is a fatal disease without cure, characterized by elevated pulmonary vascular resistance due to vascular smooth muscle cell proliferation in precapillary arteries, perivascular inflammation, and asymmetric neointimal hyperplasia. Here, we show that human PAH is characterized by overexpression of the NOTCH ligand JAGGED-1 (JAG-1) in small pulmonary artery smooth muscle cells and that JAG-1 selectively controls NOTCH3 signaling and cellular proliferation in an autocrine fashion. In contrast, the NOTCH ligand DELTA-LIKE 4 is minimally expressed in small pulmonary artery smooth muscle cells from individuals with PAH, inhibits NOTCH3 cleavage and signaling, and retards vascular smooth muscle cell proliferation. A new monoclonal antibody for the treatment of PAH, which blocks JAG-1 cis- and trans-induced cleavage of the NOTCH3 receptor in the pulmonary vasculature, was developed. Inhibition of JAG-1-induced NOTCH3 signaling in the lung reverses clinical and pathologic pulmonary hypertension in two rodent models of disease, without toxic side effects associated with nonspecific NOTCH inhibitors. Our data suggest opposing roles of NOTCH ligands in the pulmonary vasculature in pulmonary hypertension. We propose that selectively targeting JAG-1 activation of NOTCH3 may be an effective, safe strategy to treat PAH.