Brain ischemia causes systemic Notch1 activity in endothelial cells to drive atherosclerosis.

Stroke leads to persistently high risk for recurrent vascular events caused by systemic atheroprogression that is driven by endothelial cell (EC) activation. However, whether and how stroke induces sustained pro-inflammatory and proatherogenic endothelial alterations in systemic vessels remain poorly understood. We showed that brain ischemia induces persistent activation, the upregulation of adhesion molecule VCAM1, and increased senescence in peripheral ECs until 4 weeks after stroke onset. This aberrant EC activity resulted from sustained Notch1 signaling, which was triggered by increased circulating Notch1 ligands DLL1 and Jagged1 after stroke in mice and humans. Consequently, this led to increased myeloid cell adhesion and atheroprogression by generating a senescent, pro-inflammatory endothelium. Notch1- or VCAM1-blocking antibodies and the genetic ablation of endothelial Notch1 reduced atheroprogression after stroke. Our findings revealed a systemic machinery that induces the persistent activation of peripheral ECs after stroke, which paves the way for therapeutic interventions or the prevention of recurrent vascular events following stroke.

A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity.

In obesity, inflammation of white adipose tissue (AT) is associated with diminished generation of beige adipocytes ('beige adipogenesis'), a thermogenic and energy-dissipating function mediated by beige adipocytes that express the uncoupling protein UCP1. Here we delineated an inflammation-driven inhibitory mechanism of beige adipogenesis in obesity that required direct adhesive interactions between macrophages and adipocytes mediated by the integrin α4 and its counter-receptor VCAM-1, respectively; expression of the latter was upregulated in obesity. This adhesive interaction reciprocally and concomitantly modulated inflammatory activation of macrophages and downregulation of UCP1 expression dependent on the kinase Erk in adipocytes. Genetic or pharmacological inactivation of the integrin α4 in mice resulted in elevated expression of UCP1 and beige adipogenesis of subcutaneous AT in obesity. Our findings, established in both mouse systems and human systems, reveal a self-sustained cycle of inflammation-driven impairment of beige adipogenesis in obesity.

Metformin synergizes with PD-1 blockade to promote normalization of tumor vessels via CD8T cells and IFNγ.

Tumor blood vessels are highly leaky in structure and have poor blood perfusion, which hampers infiltration and function of CD8T cells within tumor. Normalizing tumor vessels is thus thought to be important in promoting the flux of immune T cells and enhancing ant-tumor immunity. However, how tumor vasculature is normalized is poorly understood. Metformin (Met) combined with ant-PD-1 therapy is known to stimulate proliferation of and to produce large amounts of IFNγ from tumor-infiltrating CD8T lymphocytes (CD8TILs). We found that the combination therapy promotes the pericyte coverage of tumor vascular endothelial cells (ECs) to improve blood perfusion and that it suppresses the hyperpermeability through the increase of VE-cadherin. Peripheral node addressin(PNAd) and vascular cell adhesion molecule (VCAM)-1, both implicated to promote tumor infiltration of CD8T cells, were also increased. Importantly, tumor vessel normalization, characterized as the reduced 70-kDa dextran leakage and the enhancement of VE-cadherin and VCAM-1, were canceled by anti-CD8 Ab or anti-IFNγ Ab injection to mice. The increased CD8TILs were also abrogated by anti-IFNγ Ab injection. In vascular ECs, flow cytometry analysis revealed that pSTAT1 expression was found to be associated with VE-cadherin expression. Moreover, in vitro treatment with Met and IFNγ enhanced VE-cadherin and VCAM-1 on human umbilical vein endothelial cells (HUVECs). The Kaplan-Meier method revealed a correlation of VE-cadherin or VCAM-1 levels with overall survival in patients treated with immune checkpoint inhibitors. These data indicate that IFNγ-mediated cross talk of CD8TILs with tumor vessels is important for creating a better tumor microenvironment and maintaining sustained antitumor immunity.

cellPLATO - an unsupervised method for identifying cell behaviour in heterogeneous cell trajectory data.

Advances in imaging, segmentation and tracking have led to the routine generation of large and complex microscopy datasets. New tools are required to process this 'phenomics' type data. Here, we present 'Cell PLasticity Analysis Tool' (cellPLATO), a Python-based analysis software designed for measurement and classification of cell behaviours based on clustering features of cell morphology and motility. Used after segmentation and tracking, the tool extracts features from each cell per timepoint, using them to segregate cells into dimensionally reduced behavioural subtypes. Resultant cell tracks describe a 'behavioural ID' at each timepoint, and similarity analysis allows the grouping of behavioural sequences into discrete trajectories with assigned IDs. Here, we use cellPLATO to investigate the role of IL-15 in modulating human natural killer (NK) cell migration on ICAM-1 or VCAM-1. We find eight behavioural subsets of NK cells based on their shape and migration dynamics between single timepoints, and four trajectories based on sequences of these behaviours over time. Therefore, by using cellPLATO, we show that IL-15 increases plasticity between cell migration behaviours and that different integrin ligands induce different forms of NK cell migration.

DOCK2 Promotes Atherosclerosis by Mediating the Endothelial Cell Inflammatory Response.

The pathology of atherosclerosis, a leading cause of mortality in patients with cardiovascular disease, involves inflammatory phenotypic changes in vascular endothelial cells. This study explored the role of the dedicator of cytokinesis (DOCK)-2 protein in atherosclerosis. Mice with deficiencies in low-density lipoprotein receptor and Dock2 (Ldlr-/-Dock2-/-) and controls (Ldlr-/-) were fed a high-fat diet (HFD) to induce atherosclerosis. In controls, Dock2 was increased in atherosclerotic lesions, with increased intercellular adhesion molecule (Icam)-1 and vascular cell adhesion molecule (Vcam)-1, after HFD for 4 weeks. Ldlr-/-Dock2-/- mice exhibited significantly decreased oil red O staining in both aortic roots and aortas compared to that in controls after HFD for 12 weeks. In control mice and in humans, Dock2 was highly expressed in the ECs of atherosclerotic lesions. Dock2 deficiency was associated with attenuation of Icam-1, Vcam-1, and monocyte chemoattractant protein (Mcp)-1 in the aortic roots of mice fed HFD. Findings in human vascular ECs in vitro suggested that DOCK2 was required in TNF-α-mediated expression of ICAM-1/VCAM-1/MCP-1. DOCK2 knockdown was associated with attenuated NF-κB phosphorylation with TNF-α, partially accounting for DOCK2-mediated vascular inflammation. With DOCK2 knockdown in human vascular ECs, TNF-α-mediated VCAM-1 promoter activity was inhibited. The findings from this study suggest the novel concept that DOCK2 promotes the pathogenesis of atherosclerosis by modulating inflammation in vascular ECs.

Targeting lipid nanoparticles to the blood-brain barrier to ameliorate acute ischemic stroke.

Effective delivery of mRNA or small molecule drugs to the brain is a significant challenge in developing treatment for acute ischemic stroke (AIS). To address the problem, we have developed targeted nanomedicine to increase drug concentrations in endothelial cells of the blood-brain barrier (BBB) of the injured brain. Inflammation during ischemic stroke causes continuous neuronal death and an increase in the infarct volume. To enable targeted delivery to the inflamed BBB, we conjugated lipid nanocarriers (NCs) with antibodies that bind cell adhesion molecules expressed at the BBB. In the transient middle cerebral artery occlusion mouse model, NCs targeted to vascular cellular adhesion molecule-1 (VCAM) achieved the highest level of brain delivery, nearly two orders of magnitude higher than untargeted ones. VCAM-targeted lipid nanoparticles with luciferase-encoding mRNA and Cre-recombinase showed selective expression in the ischemic brain. Anti-inflammatory drugs administered intravenously after ischemic stroke reduced cerebral infarct volume by 62% (interleukin-10 mRNA) or 35% (dexamethasone) only when they were encapsulated in VCAM-targeted NCs. Thus, VCAM-targeted lipid NCs represent a new platform for strongly concentrating drugs within the compromised BBB of penumbra, thereby ameliorating AIS.

Activation of heme oxygenase-1 by laminar shear stress ameliorates high glucose-induced endothelial cell and smooth muscle cell dysfunction.

High glucose (HG)-induced endothelial cell (EC) and smooth muscle cell (SMC) dysfunction is critical in diabetes-associated atherosclerosis. However, the roles of heme oxygenase-1 (HO-1), a stress-response protein, in hemodynamic force-generated shear stress and HG-induced metabolic stress remain unclear. This investigation examined the cellular effects and mechanisms of HO-1 under physiologically high shear stress (HSS) in HG-treated ECs and adjacent SMCs. We found that exposure of human aortic ECs to HSS significantly increased HO-1 expression; however, this upregulation appeared to be independent of adenosine monophosphate-activated protein kinase, a regulator of HO-1. Furthermore, HSS inhibited the expression of HG-induced intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and reactive oxygen species (ROS) production in ECs. In an EC/SMC co-culture, compared with static conditions, subjecting ECs close to SMCs to HSS and HG significantly suppressed SMC proliferation while increasing the expression of physiological contractile phenotype markers, such as α-smooth muscle actin and serum response factor. Moreover, HSS and HG decreased the expression of vimentin, an atherogenic synthetic phenotypic marker, in SMCs. Transfecting ECs with HO-1-specific small interfering (si)RNA reversed HSS inhibition on HG-induced inflammation and ROS production in ECs. Similarly, reversed HSS inhibition on HG-induced proliferation and synthetic phenotype formation were observed in co-cultured SMCs. Our findings provide insights into the mechanisms underlying EC-SMC interplay during HG-induced metabolic stress. Strategies to promote HSS in the vessel wall, such as continuous exercise, or the development of HO-1 analogs and mimics of the HSS effect, could provide an effective approach for preventing and treating diabetes-related atherosclerotic vascular complications.

VCAM-1 mediates proximal tubule-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

Studies in animal models have suggested a linkage between the inflammatory response to injury and subsequent nephron loss during the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Failure of normal repair during the CKD transition correlates with de novo expression of vascular cell adhesion protein-1 (VCAM-1) by a subset of injured proximal tubule cells. This study identified the role of VCAM-1 expression in promoting the failed repair state. Single-cell transcriptome analysis of patients with AKI and CKD and whole kidney RNA and protein analyses of mouse models of CKD confirmed a marked increase of VCAM-1 expression in the proximal tubules of injured kidneys. In immortalized mouse proximal tubular cells and primary cultured renal cells (PCRCs), VCAM-1 expression was induced by proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. Analyses of bulk RNA sequencing of TNF-α-treated primary cultured renal cells or pseudo-bulk RNA sequencing of biopsies from Kidney Precision Medicine Project datasets indicated activation of NF-κB and an enrichment of inflammatory response and cell adhesion pathways in VCAM-1-positive cells. Pharmacological inhibition of NF-κB signaling or genetic deletion of myeloid differentiation factor 88 and TIR domain-containing adapter-inducing interferon-ß suppressed TNF-α- and IL-1ß-induced VCAM-1 expression in vitro. TNF-α stimulation or overexpression of VCAM-1 significantly increased splenocyte adhesion to the mouse proximal tubular monolayer in culture. These results demonstrate that persistence of proinflammatory cytokines after AKI can induce NF-κB-dependent VCAM-1 expression by proximal tubule cells, mediating increased immune cell adhesion to the tubule and thus promoting further tubule injury and greater risk of progression from AKI to CKD.NEW & NOTEWORTHY We demonstrated the induction of VCAM-1 and its biological function in proximal tubules. We found that proinflammatory cytokines (TNF-α and IL-1ß) significantly induced VCAM-1 expression via NF-κB signaling pathway. TNF-α treatment or overexpression of VCAM-1 in immortalized MPT cells increased CD45+ splenocyte adhesion. Pharmacological inhibition of NF-κB or genetic deletion of Vcam1 suppressed TNF-α-induced splenocyte adhesion in vitro, suggesting that VCAM-1 mediates proximal tubular-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

Acid-sensing receptor GPR4 plays a crucial role in lymphatic cancer metastasis.

Cancer tissues exhibit an acidic microenvironment owing to the accumulation of protons and lactic acid produced by cancer and inflammatory cells. To examine the role of an acidic microenvironment in lymphatic cancer metastasis, gene expression profiling was conducted using human dermal lymphatic endothelial cells (HDLECs) treated with a low pH medium. Microarray and gene set enrichment analysis revealed that acid treatment induced the expression of inflammation-related genes in HDLECs, including genes encoding chemokines and adhesion molecules. Acid treatment-induced chemokines C-X3-C motif chemokine ligand 1 (CX3CL1) and C-X-C motif chemokine ligand 6 (CXCL6) autocrinally promoted the growth and tube formation of HDLECs. The expression of vascular cell adhesion molecule 1 (VCAM-1) increased in HDLECs after acid treatment in a time-dependent manner, which, in turn, enhanced their adhesion to melanoma cells. Among various acid-sensing receptors, HDLECs basally expressed G protein-coupled receptor 4 (GPR4), which was augmented under the acidic microenvironment. The induction of chemokines or VCAM-1 under acidic conditions was attenuated by GPR4 knockdown in HDLECs. In addition, lymph node metastases in a mouse melanoma model were suppressed by administering an anti-VCAM-1 antibody or a GPR4 antagonist. These results suggest that an acidic microenvironment modifies the function of lymphatic endothelial cells via GPR4, thereby promoting lymphatic cancer metastasis. Acid-sensing receptors and their downstream molecules might serve as preventive or therapeutic targets in cancer.

Muscone inhibits the progression of atherosclerotic plaques in mice aorta by inhibiting the NF-κB/p65 pathway.

Atherosclerosis (AS) is considered to be one of the main pathogenic factors of coronary heart disease, cerebral infarction and peripheral vascular disease. Oxidative stress and inflammation run through the occurrence and development of atherosclerosis and related cardiovascular events. Muscone is a natural extract of deer musk and also the main physiological active substance of musk. This study investigated the impact of muscone on atherosclerosis. ApoE-/- mice were used to establised AS model and injected with low-dose (4 mg/kg/day) or high-dose (8 mg/kg/day) of muscone intraperitoneally for 4 weeks. Then aortic tissues were collected, and pathological sections of the aorta were prepared for oil red staining, HE and masson staining. The changes of MDA, SOD, VCAM-1, NF-κB, and TNF-α were observed by Western blotting or immunofluorescence staining. The results showed that high-dose muscone could effectively reduce the plaque area/aortic root area and relative atherosclerotic area, reduce the collagen composition in plaque tissue. In addition, we also found that high-dose muscone can effectively increase MDA level, reduce the level of SOD, and inhibit the expression of VCAM-1, NF-κB/p65, TNF-α in arterial plaques. Our results indicate that the administration of muscone has the benefit of inhibiting atherosclerosis. The potential mechanisms may be associated with antioxidant effect and inhibition of inflammatory reaction in arterial plaques. With the increasing understanding of the relationship between muscone and atherosclerosis, muscone has high potential value as a new drug to treat atherosclerosis.

Amiodarone inhibits the Toll-like receptor 3-mediated nuclear factor κB signaling pathway by blocking organelle acidification.

Toll-like receptor (TLR) agonists or pro-inflammatory cytokines converge to activate the nuclear factor κB (NF-κB) signaling pathway, which provokes inflammatory responses. In the present study, we identified amiodarone hydrochloride as a selective inhibitor of the TLR3-mediated NF-κB signaling pathway by screening the RIKEN NPDepo Chemical Library. In human umbilical vein endothelial cells (HUVEC), amiodarone selectively inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) induced by polyinosinic-polycytidylic acid (Poly(I:C)), but not tumor necrosis factor-α, interleukin-1α, or lipopolysaccharide. In response to a Poly(I:C) stimulation, amiodarone at 20 µM reduced the up-regulation of mRNA expression encoding ICAM-1, vascular cell adhesion molecule-1, and E-selectin. The nuclear translocation of the NF-κB subunit RelA was inhibited by amiodarone at 15-20 µM in Poly(I:C)-stimulated HUVEC. Amiodarone diminished the fluorescent dots of LysoTracker® Red DND-99 scattered over the cytoplasm of HUVEC. Therefore, the present study revealed that amiodarone selectively inhibited the TLR3-mediated NF-κB signaling pathway by blocking the acidification of intracellular organelles.

Endothelial IL17RD promotes Western diet-induced aortic myeloid cell infiltration.

The Interleukin-17 (IL17) family is a group of cytokines implicated in the etiology of several inflammatory diseases. Interleukin-17 receptor D (IL17RD), also known as Sef (similar expression to fibroblast growth factor) belonging to the family of IL17 receptors, has been shown to modulate IL17A-associated inflammatory phenotypes. The objective of this study was to test the hypothesis that IL17RD promotes endothelial cell activation and consequent leukocyte adhesion. We utilized primary human aortic endothelial cells and demonstrated that RNAi targeting of IL17RD suppressed transcript levels by 83 % compared to non-targeted controls. Further, RNAi knockdown of IL17RD decreased the adhesion of THP-1 monocytic cells onto a monolayer of aortic endothelial cells in response to IL17A. Additionally, we determined that IL17A did not significantly enhance the activation of canonical MAPK and NFκB pathways in endothelial cells, and further did not significantly affect the expression of VCAM-1 and ICAM-1 in aortic endothelial cells, which is contrary to previous findings. We also determined the functional relevance of our findings in vivo by comparing the expression of endothelial VCAM-1 and ICAM-1 and leukocyte infiltration in the aorta in Western diet-fed Il17rd null versus wild-type mice. Our results showed that although Il17rd null mice do not have significant alteration in aortic expression of VCAM-1 and ICAM-1 in endothelial cells, they exhibit decreased accumulation of proinflammatory monocytes and neutrophils, suggesting that endothelial IL17RD induced in vivo myeloid cell accumulation is not dependent on upregulation of VCAM-1 and ICAM-1 expression. We further performed proteomics analysis to identify potential molecular mediators of the IL17A/IL17RD signaling axis. Collectively, our results underscore a critical role for Il17rd in the regulation of aortic myeloid cell infiltration in the context of Western diet feeding.

Pharmacological inhibition of histone deacetylase alleviates chronic unpredictable stress induced atherosclerosis and endothelial dysfunction via upregulation of BDNF.

Long-term stress is a significant risk factor for cardiovascular diseases, including atherosclerosis and endothelial dysfunction. Moreover, prolonged stress has shown to negatively regulate central BDNF expression. The role of central BDNF in CNS disorders is well studied until recently the peripheral BDNF was also found to be involved in endothelial function regulation and atherosclerosis. The peripheral BDNF and its role in chronic stress-induced atherosclerosis and endothelial dysfunction remain unclear. Therefore, we aimed to elucidate the role of BDNF and its modulation by the HDAC inhibitor valproic acid (VA) in chronic unpredictable stress (CUS)-induced atherosclerosis and endothelial dysfunction. We demonstrated that a 10-week CUS mouse model substantially decreases central and peripheral BDNF expression, resulting in enhanced serum lipid indices, plaque deposition, fibrosis, and CD68 expression in thoracic aortas. Further, parameters associated with endothelial dysfunction such as increased levels of endothelin-1 (ET-1), adhesion molecules like VCAM-1, M1 macrophage markers, and decreased M2 macrophage markers, eNOS expression, and nitrite levels in aortas, were also observed. VA (50 mg/kg, 14 days, i. p.) was administered to mice following 8 weeks of CUS exposure until the end of the experimental procedure. VA significantly prevented the decrease in BDNF, eNOS and nitrite levels, reduced lesion formation and fibrosis in thoracic aortas and increased ET-1, and VCAM-1 followed by M2 polarization in VA-treated mice. The study highlights the potential of epigenetic modulation of BDNF as a therapeutic target, in stress-induced cardiovascular pathologies and suggests that VA could be a promising agent for mitigating CUS-induced endothelial dysfunction and atherosclerosis by BDNF modulation.

Th1 cells contribute to retinal ganglion cell loss in glaucoma in a VCAM-1-dependent manner.

Glaucoma is a complex neurodegenerative disorder characterized by the progressive loss of retinal ganglion cells (RGC) and optic nerve axons, leading to irreversible visual impairment. Despite its clinical significance, the underlying mechanisms of glaucoma pathogenesis remain poorly understood. In this study, we aimed to unravel the multifaceted nature of glaucoma by investigating the interaction between T cells and retinas. By utilizing clinical samples, murine glaucoma models, and T cell transfer models, we made several key findings. Firstly, we observed that CD4+ T cells from glaucoma patients displayed enhanced activation and a bias towards T helper (Th) 1 responses, which correlated with visual impairment. Secondly, we identified the infiltration of Th1 cells into the retina, where they targeted RGC and integrated into the pro-inflammatory glial network, contributing to progressive RGC loss. Thirdly, we discovered that circulating Th1 cells upregulated vascular cell adhesion protein 1 (VCAM-1) on retinal microvessels, facilitating their entry into the neural retina. Lastly, we found that Th1 cells underwent functional reprogramming before reaching the retina, acquiring a phenotype associated with lymphocyte migration and neurodegenerative diseases. Our study provides novel insights into the role of peripheral CD4+ T cells in glaucoma pathogenesis, shedding light on the mechanisms underlying their infiltration into the retina and offering potential avenues for innovative therapeutic interventions in this sight-threatening disease.

Targeted deletion of NR2F2 and VCAM1 in theca cells impacts ovarian follicular development: insights into polycystic ovary syndrome?†.

Defining features of polycystic ovary syndrome (PCOS) include elevated expression of steroidogenic genes, theca cell androgen biosynthesis, and peripheral levels of androgens. In previous studies, we identified vascular cell adhesion molecule 1 (VCAM1) as a selective androgen target gene in specific NR2F2/SF1 (+/+) theca cells. By deleting NR2F2 and VCAM1 selectively in CYP17A1 theca cells in mice, we documented that NR2F2 and VCAM1 impact distinct and sometimes opposing theca cell functions that alter ovarian follicular development in vivo: including major changes in ovarian morphology, steroidogenesis, gene expression profiles, immunolocalization images (NR5A1, CYP11A1, NOTCH1, CYP17A1, INSL3, VCAM1, NR2F2) as well as granulosa cell functions. We propose that theca cells impact follicle integrity by regulating androgen production and action, as well as granulosa cell differentiation/luteinization in response to androgens and gonadotropins that may underlie PCOS.

ANGPTL3 is a novel HDL component that regulates HDL function.

BACKGROUND: Angiopoietin-like protein 3 (ANGPTL3) is secreted by hepatocytes and inhibits lipoprotein lipase and endothelial lipase activity. Previous studies reported the correlation between plasma ANGPTL3 levels and high-density lipoprotein (HDL). Recently ANGPTL3 was found to preferentially bind to HDL in healthy human circulation. Here, we examined whether ANGPTL3, as a component of HDL, modulates HDL function and affects HDL other components in human and mice with non-diabetes or type 2 diabetes mellitus. METHODS: HDL was isolated from the plasma of female non-diabetic subjects and type-2 diabetic mellitus (T2DM) patients. Immunoprecipitation, western blot, and ELISA assays were used to examine ANGPTL3 levels in HDL. Db/m and db/db mice, AAV virus mediated ANGPTL3 overexpression and knockdown models and ANGPTL3 knockout mice were used. The cholesterol efflux capacity induced by HDL was analyzed in macrophages preloaded with fluorescent cholesterol. The anti-inflammation capacity of HDL was assessed using flow cytometry to measure VCAM-1 and ICAM-1 expression levels in TNF-α-stimulated endothelial cells pretreated with HDL. RESULTS: ANGPTL3 was found to bind to HDL and be a component of HDL in both non-diabetic subjects and T2DM patients. Flag-ANGPTL3 was found in the HDL of transgenic mice overexpressing Flag-ANGPTL3. ANGPLT3 of HDL was positively associated with cholesterol efflux in female non-diabetic controls (r = 0.4102, p = 0.0117) but not in female T2DM patients (r = - 0.1725, p = 0.3224). Lower ANGPTL3 levels of HDL were found in diabetic (db/db) mice compared to control (db/m) mice and were associated with reduced cholesterol efflux and inhibition of VCAM-1 and ICAM-1 expression in endothelial cells (p < 0.05 for all). Following AAV-mediated ANGPTL3 cDNA transfer in db/db mice, ANGPTL3 levels were found to be increased in HDL, and corresponded to increased cholesterol efflux and decreased ICAM-1 expression. In contrast, knockdown of ANGPTL3 levels in HDL by AAV-mediated shRNA transfer led to a reduction in HDL function (p < 0.05 for both). Plasma total cholesterol, total triglycerides, HDL-c, protein components of HDL and the cholesterol efflux function of HDL were lower in ANGPTL3-/- mice than ANGPTL3+/+ mice, suggesting that ANGPTL3 in HDL may regulate HDL function by disrupting the balance of protein components in HDL. CONCLUSION: ANGPTL3 was identified as a component of HDL in humans and mice. ANGPTL3 of HDL regulated cholesterol efflux and the anti-inflammatory functions of HDL in T2DM mice. Both the protein components of HDL and cholesterol efflux capacity of HDL were decreased in ANGPTL3-/- mice. Our findings suggest that ANGPTL3 in HDL may regulate HDL function by disrupting the balance of protein components in HDL. Our study contributes to a more comprehensive understanding of the role of ANGPTL3 in lipid metabolism.

Removal of endothelial surface-associated von villebrand factor suppresses accelerate datherosclerosis after myocardial infarction.

BACKGROUND: Thromboinflammation involving platelet adhesion to endothelial surface-associated von Willebrand factor (VWF) has been implicated in the accelerated progression of non-culprit plaques after MI. The aim of this study was to use arterial endothelial molecular imaging to mechanistically evaluate endothelial-associated VWF as a therapeutic target for reducing remote plaque activation after myocardial infarction (MI). METHODS: Hyperlipidemic mice deficient for the low-density lipoprotein receptor and Apobec-1 underwent closed-chest MI and were treated chronically with either: (i) recombinant ADAMTS13 which is responsible for proteolytic removal of VWF from the endothelial surface, (ii) N-acetylcysteine (NAC) which removes VWF by disulfide bond reduction, (iii) function-blocking anti-factor XI (FXI) antibody, or (iv) no therapy. Non-ischemic controls were also studied. At day 3 and 21, ultrasound molecular imaging was performed with probes targeted to endothelial-associated VWF A1-domain, platelet GPIbα, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) at lesion-prone sites of the aorta. Histology was performed at day 21. RESULTS: Aortic signal for P-selectin, VCAM-1, VWF, and platelet-GPIbα were all increased several-fold (p < 0.01) in post-MI mice versus sham-treated animals at day 3 and 21. Treatment with NAC and ADAMTS13 significantly attenuated the post-MI increase for all four molecular targets by > 50% (p < 0.05 vs. non-treated at day 3 and 21). On aortic root histology, mice undergoing MI versus controls had 2-4 fold greater plaque size and macrophage content (p < 0.05), approximately 20-fold greater platelet adhesion (p < 0.05), and increased staining for markers of platelet transforming growth factor-ß1 signaling. Accelerated plaque growth and inflammatory activation was almost entirely prevented by ADAMTS13 and NAC. Inhibition of FXI had no significant effect on molecular imaging signal or plaque morphology. CONCLUSIONS: Plaque inflammatory activation in remote arteries after MI is strongly influenced by VWF-mediated platelet adhesion to the endothelium. These findings support investigation into new secondary preventive therapies for reducing non-culprit artery events after MI.

Long-term abuse of caffeine sodium benzoate induces endothelial cells injury and leads to coagulation dysfunction.

Our hospital admitted a patient who had difficulty in coagulation even after blood replacement, and the patient had abused caffeine sodium benzoate (CSB) for more than 20 years. Hence, we aimed to explore whether CSB may cause dysfunction in vascular endothelial cells and its possible mechanism. Low, medium, and high concentrations of serum of long-term CSB intake patients were used to treat HUVECs, with LPS as the positive control. MTT and CCK8 were performed to verify CSB's damaging effect on HUVECs. The expression of ET-1, ICAM-1, VCAM-1, and E-selectin were measured by ELISA. TUNEL assay and Matrigel tube formation assay were carried out to detect apoptosis and angiogenesis of HUVECs. Flow cytometry was applied to analyze cell cycles and expression of CD11b, PDGF, and ICAM-1. Expression of PDGF-BB and PCNA were examined by western blot. The activation of MAPK signaling pathway was detected by qRT-PCR and western blot. Intracellular Ca2+ density was detected by fluorescent probes. CCK8 assay showed high concentration of CSB inhibited cell viability. Cell proliferation and angiogenesis were inhibited by CSB. ET-1, ICAM-1, VCAM-1, and E-selectin upregulated in CSB groups. CSB enhanced apoptosis of HUVECs. CD11b, ICAM-1 increased and PDGF reduced in CSB groups. The expression level and phosphorylation level of MEK, ERK, JUN, and p38 in MAPK pathway elevated in CSB groups. The expression of PCNA and PDGF-BB was suppressed by CSB. Intracellular Ca2+ intensity was increased by CSB. Abuse of CSB injured HUVECs and caused coagulation disorders.

The Effects of Hyperglycemia on Early Endothelial Activation and the Initiation of Atherosclerosis.

It is well established that patients with diabetes have an increased risk of developing atherosclerotic cardiovascular disease. The earliest detectable sign of atherosclerosis initiation is endothelial cell activation. Activated endothelial cells express adhesion proteins, P-selectin, E-selectin, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1, which function to recruit monocytes to the subendothelial layer. This study examines the effect of hyperglycemia on endothelial cell activation and the initiation and progression of atherosclerosis. In vitro studies revealed that exposure of human aortic endothelial cells to elevated (30 mmol/L) glucose concentrations significantly increased the expression of P-selectin, E-selectin, and vascular cell adhesion molecule-1. In vivo studies showed that, before lesion development, 5-week-old hyperglycemic ApoE-/-Ins2+/akita mice had significantly increased expression of these adhesion proteins in the aortic sinus and increased macrophage infiltration, compared with normoglycemic ApoE-/- controls. At 25 weeks of age, ApoE-/-Ins2+/akita mice had significantly larger atherosclerotic plaques than ApoE-/- controls (0.022 ± 0.004 versus 0.007± 0.001 mm3; P < 0.05). Similar endothelial activation was observed in heterozygous ApoE+/-Ins2+/akita mice; however, detectable atherosclerotic lesions did not develop in the absence of dyslipidemia. Lowering blood glucose levels (by 55%) using a sodium-glucose cotransporter 2 inhibitor reduced endothelial activation. Together, these findings support a causative role for hyperglycemia in atherogenesis and highlight the importance of blood glucose regulation in preventing atherosclerotic cardiovascular disease.

Long non-coding RNA H19X as a regulator of mononuclear cell adhesion to the endothelium in systemic sclerosis.

OBJECTIVE: To define the functional relevance of H19 X-linked (H19X) co-expressed long non-coding RNA (lncRNA) in endothelial cell (EC) activation as a key process in SSc vasculopathy. METHODS: H19X expression in SSc skin biopsies was analysed from single-cell RNA sequencing (scRNA-seq) data. Differential expression and pathway enrichment analysis between cells expressing (H19Xpos) and non-expressing H19X (H19Xneg) cells was performed. H19X function was investigated in human dermal microvascular ECs (HDMECs) by silencing. H19X and EC adhesion molecule levels were analysed by real-time quantitative PCR and western blot after stimulation with pro-inflammatory cytokines. Cytoskeletal rearrangements were analysed by fluorescent staining. Endothelial adhesion was evaluated by co-culture of HDMECs and fluorescent-labelled peripheral blood mononuclear cells (PBMCs). Shedding vascular cell adhesion protein 1 (VCAM1) was evaluated by ELISA on HDMEC supernatant. RESULTS: The scRNA-seq data showed significant upregulation of H19X in SSc compared with healthy ECs. In HDMECs, H19X was consistently induced by IFN type I and II. H19X knockdown lead to a significant decrease in the mRNA of several adhesion molecules. In particular, VCAM1 was significantly reduced at the protein and mRNA levels. Co-expression analysis of the scRNA-seq data confirmed higher expression of VCAM1 in H19Xpos ECs. ECs were also strongly associated with the 'cell adhesion molecule' pathway. Moreover, the VCAM1 downstream pathway displayed less activation following H19X knockdown. Contractility of HDMECs, PBMC adhesion to HDMECs and VCAM1 shedding were also reduced following H19X knockdown. CONCLUSIONS: lncRNA H19X may contribute to EC activation in SSc vasculopathy, acting as a regulator of expression of adhesion molecules in ECs.