scholarly journals RIPK1 Expression Associates with Inflammation in Early Atherosclerosis in Humans and Can be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice

Circulation ◽  
2020 ◽  
Author(s):  
Denuja Karunakaran ◽  
My-Anh Nguyen ◽  
Michele Geoffrion ◽  
Dianne Vreeken ◽  
Zachary Lister ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Activation ◽  
Inflammatory Responses ◽  
Early Stage ◽  
Oxidized Ldl ◽  
Loss Of Function ◽  
Aortic Sinus ◽  
Atherosclerotic Lesions

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. Previously, we showed that macrophages in the atherogenic plaque undergo RIPK3-MLKL-dependent programmed necroptosis in response to sterile ligands such as oxidized LDL and damage-associated patterns (DAMPs) and necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1, which acts as a master switch that controls whether the cell undergoes NFκB-dependent inflammation, caspase-dependent apoptosis or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is largely driven by NFκB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NFκB-dependent inflammation in early atherogenic lesions and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and using loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 anti-sense oligonucleotides (ASO) to Apoe -/- mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 ASOs led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, p<0.01) and plasma inflammatory cytokines (IL-1α, IL-17A, p<0.05) compared to controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NFκB, TNFα, IL-1α) and in vivo LPS- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin and monocyte attachment. Conclusions: We have identified RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Helong Zhao ◽  
Appakkudal Anand ◽  
Ramesh Ganju
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Model Studies ◽  
Endothelial Inflammation ◽  
Anti Inflammatory ◽  
Whole Heart

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.

Disruption of endothelial Pfkfb3 ameliorates diet-induced murine insulin resistance

2021 ◽  
Author(s):  
Qiuhua Yang ◽  
Jiean Xu ◽  
Qian Ma ◽  
Zhiping Liu ◽  
Yaqi Zhou ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Endothelial Cells ◽  
High Fat Diet ◽  
Inflammatory Responses ◽  
Critical Role ◽  
Endothelial Inflammation ◽  
Glucose Clearance ◽  
Deficient Mice

Overnutrition-induced endothelial inflammation plays a crucial role in high fat diet (HFD)-induced insulin resistance in animals. Endothelial glycolysis plays a critical role in endothelial inflammation and proliferation, but its role in diet-induced endothelial inflammation and subsequent insulin resistance has not been elucidated. PFKFB3 is a critical glycolytic regulator, and its increased expression has been observed in adipose vascular endothelium of C57BL/6J mice fed with HFD in vivo, and in palmitate (PA)-treated primary human adipose microvascular endothelial cells (HAMECs) in vitro. We generated mice with Pfkfb3 deficiency selective for endothelial cells to examine the effect of endothelial Pfkfb3 in endothelial inflammation in metabolic organs and in the development of HFD-induced insulin resistance. EC Pfkfb3-deficient mice exhibited mitigated HFD-induced insulin resistance, including decreased body weight and fat mass, improved glucose clearance and insulin sensitivity, and alleviated adiposity and hepatic steatosis. Mechanistically, cultured PFKFB3 knockdown HAMECs showed decreased NF-κB activation induced by PA, and consequent suppressed adhesion molecule expression and monocyte adhesion. Taken together, these results demonstrate that increased endothelial PFKFB3 expression promotes diet-induced inflammatory responses and subsequent insulin resistance, suggesting that endothelial metabolic alteration plays an important role in the development of insulin resistance.

scholarly journals Gga3 deletion and a GGA3 rare variant associated with late onset Alzheimer’s disease trigger BACE1 accumulation in axonal swellings

2020 ◽  
Vol 12 (570) ◽  
pp. eaba1871
Author(s):  
Selene Lomoio ◽  
Rachel Willen ◽  
WonHee Kim ◽  
Kevin Z. Ho ◽  
Edward K. Robinson ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Rare Variant ◽  
Late Onset ◽  
Early Stage ◽  
Loss Of Function ◽  
Axonal Trafficking ◽  
Axonal Swellings

Axonal dystrophy, indicative of perturbed axonal transport, occurs early during Alzheimer’s disease (AD) pathogenesis. Little is known about the mechanisms underlying this initial sign of the pathology. This study proves that Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) loss of function, due to Gga3 genetic deletion or a GGA3 rare variant that cosegregates with late-onset AD, disrupts the axonal trafficking of the β-site APP-cleaving enzyme 1 (BACE1) resulting in its accumulation in axonal swellings in cultured neurons and in vivo. We show that BACE pharmacological inhibition ameliorates BACE1 axonal trafficking and diminishes axonal dystrophies in Gga3 null neurons in vitro and in vivo. These data indicate that axonal accumulation of BACE1 engendered by GGA3 loss of function results in local toxicity leading to axonopathy. Gga3 deletion exacerbates axonal dystrophies in a mouse model of AD before β-amyloid (Aβ) deposition. Our study strongly supports a role for GGA3 in AD pathogenesis, where GGA3 loss of function triggers BACE1 axonal accumulation independently of extracellular Aβ, and initiates a cascade of events leading to the axonal damage distinctive of the early stage of AD.

scholarly journals The cerebral cavernous malformation proteins CCM2L and CCM2 prevent the activation of the MAP kinase MEKK3

2015 ◽  
Vol 112 (46) ◽  
pp. 14284-14289 ◽  
Author(s):  
Xavier Cullere ◽  
Eva Plovie ◽  
Paul M. Bennett ◽  
Calum A. MacRae ◽  
Tanya N. Mayadas
Keyword(s):  
Endothelial Cells ◽  
Cavernous Malformation ◽  
Body Axis ◽  
Cerebral Cavernous Malformations ◽  
Loss Of Function ◽  
Cavernous Malformations ◽  
Downstream Target ◽  
Density Flow

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe “big heart” and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

β2-Glycoprotein I as a ‘Cofactor’ for anti-phospholipid reactivity with endothelial cells

Lupus ◽  
1998 ◽  
Vol 7 (2_suppl) ◽  
pp. 44-47 ◽  
Author(s):  
PL Meroni ◽  
N Del Papa ◽  
E Raschi ◽  
P Panzeri ◽  
MO Borghi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Site ◽  
Synthetic Peptide ◽  
Cell Activation ◽  
Heparan Sulphate ◽  
Membrane Structures ◽  
Glycoprotein I ◽  
Endothelial Growth Factor

β2-glycoprotein I (β2GPI) is a cofactor for anti-phospholipid (aPL) binding to cardiolipin (CL)-coated plates. β2GPI is also able to bind to endothelial cell (EC) membranes as supported by in-vivo as well as by in-vitro studies. The PL-binding site in the fifth domain of the molecule is involved in the adhesion to endothelium. Actually, specific mutations in this molecular portion abolish endothelium binding and a synthetic peptide spanning the sequence Glu274 –Cys288 of the CL-binding site displays comparable adhesion to EC monolayers. Heparan sulphate appears to be one of the anionic EC membrane structures with which cationic β2GPI interacts, as supported by studies with heparitinase-treated EC. β2GPI binding to EC might be related to its activity as endothelial growth factor or as a lipid-carrying glycoprotein. Adhesion of β2GPI to endothelial membranes offers suitable epitopes for circulating aPL that, once bound, can induce cell activation

scholarly journals The Role of Methionine Aminopeptidase 2 in Lymphangiogenesis

2020 ◽  
Vol 21 (14) ◽  
pp. 5148
Author(s):  
Rawnaq Esa ◽  
Eliana Steinberg ◽  
Dvir Dror ◽  
Ouri Schwob ◽  
Mehrdad Khajavi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Function ◽  
Loss Of Function ◽  
Methionine Aminopeptidase ◽  
Zebrafish Model ◽  
Lymphatic Capillary ◽  
Intracellular Enzyme

During the metastasis process, tumor cells invade the blood circulatory system directly from venous capillaries or indirectly via lymphatic vessels. Understanding the relative contribution of each pathway and identifying the molecular targets that affect both processes is critical for reducing cancer spread. Methionine aminopeptidase 2 (MetAp2) is an intracellular enzyme known to modulate angiogenesis. In this study, we investigated the additional role of MetAp2 in lymphangiogenesis. A histological staining of tumors from human breast-cancer donors was performed in order to detect the level and the localization of MetAp2 and lymphatic capillaries. The basal enzymatic level and activity in vascular and lymphatic endothelial cells were compared, followed by loss of function studies determining the role of MetAp2 in lymphangiogenesis in vitro and in vivo. The results from the histological analyses of the tumor tissues revealed a high MetAp2 expression, with detectable sites of co-localization with lymphatic capillaries. We showed slightly reduced levels of the MetAp2 enzyme and MetAp2 mRNA expression and activity in primary lymphatic cells when compared to the vascular endothelial cells. The genetic and biochemical manipulation of MetAp2 confirmed the dual activity of the enzyme in both vascular and lymphatic remodulation in cell function assays and in a zebrafish model. We found that cancer-related lymphangiogenesis is inhibited in murine models following MetAp2 inhibition treatment. Taken together, our study provides an indication that MetAp2 is a significant contributor to lymphangiogenesis and carries a dual role in both vascular and lymphatic capillary formation. Our data suggests that MetAp2 inhibitors can be effectively used as anti-metastatic broad-spectrum drugs.

scholarly journals Modeling early stage atherosclerosis in a primary human vascular microphysiological system

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xu Zhang ◽  
Muath Bishawi ◽  
Ge Zhang ◽  
Varun Prasad ◽  
Ellen Salmon ◽  
...  
Keyword(s):  
Cell Activation ◽  
Foam Cell ◽  
Early Stage ◽  
Cell Formation ◽  
Density Lipoprotein ◽  
Foam Cell Formation ◽  
No Production ◽  
Endothelial Cell Activation

Abstract Novel atherosclerosis models are needed to guide clinical therapy. Here, we report an in vitro model of early atherosclerosis by fabricating and perfusing multi-layer arteriole-scale human tissue-engineered blood vessels (TEBVs) by plastic compression. TEBVs maintain mechanical strength, vasoactivity, and nitric oxide (NO) production for at least 4 weeks. Perfusion of TEBVs at a physiological shear stress with enzyme-modified low-density-lipoprotein (eLDL) with or without TNFα promotes monocyte accumulation, reduces vasoactivity, alters NO production, which leads to endothelial cell activation, monocyte accumulation, foam cell formation and expression of pro-inflammatory cytokines. Removing eLDL leads to recovery of vasoactivity, but not loss of foam cells or recovery of permeability, while pretreatment with lovastatin or the P2Y11 inhibitor NF157 reduces monocyte accumulation and blocks foam cell formation. Perfusion with blood leads to increased monocyte adhesion. This atherosclerosis model can identify the role of drugs on specific vascular functions that cannot be assessed in vivo.

Abstract 464: Membrane Microparticles From Ischemic Muscle Induce In Vitro Progenitor Cells Differentiation And In Vivo Neovascularization.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Aurelie S Leroyer ◽  
Teni G Ebrahimian ◽  
Jose Vilar ◽  
Bernard I Levy ◽  
Alain Tedgui ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Artery Ligation ◽  
Control Muscle ◽  
Tissue Ischemia ◽  
Subunit Expression

Postischemic neovascularization is mediated, at least in part, by the homing of progenitor cells to sites of injury and their differentiation into endothelial cells (EC). However, the mechanisms of in situ progenitor differentiation remain for the most part unknown. We hypothesized that miproparticles (MPs) released following ischemia-induced cell activation or apoptosis are the endogenous signal of postischemic vasculogenesis. MPs were detected by electron microscopy as vesicles of 0.1 to 1 μm in diameter in mice ischemic hindlimb muscle, 48hrs after unilateral femoral artery ligation. After isolation by sequential centrifugations, flow cytometry analyses showed that AnnexinV+ MPs concentration in ischemic calf was 3.5-fold higher than in control muscle (1392±406 vs. 394±180 AnnV+MPs/mg, p<0.005) and mainly originated from endothelial cells (47% of MPs are CD144+). MPs isolated from ischemic muscles induced a more potent in vitro bone marrow-mononuclear cells (BM-MNC) differentiation into EPC, than those isolated from control muscle (6.1±1.0% vs. 3.5±0.7% Dil-LDL/BS1lectin+ cells/field, p<0.05). Moreover, MPs isolated from GFP+ ischemic muscles colocalized with differentiated BM-MNC. MPs isolated from atherosclerotic plaque were uneffective whereas those isolated from apoptotic or IL-1 activated EC also promoted BM-MNC differentiation. Interestingly, MPs from ischemic muscles produced more reactive oxygen species and expressed significantly higher levels of NADPH oxidase p47 (6 fold ; p<0.05) and p67 subunits (16 fold ; p<0.005) than those from control, whereas gp91 subunit expression was unchanged. The MPs-induced BM-MNC differentiation was reduced by two fold with MPs isolated from gp91-deficient mice (p<0.05). MPs effect on post-ischemic vasculogenesis and revascularization was examined in the ischemic hindlimb model. MPs isolated from ischemic muscles were injected into ischemic legs in parallel with venous injection of BM-MNC. MPs increased the pro-angiogenic effect of BM-MNC transplantation and this effect was blunted with MPs from gp91−/− mice. These results demonstrate that MPs produced after tissue ischemia stimulate progenitor cell differentiation and subsequently promote postnatal neovascularization.

scholarly journals C-Reactive Protein Induces Release of Both Endothelial Microparticles and Circulating Endothelial Cells In Vitro and In Vivo: Further Evidence of Endothelial Dysfunction

2011 ◽  
Vol 57 (12) ◽  
pp. 1757-1761 ◽  
Author(s):  
Sridevi Devaraj ◽  
Pappanaicken R Kumaresan ◽  
Ishwarlal Jialal
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Oxidized Ldl ◽  
Circulating Endothelial Cells ◽  
Early Event ◽  
C Reactive Protein ◽  
Endothelial Microparticles ◽  
Reactive Protein

BACKGROUND Inflammation is pivotal in atherosclerosis. A key early event in atherosclerosis is endothelial dysfunction. C-reactive protein (CRP), the prototypic marker of inflammation in humans, is a risk marker for cardiovascular disease, and there is mounting evidence to support its role in atherothrombosis. CRP has been shown to promote endothelial dysfunction both in vitro and in vivo. Emerging biomarkers of endothelial dysfunction include circulating endothelial cells (CECs) and endothelial microparticles (EMPs). However, there is a paucity of data examining the effect of CRP on CEC and EMP production in vitro and in vivo. METHODS In this report, we treated human aortic endothelial cells (HAECs) with increasing concentrations of CRP (0–50 μg/mL) or boiled CRP. We counted CECs and EMPs by flow cytometry. RESULTS Although CRP treatment resulted in a significant increase in release of both CECs and EMPs, boiled CRP failed to have an effect. Pretreatment of HAECs with sepiapterin or diethylenetriamine NONOate, both of which preserve nitric oxide (NO), resulted in attenuation of CRP's effects on CECs and EMPs. CD32 and CD64 blocking antibodies but not CD16 antibody or lectin-like oxidized LDL receptor 1 small interfering RNA (LOX-1 siRNA) prevented CRP-induced production of CECs and EMPs. Furthermore, delivery of human CRP to Wistar rats compared with human serum albumin resulted in significantly increased CECs and EMPs, corroborating the in vitro findings. CONCLUSIONS We provide novel data that CRP, via NO deficiency, promotes endothelial dysfunction by inducing release of CECs and EMPs, which are biomarkers of endothelial dysfunction.

scholarly journals Serine Lipids of Porphyromonas gingivalis Are Human and Mouse Toll-Like Receptor 2 Ligands

2013 ◽  
Vol 81 (9) ◽  
pp. 3479-3489 ◽  
Author(s):  
Robert B. Clark ◽  
Jorge L. Cervantes ◽  
Mark W. Maciejewski ◽  
Vahid Farrokhi ◽  
Reza Nemati ◽  
...  
Keyword(s):  
Cell Activation ◽  
Inflammatory Responses ◽  
Periodontal Pathogen ◽  
Toll Like Receptor ◽  
Dendritic Cell Activation ◽  
Content Type ◽  
Hek Cells ◽  
Toll Like Receptor 2

ABSTRACTThe total cellular lipids ofPorphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids ofP. gingivalisand define which lipid classes account for the TLR2 engagement, based on bothin vitrohuman cell assays andin vivostudies in mice. Specific serine-containing lipids ofP. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods.In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2−/−) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2−/−mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced byP. gingivalisthat likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

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