Endothelial Cell Inflammatory Responses Research Articles

EOLA1 protects lipopolysaccharide induced IL-6 production and apoptosis by regulation of MT2A in human umbilical vein endothelial cells.

Endothelial cell (EC) injury or dysfunction is believed to be mediated at least in part by lipopolysaccharide (LPS). Recent studies have shown that LPS induces apoptosis in different types of endothelium, including HUVEC. Previously we used EOLA1 (endothelial-overexpressed LPS-associated factor 1) cDNA as a bait and performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2a (MT2a) as the associated protein. EOLA1 protein plays a role as a signal transduction factor. But the mechanism of EOLA1 mediated the protection of cell production of IL-6 and apopotosis in HUVEC is not known. MT2a is expressed in many kinds of cells and plays a role in inflammation. In this study, we demonstrated that LPS could induce EOLA1 expression in time-dependent and apparently contributed to the inhibition of IL-6 production and apoptosis induced by LPS treatment. We also found that deletion of EOLA1 promoted IL-6 production and apoptosis in the treatment of LPS in HUVEC. Furthermore, we demonstrated that MT2a was activated by LPS, and played a key role in LPS-induced IL-6 expression in HUVEC. We further provided the evidence that EOLA1 functioned as a negative regulator for LPS response by regulation of MT2a. These findings suggest that EOLA1 may have an important regulatory role during EC inflammatory responses.

Combined incubation of platelets and endothelial cells with glycated albumin: Altered thrombogenic and inflammatory responses.

Diabetes mellitus is a salient risk factor for the development of cardiovascular diseases (CVDs). Part of this risk is associated with the presence of advanced glycation end products (AGEs), which have been shown to up-regulate platelet or endothelial cell inflammatory and thrombogenic responses that are associated with CVDs. However, platelets perform mechanisms that alter endothelial cell inflammatory and thrombogenic responses, and endothelial cells perform similar mechanisms on platelets. Thus, our goal was to evaluate platelet and endothelial cell inflammatory and thrombogenic reactions that AGEs elicit during concurrent exposure. Endothelial cells were incubated with AGEs for 5 days, after which platelets were added. A time course for CVD inflammatory and thrombogenic responses was quantified as a function of extent of glycation. In general, the presence of platelets reduced AGE-induced endothelial cell responses associated with CVD progression and the presence of endothelial cells reduced platelet adhesion and activation responses, as compared with individual exposures. In general, the presence of irreversibly glycated albumin promoted CVD development to a greater extent than reversibly glycated albumin. This suggests that under diabetic conditions, platelets and endothelial cells can negatively feedback on each other, likely via enhanced adhesion, to elicit a reduced response associated with CVD progression.

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine (NADA) and Synthetic Cannabinoid WIN55,212-2 Abate the Inflammatory Activation of Human Endothelial Cells

Although cannabinoids, such as Δ(9)-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB1R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB2R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB1R/CB2R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.

Monophosphoryl lipid A alters the inflammatory response of endothelial cells challenged with lipopolysaccharide (855.4)

Monophosphoryl lipid A (MPLA) is a lipid A mimetic that induces a state of endotoxin tolerance when given prior to lipopolysaccharide (LPS) challenge. As endothelial cells regulate inflammation, we hypothesized that MPLA would downregulate the inflammatory response of endothelial cells to LPS in a serum dependent manner. We cultured human umbilical vein endothelial cells (HUVECs) and exposed them to MPLA or a vehicle control for 6 hours. Afterwards, HUVECs were challenged with TNFα or LPS or saline for 24 hours. We assessed activation by quantifying IL‐6 levels in the culture media. In the presence of 2% serum, MPLA alone enhanced IL‐6 production compared to controls. LPS increased IL‐6 production much more potently, but this effect was reduced by 47 ± 5% when pre‐exposed to MPLA (p<0.05). MPLA had no effect on TNFα‐induced IL‐6 production. In the absence of serum, LPS induction of IL‐6 was nearly abolished secondary to lack of serum co‐factors. However, MPLA enhancement of IL‐6 production persisted in a serum free environment (p<0.05). These studies demonstrate that MPLA alters the inflammatory response of endothelial cells after LPS challenge and that the effects of MPLA are serum independent.

G protein‐coupled receptor kinase 2 moderates recruitment of THP‐1 cells to the endothelium by limiting histamine‐invoked Weibel‐Palade body exocytosis

BackgroundG protein-coupled receptors (GPCRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory.ObjectivesTo determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory.MethodssiRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells.ResultsG protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization.ConclusionsG protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.

Inflammatory responses of endothelial cells are differentially modulated by monocyte subsets

Inflammatory responses of endothelial cells are differentially modulated by monocyte subsets

EphA2 knockdown attenuates atherosclerotic lesion development in ApoE−/− mice

The inflammatory response of vascular endothelial cells plays important roles in the initiation and progression of atherosclerotic lesions. EphA2 receptor activation promotes the endothelial cell inflammatory response, and its expression is increased in the endothelial cell layer of atherosclerotic plaques. However, the association between EphA2 and atherosclerosis has not been determined. Eight-week-old male ApoE(-/-) mice were systemically infected with adenoassociated virus serotype 9 carrying a small hairpin RNA specifically targeting the EphA2 gene to knock down EphA2 expression in aortic endothelial cells. These mice were then fed a high-cholesterol diet for 12 weeks. Blood was collected for the measurement of plasma lipids. The aortas were harvested to evaluate the atherosclerotic lesion size, macrophage components, and expression of proinflammatory genes using Oil Red O staining, immunofluorescence staining, and molecular biology analysis. The lesions formed in the entire aorta and aortic sinus of the ApoE(-/-) mice with EphA2 knockdown were significantly smaller than those in the control mice (10.7%±3.1% versus 25.1%±4.2%; 0.51±0.02mm(2) versus 0.85±0.03mm(2); n=10; P<.05). Furthermore, the lesions in the ApoE(-/-) mice with EphA2 knockdown displayed reduced inflammation compared with the control mice, as reflected by the decreased macrophage infiltration (8.2%±2.9% versus 22.7%±4%; n=10; P<.05); decreased nuclear factor-κβ activation; and diminished expression of vascular cell adhesion molecule-1, E-selectin, and monocyte chemotactic protein-1 (all P<.05). Our data demonstrate that the EphA2 receptor silencing attenuates the extent and inflammation of atherosclerotic lesions in ApoE(-/-) mice. Thus, EphA2 knockdown in endothelial cells represents a novel therapeutic strategy for patients with atherosclerosis.

No signs of inflammation during knee surgery with ischemia: a study involving inhaled nitric oxide.

Nitric oxide donors and inhaled nitric oxide (iNO) may decrease ischemia/reperfusion injury as reported in animal and human models. We investigated whether the attenuation of reperfusion injury, seen by others, in patients undergoing knee arthroplasty could be reproduced when patients had spinal anesthesia. 45 consecutive patients were randomized into three groups (n = 15). Groups 1 and 3 were receiving iNO 80 ppm or placebo (nitrogen, N2) throughout the entire operation, and group 2 only received iNO in the beginning and at the end of the operation. Blood samples were collected before surgery, at the end of the surgery, and 2 hours postoperatively. Muscle biopsies were taken from quadriceps femoris muscle before and after ischemia. There were no increases in plasma levels of soluble adhesion molecules: ICAM, VCAM, P-selectin, E-selectin, or of HMGB1, in any of the groups. There were low numbers of CD68+ macrophages and of endothelial cells expression of ICAM, VCAM, and P-selectin in the muscle analyzed by immunohistochemistry, prior to and after ischemia. No signs of endothelial cell activation or inflammatory response neither systemically nor locally could be detected. The absence of inflammatory response questions this model of ischemia/reperfusion, but may also be related to the choice of anesthetic method EudraCTnr.

23: Synergistic effect of thrombin and bacterial LPS on human endometrial endothelial cell inflammatory cytokine response

23: Synergistic effect of thrombin and bacterial LPS on human endometrial endothelial cell inflammatory cytokine response

Combined effects of physiologically relevant disturbed wall shear stress and glycated albumin on endothelial cell functions associated with inflammation, thrombosis and cytoskeletal dynamics.

Diabetes mellitus is a major risk factor in the development of cardiovascular diseases (CVDs). The presence of advanced glycation end-products (AGEs) promotes CVDs by upregulating endothelial cell (EC) inflammatory and thrombotic responses, in a similar manner as disturbed shear stress. However, the combined effect of disturbed shear stress and AGEs on EC function has yet to be determined. Our goal was to evaluate these effects on EC responses. ECs were incubated with AGEs for 5days. ECs were then subjected to physiological or pathological shear stress. Cell metabolic activity, surface expression of intercellular adhesion molecule-1, thrombomodulin, connexin-43 and caveolin-1, and cytoskeleton organization were quantified. The results show that irreversibly glycated albumin and pathological shear stress increased EC metabolic activity, and upregulated and downregulated the EC surface expression of intercellular adhesion molecule-1 and thrombomodulin, respectively. Expression of connexin-43, caveolin-1 and cytoskeletal organization was independent of shear stress; however, the presence of irreversibly glycated AGEs markedly increased connexin-43, and decreased caveolin-1 expression and actin cytoskeletal connectivity. Our data suggest that irreversibly glycated albumin and disturbed shear stress could promote CVD pathogenesis by enhancing EC inflammatory and thrombotic responses, and through the deterioration of the cytoskeletal organization.

Inhibitory effect of Malvidin on TNF-α-induced inflammatory response in endothelial cells

Vascular inflammatory responses are key mediators of endothelial dysfunction that leads to various pathologies in many diseases including atherosclerosis and cancer. The purpose of the study was to investigate the effects and molecular mechanisms of Malvidin, a natural pigment with strong antioxidant activity, on regulating inflammatory response in endothelial cells. Our results showed that tumor necrosis factor-alpha (TNF-α) significantly increased the protein or mRNA levels of monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), whereas pretreatment with Malvidin inhibited TNF-α-induced increases of MCP-1, ICAM-1, and VCAM-1 production in a concentration-dependent manner. In addition, Malvidin could inhibit degradation of IκBα and the nuclear translocation of p65, which suggesting the anti-inflammation mechanism of Malvidin by the nuclear factor kappa B (NF-κB) pathway. These results indicate the potential role of Malvidin in preventing chronic inflammation in many diseases.

Adenosine Inhibits Pro-Inflammatory Thrombin Signaling In Endothelial Cells

Adenosine is an important regulatory metabolite which attenuates inflammation when it binds and activates the adenosine A2A receptor subtype on immune cells. However, the effect of adenosine on inflammatory responses in endothelial cells is mostly unknown. Thrombin as a known pro-inflammatory protease is involved in a variety of pathophysiological processes associated with inflammation in stimulated endothelial cells. The present study investigated the effect of adenosine on thrombin-mediated modulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs). Adenosine, in a concentration-dependent manner (1-100µM), inhibited the barrier disruptive effect of thrombin (20nM) on endothelial monolayer. The expression level of adenosine receptors, A1, A2A, A2B and A3 was examined in HUVECs and it was found that A2A and A2B are the highest expressing receptors among the four subtypes (A2B> A2A>A1>A3) in endothelial cells. Further studies revealed that the barrier protective effect of adenosine on thrombin-induced hyperpermeability in HUVECs was abrogated by the A2A receptor specific siRNA or the A2A receptor specific antagonist, ZM-241385, but not by siRNAs targeting the other adenosine receptor subtypes. To further determine the molecular mechanisms of the barrier protective effect of adenosine, its effect on thrombin-induced RhoA activation was assessed. Pretreatment of endothelial cells with adenosine prevented both thrombin-induced RhoA activation (Rho-GTP) and its membrane translocation as evidenced by cell fractionation and Pull-down assays, respectively. Moreover, preincubation of endothelial cells with adenosine down-regulated the expression of cell surface adhesion molecules (VCAM-1 and ICAM-1) and thrombin-mediated activation of nuclear factor-kappaB (NF-kB) pathway. Adenosine also inhibited secretion of the early chemokine, MCP-1, and the late-acting pro-inflammatory cytokine, HMGB-1, by thrombin-stimulated endothelial cells. Taken together, these results suggest that adenosine can inhibit pro-inflammatory thrombin signaling responses in endothelial cells by specifically activating the A2A receptor subtype, thereby protecting endothelium during the activation of coagulation and inflammatory pathways. Disclosures:No relevant conflicts of interest to declare.

NADPH oxidase and nitric oxide synthase-dependent superoxide production is increased in proliferative lupus nephritis.

Lupus nephritis (LN) is an immune complex-mediated glomerulonephritis. Proliferative LN (PLN, ISN/RPS classes III and IV)) often leads to renal injury or failure despite traditional induction and maintenance therapy. Successful targeted therapeutic development requires insight into mediators of inflammation in PLN. Superoxide (SO) and its metabolites are mediators of the innate immune response through their ability to mediate reduction-oxidation signaling. Endothelial nitric oxide synthase (eNOS) modulates inflammatory responses in endothelial cells. We hypothesized that markers of SO production would be increased in active PLN and that SO production would be dependent on the activity of select enzymes in the renal cortex. Patients with systemic lupus erythematosus were enrolled at the time of renal biopsy for active LN of all classes. Serum collected at baseline was analyzed by HPLC with electrochemical detection for markers of SO production (durable modifications of serum protein Tyr ultimately requiring SO as a substrate). Renal cortex from MRL/MpJ-FAS(lpr) (MRL/lpr) mice with and without functional eNOS was analyzed during active disease for superoxide (SO) production with and without inhibitors of SO-producing enzymes. Serum protein modifications indicative of total SO production were significantly higher in patients with PLN. These markers were increased in association with more active, inflammatory PLN. Mice lacking functional eNOS had 80% higher levels of renal cortical SO during active disease, and inhibitors of nitric oxide synthase and NADPH oxidase reduced these levels by 60% and 77%, respectively. These studies demonstrate that SO production is unique to active PLN in a NOS and NADPH oxidase-dependent fashion. These findings suggest the emulating or augmenting eNOS activity or inhibiting NADPH oxidase SO production may be targets of therapy in patients with PLN. The markers of SO production used in this study could rationally be used to select SO-modulating therapies and serve as pharmacodynamic indicators for dose titration.

The Small GTPase RhoB Regulates TNFα Signaling in Endothelial Cells

The inflammatory response of endothelial cells triggered by cytokines such as TNFα and IL1β plays a pivotal role in innate immunity. Upon pro-inflammatory cytokine stimulation, endothelial cells produce chemokines and cytokines that attract and activate leukocytes, and express high levels of leukocyte adhesion molecules. This process is mediated by intracellular signaling cascades triggered by activation of e.g. the TNFα receptor (TNFR) that lead to the activation of the NFκB transcription factor and of MAP kinases, which in turn activate inflammatory gene transcription. We found that the small GTPase RhoB was strongly and rapidly upregulated in primary human endothelial cells by TNFα, IL1β and LPS. We subsequently investigated the role of RhoB in the regulation of TNFR signaling in endothelial cells by silencing RhoB expression with siRNA. We provide evidence that the TNFα-induced activation of p38 MAP kinase is strongly dependent on RhoB, but not on RhoA, while JNK activation is regulated by both RhoB and RhoA. Consistent with the important role of p38 MAP kinase in inflammation, we demonstrate that loss of RhoB impairs TNFα-induced ICAM-1 expression and reduces cell production of IL6 and IL8. In addition, we show that RhoB silencing alters the intracellular traffic of TNFα after endocytosis. Since RhoB is a known regulator of the intracellular traffic of membrane receptors, our data suggest that RhoB controls TNFα signaling through the regulation of the TNFR traffic.

Aspirin attenuates angiotensin II-induced inflammation in bone marrow mesenchymal stem cells via the inhibition of ERK1/2 and NF-κB activation

Angiotensin II (Ang II) is a peptide hormone that plays a critical role in numerous physiological and pathophysiological processes. It is also commonly used as an inducer for the directional differentiation of bone marrow mesenchymal stem cells (bmMSCs). Previous studies demonstrated that Ang II induces inflammatory responses in endothelial cells, smooth muscle cells and fibroblasts. Aspirin is generally used as analgesic, antipyretic and occasionally anti-inflammatory medication. Whether aspirin suppresses inflammatory responses in bmMSCs has not been elucidated. In this study, we investigated the effect of aspirin on Ang II-induced inflammation in bmMSCs. Our results demonstrated that Ang II (10 nM-10 μM) increased the secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-6 from bmMSCs in a dose-dependent manner. This result was further confirmed by a reverse transcription-polymerase chain reaction (RT-PCR) assay, which demonstrated a dose-dependent increase in the mRNA expression of TNF-α, IL-6, IL-1β and monocyte chemotactic protein-1 (MCP-1) in bmMSCs following exposure to Ang II. Furthermore, it was also observed that Ang II increased the expression of phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-p65 in bmMSCs. The application of aspirin (0.1 mM) significantly inhibited the activation of ERK1/2 and NF-κB, the expression of TNF-α, IL-6, IL-1β and MCP-1 genes and the secretion of TNF-α and IL-6. Our findings indicated that aspirin may attenuate Ang II-induced inflammation in bmMSCs via the inhibition of ERK1/2 and NF-κB activation.

Bone morphogenetic protein (BMP-2) is involved in mononuclear cell function and their interaction with endothelial cells

Background: Mononuclear cells play a crucial role in vascular remodelling and cardiovascular disease. Monocytes are recruited to sites of collateral growth where they contribute to arteriogenesis. However, prolonged presence or increased accumulation of these cells can compromise tissue integrity and lead to atherosclerosis. Bone morphogenetic proteins (BMPs) are members of the Transforming growth factor (TGF)-β family of cytokines and play important roles in vascular remodelling and function. It was shown that BMP-2 is up-regulated in calcified atherosclerotic plaques. BMP-2 contributes to calcification and inflammation of the vascular wall. It is unknown whether these effects of BMP-2 are also due to its effects on monocyte function or/and on the monocyte-endothelial cell interactions. Methods: Human monocytes were isolated from peripheral blood through gradient centrifugation and subsequent negative immunological magnet isolation. Monocyte motility was analysed by the modified Boyden chamber migration assay. Protein phosphorylation was assessed by immunoblotting. For endothelial cell adhesion assays, mouse endothelial cells were stimulated with different ligands and mononuclear cell adhesion was analysed. The involvement of different signalling cascades was analysed using specific kinase inhibitors. Results: Here we show that stimulation of primary monocytes with BMP-2 results in monocyte polarization. BMP-2 stimulated monocyte chemotaxis in a dose-dependent manner, while addition of Noggin, a natural BMP-2/4/7 antagonist, hindered monocyte chemotaxis towards BMP-2. Additionally BMP2 induces mononuclear cell adhesion on fibronectin. BMPs signal into the cells by activating both Smad and non-Smad signalling pathways such as PI3K and p38 MAPK pathway. Our results suggest that both PI3K and p38 pathways are involved in BMP-2-induced monocyte migration. Moreover, BMP-2 stimulated monocyte adhesion on endothelial cells by inducing inflammatory responses in endothelial cells in a PI3K and p38 dependent manner. Conclusions: Our results reveal that BMP-2 is a novel stimulator of human monocyte function and suggest that BMP-2 may exert its pro-inflammatory effects and pro-calcification action in atherosclerosis by activating the endothelium and in addition by recruitment of monocytes.

MicroRNAs in Cardiometabolic Diseases

BACKGROUND: MicroRNAs (miRNAs) are ~22-nucleotide noncoding RNAs with critical functions in multiple physiological and pathological processes. An explosion of reports on the discovery and characterization of different miRNA species and their involvement in almost every aspect of cardiac biology and diseases has established an exciting new dimension in gene regulation networks for cardiac development and pathogenesis.CONTENT: Alterations in the metabolic control of lipid and glucose homeostasis predispose an individual to develop cardiometabolic diseases, such as type 2 diabetes mellitus and atherosclerosis. Work over the last years has suggested that miRNAs play an important role in regulating these physiological processes. Besides a cell-specific transcription factor profile, cell-specific miRNA-regulated gene expression is integral to cell fate and activation decisions. Thus, the cell types involved in atherosclerosis, vascular disease, and its myocardial sequelae may be differentially regulated by distinct miRNAs, thereby controlling highly complex processes, for example, smooth muscle cell phenotype and inflammatory responses of endothelial cells or macrophages. The recent advancements in using miRNAs as circulating biomarkers or therapeutic modalities, will hopefully be able to provide a strong basis for future research to further expand our insights into miRNA function in cardiovascular biology.SUMMARY: MiRNAs are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. They are potent modulators of diverse biological processes and pathologies. Recent findings demonstrated the importance of miRNAs in the vasculature and the orchestration of lipid metabolism and glucose homeostasis. MiRNA networks represent an additional layer of regulation for gene expression that absorbs perturbations and ensures the robustness of biological systems. A detailed understanding of the molecular and cellular mechanisms of miRNA-mediated effects on metabolism and vascular pathophysiology could pave the way for the development of novel diagnostic markers and therapeutic approaches.KEYWORDS: microRNA, lipid metabolism, glucose homeostasis, vascular endothelium, vascular smooth muscle, atherosclerosis

HMGB1 induces an inflammatory response in endothelial cells via the RAGE-dependent endoplasmic reticulum stress pathway

The high mobility group 1B protein (HMGB1) mediates chronic inflammatory responses in endothelial cells, which play a critical role in atherosclerosis. However, the underlying mechanism is unknown. The goal of our study was to identify the effects of HMGB1 on the RAGE-induced inflammatory response in endothelial cells and test the possible involvement of the endoplasmic reticulum stress pathway. Our results showed that incubation of endothelial cells with HMGB1 (0.01–1μg/ml) for 24h induced a dose-dependent activation of endoplasmic reticulum stress transducers, as assessed by PERK and IRE1 protein expression. Moreover, HMGB1 also promoted nuclear translocation of ATF6. HMGB1-mediated ICAM-1 and P-selectin production was dramatically suppressed by PERK siRNA or IRE1 siRNA. However, non-targeting siRNA had no such effects. HMGB1-induced increases in ICAM-1 and P-selectin expression were also inhibited by a specific eIF2α inhibitor (salubrinal) and a specific JNK inhibitor (SP600125). Importantly, a blocking antibody specifically targeted against RAGE (anti-RAGE antibody) decreased ICAM-1, P-selectin and endoplasmic reticulum stress molecule (PERK, eIF2α, IRE1 and JNK) protein expression levels. Collectively, these novel findings suggest that HMGB1 promotes an inflammatory response by inducing the expression of ICAM-1 and P-selectin via RAGE-mediated stimulation of the endoplasmic reticulum stress pathway.

Induction of ATF3 Gene Network by Triglyceride-Rich Lipoprotein Lipolysis Products Increases Vascular Apoptosis and Inflammation

Elevation of triglyceride-rich lipoproteins (TGRLs) contributes to the risk of atherosclerotic cardiovascular disease. Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated. We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot respectively. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of phosphorylated c-Jun N-terminal kinase (JNK), previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. Small interfering RNA (siRNA)-mediated inhibition of ATF3 blocked lipolysis products-induced transcription of E-selectin and interleukin-8, but not interleukin-6 or nuclear factor-κB. c-Jun, a downstream protein in the JNK pathway, was phosphorylated, whereas expression of nuclear factor-κB-dependent JunB was downregulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression, suggesting that JNK is upstream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild-type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3(-/-) mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared with fasting monocytes. This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.

Anti-inflammatory effect of ubiquinol-10 on young and senescent endothelial cells via miR-146a modulation

Clinical evidence demonstrates that ubiquinol-10, the reduced active form of coenzyme Q10 (CoQ10H2), improves endothelial function through its antioxidant and probably its anti-inflammatory properties. We previously reported that a biomarker combination including miR-146a, its target protein IL-1 receptor-associated kinase (IRAK-1), and released interleukin (IL)-6, here collectively designated as MIRAKIL, indicates senescence-associated secretory phenotype (SASP) acquisition by primary human umbilical vein endothelial cells (HUVECs). We explore the ability of short- and long-term CoQ10H2 supplementation to affect MIRAKIL in HUVECs, used as a model of vascular aging, during replicative senescence in the absence/presence of lipopolysaccharide (LPS), a proinflammatory stimulus. Senescent HUVECs had the same ability as young cells to internalize CoQ10 and exhibit an improved oxidative status. LPS-induced NF-κB activation diminished after CoQ10H2 pretreatment in both young and senescent cells. However, short-term CoQ10H2 supplementation attenuated LPS-induced MIRAKIL changes in young cells; in senescent cells CoQ10H2 supplementation significantly attenuated LPS-induced miR-146a and IRAK-1 modulation but failed to curb IL-6 release. Similar results were obtained with long-term CoQ10H2 incubation. These findings provide new insights into the molecular mechanisms by which CoQ10H2 stems endothelial cell inflammatory responses and delays SASP acquisition. These phenomena may play a role in preventing the endothelial dysfunction associated with major age-related diseases.