MicroRNA-328 ameliorates oxidized low-density lipoprotein-induced endothelial cells injury through targeting HMGB1 in atherosclerosis

Atherosclerosis has been recognized as a chronic inflammatory disease, which can harden the vessel wall and narrow the arteries. MicroRNAs exhibit crucial roles in various diseases including atherosclerosis. However, so far, the role of miR-328 in atherosclerosis remains barely explored. Therefore, our study concentrated on the potential role of miR-328 in vascular endothelial cell injury during atherosclerosis. In our current study, we observed that oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptosis and inhibited cell viability dose-dependently and time-dependently. In addition, indicated dosage of ox-LDL obviously triggered HUVECs inflammation and oxidative stress process. Then, it was found that miR-328 in HUVECs was reduced by ox-LDL. HUVECs apoptosis was greatly repressed and cell survival was significantly upregulated by overexpression of miR-328. Furthermore, mimics of miR-328 rescued cell inflammation and oxidative stress process induced by ox-LDL. Oppositely, inhibitors of miR-328 strongly promoted ox-LDL-induced endothelial cells injury in HUVECs. By using bioinformatics analysis, high-mobility group box-1 (HMGB1) was predicted as a downstream target of miR-328. HMGB1 has been reported to be involved in atherosclerosis development. The correlation between miR-328 and HMGB1 was validated in our current study. Taken these together, it was implied that miR-328 ameliorated ox-LDL-induced endothelial cells injury through targeting HMGB1 in atherosclerosis.     

EETs Attenuate Ox-LDL-Induced LTB4 Production and Activity by Inhibiting p38 MAPK Phosphorylation and 5-LO/BLT1 Receptor Expression in Rat Pulmonary Arterial Endothelial Cells

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B₄ (LTB₄), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB₄ production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB₄ production and activity. We found that treatment with ox-LDL increased the production of LTB₄ and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB₄ receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB₄ receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB₄ production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.     

Low Shear Stress Induced HMGB1 Translocation and Release via PECAM-1/PARP-1 Pathway to Induce Inflammation Response

Low shear stress (LSS) plays a critical role in the site predilection of atherosclerosis through activation of cellular mechanosensors, such as platelet endothelial cell adhesion molecule 1 (PECAM-1). Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that regulates the expression of various inflammatory cytokines. The nuclear enzyme high mobility group box 1 (HMGB1) can induce inflammation response by binding to toll-like receptor 4 (TLR4). In the present study, we aimed to investigate the role and mechanism of HMGB1 in LSS induced inflammation in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated by undisturbed shear stress (USS, 1 Pa) and LSS (0.4 Pa) in our experiments. Gene expression was inhibited by small interfering RNA (siRNA). ICAM-1 expression was regulated by LSS in a time dependent manner. LSS can induce HMGB1 translocation from nucleus to cytoplasm and release. Compared with the USS, LSS could increase the protein expression of PECAM-1 and PARP-1 as well as the secretion of TNF-α and IL-1β. LSS induced the translocation of HMGB1 from nucleus to cytoplasm. Inhibition of HGMB1 reduced LSS-induced inflammatory response. Inhibition of PARP-1 suppressed inflammatory response through inhibiting TLR4 expression and HMGB1 translocation. PECAM-1 inhibition reduced LSS-induced ICAM-1 expression, TNF-α and IL-1β secretion, and monocytes adhesion. LSS can induce inflammatory response via PECAM-1/PARP-1/HMGB1 pathway. PARP-1 plays a fundamental role in HMGB1 translocation and TLR4 expression. Inhibition of PARP-1 may shed light on the treatment of HMGB1 involved inflammation during atherosclerosis.     

A fermented bean flour extract downregulates LOX-1, CHOP and ICAM-1 in HMEC-1 stimulated by ox-LDL

This study focused on an extract from fermented flour from the Lady Joy variety of the common bean Phaseolus vulgaris. The extract, Lady Joy lysate (Lys LJ), is enriched in antioxidant compounds during the fermentation. We assessed it for its protective effect on endothelial cells treated with oxidized-LDL (ox-LDL). The oxidative stress was determined by measuring the contents of thiobarbituric acid-reactive substances and reactive oxygen metabolites. ICAM-1, ET-1 and IL-6 concentrations were assessed using ELISA. LOX-1 and CHOP expression were analyzed using both quantitative RT-PCR and ELISA or western blotting. Ox-LDL treatment induced significant oxidative stress, which was strongly reduced by pre-treatment with the extract. The ox-LDL exposure significantly enhanced ICAM-1, IL-6 and ET-1 levels over basal levels. Lys LJ pre-treatment exerted an inhibitory effect on ox-LDL-induced endothelial activation with ICAM-1 levels comparable to those for the untreated cells. IL-6 and ET-1 production, although reduced, was still significantly higher than for the control. Both LOX-1 and CHOP expression were upregulated after ox-LDL exposure, but this effect was significantly decreased after Lys LJ pre-treatment. Lys LJ alone did not alter the ICAM-1, IL-6 and ET-1 concentrations or CHOP expression, but it did significantly lower the LOX-1 protein level. Our data suggest that Lys LJ is an effective antioxidant that is able to inhibit the oxidation process, but that it is only marginally active against inflammation and ET-1 production in HMEC-1 exposed to ox-LDL.     

PCSK9 siRNA inhibits HUVEC apoptosis induced by ox-LDL via Bcl/Bax–caspase9–caspase3 pathway

This paper investigated the effects of ox-LDL on PCSK9, and the molecular mechanisms of PCSK9 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cells (HUVECs), to clarify the role of PCSK9 in atherosclerogenesis. HUVECs were incubated with ox-LDL for 24?h. The apoptosis was observed by Hoechst 33258 staining. The expression of PCSK9, LOX-1 mRNAs and proteins was detected by RT-PCR, western blot, respectively. The PCSK9 siRNAs labeled with fluorescence were transfected into HUVECs by Lipofectamine 2000. After transfection for 24?h, cells were treated with ox-LDL for 24?h, HUVECs apoptosis transfected siRNA was detected by Hoechst 33258 staining and flow cytometer. The expression of Bcl-2, Bax, caspase3, 8, 9 was detected by western blot. The activity of caspase3, 9 was detected by kits. Our results showed that apoptosis of HUVECs and the expressions of PCSK9 and LOX-1 were upregulated secondary to induction by ox-LDL in a concentration-dependent manner. However, ox-LDL-induced HUVEC apoptosis and PCSK9 expression, but not LOX-1 expression, were significantly reduced by PCSK9 siRNA. These results demonstrate a linkage between HUVEC apoptosis and PCSK9 expression. Furthermore, we detected the possible pathway involved in apoptotic regulation by PCSK9 siRNA; our results showed that the expression of Bcl-2 decreased, whereas that of Bax increased. In addition, ox-LDL enhanced the activity of caspase9 and then caspase3. Pretreatment of HUVECs with PCSK9 siRNA blocked these effects of ox-LDL. These findings suggest that ox-LDL-induced HUVECs apoptosis could be inhibited by PCSK9 siRNA, in which Bcl/Bax-caspase9-caspase3 pathway maybe was involved through reducing the Bcl-2/Bax ratio and inhibited the activation of both caspase9 and 3.     

Probenecid Protects Against Transient Focal Cerebral Ischemic Injury by Inhibiting HMGB1 Release and Attenuating AQP4 Expression in Mice

Stroke results in inflammation, brain edema, and neuronal death. However, effective neuroprotectants are not available. Recent studies have shown that high mobility group box-1 (HMGB1), a proinflammatory cytokine, contributes to ischemic brain injury. Aquaporin 4 (AQP4), a water channel protein, is considered to play a pivotal role in ischemia-induced brain edema. More recently, studies have shown that pannexin 1 channels are involved in cerebral ischemic injury and the cellular inflammatory response. Here, we examined whether the pannexin 1 channel inhibitor probenecid could reduce focal ischemic brain injury by inhibiting cerebral inflammation and edema. Transient focal ischemia was induced in C57BL/6J mice by middle cerebral artery occlusion (MCAO) for 1 h. Infarct volume, neurological score and cerebral water content were evaluated 48 h after MCAO. Immunostaining, western blot analysis and ELISA were used to assess the effects of probenecid on the cellular inflammatory response, HMGB1 release and AQP4 expression. Administration of probenecid reduced infarct size, decreased cerebral water content, inhibited neuronal death, and reduced inflammation in the brain 48 h after stroke. In addition, HMGB1 release from neurons was significantly diminished and serum HMGB1 levels were substantially reduced following probenecid treatment. Moreover, AQP4 protein expression was downregulated in the cortical penumbra following post-stroke treatment with probenecid. These results suggest that probenecid, a powerful pannexin 1 channel inhibitor, protects against ischemic brain injury by inhibiting cerebral inflammation and edema.     

LncRNA OIP5-AS1 facilitates ox-LDL-induced endothelial cell injury through the miR-98-5p/HMGB1 axis

Cerebrovascular diseases have a high mortality and disability rate in developed countries. Endothelial cell injury is the main cause of atherosclerosis and cerebrovascular disease. Long non-coding RNA (lncRNA) has been proved to participate in the progression of endothelial cell. Our study aimed to develop the function of lncRNA opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in oxidative low-density lipoprotein (ox-LDL)-induced endothelial cell injury. The expression of OIP5-AS1, miR-98-5p and High-mobility group protein box-1 (HMGB1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry were used to detect the cell proliferation and apoptosis. The levels of cyclinD1, Bcl-2 Associated X Protein (Bax), Cleaved-caspase-3, Toll like receptors 4 (TLR4), phosphorylation of p65 (p-P65), phosphorylation of nuclear factor-kappa B inhibitor α (p-IκB-α) and HMGB1 were measured by Western blot. The concentrations of Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and Tumor necrosis factor-α (TNF-α) were detected by Enzyme-linked immunosorbent assay (ELISA). The production of Reactive oxygen species (ROS), Superoxide Dismutase (SOD) and malondialdehyde (MDA) was detected by the corresponding kit. The targets of OIP5-AS and miR-98-5p were predicted by starBase 3.0 and TargetScan and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The expression of OIP5-AS1 was upregulated, while miR-98-5p was downregulated in ox-LDL-induced human umbilical vein endothelial cells (HUVECs). Functionally, knockdown of OIP5-AS1 induced proliferation and inhibited apoptosis, inflammatory injury and oxidative stress injury in ox-LDL-induced HUVEC cells. Interestingly, miR-98-5p was a target of OIP5-AS1 and miR-98-5p inhibition abolished the effects of OIP5-AS1 downregulation on ox-LDL-induced HUVECs injury. More importantly, miR-98-5p directly targeted HMGB1, and OIP5-AS1 regulated the expression of HMGB1 by sponging miR-98-5p. Finally, OIP5-AS1 regulated the TLR4/nuclear factor-kappa B (NF-κB) signaling pathway through miR-98-5p/HMGB1 axis. LncRNA OIP5-AS1 accelerates ox-LDL-induced endothelial cell injury through regulating HMGB1 mediated by miR-98-5p via the TLR4/NF-κB signaling pathway.

     

Intercellular adhesion molecule-1 and vascular endothelial growth factor expression kinetics in macrophage-derived foam cells

Macrophage-derived foam cells seem to play an important role during inflammatory response of atherosclerosis, in which the overexpression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF) are associated with the early and later pathological changes in foam cell formation. In this study, we investigated the expression kinetics of ICAM-1 and VEGF in macrophage-derived foam cells. The foam cell model was established through incubating the human monocyte line (U937 cells) with oxidized-low density lipoprotein (ox-LDL). Up-regulated expressions of ICAM-1 and VEGF were analyzed in protein and mRNA levels in U937 foam cells by flow cytometry, ELISA, and Northern blot. Kinetic studies showed the deferent kinds of expression curves in dose response and time course. The expression dose-kinetics demonstrated that the ICAM-1 showed the peak expression induced by ox-LDL 50 mg/L, while VEGF levels increased in a dose-dependent manner with the maximum level induced by ox-LDL 200 mg/L. Time-kinetic studies revealed that the ICAM-1 levels showed the peak expression in 12 h while VEGF expression increased in a time-dependent manner with the maximum level in 48 h. These results proved that both ICAM-1 and VEGF expressions were enhanced in the macrophage-derived foam cells, but ICAM-1 expression increased earlier than the up-regulation of VEGF; low dose of ox-LDL mainly up regulated ICAM-1 expression, while high dose mainly increased the VEGF expression.     

Galectin-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin β1-RhoA-JNK signaling activation

Oxidized low-density lipoprotein (Ox-LDL)-induced endothelial cell injury plays a crucial role in the pathogenesis of atherosclerosis (AS). Plasma galectin-3 (Gal-3) is elevated inside and drives diverse systemic inflammatory disorders, including cardiovascular diseases. However, the exact role of Gal-3 in ox-LDL-mediated endothelial injury remains unclear. This study explores the effects of Gal-3 on ox-LDL-induced endothelial dysfunction and the underlying molecular mechanisms. In this study, Gal-3, integrin β1, and GTP-RhoA in the blood and plaques of AS patients were examined by ELISA and western blot respectively. Their levels were found to be obviously upregulated compared with non-AS control group. CCK8 assay and flow cytometry analysis showed that Gal-3 significantly decreased cell viability and promoted apoptosis in ox-LDL-treated human umbilical vascular endothelial cells (HUVECs). The upregulation of integrinβ1, GTP-RhoA, p-JNK, p-p65, p-IKKα, and p-IKKβ induced by ox-LDL was further enhanced by treatment with Gal-3. Pretreatment with Gal-3 increased expression of inflammatory factors (interleukin [IL]-6, IL-8, and IL-1β), chemokines(CXCL-1 and CCL-2) and adhesion molecules (VCAM-1 and ICAM-1). Furthermore, the promotional effects of Gal-3 on NF-κB activation and inflammatory factors in ox-LDL-treated HUVECs were reversed by the treatments with integrinβ1-siRNA or the JNK inhibitor. We also found that integrinβ1-siRNA decreased the protein expression of GTP-RhoA and p-JNK, while RhoA inhibitor partially reduced the upregulated expression of p-JNK induced by Gal-3. In conclusion, our finding suggests that Gal-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin β1-RhoA-JNK signaling activation.     

ClC-2 inhibition prevents macrophage foam cell formation by suppressing Nlrp3 inflammasome activation

ABSTRACT

Macrophage foam cell formation and inflammation are a pathological hallmark of atherosclerosis. ClC-2 has been implicated in various pathological processes, including inflammation and lipid metabolic disorder. However, the functional role of ClC-2 in macrophage foam cell formation and inflammation is unclear. Here, we found that ClC-2 was dominantly expressed in macrophages of atherosclerotic plaque and increased in atherogenesis. Knockdown of ClC-2 inhibited ox-LDL -induced lipid uptake and deposition in macrophages. The increase in CD36 expression and the decrease in ABCA1 expression induced by ox-LDL were alleviated by ClC-2 downregulation. Further, ClC-2 lacking limited the ox-LDL-induced secretion of inflammatory cytokines and chemokine, and suppressed Nlrp3 inflammasome activation. Restoration of Nlrp3 expression reversed the effect of ClC-2 downregulation on macrophage lipid accumulation and inflammation. Collectively, our study demonstrates that ClC-2 knockdown ameliorates ox-LDL-induced macrophage foam cell formation and inflammation by inhibiting Nlrp3 inflammasome activation.     

香鱼 HMGB1克隆、序列分析及表达特征

高迁移率族蛋白B1（High mobility group box protein 1, HMGB1）属于晚期细胞因子,与炎症反应相关。为研究香鱼HMGB1在鳗利斯顿氏菌感染引起的炎症反应中的作用,采用随机测序从肝脏组织cDNA文库中获得HMGB1基因,全长1233 bp,编码一个由204个氨基酸组成的23.3 kDa的蛋白。分析表明,该蛋白与胡瓜鱼HMGB1蛋白同源性最高。健康香鱼HMGB1基因在肌肉、脑和肝脏组织中表达量最高,而在鳃和肠中几乎不表达。 qRT-PCR表明,鳗利斯顿氏菌感染过程中,香鱼肌肉、脑和肝脏组织中HMGB1基因表达显著上调,分别在72、48 h达到峰值。原核表达香鱼HMGB1重组蛋白并制备抗血清,Western blot显示,抗血清能与目的蛋白起特异性反应,能用于研究HMGB1蛋白表达变化,进一步揭示HMGB1基因与鳗利斯顿氏菌感染引起的炎症晚期反应相关性。     

Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-gamma

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in osteoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1.     

Cryptotanshinone inhibits oxidized LDL-induced adhesion molecule expression via ROS dependent NF-κB pathways

Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, play important roles in the initial stage of atherosclerosis. Cryptotanshinone (CPT), a natural compound isolated from Salvia miltiorrhiza Bunge, exhibits anti-atherosclerotic activity although the underlying mechanisms remain elusive. In this study, the protective effect of CPT against oxidized low-density lipoprotein (ox-LDL)-induced adhesion molecule expression was investigated in human umbilical vein endothelial cells. Ox-LDL significantly induced ICAM-1, VCAM-1, and E-selectin expression at the mRNA and protein levels but reduced eNOS phosphorylation and NO generation, which were reversed by CPT pretreatment. Sodium nitroprusside, a NO donor, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, and BAY117082, a NF-κB inhibitor, inhibited ox-LDL-induced ICAM-1, VCAM-1, and E-selectin expression. Ox-LDL-induced ROS production was significantly inhibited by CPT and NAC. Furthermore, ox-LDL activated the NF-κB signaling pathway by inducing phosphorylation of IKKβ and IκBα, promoting the interaction of IKKβ and IκBα, and increasing p65 nuclear translocation, which were significantly inhibited by CPT. In addition, CPT, NAC, and BAY117082 inhibited ox-LDL-induced membrane expression of ICAM-1, VCAM-1, E-selectin, and endothelial–monocyte adhesion and restored eNOS phosphorylation and NO generation. Results suggested that CPT inhibited ox-LDL-induced adhesion molecule expression by decreasing ROS and inhibiting the NF-κB pathways, which provides new insight into the anti-atherosclerotic mechanism of CPT.     

Rac3, but not Rac1, promotes ox-LDL induced endothelial dysfunction by downregulating autophagy

The endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis, which can lead to oxidative stress and inflammation. The role of autophagy in the process of atherosclerosis has drawn increasing attention. The human umbilical vein endothelial cells (HUVECs), whose Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rac3 was knockdown, were used to detect whether the possible molecular mechanisms of Rac1 and Rac3 for anti-inflammatory in endothelial cells was effected by downregulation of autophagy. The HUVECs were incubated with ox-LDL. The inflammatory factors and autophagy proteins were evaluated to ascertain and compare the effect of Rac1 and Rac3 on autophagy. Then, 3-methyladenine (3-MA) as an inhibiter of autophagy was used to detect whether the effect of Rac1 and Rac3 was related to autophagy. ox-LDL-induced cell dysfunction in HUVECs was determined by testing the formation of foam cells, the expression of nuclear factor (NF)-κB and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 and NF-κB p65 and other inflammatory factors, the release of reactive oxygen species by oxidative stress and the dysfunction of the cytomembrane. And ApoE^−/− mice on a high-fat diet were used as an animal model to detect the effect of Rac1 and Rac3 in vivo. The results showed that when Rac1 and Rac3 were decreased in HUVECs, the cell dysfunction caused by ox-LDL was inhibited. If 3-MA was used to inhibit autophagy in Rac1 and Rac3 knockdown cells, the injury induced by ox-LDL on the cells was recovered. These results indicated that the effect of Rac1 and Rac3 was combined with ox-LDL, which was related to inhibition of autophagy. The effect of Rac3 was more significant than that of Rac1.     

Modulation of diabetes-related liver injury by the HMGB1/TLR4 inflammatory pathway

Chronic inflammation plays an essential role in the development of diabetic complications. Understanding the molecular mechanisms that support inflammation is a prerequisite for the design of novel anti-inflammatory therapies. These would take into consideration circulating levels of cytokines and damage-associated molecular patterns (DAMPs) that include the high mobility group box 1 (HMGB1) protein which, in part, promotes the inflammatory response through TLR4 signaling. The liver, as the source of circulating cytokines and acute-phase proteins, contributes to the control of systemic inflammation. We previously found that liver injury in streptozotocin-induced diabetic rats correlated with the level of oxidative stress, increased expression of HMGB1, and with the activation of TLR4-mediated cell death pathways. In the present work, we examined the effects of ethyl pyruvate (EP), an inhibitor of HMGB1 release/expression, on the modulation of activation of the HMGB1/TLR4 inflammatory cascade in diabetic liver. We observed that increased expression of inflammatory markers, TNF-α, IL-6, and haptoglobin in diabetic liver was associated with increased HMGB1/TLR4 interaction, activation of MAPK (p38, ERK, JNK)/NF-κB p65 and JAK1/STAT3 signaling pathways, and with decreased expression of Nrf2-regulated antioxidative enzymes. The reduction in HMGB1 expression as the result of EP administration reduced the pro-inflammatory activity of HMGB1 and exerted a protective effect on diabetic liver, which was observed as improved liver histology and antioxidant and inflammatory statuses. Our results suggest that prevention of HMGB1 release and blockage of the HMGB/TLR4 axis represents a potentially effective therapeutic strategy aimed at ameliorating diabetes-induced inflammation and ensuing liver injury.     

Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations

Introduction

Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity.

Methods

The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies.

Results

Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4.

Conclusion

Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation.     

Expression of ICAM-1 on intact cartilage and isolated chondrocytes

Summary A major factor in cellular cytotoxicity is the interaction between LFA-1 on leukocytes and ICAM-1 on targets. Because several inflammatory cartilage diseases are characterized by the presence of leukocyte infiltrates, the expression of ICAM-1 on human cartilage, cultured chondrocytes, and transplanted cartilage was investigated using monoclonal antibodies. Frozen tissue sections, chondrocytes in suspension, as well as total cellular mRNA were prepared from human cartilage samples. ICAM-1 expression was studied with two different monoclonal antibodies directed against ICAM-1 by immunohistochemical APAAP-staining and additional flow cytometric analyses. The expression of ICAM-1-mRNA in cartilage tissue was analyzed using the northern blot hybridization technique. Furthermore, chondrocytes were treated in culture with interleukin-1 (IL-1) and gamma-interferon (gamma-IFN). ICAM-1 expression after culture was quantified using flow cytometric analysis. We could detect ICAM-1 mRNA in cartilage tissue, however, the immunostaining of tissue sections using monoclonal antibodies did not give clear positive reactions. Isolated chondrocytes showed strongly positive staining patterns in comparison with adequate negative controls as assessed by flow cytometry. A dose-dependent increase of the expression of ICAM-1 on chondrocytes was observed when stimulated with IL-1 and gamma-IFN. Finally, two of the three studied transplanted autologous cartilage samples with advanced resorption showed the presence of ICAM-1 molecules as assessed by immunohistochemistry. This expression of ICAM-1 suggests that the molecule plays a role in severe cartilage inflammatory processes, where tissue damage leads to the exposure of chondrocyte surfaces.     

Toll-like Receptor 4 (TLR4) Antagonist Eritoran Tetrasodium Attenuates Liver Ischemia and Reperfusion Injury through Inhibition of High-Mobility Group Box Protein B1 (HMGB1) Signaling

Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1–TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepatocytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury.