ERK1/2-PPARγ pathway is involved in Chlamydia pneumonia-induced human umbilical vein endothelial cell apoptosis through increased LOX-1 expression.

Chlamydia pneumonia (C.pn) is a common respiratory pathogen that is involved in human cardiovascular diseases and promotes the development of atherosclerosis in hyperlipidemic animal models. C.pn reportedly up-regulated lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells. Recently, the anti-atherosclerotic activity of peroxisome proliferator-activated receptor γ (PPARγ) has been documented. In the present study, we investigated the effect of C.pn on LOX-1 expression in human umbilical vein endothelial cells (HUVECs) and identified the involvement of the PPARγ signaling pathway therein. The results showed that C.pn increased the expression of LOX-1 in HUVECs in a dose- and time-dependent manner. C.pn-induced up-regulation of LOX-1 was mediated by ERK1/2, whereas p38 MAPK and JNK had no effect on this process. C.pn induced apoptosis, inhibited cell proliferation, and decreased the expression PPARγ in HUVECs. Additionally, LOX-1 activity and cell injury caused by C.pn through activation of ERK1/2 was completely inhibited by rosiglitazone, a PPARγ agonist. In conclusion, we inferred that activation of PPARγ in HUVECs suppressed C.pn-induced LOX-1 expression and cell damage by inhibiting ERK1/2 signaling.

Interleukin-1ß Promotes Ox-LDL Uptake by Human Glomerular Mesangial Cells via LOX-1.

The aim of this study was to determine whether interleukin-1ß (IL-1ß) promotes oxidised low-density lipoprotein (Ox-LDL) uptake by human glomerular mesangial cells (HMCs) and its effect on the expression of lectin-like Ox-LDL receptor 1 (LOX-1) and to identify pathways through which IL-1ß affects lipid uptake. Confocal laser scanning microscopy and flow cytometry were used to observe the effect of IL-1ß on lipid uptake by HMCs and the pathway by which IL-1ß may mediate lipid uptake. Real-time polymerase chain reaction (PCR) and western blotting were used to evaluate the effect of IL-1ß on LOX-1 expression. Confocal laser scanning microscopy and flow cytometry revealed that IL-1ß promoted uptake of fluorescent Dil-labelled Ox-LDL(Dil-Ox-LDL) by HMCs and the enhanced uptake of Dil-Ox-LDL was partially inhibited by an anti-LOX-1 antibody evaluated by flow cytometry. Further, IL-1ß promoted LOX-1 mRNA and protein expression of HMCs in a dose- and time-dependent manner. Thus, Ox-LDL is ingested by HMCs under basic conditions. Inflammatory cytokine IL-1ß promotes Ox-LDL uptake by HMCs. Furthermore, IL-1ß promotes the mRNA and protein expression of LOX-1, a specific receptor of Ox-LDL, suggesting that the enhancement of Ox-LDL uptake may be mediated by LOX-1 pathway. Anti-LOX-1 therapy may be a promising option for treatment of glomerulosclerosis.

Gly[14]-humanin inhibits ox-LDL uptake and stimulates cholesterol efflux in macrophage-derived foam cells.

Foam cell formation, which is caused by imbalanced cholesterol influx and efflux by macrophages, plays a vital role in the occurrence and development of atherosclerosis. Humanin (HN), a mitochondria-derived peptide, can prevent the production of reactive oxygen species and death of human aortic endothelial cells exposed to oxidized low-density lipoprotein (ox-LDL) and has a protective effect on patients with in early atherosclerosis. However, the effects of HN on the regulation of cholesterol metabolism in RAW 264.7 macrophages are still unknown. This study was designed to investigate the role of [Gly14]-humanin (HNG) in lipid uptake and cholesterol efflux in RAW 264.7 macrophages. Flow cytometry and live cell imaging results showed that HNG reduced Dil-ox-LDL accumulation in the RAW 264.7 macrophages. A similar result was obtained for lipid accumulation by measuring cellular cholesterol content. Western blot analysis showed that ox-LDL treatment upregulated not only the protein expression of CD36 and LOX-1, which mediate ox-LDL endocytosis, but also ATP-binding cassette (ABC) transporter A1 and ABCG1, which mediate ox-LDL exflux. HNG pretreatment inhibited the upregulation of CD36 and LOX-1 levels, prompting the upregulation of ABCA1 and ABCG1 levels induced by ox-LDL. Therefore we concluded that HNG could inhibit ox-LDL-induced macrophage-derived foam cell formation, which occurs because of a decrease in lipid uptake and an increase in cholesterol efflux from macrophage cells.

Klotho ameliorates oxidized low density lipoprotein (ox-LDL)-induced oxidative stress via regulating LOX-1 and PI3K/Akt/eNOS pathways.

BACKGROUND: Atherosclerosis is a common cardiovascular disease that causes myocardial infarction, heart failure, and stroke. Increased oxidized low density lipoprotein (ox-LDL) in the sub-endothelium is the characteristic origin of atherogenesis. Klotho, an anti-aging protein, has been reported to protect against atherosclerosis and ameliorate endothelial dysfunction in vivo. The aim of this study is to investigatethe anti-oxidative activity of Klothoin ox-LDL-treated human umbilical vein endothelial cells (HUVECs). METHODS: After pre-treatment with 200 pMKlotho for 1 h, HUVECs were stimulated with 50 µg/ml ox-LDL for 24 h. Reactive oxygen species (ROS) and superoxide dismutase (SOD) levels were analyzed in the cells. Nitric oxide (NO) concertation was measured in the medium supernatant. Related proteins or genes were detected with Western blot or real time PCR, respectively, in the cell lysates. RESULTS: Initially, oxidative damage in HUVECs was established by adding 50 µg/mL ox-LDL, which resulted in decreased cellular viability, SOD/Cu/Zn-SOD and endothelial NO synthase (eNOS) expression and NO production, as well as increased malondialdehyde (MDA) levels, ROS production, inducible NO synthase (iNOS), phosphatidyl inositol-3 kinase (PI3K), protein kinase B (Akt), gp91 phox, and lectin-like ox-LDL receptor (LOX-1) expression in HUVECs. Pre-incubation with recombinant Klotho (200 pM) significantly prevented all of these alterations. These results suggest that Klotho can attenuate ox-LDL-induced oxidative stress in HUVECs through upregulating oxidative scavengers (SOD and NO) viaactivating the PI3K/Akt/eNOS pathway and depressing LOX-1expression. CONCLUSIONS: These results suggest that Klotho has a potential therapeutic effect on attenuating endothelial dysfunction and ameliorating atherosclerosis.

Liraglutide ameliorates oxidized LDL-induced endothelial dysfunction by GLP-1R-dependent downregulation of LOX-1-mediated oxidative stress and inflammation.

OBJECTIVE: To investigate the effects of glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide on endothelial dysfunction in LDL receptor-deficient (LDLR-KO) mice and ox-LDL-challenged human umbilical vein endothelial cells (HUVECs) and its possible mechanism. METHODS: LDLR-KO mice were randomly treated with normal saline, liraglutide, or liraglutide plus a GLP-1R antagonist exendin-9 for four weeks. In parallel, HUVECs were cultured with ox-LDL alone or combined with liraglutide, in the presence or absence of lectin-like ox-LDL receptor-1(LOX-1) overexpression or GLP-1R knockdown. Endothelial-dependent relaxation and LOX-1 protein expression of thoracic aorta, circulating levels of oxidative and inflammatory markers in mice, and cell survival, reactive oxygen species production, and expression of adhesion molecules and signal regulators in ox-LDL cultured endothelial cells were measured. RESULTS: liraglutide effectively enhanced acetylcholine-induced vasodilation, reduced LOX-1 expression in aortas, and decreased circulatory oxidative and inflammatory levels in LDLR-KO mice, which were abolished by cotreatment with exendin-9. HUVECs exposed to ox-LDL exhibited reduced cell viability, increased reactive oxygen species production and apoptosis, and elevated protein expression of ICAM-1, VCAM-1, LOX-1, NOX4, and NF-κB, which were markedly ameliorated by liraglutide treatment. The protective effects of liraglutide against ox-LDL-induced cell injury were abrogated in HUVECs overexpressing LOX-1 or silencing GLP-1R. CONCLUSIONS: Liraglutide improved oxidized LDL-induced endothelial dysfunction via GLP-1R-dependent downregulation of LOX-1-mediated oxidative stress and inflammation.

Regulation of LOX-1 on adhesion molecules and neutrophil infiltration in mouse Aspergillus fumigatus keratitis.

AIM: To determine whether lectin-like ox-LDL receptor (LOX-1) regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus (A. fumigatus) keratitis of C57BL/6 mice. METHODS: C57BL/6 mice were pretreated with a neutralizing antibody to LOX-1 (5 µg/5 µL) or control nonspecific IgG (5 µg/5 µL), LOX-1 inhibitor Poly-I (2 µg/5 µL) or PBS by subconjunctival injection. Fungal keratitis (FK) mouse models of C57BL/6 mice were established by scraping corneal central epithelium, smearing A. fumigatus on the corneal surface and covering the eye with contact lenses. The corneal response to infection was assessed via clinical score. The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin were tested in control and infected corneas by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of ICAM-1 were evaluated by immunofluorescence (IF) and Western blot. Neutrophils were extracted from the abdominal cavity of C57BL/6 mice followed by pretreatment using antibody to LOX-1 (10 µg/mL) or control nonspecific IgG (10 µg/mL), the Poly-I (4 µg/mL) or PBS. The cells were then stimulated with A. fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1 (LFA-1) using RT-PCR and Western blot. IF and myeloperoxidase (MPO) assays were used to assess neutrophil infiltration in mice corneas. RESULTS: Pretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS (both P<0.01). And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1, VCAM-1, P-selectin, E-selectin and LFA-1 expression compared with control groups (all P<0.01). Furthermore, pretreated with LOX-1 antibody or Poly-I, the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups (all P<0.05). Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays (both P<0.01). CONCLUSION: Inhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A. fumigatus infected corneas of C57BL/6 mice.

The role of LOX-1 on innate immunity against Aspergillus keratitis in mice.

AIM: To explore the effects of lectin-like ox-LDL receptor (LOX-1) on innate immunity against Aspergillus fumigatus (A. fumigatus ) in mice cornea. METHODS: The mRNA levels of LOX-1 were tested in normal and A. fumigatus infected corneas of C57BL/6 and BALB/c mice. The expression of LOX-1, pro-inflammatory cytokines TNF-α, CXCL1 and IL-6, anti-inflammatory cytokines IL-10, and matrix metalloproteinase 9 (MMP9) were tested with treatment with LOX-1 neutralizing antibody or control IgG in A. fumigatus infected corneas of C57BL/6. Macrophages and neutrophils were extracted from susceptible C57BL/6 mice, and pretreated with LOX-1 neutralizing antibody or IgG, then stimulated with A. fumigatus. The mRNA levels of LOX-1, TNF-α, CXCL1, IL-6, IL-10 and MMP9 were evaluated by polymerase chain reaction. RESULTS: The expression of LOX-1 was significantly increased in C57BL/6 mice corneas after A. fumigatus infection compared with BABL/c mice. After treatment with LOX-1 neutralizing antibody, the expression of LOX-1, TNF-α, CXCL1, IL-6, MMP9 and IL-10 in C57BL/6 corneas were significantly decreased compared with treatment with control IgG; the expression of LOX-1, CXCL1, IL-6 and IL-10 were significantly decreased in macrophages, while TNF-α and MMP9 expressions had no change; LOX-1, TNF-α, CXCL1, IL-6, MMP9 and IL-10 expressions were significantly decreased in neutrophils. CONCLUSION: The expression of LOX-1 can affect the expression of pro-inflammatory and anti-inflammatory cytokines in fungal infected corneas, macrophages and neutrophils of C57BL/6. LOX-1 inhibition rebalances the inflammatory response of fungal keratitis in mice.

Wnt signaling in liver fibrosis: progress, challenges and potential directions.

Liver fibrosis is a common wound-healing response to chronic liver injuries, including alcoholic or drug toxicity, persistent viral infection, and genetic factors. Myofibroblastic transdifferentiation (MTD) is the pivotal event during liver fibrogenesis, and research in the past few years has identified key mediators and molecular mechanisms responsible for MTD of hepatic stellate cells (HSCs). HSCs are undifferentiated cells which play an important role in liver regeneration. Recent evidence demonstrates that HSCs derive from mesoderm and at least in part via septum transversum and mesothelium, and HSCs express markers for different cell types which derive from multipotent mesenchymal progenitors. There is a regulatory commonality between differentiation of adipocytes and that of HSC, and the shift from adipogenic to myogenic or neuronal phenotype characterizes HSC MTD. Central of this shift is a loss of expression of the master adipogenic regulator peroxisome proliferator activated receptor γ (PPARγ). Restored expression of PPARγ and/or other adipogenic transcription genes can reverse myofibroblastic HSCs to differentiated cells. Vertebrate Wnt and Drosophila wingless are homologous genes, and their translated proteins have been shown to participate in the regulation of cell proliferation, cell polarity, cell differentiation, and other biological roles. More recently, Wnt signaling is implicated in human fibrosing diseases, such as pulmonary fibrosis, renal fibrosis, and liver fibrosis. Blocking the canonical Wnt signal pathway with the co-receptor antagonist Dickkopf-1 (DKK1) abrogates these epigenetic repressions and restores the gene PPARγ expression and HSC differentiation. The identified morphogen mediated epigenetic regulation of PPARγ and HSC differentiation also serves as novel therapeutic targets for liver fibrosis and liver regeneration. In conclusion, the Wnt signaling promotes liver fibrosis by enhancing HSC activation and survival, and we herein discuss what we currently know and what we expect will come in this field in the next future.