LOX-1 Regulation in Anti-atherosclerosis of Active Compounds of Herbal Medicine: Current Knowledge and the New Insight.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) have recently been identified to be closely related to the occurrence and development of atherosclerosis (AS). A growing body of evidence has suggested Chinese medicine takes unique advantages in preventing and treating AS. In this review, the related research progress of AS and LOX-1 has been summarized. And the anti-AS effects of 10 active components of herbal medicine through LOX-1 regulation have been further reviewed. As a potential biomarker and target for intervention in AS, LOX-1 targeted therapy might provide a promising and novel approach to atherosclerotic prevention and treatment.

LOX-1 Regulation in Anti-atherosclerosis of Active Compounds of Herbal Medicine: Current Knowledge and the New Insight / 中国结合医学杂志

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) have recently been identified to be closely related to the occurrence and development of atherosclerosis (AS). A growing body of evidence has suggested Chinese medicine takes unique advantages in preventing and treating AS. In this review, the related research progress of AS and LOX-1 has been summarized. And the anti-AS effects of 10 active components of herbal medicine through LOX-1 regulation have been further reviewed. As a potential biomarker and target for intervention in AS, LOX-1 targeted therapy might provide a promising and novel approach to atherosclerotic prevention and treatment.

C-Type Lectin-Like Receptors: Head or Tail in Cell Death Immunity.

C-type lectin-like receptors (CLRs) represent a family of transmembrane pattern recognition receptors, expressed primarily by myeloid cells. They recognize not only pathogen moieties for host defense, but also modified self-antigens such as damage-associated molecular patterns released from dead cells. Upon ligation, CLR signaling leads to the production of inflammatory mediators to shape amplitude, duration and outcome of the immune response. Thus, following excessive injury, dysregulation of these receptors leads to the development of inflammatory diseases. Herein, we will focus on four CLRs of the "Dectin family," shown to decode the immunogenicity of cell death. CLEC9A on dendritic cells links F-actin exposed by dying cells to favor cross-presentation of dead-cell associated antigens to CD8+ T cells. Nevertheless, CLEC9A exerts also feedback mechanisms to temper neutrophil recruitment and prevent additional tissue damage. MINCLE expressed by macrophages binds nuclear SAP130 released by necrotic cells to potentiate pro-inflammatory responses. However, the consequent inflammation can exacerbate pathogenesis of inflammatory diseases. Moreover, in a tumor microenvironment, MINCLE induces macrophage-induced immune suppression and cancer progression. Similarly, triggering of LOX-1 by oxidized LDL, amplifies pro-inflammatory response but promotes tumor immune escape and metastasis. Finally, CLEC12A that recognizes monosodium urate crystals formed during cell death, inhibits activating signals to prevent detrimental inflammation. Interestingly, CLEC12A also sustains type-I IFN response to finely tune immune responses in case of viral-induced collateral damage. Therefore, CLRs acting in concert as sensors of injury, could be used in a targeted way to treat numerous diseases such as allergies, obesity, tumors, and autoimmunity.

LOX-1: A potential driver of cardiovascular risk in SLE patients.

Traditional cardiovascular disease (CVD) risk factors, such as hypertension, dyslipidemia and diabetes do not explain the increased CVD burden in systemic lupus erythematosus (SLE). The oxidized-LDL receptor, LOX-1, is an inflammation-induced receptor implicated in atherosclerotic plaque formation in acute coronary syndrome, and here we evaluated its role in SLE-associated CVD. SLE patients have increased sLOX-1 levels which were associated with elevated proinflammatory HDL, oxLDL and hsCRP. Interestingly, increased sLOX-1 levels were associated with patients with early disease onset, low disease activity, increased IL-8, and normal complement and hematological measures. LOX-1 was increased on patient-derived monocytes and low-density granulocytes, and activation with oxLDL and immune-complexes increased membrane LOX-1, TACE activity, sLOX-1 release, proinflammatory cytokine production by monocytes, and triggered the formation of neutrophil extracellular traps which can promote vascular injury. In conclusion, perturbations in the lipid content in SLE patients' blood activate LOX-1 and promote inflammatory responses. Increased sLOX-1 levels may be an indicator of high CVD risk, and blockade of LOX-1 may provide a therapeutic opportunity for ameliorating atherosclerosis in SLE patients.

Galectin-3 aggravates ox-LDL-induced endothelial dysfunction through LOX-1 mediated signaling pathway.

Galectin-3, a biomarker linking oxidative stress and inflammation, participates in different mechanisms related to atherothrombosis, such as inflammation, proliferation, or macrophage chemotaxis. Accumulating evidence indicates that galectin-3 may also promote atherogenesis through inducing endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1), a receptor for oxLDL uptake, contributes to oxLDL-induced endothelial dysfunction. Whether galectin-3 induces endothelial dysfunction through modulation of LOX-1-mediated signaling remains unclear. In the present study, we explored the mechanisms underlying galectin-3 enhanced cytotoxicity of oxLDL in human umbilical vein endothelial cells (HUVECs) and the role of LOX-1. Incubation of HUVECs with galectin-3 increased the expression of LOX-1 in RNA and protein levels. In addition, the expression of LOX-1 induced by oxLDL was promoted by galectin-3. However, pretreatment of LOX-1 antibody reduced LOX-1 mRNA expression level in cells with oxLDL plus galectin-3 incubation. Compared to cells treated with oxLDL alone, reactive oxygen species (ROS) generation via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and subsequent activation of p38 mitogen-activated protein kinases followed by nuclear factor kappa B (NF-κB) activation and related inflammatory responses including adhesion molecule expression, adhesiveness of monocytic cells, and IL-8 release were also aggravated in cells treated with galectin-3 combined with oxLDL. Compared to cells treated with galectin-3 plus oxLDL group. We found that LOX-1 antibody mitigated NADPH oxidase activity, p-38 up-regulation, NF-κB activation, and proinflammatory responses in cells treated with galectin-3 combined with oxLDL. We conclude that galectin-3 enhances endothelial LOX-1 expression and propose a new mechanism by which galectin-3 may promote endothelial dysfunction by inducing inflammation via LOX-1/ROS/p38/NF-κB-mediated signaling pathway.

Deleterious role of endothelial lectin-like oxidized low-density lipoprotein receptor-1 in ischaemia/reperfusion cerebral injury.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is implicated in cardiovascular disease by modulating apoptosis and oxidative stress. We hypothesized that LOX-1 may be involved in pathophysiology of stroke by mediating ischaemia/reperfusion (I/R)-dependent cell death. Transient middle cerebral artery occlusion (tMCAO) was performed in wild-type (WT) mice, endothelial-specific LOX-1 transgenic mice (eLOX-1TG) and WT animals treated with LOX-1 silencing RNA (siRNA). In WT mice exposed to tMCAO, LOX-1 expression and function were increased in the MCA. Compared to WT animals, eLOX-1TG mice displayed increased stroke volumes and worsened outcome after I/R. Conversely, LOX-1-silencing decreased both stroke volume and neurological impairment. Similarly, in HBMVECs, hypoxia/reoxygenation increased LOX-1 expression, while LOX-1 overexpressing cells showed increased death following hypoxia reoxygenation. Increased caspase-3 activation was observed following LOX-1 overexpression both in vivo and in vitro, thus representing a likely mediator. Finally, monocytes from ischaemic stroke patients exhibited increased LOX-1 expression which also correlated with disease severity. Our data unequivocally demonstrate a key role for LOX-1 in determining outcome following I/R brain damage. Our findings could be corroborated in human brain endothelial cells and monocytes from patients, underscoring their translational relevance and suggesting siRNA-mediated LOX-1 knockdown as a novel therapeutic strategy for stroke patients.

Toll-like receptor 2 activation primes and upregulates osteoclastogenesis via lox-1.

BACKGROUND: Lectin-like oxidized low-density-lipoprotein receptor 1 (Lox-1) is the receptor for oxidized low-density lipoprotein (oxLDL), a mediator in dyslipidemia. Toll-like receptor (TLR)-2 and - 4 are receptors of lipopolysaccharide (LPS) from Porphyromonas gingivalis, a major pathogen of chronic periodontitis. Although some reports have demonstrated that periodontitis has an adverse effect on dyslipidemia, little is clear that the mechanism is explained the effects of dyslipidemia on osteoclastogenesis. We have hypothesized that osteoclast oxLDL has directly effect on osteoclasts (OCs), and therefore alveolar bone loss on periodontitis may be increased by dyslipidemia. The present study aimed to elucidate the effect of Lox-1 on osteoclastogenesis associated with TLRs in vitro. METHODS: Mouse bone marrow cells (BMCs) were stimulated with macrophage colony-stimulating factor into bone marrow macrophages (BMMs). The cells were also stimulated with synthetic ligands for TLR2 (Pam3CSK4) or TLR4 (Lipid A), with or without receptor activator of nuclear factor kappa-B ligand (RANKL), and assessed for osteoclastogenesis by tartrate-resistant acid phosphatase (TRAP) staining, immunostaining, western blotting, flow activated cell sorting (FACS) analysis, real-time polymerase chain reaction (PCR), and reverse transcription PCR. RESULTS: Lox-1 expression was significantly upregulated by Pam3CSK4 and Lipid A in BMCs (p < 0.05), but not in BMMs. FACS analysis identified that Pam3CSK4 upregulated RANK and Lox-1 expression in BMCs. TRAP-positive cells were not increased by stimulation with Pam3CSK4 alone, but were increased by stimulation with combination combined Pam3CSK and oxLDL. Expression of both Lox-1 and myeloid differentiation factor 88 (MyD88), an essential adaptor protein in the TLR signaling pathway, were suppressed by inhibitors of TLR2, TLR4 and mitogen-activated protein kinase (MAPK). CONCLUSIONS: This study supports that osteoclastogenesis is promoted under the coexistence of oxLDL by TLR2-induced upregulation of Lox-1 in BMCs. This indicates that periodontitis could worsen with progression of dyslipidemia.

Role of sLOX-1 in intracranial artery stenosis and in predicting long-term prognosis of acute ischemic stroke.

Objective: The role of sLOX-1 in acute ischemic stroke still remains unclear. This study aims to demonstrate the value of sLOX-1 in evaluating degrees of intracranial artery stenosis and to predict prognosis in stroke. Methods: Two hundred and seventy-two patients were included in this study and basic data were collected within 72 hr on admission. We assessed the association between sLOX-1 levels and stroke conditions in one-year duration. After adjusting for potential confounders, regression analyses were performed. Results: We found that sLOX-1 levels were increased significantly in severe patients compared to the mild stroke group (p = .011). After adjusting confounders, sLOX-1 was associated with a poor functional outcome in patients with an adjusted OR of 2. 946 (95% CI, 1.788-4.856, p < .001). There was also positive correlation between sLOX-1 levels and the degrees of intracranial artery stenosis in the different groups (p = .029). Conclusions: Our study demonstrated that sLOX-1 levels could be used to evaluate the severity of stroke and the degrees of intracranial artery stenosis. Furthermore, sLOX-1 could be exploited to predict the long-term functional outcome of stroke.

Oxidized low-density lipoprotein stimulates epithelial sodium channels in endothelial cells of mouse thoracic aorta.

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) is expressed in endothelial cells and acts as a negative modulator of vasodilatation. Oxidized LDL (ox-LDL) is a key pathological factor in endothelial dysfunction. In the present study we examined the role of ENaC in ox-LDL-induced endothelial dysfunction and its associated signal transduction pathway. EXPERIMENTAL APPROACH: Patch clamp techniques combined with pharmacological approaches were used to examine ENaC activity in the endothelial cells of a split-open mouse thoracic aorta. Western blot analysis was used to determine ENaC expression in the aorta. The aorta relaxation was measured using a wire myograph assay. KEY RESULTS: Ox-LDL, but not LDL, significantly increased ENaC activity in the endothelial cells attached to split-open thoracic aortas, and the increase was inhibited by a lectin-like ox-LDL receptor-1 (LOX-1) antagonist (κ-carrageenan), an NADPH oxidase inhibitor (apocynin), and a scavenger of ROS (TEMPOL). Sodium nitroprusside, an NO donor, diminished the ox-LDL-mediated activation of ENaC, and this effect was abolished by inhibiting soluble guanylate cyclase (sGC) and PKG. Ox-LDL reduced the endothelium-dependent vasodilatation of the aorta pectoralis induced by ACh, and this reduction was partially restored by blocking ENaC. CONCLUSION AND IMPLICATIONS: Ox-LDL stimulates ENaC in endothelial cells through LOX-1 receptor-mediated activation of NADPH oxidase and accumulation of intracellular ROS. Since the stimulation of ENaC can be reversed by elevating NO, we suggest that both inhibition of ENaC and an elevation of NO may protect the endothelium from ox-LDL-induced dysfunction. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Syncytiotrophoblast extracellular vesicles impair rat uterine vascular function via the lectin-like oxidized LDL receptor-1.

Syncytiotrophoblast extracellular vesicles (STBEVs) are placenta derived particles that are released into the maternal circulation during pregnancy. Abnormal levels of STBEVs have been proposed to affect maternal vascular function. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a multi-ligand scavenger receptor. Increased LOX-1 expression and activation has been proposed to contribute to endothelial dysfunction. As LOX-1 has various ligands, we hypothesized that, being essentially packages of lipoproteins, STBEVs are able to activate the LOX-1 receptor thereby impairing vascular function via the production of superoxide and decreased nitric oxide bioavailability. Uterine arteries were obtained in late gestation from Sprague-Dawley rats and incubated for 24h with or without human STBEVs (derived from a normal pregnant placenta) in the absence or presence of a LOX-1 blocking antibody. Vascular function was assessed using wire myography. Endothelium-dependent maximal vasodilation to methylcholine was impaired by STBEVs (MCh Emax: 57.7±5.9% in STBEV-incubated arteries vs. 77.8±2.9% in controls, p<0.05). This was prevented by co-incubation of STBEV-incubated arteries with LOX-1 blocking antibodies (MCh Emax: 78.8±4.3%, p<0.05). Pre-incubation of the vessels with a nitric oxide synthase inhibitor (L-NAME) demonstrated that the STBEV-induced impairment in vasodilation was due to decreased nitric oxide contribution (ΔAUC 12.2±11.7 in STBEV-arteries vs. 86.5±20 in controls, p<0.05), which was abolished by LOX-1 blocking antibody (ΔAUC 98.9±17, p<0.05). In STBEV-incubated vessels, LOX-1 inhibition resulted in an increased endothelial nitric oxide synthase expression (p<0.05), to a level similar to control vessels. The oxidant scavenger, superoxide dismutase, did not improve this impairment, nor were vascular superoxide levels altered. Our data support an important role for STBEVs in impairment of vascular function via activation of LOX-1 and reduced nitric oxide mediated vasodilation. Moreover, we postulate that the LOX-1 pathway could be a potential therapeutic target in pathologies associated with vascular dysfunction during pregnancy.

Statins Attenuate Activation of the NLRP3 Inflammasome by Oxidized LDL or TNFα in Vascular Endothelial Cells through a PXR-Dependent Mechanism.

Excessive activation of the NLRP3 inflammasome is implicated in cardiovascular diseases. Statins exert an anti-inflammatory effect independent of their cholesterol-lowering effect. This study investigated the potential role of statins in the activation of the NLRP3 inflammasome in endothelial cells (ECs). Western blotting and quantitative reverse-transcription polymerase chain reaction showed that oxidized low-density lipoprotein (ox-LDL) or tumor necrosis factor α (TNFα) activated the NLRP3 inflammasome in ECs. Simvastatin or mevastatin significantly suppressed the effects of ox-LDL or TNFα Promoter reporter assays and small interfering RNA knockdown revealed that statins inhibit ox-LDL-mediated NLRP3 inflammasome activation via the pregnane X receptor (PXR). In addition, PXR agonists (rifampicin and SR12813) or overexpression of a constitutively active PXR markedly suppressed the NLRP3 inflammasome activation. Conversely, PXR knockdown abrogated the suppressive effect of rifampicin on NLRP3 inflammasome activation. Knockdown of lectin-like ox-LDL receptor or overexpression of IκBα-attenuated ox-LDL- or TNFα-triggered activation of the NLRP3 inflammasome. Chromatin immunoprecipitation assays indicated that mevastatin inhibited nuclear factor-κB binding to the promoter regions of the human NLRP3 gene. Collectively, these results demonstrate that the statin activation of PXR inhibits the activation of NLRP3 inflammasome in response to atherogenic stimuli such as ox-LDL and TNFα in ECs, providing a new mechanism for the cardiovascular benefit of statins.

Carbamylated Low-Density Lipoproteins Induce a Prothrombotic State Via LOX-1: Impact on Arterial Thrombus Formation In Vivo.

BACKGROUND: Carbamylation alters low-density lipoprotein (LDL) structure and is thought to promote vascular inflammation and dysfunction in patients with chronic kidney disease (CKD). OBJECTIVES: This study sought to determine whether carbamylated LDL (cLDL) exerts prothrombotic effects in vascular cells and platelets and whether cLDL enhances arterial thrombus formation in vivo. METHODS: LDL was isolated from healthy subjects or patients with CKD by sequential ultracentrifugation. Ex vivo carbamylation of LDL from healthy subjects was induced with potassium cyanate. Arterial thrombus formation was analyzed in a murine carotid artery photochemical injury model. Protein expression and mRNA levels were analyzed by Western blotting, flow cytometry, and real-time PCR. Platelet aggregation was measured by impedance aggregometry. RESULTS: Intravenous administration of cLDL in mice accelerated arterial thrombus formation compared to treatment with native LDL (nLDL) or vehicle. Tissue lysates of mouse carotid arteries revealed that cLDL induced the expression of TF, PAI-1, and LOX-1 mRNA in vascular cells. In human aortic smooth muscle and endothelial cells, cLDL induced TF and PAI-1 expression. In contrast, nLDL had no effect on either cell type. While nLDL and cLDL had no aggregatory effect on resting platelets, cLDL enhanced platelet aggregation in response to different agonists. This effect was mediated by mitogen-activated protein kinase p38 phosphorylation and LOX-1 translocation to the surface. LDL isolated from patients with CKD mimicked the prothrombotic effects of cLDL on vascular cells, platelets, and thrombus formation in vivo. CONCLUSIONS: We found that cLDL induces prothrombotic effects in vascular cells and platelets by activation of the LOX-1 receptor and enhances thrombus formation in vivo. This observation reveals a new mechanism underlying the increased incidence of acute thrombotic events observed in patients with CKD and may lead to the development of new lipid-targeting therapies in this population.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy.

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications.

Deletion of LOX-1 Protects against Heart Failure Induced by Doxorubicin.

Oxidative stress is one of the major factors in doxorubicin (DOX)-induced cardiomyopathy. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1) plays an important role to regulate cardiac remodeling and oxidative stress after ischemia-reperfusion. Therefore, we examined whether or not LOX-1 contributes to the pathogenesis of DOX-induced cardiomyopathy. Cardiomyopathy was induced by a single intraperitoneal injection of DOX into wild-type (WT) mice and LOX-1 knockout (KO) mice. Echocardiography and catheter-based hemodynamic assessment apparently revealed preserved left ventricular (LV) fractional shortening (FS) and cavity size of LOX-1 KO mice compared with those of WT mice after DOX administration. Less production of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1ß) was observed in LOX-1 KO mice than WT mice after DOX administration. Western blotting analysis also showed lower activation of nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) in LOX-1 KO mice treated with DOX than WT mice treated with DOX. In fact, NF-κB-dependent gene expressions of LOX-1 and vascular cell adhesion molecule-1 (VCAM-1) were suppressed in LOX-1 KO mice treated with DOX compared with WT mice treated with DOX. Therefore, histological analyses showed attenuation of leukocyte infiltration and cardiac fibrosis in LOX-1 KO mice compared with WT mice. Meanwhile, extracellular signal-regulated kinase MAPK (ERK) inactivation and decreased expression of sarcomeric proteins and related transcription factor GATA-4 in WT mice treated with DOX administration were not seen in LOX-1 KO mice treated with DOX administration and WT and LOX-1 KO mice treated with vehicle. Decreased expression of sarcometric proteins resulted in smaller diameters of cardiomyocytes in WT mice than in LOX-1 KO mice after DOX treatment. The expression of LOX-1 in cardiomyocytes was much more abundant than that in endothelial cells, fibroblasts and inflammatory cells. Endothelial cells, fibroblasts and inflammatory cells treated with DOX showed no elevated LOX-1 expression compared with those treated with vehicle. However, cardiomyocytes treated with DOX showed much more expression of LOX-1 than those treated with vehicle. Immunohistochemistry study also showed that LOX-1 expression was strongly elevated in cardiomyocytes in the heart tissue of mice treated with DOX in vivo. We conclude that LOX-1 in cardiomyocytes plays the most important roles in the pathology of DOX-induced cardiomyopathy. LOX-1 deletion altered the LOX-1-related signaling pathway, which led to improvements in cardiac function, myocardial inflammation, fibrosis and degenerative changes after DOX treatment.

MiR-590-5p Inhibits Oxidized- LDL Induced Angiogenesis by Targeting LOX-1.

Oxidized low-density lipoprotein (ox-LDL) is, at least in part, responsible for angiogenesis in atherosclerotic regions. This effect of ox-LDL has been shown to be mediated through a specific receptor LOX-1. Here we describe the effect of miR-590-5p on ox-LDL-mediated angiogenesis in in vitro and in vivo settings. Human umbilical vein endothelial cells (HUVECs) were transfected with miR-590-5p mimic or inhibitor followed by treatment with ox-LDL. In other experiments, Marigel plugs were inserted in the mice subcutaneous space. Both in vitro and in vivo studies showed that miR-590-5p mimic (100 nM) inhibited the ox-LDL-mediated angiogenesis (capillary tube formation, cell proliferation and migration as well as pro-angiogenic signals- ROS, MAPKs, pro-inflammatory cytokines and adhesion-related proteins). Of note, miR-590-5p inhibitor (200 nM) had the opposite effects. The inhibitory effect of miR-590-5p on angiogenesis was mediated by inhibition of LOX-1 at translational level. The inhibition of LOX-1 by miR-590-5p was confirmed by luciferase assay. In conclusion, we show that MiR-590-5p inhibits angiogenesis by targeting LOX-1 and suppressing redox-sensitive signals.

The Role of LOX-1 in Innate Immunity to Aspergillus fumigatus in Corneal Epithelial Cells.

PURPOSE: To determine the role of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) in corneal epithelial cells exposed to fungus. METHODS: Thirteen corneas with fungal keratitis composed the experimental group. Ten healthy donor corneas were the control group. Corneal epithelium was scraped and LOX-1 mRNA tested. Immunostaining was used to detect LOX-1 protein. Human corneal epithelial cells (HCECs) were treated with 75% ethanol-killed Aspergillus fumigatus. LOX-1 was detected, and CXCL1, TNF-α, IL-6, and dectin-1 levels were detected with or without neutralization of LOX-1. Rat fungal keratitis was established and LOX-1 was detected by PCR, immunostaining, and Western blot. Phosphorylated-p38MAPK (p-p38MAPK) protein; CXCL1, TNF-α, and IL-6 mRNA levels; and myeloperoxidase (MPO) were tested before and after LOX-1 neutralization. RESULTS: LOX-1 was expressed in corneal epithelium. In vitro cellular experiment showed that LOX-1 was detected in normal HCECs, and LOX-1 mRNA increased after stimulation of A. fumigatus and peaked at 8 hours; LOX-1 protein expression increased after stimulation at 24 and 48 hours. Neutralization of LOX-1 decreased expression of CXCL1, TNF-α, but did not change IL-6 or dectin-1 expression. Aspergillus fumigatus keratitis developed in rats activated p38MAPK and elevated the expression of CXCL1, TNF-α, and IL-6 through LOX-1. LOX-1 neutralization reduced MPO levels. CONCLUSIONS: LOX-1 expressed in normal corneal epithelium and HCECs and A. fumigatus elevated the expression of CXCL1 and TNF-α through LOX-1. Rat A. fumigatus keratitis activated p38MAPK and elevated the expression of CXCL1, TNF-α, and IL-6 through LOX-1.

Hemodynamic shear stress modulates endothelial cell autophagy: Role of LOX-1.

OBJECTIVES: Shear stress, autophagy and LOX-1 are important players in atherogenesis. Direct impact of shear stress on autophagy development in endothelial cells and role of LOX-1 therein are undelineated. METHODS AND RESULTS: A parallel-plate flow chamber was used to vary shear stress (3 to 30 dyn/cm(2)), and determine autophagy in endothelial cells. We observed that low shear stress (3 dyn/cm(2)) enhanced autophagy (expression of LC3-II) 2-3 fold, and increasing shear stress (15 to 30 dyn/cm(2)) resulted in a gradual decline. Autophagy increased when cells were treated with an inflammatory stimulus lipopolysaccharide (LPS). LOX-1 expression paralleled autophagy development. The in vitro observations were confirmed in the in vivo setting by studying autophagy (LC3-II and Beclin-1) and LOX-1 expression in wild-type mice given LPS. Expression of both autophagy and LOX-1 was most pronounced in aorto-iliac bifurcation region where shear stress is lower compared with aortic arch, thoracic aorta and iliac artery. To define the role of LOX-1 in the development of autophagy, we studied LOX-1 knockout mice. These mice despite LPS administration exhibited less autophagy (vs. wild-type mice). Role of LOX-1 in the regulation of autophagy was further established with LOX-1 inhibition (siRNA transfection and use of antibody) or overexpression (cDNA transfection), which showed that LOX-1 knockdown reduced while LOX-1 overexpression enhanced LC3-II expression in endothelial cells. CONCLUSIONS: These observations suggest that low shear stress is a powerful regulator on autophagy, particularly in state of inflammation, and LOX-1 plays an important role in shear stress induced autophagy.

LOX-1 in atherosclerotic disease.

Oxidized low-density lipoprotein (LDL) exhibits various biological activities and accumulates in atheromas. LOX-1 (lectin-like oxidized LDL receptor) is the receptor that mediates oxidized LDL activity in vascular endothelial cells. Activation of LOX-1 results in oxidized LDL-induced endothelial dysfunction and hyperlipidemia-induced vascular lipid deposition. We hypothesized that LOX-1 is a candidate risk factor beyond LDL cholesterol (LDLC) and developed a novel assay to quantify LOX-1 ligand containing apoB (LAB). In men from the United States, serum LAB showed a significant positive association with carotid intima-media thickness, independent of LDLC. LAB and the LOX index (obtained by multiplying LAB by sLOX-1) were significantly associated with the incidence of coronary artery disease and ischemic stroke after adjusting for confounding factors, including non-HDL cholesterol. sLOX-1 is thought to be a better biomarker for early diagnosis of acute coronary syndrome than traditional biomarkers, including troponin T. LAB was associated with various atherosclerotic risk factors such as smoking, obesity, diabetes, diastolic hypertension, hypertriglyceridemia, and metabolic syndrome. Measurement of the soluble form of LOX-1 (sLOX-1) and LAB seems to be useful for evaluating the state and risk of atherosclerosis and atherosclerosis-related diseases. Further prospective studies using large populations and randomized clinical trials on sLOX-1, LAB, and the LOX index are needed.

C-reactive protein specifically enhances platelet-activating factor-induced inflammatory activity in vivo.

Platelet-activating factor (PAF) is a potent lipid mediator that is implicated in numerous inflammatory diseases. C-reactive protein (CRP) is an acute-phase plasma protein that increases rapidly and dramatically in response to inflammation. In this study, we investigated the effect of the interaction between CRP and PAF on inflammatory responses in vivo. From binding analysis using a time-resolved fluorometric assay, CRP bound to PAF and its precursor/metabolite lyso-PAF in a concentration-dependent manner. In addition, CRP bound to several phospholipids containing lysophosphatidylcholine, which bears structural resemblance to PAF and lyso-PAF, sphingosylphosphorylcholine, and lysophosphatidylethanolamine more readily than to lysophosphatidic acid and lysophosphatidylserine. In in vivo experiments using a rat model of hind paw oedema, CRP increased PAF-induced rat paw oedema in a dose-dependent manner, without causing the oedema itself, but it did not increase histamine and serotonin-induced paw oedema. Furthermore, the receptor for CRP, lectin-like oxidized low-density lipoprotein receptor 1 was not involved in the increase in PAF-induced inflammatory responses caused by CRP. These results indicate that CRP can specifically enhance PAF-induced inflammatory activity through binding to PAF and lyso-PAF. Therefore, CRP may accelerate the pathogenesis of numerous inflammatory diseases caused by PAF.

LOX-1 - dependent mitochondrial DNA damage and NLRP3 activation during systemic inflammation in mice.

BACKGROUND: Lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) is known to be involved in many pathophysiological events, such as inflammation. METHODS: To clarify the role of LOX-1 in mtDNA damage and NLRP3 inflammasome activation, we studied wild-type (WT) and LOX-1 knockout (KO) mice given thioglycollate, an inflammatory stimulus. RESULTS: We observed intense inflammatory response (CD45 and CD68 expression) and mtDNA damage in spleen and kidneys of WT mice given thioglycollate. The abrogation of LOX-1 (use of LOX-1 knockout mice) reduced the inflammatory response as well as mtDNA damage (P<0.05 vs. WT mice). We also observed that mice with LOX-1 deletion had markedly reduced expression of caspase-1 (P10 and P20 subunits) as well as cleaved IL-1ß and IL-18. These mice also had much less mtDNA damage and only limited NLRP3 inflammasome expression. CONCLUSIONS: These in vivo observations indicate that LOX-1 plays a key role in mtDNA damage which then leads to NLRP3 inflammasome activation during inflammation.