Characterization of low-density granulocytes in COVID-19.

Severe COVID-19 is characterized by extensive pulmonary complications, to which host immune responses are believed to play a role. As the major arm of innate immunity, neutrophils are one of the first cells recruited to the site of infection where their excessive activation can contribute to lung pathology. Low-density granulocytes (LDGs) are circulating neutrophils, whose numbers increase in some autoimmune diseases and cancer, but are poorly characterized in acute viral infections. Using flow cytometry, we detected a significant increase of LDGs in the blood of acute COVID-19 patients, compared to healthy controls. Based on their surface marker expression, COVID-19-related LDGs exhibit four different populations, which display distinctive stages of granulocytic development and most likely reflect emergency myelopoiesis. Moreover, COVID-19 LDGs show a link with an elevated recruitment and activation of neutrophils. Functional assays demonstrated the immunosuppressive capacities of these cells, which might contribute to impaired lymphocyte responses during acute disease. Taken together, our data confirms a significant granulocyte activation during COVID-19 and suggests that granulocytes of lower density play a role in disease progression.

Low level of stromal lectin-like oxidized LDL receptor 1 and CD8+ cytotoxic T-lymphocytes indicate poor prognosis of colorectal cancer.

BACKGROUND: Lectin-like oxidized LDL receptor-1 (LOX-1) has been identified as a new marker for functional myeloid-derived suppressor cells (MDSCs) that exhibit an immunosuppressive phenotype in the tumor microenvironment (TME). However, the role of LOX-1+ cells in the TME of colorectal cancer (CRC) remains unknown. AIM: This study aimed to determine the expression and significance of LOX-1 in the TME of clinical CRC specimens. METHODS AND RESULTS: We performed immunohistochemical and genetic analyses of LOX-1, CD8, KRAS, and BRAF in 128 resected CRC specimens and determined the expression of IFN-γ and IL-10 using real-time reverse transcription-polymerase chain reaction. We analyzed the correlation between LOX-1, TME factors, gene alteration, clinicopathological factors, and disease prognosis. The co-expression pattern of LOX-1, hematopoietic markers, and a fibroblast marker was evaluated using multiplex immunofluorescence staining. Low stromal LOX-1 expression and low intratumoral CD8+ cytotoxic T-lymphocyte (CTL) status correlated with poor prognosis. Moreover, stromal LOX-1-low/CD8+ CTL-low status was the most important independent prognostic factor of poor overall survival. Most of the LOX-1+ stromal cells were positive for CD163+ , indicating they were CD163+ M2 macrophages. CONCLUSIONS: The MDSC marker, LOX-1, was mainly expressed by M2 macrophages in CRC tissues. LOX-1+ macrophages and CD8+ CTLs may serve as useful biomarkers for predicting the prognosis of CRC.

Role of Plasma Soluble Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 and microRNA-98 in Severity and Risk of Coronary Artery Disease.

BACKGROUND: Coronary artery diseases are the most important cause of premature death, and these diseases are predominantly related to atherosclerosis. Soluble lectin-like oxidized low-density lipoprotein receptor-1 and microRNAs are closely associated with atherosclerotic coronary heart diseases. AIMS: To investigate the relationship between the severity and risk of coronary artery disease and plasma soluble lectin-like oxidized low-density lipoprotein receptor-1 and miR-98. STUDY DESIGN: Case-control study. METHODS: Angiographically documented patients with 38 single-vessel, 75 double-vessel, and 62 multi-vessel coronary artery disease; 62 healthy control participants; and 24-hour hypoxic (1% oxygen) human umbilical vein endothelial cells were included in this study. Circulating soluble lectin-like oxidized low-density lipoprotein receptor-1 concentrations were determined through enzyme-linked immunosorbent assays, and miR-98 expressions were measured by quantitative real-time polymerase chain reaction. RESULTS: The expressions of plasma soluble lectin-like oxidized low-density lipoprotein receptor-1 levels were progressively and significantly higher in patients with single-vessel, double-vessel, and multi-vessel coronary artery disease than in healthy controls (p<0.001). Circulating soluble lectin-like oxidized low-density lipoprotein receptor-1 concentrations in female patients with multi-vessel, double-vessel, and single-vessel coronary artery disease had evidently elevated compared with that in male patients (p<0.001). Plasma soluble lectin-like oxidized low-density lipoprotein receptor-1 levels were remarkably increased in females with coronary artery disease in different age groups compared with the males in the same age groups (p<0.001). Patients with single-vessel (areas under the curve=0.879), double-vessel (area under the curve=0.928), and multi-vessel (area under the curve=0.943) coronary artery disease have been clearly differentiated from healthy participants with respect to high sensitivity and specificity. The expression of miR-98 was noticeably downregulated in patients with single-, double- and multi-vessel occluded coronary artery disease and in hypoxic human umbilical vein endothelial cell compared with controls (p<0.001). Significantly elevated lectin-like oxidized low-density lipoprotein receptor-1 and caspase-3 activity and remarkably decreased cellular viability in hypoxic injured human umbilical vein endothelial cell. On the contrary, mimic of miR-98 markedly reduced caspase-3 and lectin-like oxidized low-density lipoprotein receptor-1 levels and highly increased cellular viability. CONCLUSION: Elevated circulating plasma soluble lectin-like oxidized low-density lipoprotein receptor-1 levels have a potential impact to identify the severity of coronary artery disease and have a strong correlation with aging as well as the female gender. Reduced plasma miR-98 level is possibly considered a risk factor for coronary artery disease, and agomiR-98 prevents atherosclerosis and cellular injury by targeting lectin-like oxidized low-density lipoprotein receptor-1.

Chia (Salvia hispanica L.) Seed Total Protein and Protein Fractions Digests Reduce Biomarkers of Inflammation and Atherosclerosis in Macrophages In Vitro.

SCOPE: The objectives are to evaluate the anti-inflammatory and anti-atherosclerotic effects of digested total protein and digested protein fractions from chia seed in macrophages in vitro. METHODS AND RESULTS: Total protein and protein fractions (albumin, globulin, glutelin, and prolamin) are isolated from chia seed and digested using simulated gastrointestinal conditions, resulting in digested total protein (DTP) and digested protein fractions (DPF). DTP and DPF are applied (1.0 mg mL-1 ) in RAW 264.4 macrophages stimulated with LPS (1 µg mL-1 ) for inflammation or ox-LDL (80 µg mL-1 ) for atherosclerosis. In the inflammatory process, DTP and DPF reduce p-NF-κB, iNOS, p-JNK, and AP-1. Digested glutelin reduces the secretion of nitric oxide (65.1%), reactive oxygen species (19.7%), prostaglandins (34.6%), TNF-α (24.1%), MCP-1 (18.9%), IL-6 (39.6%), and IL-10 (68.7%). DTP and DPF reduce the NF-κB translocation to nuclei. DTP and digested glutelin reduce iCAM expression (86.4%, 80.8%), LOX-1 (37.3%, 35.7%), iNOS (67.0%, 42.2%), and NF-κB (57.5%, 71.1%). DTP is effective in reducing secretion of nitric oxide (43.4%), lipid accumulation (41.9%), prostaglandins (41.9%), TNF-α (43.3%), MCP-1 (47.6%), and IL-6 (50.5%). Peptides from chia DTP and DPF are also characterized. CONCLUSION: DTP and digested glutelin from chia seed reduce expression and secretion of markers related to inflammation and atherosclerosis pathways.

In vivo MRI detection of carotid atherosclerotic lesions and kidney inflammation in ApoE-deficient mice by using LOX-1 targeted iron nanoparticles.

Lectin-like Oxidized Low-Density Lipoprotein Receptor 1 (LOX-1) plays a key role in atherosclerotic plaque initiation, formation and rupture, as well as in hyperlipidemia-induced glomerular disease. Here we report a sensitive, specific and biocompatible LOX-1-targeted-USPIO for the noninvasive MR imaging of LOX-1 within carotid atherosclerotic lesions and glomerular disease in apoE-deficient mice. In vitro analysis showed the highest uptake of targeted USPIOs in only activated RAW264.7 macrophages, and in vivo MRI studies showed signal loss in carotid atherosclerotic lesions after administration of targeted USPIOs at 8h and 24h. These areas of signal loss were correlated with the presence of nanoparticles in the atherosclerotic lesions, and immunohistochemistry and Perl's staining confirmed the co-localization of the LOX-1/macrophages/MMP-9 and targeted nanoparticles. Finally, additional studies suggest that this targeted probe may have potential to noninvasively image early glomerular disease. This finding may provide important methods for characterizing vulnerable atherosclerotic plaques and hyperlipidemia-induced glomerular diseases. FROM THE CLINICAL EDITOR: A functionalized USPIO-based negative contrast material was used in this study, demonstrating feasibility of sensitive MRI-based detection of atherosclerotic plaque formation in the carotid arteries and in the renal cortex, paving the way to potential future clinical applications.

Soluble lectin-like oxidized low-density lipoprotein receptor-1 levels in synovial fluid are correlated with disease severity of knee osteoarthritis.

OBJECTIVES: This study was aimed to measure soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) levels in serum and synovial fluid (SF) of knee osteoarthritis (OA) patients and to investigate the correlation between sLOX-1 levels and the disease severity. DESIGN AND METHODS: Two hundred and sixteen OA patients and 83 healthy controls were enrolled in this study. All OA patients were scored for Kellgren-Lawrence (KL) grade (0-4). The sLOX-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: In OA patients, the mean sLOX-1 levels in SF were markedly lower than those in paired serum samples (P<0.01). The SF sLOX-1 levels increased with higher KL grade (P<0.01) and were significantly correlated with disease severity (r = 0.324, P<0.01). SF sLOX-1 level was the independent factor for predicting the disease severity of OA (ß: 0.281, 95%CI: 0.145-0.396). CONCLUSIONS: SF sLOX-1 levels were independently and positively associated with disease severity in knee OA.

Uterine artery notch is associated with increased placental endothelial nitric oxide synthase and heat shock protein.

OBJECTIVE: This study was undertaken to evaluate whether uterine artery notch is associated with placental expression of endothelial nitric oxide synthase (eNOS), Heat shock protein 70 (Hsp70), and Lectin-like oxidized low-density lipoprotein receptor (Lox) in pregnant women. STUDY DESIGN: Uterine artery Doppler was performed in 30 pregnant women divided into two groups with or without the presence of uterine artery notches. After delivery, placental expression of eNOS, Hsp70, and Lox were evaluated with western blot analysis. RESULTS: Higher levels of placental eNOS (p = 0.02) and Hsp70 (p = 0.001) were expressed in the group with uterine artery notch compared with the group without uterine artery notch. There was no significant difference for placental Lox expression between group with uterine artery notch and without uterine artery notch (p = 0.17). CONCLUSION: Our results suggest that uterine artery notch is associated with increased placental expression of eNOS and Hsp in pregnant women.

Relationship between lectin-like oxidized low-density lipoprotein receptor 1 expression and preoperative echogenic findings of vulnerable carotid plaque.

PURPOSE: Lectin-like oxidized low-density lipoprotein 1 (LOX1) is an important cell surface receptor for the progression of atherosclerosis. Our purpose is to clarify the relationships of LOX1 and atherosclerotic factors for the vulnerability of carotid plaque and preoperative echogenic findings. METHODS: We examined LOX1 expression, matrix metalloproteinase (MMP)-2,9, and tissue inhibitor of MMP (TIMP)-2 by immunohistochemical analysis using carotid endarterectomy specimens obtained from 14 patients. Groups were divided into stable plaque group A and vulnerable plaque group B by preoperative echogenic findings of carotid plaques. Endothelial immunoreactivity was calculated, and the immunohistochemical findings were compared. RESULTS: LOX1 was remarkably expressed, especially in smooth muscle cells in vulnerable plaque and colocalized in MMP-9 positive cells and apoptotic cells. All LOX1, MMP-2,9, and TIMP2 were remarkably expressed in the subendothelial layer in group B compared with group A. The endothelial LOX1 index was 63.75 +/- 4.92 in group A and 83.0 +/- 5.02 in group B (p = 0.02). The endothelial MMP-2 index was 24.38 +/- 5.50 in group A and 32.83 +/- 6.79 in group B (p = 0.01). The endothelial MMP-9 index was 46.13 +/- 6.31 in group A and 59.17 +/- 2.14 in group B (p = 0.002). The endothelial TIMP-2 index had no significant difference between two groups (p = 0.14). CONCLUSION: LOX-1 may play an important role in the progression of vulnerable carotid plaque and might regulate vulnerable plaque formation in cooperation with MMPs and TIMP-2. Endothelial MMP-2 might suppress TIMP-2 activation in vulnerable plaques.

Modulation of placental protein expression of OLR1: implication in pregnancy-related disorders or pathologies.

The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR1) is a newly described receptor for oxidatively modified LDL. The human pregnancy is associated with hyperlipidemia and oxidative stress. It has been reported that modification in maternal lipid profile can induce disturbance during pregnancy. In this study, we have evaluated the expression protein level of OLR1 in human term placenta of women having plasma cholesterol level lower to 7 mM or higher to 8 mM and women of gestational diabetes mellitus (GDM) by western blot analysis. The present study demonstrates that the maternal lipid profile is associated with placental protein expression of OLR1. A significant increase in the protein expression of OLR1 was observed in placenta of women with elevated plasmatic total cholesterol level (>8 mM). In addition, the placental protein expression of OLR1 is increased in mothers having the highest pre-pregnancy body mass index (BMI) and low (<7 mM) plasmatic total cholesterol level at term. Interestingly, the placental protein expression of OLR1 is increased in the presence of GDM pregnancies compared with normal lipids level pregnancies, without the modification of mRNA expression. In conclusion, placental OLR1 protein expression is associated with maternal lipid profile, pre-pregnancy BMI, and pathology of GDM.

Lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) expression in human articular chondrocytes.

OBJECTIVE: To investigate the involvement of oxidized low density lipoprotein (ox-LDL) and the expression of its receptor lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) in osteoarthritis, by determining the ox-LDL in synovial fluid and the expression of LOX-1 mRNA and protein in osteoarthritic as well as normal cartilage. In addition, the effect of ox-LDL on chondrocyte viability and the effect of ascorbic acid (a well-known anti-oxidant) on LOX-1 expression were studied. METHODS: Fifteen patients were included in the study. Osteoarthritic articular cartilage was obtained from two distinct locations in the knee (n = 10) and hip (n = 5), specifically from weight-bearing and non-weight-bearing areas of the same joints. Five individuals were used as controls. mRNA and protein expression were studied by RT-PCR and immunofluorescence, respectively. Ox-LDL was measured in the synovial fluid and in paired serum samples from the patients using the ELISA method. RESULTS: Ox-LDL was detected in the synovial fluid and its receptor LOX-1 was detected in cartilage from both weight-bearing and non-weight-bearing areas, whereas no LOX-1 expression was found in normal cartilage. Ox-LDL reduced chondrocyte viability in cell cultures, while the addition of ascorbic acid to osteoarthritic chondrocytes resulted in a decrease in LOX-1 mRNA expression. CONCLUSION: The detection of LOX-1 mRNA and protein expression in osteoarthritic cartilage drawn from both weight-bearing and non-weight-bearing regions of the same patients suggest that LOX-1 may be involved in the progression and pathogenesis of osteoarthritis.

Modulation of ADP-induced platelet activation by aspirin and pravastatin: role of lectin-like oxidized low-density lipoprotein receptor-1, nitric oxide, oxidative stress, and inside-out integrin signaling.

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a receptor for oxidized-LDL, is up-regulated in activated endothelial cells, and it plays a role in atherothrombosis. However, its role in platelet aggregation is unclear. Both aspirin and HMG CoA reductase inhibitors (statins) reduce LOX-1 expression in endothelial cells. In this study, we investigated the effect of aspirin and pravastatin on LOX-1 expression on plate-lets. After ADP stimulation, mean fluorescence intensity of LOX-1 expression on platelets increased 1.5- to 2.0-fold. Blocking LOX-1 inhibited ADP-induced platelet aggregation in a concentration- and time-dependent manner. We also established that LOX-1 is important for ADP-stimulated inside-out activation of platelet alpha(IIb)beta(3) and alpha(2)beta(1) integrins (fibrinogen receptors). The specificity of this interaction was determined by arginine-glycine-aspartate-peptide inhibition. Furthermore, we found that LOX-1 inhibition of integrin activation is mediated by inhibition of protein kinase C activity. In other experiments, treatment with aspirin (1-10 mM) and pravastatin (1-5 microM) reduced platelet LOX-1 expression, with a synergistic effect of the combination of aspirin and pravastatin. Aspirin and pravastatin both reduced reactive oxygen species (ROS) released by activated platelets measured as malonyldialdehyde (MDA) release and nitrate/nitrite ratio. Aspirin and pravastatin also enhanced nitric oxide (NO) release measured as nitrite/nitrite + nitrate (NOx) ratio in platelet supernates. Small concentrations of aspirin and pravastatin had a synergistic effect on the inhibition of MDA release and enhancement of nitrite/NOx. Thus, LOX-1 is important for ADP-mediated platelet integrin activation, possibly through protein kinase C activation. Furthermore, aspirin and pravastatin inhibit LOX-1 expression on platelets in part by favorably affecting ROS and NO release from activated platelets.

Short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases vascular endothelial growth factor expression in rat retina.

Hyperhomocysteinemia is associated with an increase in the incidence of vascular diseases, including retinal vascular diseases. We examined the effects of high plasma levels of homocysteine on retinal glial cells and vascular endothelial growth factor (VEGF) expression. Male Sprague-Dawley rats were fed either a 3.0 g/kg homocystine diet or a control diet for 2 week. The homocystine-diet group had higher plasma levels of homocysteine and thiobarbituric acid reactive substances (TBARSs) and lower plasma levels of folate, retinol, alpha-tocopherol, and retinal expression of CuZn superoxide dismutase (SOD) than the controls. The rats fed the homocystine-diet showed an increase in vimentin, glial fibrillary acidic protein (GFAP), and VEGF immunoreactivity in the retina as compared to the controls. The increase in vimentin immunoreactivity in the hyperhomocysteinemic rats was correlated with changes in GFAP immunoreactivity in astrocytes within the ganglion cell layer. We found for the first time that short-term hyperhomocysteinemia-induced oxidative stress activates retinal glial cells and increases VEGF expression in the retina.

Metformin reduces endothelial cell expression of both the receptor for advanced glycation end products and lectin-like oxidized receptor 1.

Beyond its antihyperglycemic action, the antidiabetic oral drug metformin possesses antioxidant properties that may contribute to improve the cardiovascular deleterious effects of the diabetic disease. We explored whether metformin could modulate the redox-sensible expression of receptor for advanced glycation end products (RAGE) and lectin-like oxidized receptor 1 (LOX-1), 2 endothelial membrane receptors involved in the arterial endothelial dysfunction observed in diabetes. Bovine aortic endothelial cells, either unstimulated or activated by high levels of glucose (30 mmol/L) or advanced glycation end products, were incubated for 72 hours with metformin at therapeutically relevant concentrations (10(-5) to 5 x 10(-4) mol/L). The expressions of RAGE and LOX-1 were evaluated on cell extracts by Western blot analysis. Metformin was shown to reduce, in dose-dependent manner, such expression of the 2 receptors, both in stimulated (by either glucose or advanced glycation end products) and in unstimulated cells. The effect of metformin was associated with a decrease in intracellular reactive oxygen species as assessed using the 2',7'-dichlorodihydrofluorescein diacetate fluoroprobe. Taken together, our results suggest that the intracellular antioxidant properties of metformin may result in the inhibition of cell expression of both RAGE and LOX-1, possibly through a modulation of redox-sensible nuclear factors such as nuclear factor kappaB, that were shown to be involved in such receptor cell expression.

Inhibitory effects of procyanidin B(2) dimer on lipid-laden macrophage formation.

A proteomic analysis of procyanidin B(2) isolated from cocoa against oxidized low-density lipoprotein-induced lipid-laden macrophage formation was performed. Of approximately 400 detected proteins, 12 were differentially expressed as a result of B(2) treatment. They were subsequently identified by liquid chromatography-electrospray ionization-tandem mass spectrometry and the SWISS-PROT database. Further reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that B(2) strongly inhibited arachidonic acid inflammatory reactions, apoptosis, and their coupled mitogen-activated protein kinase and NF-kappaB pathways. To highlight proteins or genes with similar expressed patterns and similarly biological function induced by B(2) in lipid-laden macrophages, a cluster and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed. The data were mapped to multiple pathways. Further validation of the bioinformatic results revealed that activation of Wnt signaling may contribute to the cardioprotection of B(2). The differentially expressed genes and proteins mentioned above induced by B(2) are through regulating nuclear transcription factors, activating peroxisome proliferator-activated receptor-gamma and inhibiting AP-1 mRNA expressions. These in vitro data help to interpret the beneficial effects of B(2) in reducing the risk of atherosclerosis after consumption of flavonoid-rich foods. Many differentially expressed genes induced by B(2) help to uncover novel targets and may help to target disease interactions in atherosclerosis in the future.

Atherosclerosis and the Lectin-like OXidized low-density lipoprotein scavenger receptor.

The Lectin-like OXidized low-density lipoprotein scavenger receptor (LOX-1) is implicated in vascular inflammation and atherosclerotic plaque initiation, progression, and destabilization. LOX-1 levels are elevated upon recognition of oxidized low-density lipoprotein, a key pro-atherogenic substance in the vasculature. Recent evidence indicates this gene product is a biomarker of inflammation and disease status. We review and assess the role of LOX-1 in atherosclerotic plaque formation, physiologic regulation, and as a biomarker and target in cardiovascular disease diagnosis and prevention.

Lectin-like, oxidized low-density lipoprotein receptor-1 (LOX-1): a critical player in the development of atherosclerosis and related disorders.

LOX-1, a lectin-like 52-kD receptor for oxidized low-density lipoproteins (ox-LDL), is present primarily on endothelial cells. This receptor is upregulated by ox-LDL itself and by angiotensin II, endothelin, cytokines, and shear stress, all participants in atherosclerosis. This receptor is upregulated in the arteries of hypertensive, dyslipidemic, and diabetic animals. Upregulation of LOX-1 has been identified in atherosclerotic arteries of several animal species and humans, not only on the endothelial lining, but also in the neovasculature of the atherosclerotic plaque, and this receptor is often co-localized with apoptotic cells. Recent studies show upregulation of LOX-1 in the ischemic-reperfused myocardium. LOX-1 inhibition is associated with attenuation of atherosclerosis and associated ischemic injury. LOX-1 may be a novel, exciting target for drug therapy.

Oxidized LDL downregulates ATP-binding cassette transporter-1 in human vascular endothelial cells via inhibiting liver X receptor (LXR).

OBJECTIVE: ATP-binding cassette transporter-1 (ABCA1) mediates the active efflux of cholesterol and phospholipids, playing an important role in cholesterol homeostasis and atherogenesis. Oxidized low density lipoprotein (oxLDL) is an atherogenic molecule associated with the vascular endothelial dysfunction and development of atherosclerotic plaque. This report describes the effect of copper-catalyzed oxLDL on the regulation of ABCA1 in human endothelial cells (ECs). METHODS AND RESULTS: oxLDL downregulated ABCA1 at both mRNA and protein levels in a dose-dependent manner. This inhibitory effect of oxLDL was observed with both minimally and extensively oxLDL. Transfection of the ABCA1 promoter luciferase revealed oxLDL to substantially decrease ABCA1 promoter activity at basal conditions and after stimulation by overexpressing the liver X receptor LXRalpha and retinoid X receptor RXRalpha. oxLDL also attenuated LXR activation by blocking LXR ligand binding and interfering with the generation of 27-hydroxycholesterol, an LXR endogenous ligand. Furthermore, oxLDL inhibited exogenous cholesterol- and oxysterol-induced endothelial ABCA1 induction. CONCLUSION: oxLDL downregulated ABCA1 by inhibiting LXR activation in endothelial cells. Such an effect may contribute to endothelial dysfunction and plaque formation.