Increased expression of CD38 on endothelial cells in SARS-CoV-2 infection in cynomolgus macaques.

SARS-CoV-2 infection causes activation of endothelial cells (ECs), leading to dysmorphology and dysfunction. To study the pathogenesis of endotheliopathy, the activation of ECs in lungs of cynomolgus macaques after SARS-CoV-2 infection and changes in nicotinamide adenine dinucleotide (NAD) metabolism in ECs were investigated, with a focus on the CD38 molecule, which degrades NAD in inflammatory responses after SARS-CoV-2 infection. Activation of ECs was seen from day 3 after SARS-CoV-2 infection in macaques, with increases of intravascular fibrin and NAD metabolism-associated enzymes including CD38. In vitro, upregulation of CD38 mRNA in human ECs was detected after interleukin 6 (IL-6) trans-signaling induction, which was increased in the infection. In the presence of IL-6 trans-signaling stimulation, however, CD38 mRNA silencing induced significant IL-6 mRNA upregulation in ECs and promoted EC apoptosis after stimulation. These results suggest that upregulation of CD38 in patients with COVID-19 has a protective role against IL-6 trans-signaling stimulation induced by SARS-CoV-2 infection.

Lectin-like oxidized low-density lipoprotein receptor 1 attenuates pneumonia-induced lung injury.

Identifying host factors that contribute to pneumonia incidence and severity are of utmost importance to guiding the development of more effective therapies. Lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1, encoded by OLR1) is a scavenger receptor known to promote vascular injury and inflammation, but whether and how LOX-1 functions in the lung are unknown. Here, we provide evidence of substantial accumulation of LOX-1 in the lungs of patients with acute respiratory distress syndrome and in mice with pneumonia. Unlike previously described injurious contributions of LOX-1, we found that LOX-1 is uniquely protective in the pulmonary airspaces, limiting proteinaceous edema and inflammation. We also identified alveolar macrophages and recruited neutrophils as 2 prominent sites of LOX-1 expression in the lungs, whereby macrophages are capable of further induction during pneumonia and neutrophils exhibit a rapid, but heterogenous, elevation of LOX-1 in the infected lung. Blockade of LOX-1 led to dysregulated immune signaling in alveolar macrophages, marked by alterations in activation markers and a concomitant elevation of inflammatory gene networks. However, bone marrow chimeras also suggested a prominent role for neutrophils in LOX-1-mediated lung protection, further supported by LOX-1+ neutrophils exhibiting transcriptional changes consistent with reparative processes. Taken together, this work establishes LOX-1 as a tissue-protective factor in the lungs during pneumonia, possibly mediated by its influence on immune signaling in alveolar macrophages and LOX-1+ airspace neutrophils.

Adiponectin forms a complex with atherogenic LDL and inhibits its downstream effects.

Adiponectin, an adipocyte-derived protein, has antiatherogenic and antidiabetic effects, but how it confers the atherogenic effects is not well known. To study the antiatherogenic mechanisms of adiponectin, we examined whether it interacts with atherogenic low density lipoprotein (LDL) to attenuate LDL's atherogenicity. L5, the most electronegative subfraction of LDL, induces atherogenic responses similarly to copper-oxidized LDL (oxLDL). Unlike the native LDL endocytosed via the LDL receptor, L5 and oxLDL are internalized by cells via the lectin-like oxidized LDL receptor-1 (LOX-1). Using enzyme-linked immunosorbent assays (ELISAs), we showed that adiponectin preferentially bound oxLDL but not native LDL. In Chinese hamster ovary (CHO) cells transfected with the LOX-1 or LDL receptor, adiponectin selectively inhibited the uptake of oxLDL but not of native LDL, respectively. Furthermore, adiponectin suppressed the internalization of oxLDL in human coronary artery endothelial cells (HCAECs) and THP-1-derived macrophages. Western blot analysis of human plasma showed that adiponectin was abundant in L5 but not in L1, the least electronegative subfraction of LDL. Sandwich ELISAs with anti-adiponectin and anti-apolipoprotein B antibodies confirmed the binding of adiponectin to L5 and oxLDL. In LOX-1-expressing CHO cells, adiponectin inhibited cellular responses to oxLDL and L5, including nuclear factor-κB activation and extracellular signal-regulated kinas phosphorylation. In HCAECs, adiponectin inhibited oxLDL-induced endothelin-1 secretion and extracellular signal-regulated kinase phosphorylation. Conversely, oxLDL suppressed the adiponectin-induced activation of adenosine monophosphate-activated protein kinase in COS-7 cells expressing adiponectin receptor AdipoR1. Our findings suggest that adiponectin binds and inactivates atherogenic LDL, providing novel insight into the antiatherogenic mechanisms of adiponectin.

Detection of a High Ratio of Soluble to Membrane-Bound LOX-1 in Aspirated Coronary Thrombi From Patients With ST-Segment-Elevation Myocardial Infarction.

Background The circulating level of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) is a valuable biomarker of acute myocardial infarction (AMI). The most electronegative low-density lipoprotein, L5, signals through LOX-1 to trigger atherogenesis. We examined the characteristics of LOX-1 and the role of L5 in aspirated coronary thrombi of AMI patients. Methods and Results Intracoronary thrombi were aspirated by performing interventional thrombosuction in patients with ST-segment-elevation myocardial infarction (STEMI; n=32) or non-ST-segment-elevation myocardial infarction (n=12). LOX-1 level and the ratio of sLOX-1 to membrane-bound LOX-1 were higher in thrombi of STEMI patients than in those of non-ST-segment-elevation myocardial infarction patients. In all aspirated thrombi, LOX-1 colocalized with apoB100. When we explored the role of L5 in AMI, deconvolution microscopy showed that particles of L5 but not L1 (the least electronegative low-density lipoprotein) quickly formed aggregates prone to retention in thrombi. Treating human monocytic THP-1 cells with L5 or L1 showed that L5 induced cellular adhesion and promoted the differentiation of monocytes into macrophages in a dose-dependent manner. In a second cohort of AMI patients, the L5 percentage and plasma concentration of sLOX-1 were higher in STEMI patients (n=33) than in non-ST-segment-elevation myocardial infarction patients (n=25), and sLOX-1 level positively correlated with L5 level in AMI patients. Conclusions The level of LOX-1 and the ratio of sLOX-1 to membrane-bound LOX-1 in aspirated thrombi, as well as the circulating level of sLOX-1 were higher in STEMI patients than in non-ST-segment-elevation myocardial infarction patients. L5 may play a role in releasing a high level of sLOX-1 into the circulation of STEMI patients.

Human electronegative LDL induces mitochondrial dysfunction and premature senescence of vascular cells in vivo.

Dysregulation of plasma lipids is associated with age-related cardiovascular diseases. L5, the most electronegative subfraction of chromatographically resolved low-density lipoprotein (LDL), induces endothelial dysfunction, whereas the least electronegative subfraction, L1, does not. In this study, we examined the effects of L5 on endothelial senescence and its underlying mechanisms. C57B6/J mice were intravenously injected with L5 or L1 (2 mg kg-1  day-1 ) from human plasma. After 4 weeks, nuclear γH2AX deposition and senescence-associated ß-galactosidase staining indicative of DNA damage and premature senescence, respectively, were increased in the aortic endothelium of L5-treated but not L1-treated mice. Similar to that, in Syrian hamsters with elevated serum L5 levels induced by a high-fat diet, nuclear γH2AX deposition and senescence-associated ß-galactosidase staining were increased in the aortic endothelium. This phenomenon was blocked in the presence of N-acetyl-cysteine (free-radical scavenger) or caffeine (ATM blocker), as well as in lectin-like oxidized LDL receptor-1 (LOX-1) knockout mice. In cultured human aortic endothelial cells, L5 augmented mitochondrial oxygen consumption and mitochondrial free-radical production, which led to ATM activation, nuclear γH2AX deposition, Chk2 phosphorylation, and TP53 stabilization. L5 also decreased human telomerase reverse transcriptase (hTERT) protein levels and activity. Pharmacologic or genetic manipulation of the reactive oxygen species (ROS)/ATM/Chk2/TP53 pathway efficiently blocked L5-induced endothelial senescence. In conclusion, L5 may promote mitochondrial free-radical production and activate the DNA damage response to induce premature vascular endothelial senescence that leads to atherosclerosis. Novel therapeutic strategies that target L5-induced endothelial senescence may be used to prevent and treat atherosclerotic vascular disease.

Ecrg4 peptide is the ligand of multiple scavenger receptors.

Esophageal cancer-related gene 4 (Ecrg4) encodes a hormone-like peptide that is believed to be involved in a variety of physiological phenomena, including tumour suppression. Recent progress in the study of Ecrg4 has shown that Ecrg4 is a proinflammatory factor and induces the expression of several cytokines and chemokines in macrophages/microglia. However, the detailed molecular mechanisms of Ecrg4 signalling, especially the Ecrg4 receptors, remain poorly understood. Here, using retrovirus-mediated expression cloning, we identified lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) as a membrane protein that binds amino acid residues 71-132 of Ecrg4 (Ecrg4(71-132)). Moreover, in addition to LOX-1, several scavenger receptors, such as Scarf1, Cd36 and Stabilin-1, facilitated the efficient internalisation of Ecrg4(71-132) into cells. A broad competitive inhibitor of scavenger receptors, polyinosinic acid, reduced both the binding of Ecrg4(71-132) and the activation of NF-κB in microglia. This activation was dependent on MyD88, an adaptor protein that recruits signalling proteins to Toll-like receptors (TLRs), with the consequent induction of various immune responses. These data suggest that multiple scavenger receptors recognise Ecrg4(71-132) and transduce its signals, together with TLRs, in microglia.

Smoking cessation reduces the lectin-like low-density lipoprotein receptor index, an independent cardiovascular risk marker of vascular inflammation.

Vessel wall inflammation promotes the destabilization of atherosclerotic plaques. The lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) expressed by vascular cells and monocytes. LOX index is calculated by multiplying LOX-1 ligand containing apolipoprotein B level with the soluble LOX-1. A high LOX index reflects an increased risk for stroke and myocardial infarction. However, the change in LOX index after smoking cessation and the relationship between smoking-related variables and LOX index are unknown. Relation of the clinical parameters to the LOX index was examined on 180 subjects (135 males and 45 females) at the first visit to our outpatient clinic for smoking cessation. The impact of smoking cessation on the LOX index was also determined in the 94 subjects (62 males and 32 females) who successfully stopped smoking. Sex-adjusted regression analysis and multivariate analysis identified three independent determinants of the LOX index, namely, low-density lipoprotein-cholesterol (LDL-C; ß = 0.311, p < 0.001), high-sensitivity C-reactive protein (ß = 0.358, p < 0.001), and expired carbon monoxide concentration reflecting smoking heaviness (ß = 0.264, p = 0.003). Body mass index (BMI) significantly increased 3 months after the onset of smoking cessation (p < 0.001). However, the LOX index significantly decreased (p < 0.001), regardless of the rate of increase in BMI post-cessation. The LOX index is closely associated with smoking heaviness as well as dyslipidemia and an inflammation marker. Smoking cessation may induce a decrease in this cardiovascular risk marker, independently of weight gain.

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.

Serum level of LOX-1 ligand containing ApoB is associated with increased carotid intima-media thickness in Japanese community-dwelling men, especially those with hypercholesterolemia LOX-1 ligand and IMT in Japanese.

BACKGROUND: The serum level of LOX-1 ligand containing ApoB (LAB) may reflect atherogenicity better than usual lipid parameters; however, the relationship between LAB and carotid intima-media thickness (IMT) was not clear even in Asian populations. METHODS: A total of 992 community-dwelling Japanese men, aged 40 to 79 years, were enrolled in the present study. Serum LAB levels were measured by enzyme-linked immunosorbent assays (ELISAs) with recombinant LOX-1 and monoclonal anti-apolipoprotein B antibody. RESULTS: Serum LAB levels (median [interquartile range], µg cs/L) were 5341 µg cs/L (4093-7125). The mean average IMT of the common carotid artery was highest in the fourth LAB quartile (842 µm) compared with the first quartile (797 µm) after adjustment for age, high-density lipoprotein cholesterol, triglyceride, body mass index, hypertension, diabetes, high sensitivity C-reactive protein, smoking, and alcohol drinking. However, this statistically significant difference was lost after further adjustment for total cholesterol (TC). After stratification using the combination of median LAB and hypercholesterolemia (serum TC ≥ 6.21 mmol/L and/or lipid-lowering medication), the adjusted mean average IMT (standard error) in the high LAB/hypercholesterolemia group was 886 µm (12.7), 856 µm (16.7) in the low LAB/hypercholesterolemia group, and 833 µm (8.4) in the low LAB/normal cholesterol group (P = .004). After further adjustment for TC, mean average IMT in the high LAB group was significantly higher than that measured in the low LAB group in hypercholesterolemic participants not taking lipid-lowering medication. CONCLUSION: Serum LAB was associated with an increased carotid IMT in Japanese men, especially those with hypercholesterolemia.

Interplay between CRP, Atherogenic LDL, and LOX-1 and Its Potential Role in the Pathogenesis of Atherosclerosis.

BACKGROUND: Studies have shown that the classic acute-phase protein C-reactive protein (CRP) has proinflammatory effects on vascular cells and may play a causal role in the pathogenesis of coronary artery disease. A growing body of evidence has suggested that interplay between CRP, lectin-like oxidized LDL receptor-1 (LOX-1), and atherogenic LDL may underlie the mechanism of endothelial dysfunction that leads to atherosclerosis. CONTENT: We review the biochemical evidence for an association of CRP, LOX-1, and either oxidized LDL (OxLDL) or electronegative L5 LDL with the pathogenesis of coronary artery disease. Artificially oxidized OxLDL has been studied extensively for its role in atherogenesis, as has electronegative L5 LDL, which is present at increased levels in patients with increased cardiovascular risks. OxLDL and L5 have been shown to stimulate human aortic endothelial cells to produce CRP, indicating that CRP is synthesized locally in the endothelium. The ligand-binding face (B-face) of CRP has been shown to bind the LOX-1 scavenger receptor and increase LOX-1 expression in endothelial cells, thereby promoting the uptake of OxLDL or L5 by LOX-1 into endothelial cells to induce endothelial dysfunction. SUMMARY: CRP and LOX-1 may form a positive feedback loop with OxLDL or L5 in atherogenesis, whereby increased levels of atherogenic LDL in patients with cardiovascular risks induce endothelial cells to express CRP, which may in turn increase the expression of LOX-1 to promote the uptake of atherogenic LDL into endothelial cells. Further research is needed to confirm a causal role for CRP in atherogenesis.

Cross-talk between LOX-1 and PCSK9 in vascular tissues.

AIMS: Lectin-like ox-LDL receptor-1 (LOX-1) plays an important role in inflammatory diseases, such as atherosclerosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates LDL receptor degradation and influences serum LDL levels. The present study was designed to investigate the possible interaction between PCSK9 and LOX-1. METHODS AND RESULTS: In the first set of experiments, human vascular endothelial cells and smooth muscle cells were studied at baseline and after lipopolysaccharide (LPS) treatment (to create an inflammatory state). Both PCSK9 and LOX-1 were strongly induced by LPS treatment. To define the role of PCSK9 in LOX-1 expression, cells were transfected with siRNA against PCSK9, which resulted in reduced LOX-1 expression and function. On the other hand, cells exposed to recombinant hPCSK9 revealed enhanced LOX-1 expression (P < 0.05). To determine whether LOX-1 also regulates PCSK9, cultured cells in which LOX-1 was knocked down by siRNA expressed less PCSK9, whereas those transfected with hLOX-1 cDNA showed increased PCSK9 expression. The second set of experiments was carried out in wild-type (WT) and gene knockout (KO; LOX-1 and PCSK9) mice; LOX-1 KO mice showed much less PCSK9 (P < 0.05 vs. WT mice). PCSK9-KO mice showed much less LOX-1 (P < 0.05 vs. WT mice). Furthermore, we observed that mitochondrial reactive oxygen species (mtROS) plays an initiating role in the LOX-1/PCSK9 interaction, since mtROS induction enhanced and its inhibition reduced the expression of both PCSK9 and LOX-1. We also found that both LOX-1 and PCSK9 regulate adhesion molecules vascular cell adhesion molecule-1 expression. Finally, oxidized low-density lipoprotein and tumour necrosis factor-α, pro-inflammatory stimuli besides LPS, regulated PCSK9 expression that is mediated by the NF-κB signalling pathway. CONCLUSIONS: These observations suggest that LOX-1 and PCSK9 positively influence each other's expression, especially during an inflammatory reaction. mtROS appear to be important initiators of PCSK9/LOX-1 expression.

Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor.

The angiotensin II type 1 receptor (AT1) is a 7-transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the single-transmembrane oxidized LDL (oxLDL) receptor (LOX-1) resides in proximity to AT1 on cell-surface membranes and that binding of oxLDL to LOX-1 can allosterically activate AT1-dependent signaling events. oxLDL-induced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX-1, whereas little increase was observed in CHO cells expressing only LOX-1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell-surface complexes involving LOX-1 and AT1. Chimeric analysis showed that oxLDL-induced AT1 signaling events are mediated via interactions between the intracellular domain of LOX-1 and AT1 that activate AT1. oxLDL-induced impairment of endothelium-dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease.

Human serum amyloid A3 (SAA3) protein, expressed as a fusion protein with SAA2, binds the oxidized low density lipoprotein receptor.

Serum amyloid A3 (SAA3) possesses characteristics distinct from the other serum amyloid A isoforms, SAA1, SAA2, and SAA4. High density lipoprotein contains the latter three isoforms, but not SAA3. The expression of mouse SAA3 (mSAA3) is known to be up-regulated extrahepatically in inflammatory responses, and acts as an endogenous ligand for the toll-like receptor 4/MD-2 complex. We previously reported that mSAA3 plays an important role in facilitating tumor metastasis by attracting circulating tumor cells and enhancing hyperpermeability in the lungs. On the other hand, human SAA3 (hSAA3) has long been regarded as a pseudogene, which is in contrast to the abundant expression levels of the other isoforms. Although the nucleotide sequence of hSAA3 is very similar to that of the other SAAs, a single oligonucleotide insertion in exon 2 causes a frame-shift to generate a unique amino acid sequence. In the present study, we identified that hSAA3 was transcribed in the hSAA2-SAA3 fusion transcripts of several human cell lines. In the fusion transcript, hSAA2 exon 3 was connected to hSAA3 exon 1 or hSAA3 exon 2, located approximately 130kb downstream from hSAA2 exon 3 in the genome, which suggested that it is produced by alternative splicing. Furthermore, we succeeded in detecting and isolating hSAA3 protein for the first time by an immunoprecipitation-enzyme linked immune assay system using monoclonal and polyclonal antibodies that recognize the hSAA3 unique amino acid sequence. We also demonstrated that hSAA3 bound oxidized low density lipoprotein receptor (oxLDL receptor, LOX-1) and elevated the phosphorylation of ERK, the intracellular MAP-kinase signaling protein.

The Relationship between Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Ligands Containing Apolipoprotein B and the Cardio-Ankle Vascular Index in Healthy Community Inhabitants: The KOBE Study.

AIMS: Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 ligands containing apolipoprotein B (LAB) and lectin-like oxidized LDL receptor-1 (LOX-1) are known as LOX-1-related modified LDL indicators. These indicators play an important role in the early phase atherosclerosis, but the relationship between these indicators and subclinical atherosclerosis, as represented by the cardio-ankle vascular index (CAVI), has not been assessed. We herein investigated the association of LOX-1- related modified LDL indicators and the CAVI in healthy, Japanese urban community inhabitants who were considered to be at low risk for cardiovascular disease (CVD). METHODS: The participants were 515 healthy Japanese (310 men and 205 women) without a history of CVD, cancer or the use of medication for hypertension, diabetes or dyslipidaemia. To estimate the association between LOX-1-related modified LDL indicators (LAB, soluble form of LOX-1 (sLOX-1)) and the CAVI, we performed multivariable linear regression analyses with possible confounders such as the serum LDL cholesterol level. RESULTS: The plasma LAB showed a positive association with the CAVI in men (standardized coefficient: 0.11, p = 0.04). This relationship was not observed in women. On the other hand, no clear association was observed between the CAVI and the plasma sLOX-1 level in either sex. CONCLUSIONS: The plasma LAB levels may represent a useful marker for detecting potential atherosclerosis in healthy individuals considered to be at low risk for atherosclerosis and CVD. Further studies are needed to confirm the present findings.

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.

LOX-1 plays an important role in ischemia-induced angiogenesis of limbs.

LOX-1, lectin-like oxidized low-density lipoprotein (LDL) receptor-1, is a single transmembrane receptor mainly expressed on endothelial cells. LOX-1 mediates the uptake of oxidized LDL, an early step in atherosclerosis; however, little is known about whether LOX-1 is involved in angiogenesis during tissue ischemia. Therefore, we examined the role of LOX-1 in ischemia-induced angiogenesis in the hindlimbs of LOX-1 knockout (KO) mice. Angiogenesis was evaluated in a surgically induced hindlimb ischemia model using laser Doppler blood flowmetry (LDBF) and histological capillary density (CD) and arteriole density (AD). After right hindlimb ischemia, the ischemic/nonischemic hindlimb blood flow ratio was persistently lower in LOX-1 KO mice than in wild-type (WT) mice. CD and AD were significantly smaller in LOX-1 KO mice than in WT mice on postoperative day 14. Immunohistochemical analysis revealed that the number of macrophages infiltrating ischemic tissues was significantly smaller in LOX-1 KO mice than in WT mice. The number of infiltrated macrophages expressing VEGF was also significantly smaller in LOX-1 KO mice than in WT mice. Western blot analysis and ROS production assay revealed that LOX- KO mice show significant decrease in Nox2 expression, ROS production and HIF-1α expression, the phosphorylation of p38 MAPK and NF-κB p65 subunit as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and LOX-1 itself in ischemic muscles, which is supposed to be required for macrophage infiltration expressing angiogenic factor VEGF. Reduction of VEGF expression successively suppressed the phosphorylation of Akt and eNOS, which accelerated angiogenesis, in the ischemic leg of LOX-1 KO mice. Our findings indicate that LOX-1 plays an important role in ischemia-induced angiogenesis by 1) Nox2-ROS-NF-κB activation, 2) upregulated expression of adhesion molecules: VCAM-1 and LOX-1 and 3) promoting macrophage infiltration, which expresses angiogenic factor VEGF.

Prevention of export of anoxia/reoxygenation injury from ischemic to nonischemic cardiomyocytes via inhibition of endocytosis.

Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caused by export of injury signals from the infarct zone. The countermeasures to limit infarct size, therefore, should be aimed at nonselective blockade of most, if not all, injury signals from entering nonischemic cells. To test whether inhibition of endocytosis might limit infarct size, HL-1 cardiomyocytes were subjected to anoxia (6 h) and reoxygenation (1 h). Anoxic and reoxygenated cells showed a multifold increase in mitochondrial ROS production accompanied with upregulation of scavenger receptors lectin-like oxidized low-density lipoprotein receptor-1 and CD36 and stimulation of stress signals, including NADPH oxidase subunit p22(phox), SOD2, and beclin-1. Incubation of healthy cardiomyocytes in media from anoxic and reoxygenated cells (conditioned media) resulted in qualitatively similar responses, including increase in the generation of mitochondrial ROS, p22(phox), SOD2, and beclin-1. Anoxia and reoxygenation caused collapse of clathrin-mediated endocytosis and stimulation of macropinocytosis, whereas in cultures exposed to conditioned media, the activity of endocytosis was uniformly higher. Conditioned media also significantly aggravated cytotoxic effects of TNF-α and angiotensin II, and suppression of endocytosis reversed these trends, resulting in an overall increase of metabolic activity. Moreover, inhibition of endocytosis prevented binding of oxidized cellular fragments with greater efficiency than targeted neutralization of the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1. Many of the observations in HL-1 cardiomyocytes were confirmed in primary cardiomyocyte cultures. Our data suggest that endocytosis is upregulated in border zone cardiomyocytes, and inhibition of endocytosis may be an effective approach to prevent export of injury signals from the infarct zone.

Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation.

Platelet activation and aggregation underlie acute thrombosis that leads to ST-elevation myocardial infarction (STEMI). L5-highly electronegative low-density lipoprotein (LDL)-is significantly elevated in patients with STEMI. Thus, we examined the role of L5 in thrombogenesis. Plasma LDL from patients with STEMI (n = 30) was chromatographically resolved into 5 subfractions (L1-L5) with increasing electronegativity. In vitro, L5 enhanced adenosine diphosphate-stimulated platelet aggregation twofold more than did L1 and induced platelet-endothelial cell (EC) adhesion. L5 also increased P-selectin expression and glycoprotein (GP)IIb/IIIa activation and decreased cyclic adenosine monophosphate levels (n = 6, P < .01) in platelets. In vivo, injection of L5 (5 mg/kg) into C57BL/6 mice twice weekly for 6 weeks shortened tail bleeding time by 43% (n = 3; P < .01 vs L1-injected mice) and increased P-selectin expression and GPIIb/IIIa activation in platelets. Pharmacologic blockade experiments revealed that L5 signals through platelet-activating factor receptor and lectin-like oxidized LDL receptor-1 to attenuate Akt activation and trigger granule release and GPIIb/IIIa activation via protein kinase C-α. L5 but not L1 induced tissue factor and P-selectin expression in human aortic ECs (P < .01), thereby triggering platelet activation and aggregation with activated ECs. These findings indicate that elevated plasma levels of L5 may promote thrombosis that leads to STEMI.

Oxidized low density lipoprotein (LDL) affects hyaluronan synthesis in human aortic smooth muscle cells.

Thickening of the vessel in response to high low density lipoprotein(s) (LDL) levels is a hallmark of atherosclerosis, characterized by increased hyaluronan (HA) deposition in the neointima. Human native LDL trapped within the arterial wall undergoes modifications such as oxidation (oxLDL). The aim of our study is to elucidate the link between internalization of oxLDL and HA production in vitro, using human aortic smooth muscle cells. LDL were used at an effective protein concentration of 20-50 µg/ml, which allowed 80% cell viability. HA content in the medium of untreated cells was 28.9 ± 3.7 nmol HA-disaccharide/cell and increased after oxLDL treatment to 53.9 ± 5.6. OxLDL treatments doubled the transcripts of HA synthase HAS2 and HAS3. Accumulated HA stimulated migration of aortic smooth muscle cells and monocyte adhesiveness to extracellular matrix. The effects induced by oxLDL were inhibited by blocking LOX-1 scavenger receptor with a specific antibody (10 µg/ml). The cholesterol moiety of LDL has an important role in HA accumulation because cholesterol-free oxLDL failed to induce HA synthesis. Nevertheless, cholesterol-free oxLDL and unmodified cholesterol (20 µg/ml) induce only HAS3 transcription, whereas 22,oxysterol affects both HAS2 and HAS3. Moreover, HA deposition was associated with higher expression of endoplasmic reticulum stress markers (CHOP and GRP78). Our data suggest that HA synthesis can be induced in response to specific oxidized sterol-related species delivered through oxLDL.