LDL uptake-dependent phosphatidylethanolamine translocation to the cell surface promotes fusion of osteoclast-like cells.

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR∼ABCG1∼PE translocation to cell surface∼cell-cell fusion) in multinucleation of OCLs.

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.

Quantitative and qualitative lipid improvement with chronic hepatitis C virus eradication using direct-acting antivirals.

AIM: Direct-acting antivirals have revolutionized hepatitis C virus (HCV) therapy by providing a high sustained virological response (SVR) rate and subsequent favorable lipid increases. Proprotein convertase subtilisin-kexin like-9 (PCSK9) plays an important role in regulating quantitative lipid levels. This study examined the interactions between quantitative PCSK9 and lipid changes, as well as qualitative lipid changes in terms of lectin-like oxidized low-density lipoprotein (LDL) receptor-1 ligand containing apolipoprotein B (LAB) and high-density lipoprotein (HDL) cholesterol uptake capacity (HDL-CUC). METHODS: Patients with chronic HCV infection (N = 231) who achieved an SVR by direct-acting antivirals without lipid-lowering therapy were included for comparisons of PCSK9, LAB, HDL-CUC, and other clinical indices between pretreatment and SVR12 time points. RESULTS: LDL (LDL) cholesterol and HDL cholesterol levels were quantitatively increased at SVR12, along with higher PCSK9 (all p < 0.0001). PCSK9 was significantly correlated with LDL cholesterol (r = 0.244, p = 0.0003) and apolipoprotein B (r = 0.222, p = 0.0009) at SVR12. Regarding qualitative LDL changes, LAB was significantly decreased and LAB/LDL cholesterol and LAB/apolipoprotein B proportions were improved at SVR12 (all p < 0.0001). In terms of qualitative HDL changes, HDL-CUC was significantly ameliorated, along with HDL-CUC/HDL cholesterol, HDL-CUC/ apolipoprotein A1, and HDL-CUC/ apolipoprotein A2 at SVR12 (all p < 0.0001). CONCLUSIONS: HCV eradication by direct-acting antivirals may produce quantitative lipid profile changes, along with PCSK9 production recovery in addition to qualitative lipid improvement, which possibly confers the additional secondary benefits of atherosclerosis improvement and cardiovascular disease event reduction.

LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1) Deletion Has Protective Effects on Stroke in the Genetic Background of Stroke-Prone Spontaneously Hypertensive Rat.

Background and Purpose- oxLDL (oxidized low-density lipoprotein) has been known for its potential to induce endothelial dysfunction and used as a major serological marker of oxidative stress. Recently, LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a lectin-like receptor for oxLDL, has attracted attention in studies of neuronal apoptosis and stroke. We aim to investigate the impact of LOX-1-deficiency on spontaneous hypertension-related brain damage in the present study. Methods- We generated a LOX-1 deficient strain on the genetic background of stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of severe hypertension and spontaneous stroke. In this new disease model with stroke-proneness, we monitored the occurrence of brain abnormalities with and without salt loading by multiple procedures including T2 weighted magnetic resonance imaging and also explored circulatory miRNAs as diagnostic biomarkers for cerebral ischemic injury by microarray analysis. Results- Both T2 weighted magnetic resonance imaging abnormalities and physiological parameter changes could be detected at significantly delayed timing in LOX-1 knockout rats compared with wild-type SHRSP, in either case of normal rat chow and salt loading (P<0.005 in all instances; n=11-20 for SHRSP and n=13-23 for LOX-1 knockout rats). There were no significant differences in the form of magnetic resonance imaging findings between the strains. A number of miRNAs expressed in the normal rat plasma, including rno-miR-150-5p and rno-miR-320-3p, showed significant changes after spontaneous brain damage in SHRSP, whereas the corresponding changes were modest or almost unnoticeable in LOX-1 knockout rats. There appeared to be the lessening of correlation of postischemic miRNA alterations between the injured brain tissue and plasma in LOX-1 knockout rats. Conclusions- Our data show that deficiency of LOX-1 has a protective effect on spontaneous brain damage in a newly generated LOX-1-deficient strain of SHRSP. Further, our analysis of miRNAs as biomarkers for ischemic brain damage supports a potential involvement of LOX-1 in blood brain barrier disruption after cerebral ischemia. Visual Overview- An online visual overview is available for this article.

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.

Developmental Endothelial Locus-1 (Del-1) Inhibits Oxidized Low-Density Lipoprotein Activity by Direct Binding, and Its Overexpression Attenuates Atherogenesis in Mice.

BACKGROUND: Modified low-density lipoprotein (LDL) binding to scavenger receptors has been implicated in atherosclerosis. It is hypothesized that a third molecule may affect modified LDL binding, therefore, this study focuses on the soluble endogenous protein, developmental endothelial locus-1 (Del-1), as an inhibitor of oxidized LDL (oxLDL) interactions.Methods and Results:Del-1 preferentially bound oxLDL over native LDL in a cell-free binding assay. Del-1 also inhibited DiI-labeled oxLDL uptake by scavenger receptors irrespective of the receptor type (LOX-1, SR-AI, CD36, or SR-BI) expressed in COS-7 cells, and independent of cell type (human coronary artery endothelial cells (HCAECs) or THP-1-derived macrophages). Furthermore, Del-1 suppressed oxLDL-inducedMCP-1andICAM-1expression and endothelin-1 secretion in HCAECs. Then, male Del-1 transgenic (Del-1Tg) and wild-type mice (WT) mice were established and fed a Paigen diet for 20 weeks from the age of 24 weeks. While plasma lipid concentrations did not differ between WT and Del-1Tg mice, plasma LOX-1-ligand activity was significantly lower in Del-1Tg than in WT mice. Moreover, lipid accumulation in aortic roots was significantly less in the Del-1Tg mice, evaluated with Oil red-O. Taken together, Del-1 appears to block the activity of oxLDL pharmacologically by direct binding in vitro, and attenuates atherogenesis in vivo, although its role in physiological settings are yet to be resolved. CONCLUSIONS: Del-1 intercepted oxLDL before its receptor binding to reduce atherogenesis. (Circ J 2016; 80: 2541-2549).

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 signal is a potent biomarker and therapeutic target for human rheumatoid arthritis.

OBJECTIVE: To determine whether lectin-like oxidized low-density lipoprotein (ox-LDL) receptor 1 (LOX-1) and the soluble form of LOX-1 (sLOX-1) are novel target molecules for the diagnosis and treatment of rheumatoid arthritis (RA). METHODS: Expression of ox-LDL and LOX-1 proteins in human RA synovium was evaluated by immunohistochemistry. Human RA fibroblast-like synoviocytes (FLS) were assessed for ox-LDL-induced expression of LOX-1 and ox-LDL-induced production of matrix metalloproteinase 1 (MMP-1) and MMP-3. Levels of sLOX-1 in the plasma and synovial fluid of patients with RA, compared with patients with osteoarthritis (OA), were determined by a specific chemiluminescence enzyme-linked immunoassay. In animal experiments, ox-LDL was injected into the knee joints of mice, with or without an anti-LOX-1 neutralizing antibody or sLOX-1, and the severity of arthritis was analyzed by histology and immunohistochemistry. RESULTS: Oxidized LDL and LOX-1 proteins were detected in the RA synovial tissue. Levels of MMP-1 and MMP-3 were enhanced by stimulation of RA FLS with ox-LDL, and the production of both MMPs was inhibited by blockade of the ox-LDL-LOX-1 interaction with the anti-LOX-1 neutralizing antibody or sLOX-1. Levels of sLOX-1 in the plasma and synovial fluid of RA patients were significantly higher than those in OA patients and healthy controls and were positively correlated with inflammation markers and the extent of RA disease activity. In the knees of mice, blockade of the ox-LDL-LOX-1 interaction suppressed arthritic changes and reduced the expression of MMP-3 induced by ox-LDL. CONCLUSION: These findings strongly indicate that sLOX-1 is a novel biomarker that may be useful for the diagnosis of RA and for the evaluation of disease activity in RA. Furthermore, the results suggest that LOX-1 may be a potent therapeutic target for RA.

LOX-1: a multiligand receptor at the crossroads of response to danger signals.

PURPOSE OF REVIEW: LOX-1 is a multiligand receptor implicated in endothelial dysfunction and atherosclerosis, although it was originally identified as an oxidized LDL receptor. In this review, the roles of various LOX-1 ligands and their interaction with LOX-1 are discussed to understand the pathophysiological significance of LOX-1. RECENT FINDINGS: LOX-1 knockout mice showed resistance of endothelium-dependent vasorelaxation against oxidized LDL and retardation of atherosclerosis progression. LOX-1 ligand reduction in mice also attenuated atherosclerosis progression. In a human cohort study, high concentration of apoB-containing LOX-1 ligands predicted the incidence of cardiovascular disease. Furthermore, modified HDL, which existed in high concentration in the plasma of coronary artery disease patients, was found to induce impairment of endothelial nitric oxide release via LOX-1. In addition to lipoproteins, LOX-1 was found to work as a C-reactive protein receptor providing a scaffold for the activation of the complement system. SUMMARY: LOX-1 is a unique molecule among the sensors of danger signals. LOX-1 is not only sensing danger signals such as modified LDL and heat shock protein, but also scaffolding other danger sensors including C-reactive protein and C1q, and directly commanding responses to danger signals by working as a cell adhesion molecule. Via these functions, LOX-1 might work as a surveillance molecule of vascular homeostasis.

NanoSPECT imaging reveals the uptake of 123I-labelled oxidized low-density lipoprotein in the brown adipose tissue of mice via CD36.

AIMS: The liver is the major organ shown to remove oxidized low-density lipoprotein (oxLDL) from the circulation. Given increased evidence that thermogenic adipose tissue has anti-effects, we used 123I-labelled oxLDL as a tracer to reveal oxLDL accumulation in the brown adipose tissue (BAT) of mice. We also explored the mechanisms of oxLDL accumulation in BAT. METHODS AND RESULTS: We used high-resolution nanoSPECT/CT to investigate the tissue distribution of 123I-oxLDL and 123I-LDL (control) following intravenous injection into conscious mice. 123I-oxLDL distribution was discovered in BAT at an intensity equivalent to that in the liver, whereas 123I-LDL was detected mostly in the liver. Consistent with the function of BAT related to sympathetic nerve activity, administering anaesthesia in mice almost completely eliminated the accumulation of 123I-oxLDL in BAT, and this effect was reversed by administering ß3-agonist. Furthermore, exposing mice to cold stress at 4°C enhanced 123I-oxLDL accumulation in BAT. Because in 123I-oxLDL, the protein of oxLDL was labelled, we performed additional experiments with DiI-oxLDL in which the lipid phase of oxLDL was fluorescently labelled and observed similar results, suggesting that the whole oxLDL particle was taken up by BAT. To identify the receptor responsible for oxLDL uptake in BAT, we analysed the expression of known oxLDL receptors (e.g. SR-A, CD36, and LOX-1) in cultured brown adipocyte cell line and primary brown adipocytes and found that CD36 was the major receptor expressed. Treatment of cells with CD36 siRNA or CD36 neutralizing antibody significantly inhibited DiI-oxLDL uptake. Finally, CD36 deletion in mice abolished the accumulation of 123I-oxLDL and DiI-oxLDL in BAT, indicating that CD36 is the major receptor for oxLDL in BAT. CONCLUSION: We show novel evidence for the CD36-mediated accumulation of oxLDL in BAT, suggesting that BAT may exert its anti-atherogenic effects by removing atherogenic LDL from the circulation.

LOX-1 deficiency increases ruptured abdominal aortic aneurysm via thinning of adventitial collagen.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Conversely, LOX-1 deficiency has been shown to decrease inflammation and atherosclerosis, both of which have been proposed to contribute to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown. Here, we investigated the effects of Olr1 (which encodes LOX-1) deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E knockout (ApoE KO) mice to determine whether LOX-1 deficiency mitigates AAA development. To accomplish this, we used serial, non-invasive ultrasound assessment, which revealed that the incidence and expansion rate of AAA were similar regardless of Olr1 deletion. However, Olr1 deletion significantly increased severe AAAs, including ruptured AAAs resulting in death. Oil Red O staining of the harvested aortas showed that the extent of atheroma burden localized in aneurysmal lesions did not differ between LOX-1-deficient and control mice, suggesting that Olr1 deletion did not decrease atheroma burden in the aneurysmal wall. Further histopathological analysis revealed that aneurysmal lesions in LOX-1-deficient mice had fewer fibroblasts and myofibroblasts, as well as thinner adventitial collagen, although the degree of elastin fragmentation or disruption was similar between LOX-1-deficient and control mice. An in vitro study confirmed that the proliferation of adventitial fibroblasts collected from LOX-1-deficient mice was significantly attenuated despite Ang II stimulation. In conclusion, Olr1 deletion may not mitigate aneurysm development but rather increases the vulnerability of rupture by suppressing adventitial fibroblast proliferation and collagen synthesis.

Lectin-like oxidized LDL receptor 1 is involved in CRP-mediated complement activation.

BACKGROUND: C-reactive protein (CRP) is purported to be a risk factor that acts independently of LDL cholesterol in predicting all-cause mortality in patients with ischemic heart disease. Lectin-like oxidized LDL receptor 1 (LOX-1) impairs endothelial function and exacerbates myocardial injury. We recently demonstrated that CRP increased vascular permeability through direct binding to LOX-1. Here we examined, using a hypertensive rat model, whether LOX-1 is involved in CRP-induced complement activation. METHODS AND RESULTS: In the cultured LOX-1-expressing cell line hLOX-1-CHO, CRP increased complement activation, but did not do so in native CHO cells. Depleting C1q from serum abolished CRP-induced complement activation. Incubation of CRP with serum on immobilized recombinant LOX-1 similarly showed that CRP activated C1q-requiring classical complement pathway in a LOX-1-dependent manner. Interestingly, the interaction between CRP and LOX-1 was dependent on Ca²âº ion and competed with phosphocholine, suggesting that LOX-1 bound to the B-face of CRP with a phosphocholine-binding domain. This was in contrast to Fcγ receptors, to which CRP bound in A-face with complement-binding domain. In vivo, intradermal injection of CRP to hypertensive SHRSP rats induced complement activation detected by C3d deposition and leukocyte infiltration around the injected area. Anti-LOX-1 antibody reduced the extent of complement activation and leukocyte infiltration. CONCLUSIONS: LOX-1 appears to be involved in CRP-induced complement activation, and thus may serve to locate the site of CRP-induced complement activation and inflammation.

The discovery of LOX-1, its ligands and clinical significance.

LOX-1 is an endothelial receptor for oxidized low-density lipoprotein (oxLDL), a key molecule in the pathogenesis of atherosclerosis.The basal expression of LOX-1 is low but highly induced under the influence of proinflammatory and prooxidative stimuli in vascular endothelial cells, smooth muscle cells, macrophages, platelets and cardiomyocytes. Multiple lines of in vitro and in vivo studies have provided compelling evidence that LOX-1 promotes endothelial dysfunction and atherogenesis induced by oxLDL. The roles of LOX-1 in the development of atherosclerosis, however, are not simple as it had been considered. Evidence has been accumulating that LOX-1 recognizes not only oxLDL but other atherogenic lipoproteins, platelets, leukocytes and CRP. As results, LOX-1 not only mediates endothelial dysfunction but contributes to atherosclerotic plaque formation, thrombogenesis, leukocyte infiltration and myocardial infarction, which determine mortality and morbidity from atherosclerosis. Moreover, our recent epidemiological study has highlighted the involvement of LOX-1 in human cardiovascular diseases. Further understandings of LOX-1 and its ligands as well as its versatile functions will direct us to ways to find novel diagnostic and therapeutic approaches to cardiovascular disease.

Procyanidins are potent inhibitors of LOX-1: a new player in the French Paradox.

Lectin-like oxidized LDL receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL (oxLDL) and plays multiple roles in the development of cardiovascular diseases. We screened more than 400 foodstuff extracts for identifying materials that inhibit oxLDL binding to LOX-1. Results showed that 52 extracts inhibited LOX-1 by more than 70% in cell-free assays. Subsequent cell-based assays revealed that a variety of foodstuffs known to be rich in procyanidins such as grape seed extracts and apple polyphenols, potently inhibited oxLDL uptake in Chinese hamster ovary (CHO) cells expressing LOX-1. Indeed, purified procyanidins significantly inhibited oxLDL binding to LOX-1 while other ingredients of apple polyphenols did not. Moreover, chronic administration of oligomeric procyanidins suppressed lipid accumulation in vascular wall in hypertensive rats fed with high fat diet. These results suggest that procyanidins are LOX-1 inhibitors and LOX-1 inhibition might be a possible underlying mechanism of the well-known vascular protective effects of red wine, the French Paradox.

Critical role of oxidized LDL receptor-1 in intravascular thrombosis in a severe influenza mouse model.

Although coagulation abnormalities, including microvascular thrombosis, are thought to contribute to tissue injury and single- or multiple-organ dysfunction in severe influenza, the detailed mechanisms have yet been clarified. This study evaluated influenza-associated abnormal blood coagulation utilizing a severe influenza mouse model. After infecting C57BL/6 male mice with intranasal applications of 500 plaque-forming units of influenza virus A/Puerto Rico/8/34 (H1N1; PR8), an elevated serum level of prothrombin fragment 1 + 2, an indicator for activated thrombin generation, was observed. Also, an increased gene expression of oxidized low-density lipoprotein (LDL) receptor-1 (Olr1), a key molecule in endothelial dysfunction in the progression of atherosclerosis, was detected in the aorta of infected mice. Body weight decrease, serum levels of cytokines and chemokines, viral load, and inflammation in the lungs of infected animals were similar between wild-type and Olr1 knockout (KO) mice. In contrast, the elevation of prothrombin fragment 1 + 2 levels in the sera and intravascular thrombosis in the lungs by PR8 virus infection were not induced in KO mice. Collectively, the results indicated that OLR1 is a critical host factor in intravascular thrombosis as a pathogeny of severe influenza. Thus, OLR1 is a promising novel therapeutic target for thrombosis during severe influenza.

Abnormal Blood Coagulation and Kidney Damage in Aged Hamsters Infected with Severe Acute Respiratory Syndrome Coronavirus 2.

Systemic symptoms have often been observed in patients with coronavirus disease 2019 (COVID-19) in addition to pneumonia, however, the details are still unclear due to the lack of an appropriate animal model. In this study, we investigated and compared blood coagulation abnormalities and tissue damage between male Syrian hamsters of 9 (young) and over 36 (aged) weeks old after intranasal infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite similar levels of viral replication and inflammatory responses in the lungs of both age groups, aged but not young hamsters showed significant prolongation of prothrombin time and prominent acute kidney damage. Moreover, aged hamsters demonstrated increased intravascular coagulation time-dependently in the lungs, suggesting that consumption of coagulation factors causes prothrombin time prolongation. Furthermore, proximal urinary tract damage and mesangial matrix expansion were observed in the kidneys of the aged hamsters at early and later disease stages, respectively. Given that the severity and mortality of COVID-19 are higher in elderly human patients, the effect of aging on pathogenesis needs to be understood and should be considered for the selection of animal models. We, thus, propose that the aged hamster is a good small animal model for COVID-19 research.

RAGE ligands stimulate angiotensin II type I receptor (AT1) via RAGE/AT1 complex on the cell membrane.

The receptor for advanced glycation end-products (RAGE) and the G protein-coupled angiotensin II (AngII) type I receptor (AT1) play a central role in cardiovascular diseases. It was recently reported that RAGE modifies AngII-mediated AT1 activation via the membrane oligomeric complex of the two receptors. In this study, we investigated the presence of the different directional crosstalk in this phenomenon, that is, the RAGE/AT1 complex plays a role in the signal transduction pathway of RAGE ligands. We generated Chinese hamster ovary (CHO) cells stably expressing RAGE and AT1, mutated AT1, or AT2 receptor. The activation of two types of G protein α-subunit, Gq and Gi, was estimated through the accumulation of inositol monophosphate and the inhibition of forskolin-induced cAMP production, respectively. Rat kidney epithelial cells were used to assess RAGE ligand-induced cellular responses. We determined that RAGE ligands activated Gi, but not Gq, only in cells expressing RAGE and wildtype AT1. The activation was inhibited by an AT1 blocker (ARB) as well as a RAGE inhibitor. ARBs inhibited RAGE ligand-induced ERK phosphorylation, NF-κB activation, and epithelial-mesenchymal transition of rat renal epithelial cells. Our findings suggest that the activation of AT1 plays a central role in RAGE-mediated cellular responses and elucidate the role of a novel molecular mechanism in the development of cardiovascular diseases.

The endocytosis of oxidized LDL via the activation of the angiotensin II type 1 receptor.

Arrestin-dependent activation of a G-protein-coupled receptor (GPCR) triggers endocytotic internalization of the receptor complex. We analyzed the interaction between the pattern recognition receptor (PRR) lectin-like oxidized low-density lipoprotein (oxLDL) receptor (LOX-1) and the GPCR angiotensin II type 1 receptor (AT1) to report a hitherto unidentified mechanism whereby internalization of the GPCR mediates cellular endocytosis of the PRR ligand. Using genetically modified Chinese hamster ovary cells, we found that oxLDL activates Gαi but not the Gαq pathway of AT1 in the presence of LOX-1. Endocytosis of the oxLDL-LOX-1 complex through the AT1-ß-arrestin pathway was demonstrated by real-time imaging of the membrane dynamics of LOX-1 and visualization of endocytosis of oxLDL. Finally, this endocytotic pathway involving GPCR kinases (GRKs), ß-arrestin, and clathrin is relevant in accumulating oxLDL in human vascular endothelial cells. Together, our findings indicate that oxLDL activates selective G proteins and ß-arrestin-dependent internalization of AT1, whereby the oxLDL-LOX-1 complex undergoes endocytosis.

C-reactive protein uptake by macrophage cell line via class-A scavenger receptor.

BACKGROUND: C-reactive protein (CRP) increases in response to inflammation and is purported to be a risk factor for atherogenesis. We recently demonstrated that a scavenger receptor, lectin-like oxidized LDL receptor (LOX-1), is a receptor for CRP. In light of the overlapping ligand spectrum of scavenger receptors such as modified LDL, bacteria, and advanced glycation end products, we examined whether other scavenger receptors recognize CRP. METHODS: We analyzed the uptake of fluorescently labeled CRP in COS-7 cells expressing a series of scavenger receptors and in a monocytic cell line, THP-1, differentiated into macrophage with phorbol 12-myristate 13-acetate (PMA). We applied small interfering RNA (siRNA) against class-A scavenger receptor (SR-A) to THP-1 cells to suppress the expression of SR-A. We also analyzed the binding of nonlabeled CRP to immobilized recombinant LOX-1 and SR-A in vitro using anti-CRP antibody. RESULTS: COS-7 cells expressing LOX-1 and SR-A internalized fluorescently labeled CRP in a dose-dependent manner, but cells expressing CD36, SR-BI, or CD68 did not. The recombinant LOX-1 and SR-A proteins recognized nonlabeled purified CRP and native CRP in serum in vitro. THP-1 cells differentiated into macrophage-like cells by treatment with PMA-internalized fluorescently labeled CRP. siRNA against SR-A significantly and concomitantly inhibited the expression of SR-A (P < 0.01) and CRP uptake (P < 0.01), whereas control siRNA did not. CONCLUSIONS: CRP is recognized by SR-A as well as LOX-1 and taken up via SR-A in a macrophage-like cell line. This process might be of significance in the pathogenesis of atherosclerotic disease.

LOX index, a novel predictive biochemical marker for coronary heart disease and stroke.

BACKGROUND: Lectin-like oxidized LDL receptor 1 (LOX-1) is implicated in atherothrombotic diseases. Activation of LOX-1 in humans can be evaluated by use of the LOX index, obtained by multiplying the circulating concentration of LOX-1 ligands containing apolipoprotein B (LAB) times that of the soluble form of LOX-1 (sLOX-1) [LOX index = LAB x sLOX-1]. This study aimed to establish the prognostic value of the LOX index for coronary heart disease (CHD) and stroke in a community-based cohort. METHODS: An 11-year cohort study of 2437 residents age 30-79 years was performed in an urban area located in Japan. Of these, we included in the analysis 1094 men and 1201 women without history of stroke and CHD. We measured LAB and sLOX-1 using ELISAs with recombinant LOX-1 and monoclonal anti-apolipoprotein B antibody and with 2 monoclonal antibodies against LOX-1, respectively. RESULTS: During the follow-up period, there were 68 incident cases of CHD and 91 cases of stroke (with 60 ischemic strokes). Compared with the bottom quartile, the hazard ratio (HR) of the top quartile of LOX index was 1.74 (95% CI 0.92-3.30) for stroke and 2.09 (1.00-4.35) for CHD after adjusting for sex, age, body mass index, drinking, smoking, hypertension, diabetes, non-HDL cholesterol, and use of lipid-lowering agents. Compared with the bottom quartile of LOX index, the fully adjusted HRs for ischemic stroke were consistently high from the second to the top quartile: 3.39 (95% CI 1.34-8.53), 3.15 (1.22-8.13) and 3.23 (1.24-8.37), respectively. CONCLUSIONS: Higher LOX index values were associated with an increased risk of CHD. Low LOX index values may be protective against ischemic stroke.