Rheb (Ras Homolog Enriched in Brain 1) Deficiency in Mature Macrophages Prevents Atherosclerosis by Repressing Macrophage Proliferation, Inflammation, and Lipid Uptake.

OBJECTIVE: Macrophage foam cell formation is an important process in atherosclerotic plaque development. The small GTPase Rheb (Ras homolog enriched in brain 1) regulates endocytic trafficking that is critical for foam cell formation. However, it is unclear whether and how macrophage Rheb regulates atherogenesis, which are the focuses of the current study. Approach and Results: Immunofluorescence study confirmed the colocalization of Rheb in F4/80 and Mac-2 (galectin-3)-labeled lesional macrophages. Western blot and fluorescence-activated cell sorting analysis showed that Rheb expression was significantly increased in atherosclerotic lesions of atherosclerosis-prone (apoE-/- [apolipoprotein E deficient]) mice fed with Western diet. Increased Rheb expression was also observed in oxidized LDL (low-density lipoprotein)-treated macrophages. To investigate the in vivo role of macrophage Rheb, we established mature RhebmKO (macrophage-specific Rheb knockout) mice by crossing the Rheb floxed mice with F4/80-cre mice. Macrophage-specific knockout of Rheb in mice reduced Western diet-induced atherosclerotic lesion by 32%, accompanied with a decrease in macrophage content in plaque. Mechanistically, loss of Rheb in macrophages repressed oxidized LDL-induced lipid uptake, inflammation, and macrophage proliferation. On the contrary, lentivirus-mediated overexpression of Rheb in macrophages increased oxidized LDL-induced lipid uptake and inflammation, and the stimulatory effect of Rheb was suppressed by the mTOR (mammalian target of rapamycin) inhibitor rapamycin or the PKA (protein kinase A) activator forskolin. CONCLUSIONS: Macrophage Rheb plays important role in Western diet-induced atherosclerosis by promoting macrophage proliferation, inflammation, and lipid uptake. Inhibition of expression and function of Rheb in macrophages is beneficial to prevent diet-induced atherosclerosis.

LDL switches the LRP6 internalization route from flotillin dependent to clathrin dependent in hepatic cells.

Low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) was originally identified as a co-receptor of the Wnt signalling pathway and has been shown to be involved in LDL transport. In polarized hepatocytes, many apical proteins are sorted to the basolateral membrane and then internalized and transported to the apical bile canalicular membrane - a process known as transcytosis. We show that LRP6 is transcytosed to the apical membrane of polarized hepatic HepG2 cells via a flotillin-dependent manner in the absence of LDL. LRP6 formed a complex with Niemann-Pick type C1-like 1 (NPC1L1), which is localized to the bile canalicular membrane of the liver and is involved in cholesterol absorption from the bile. LRP6 was required for apical membrane localization of NPC1L1 in the absence of LDL. Clathrin-dependent LRP6 internalization occurred in the presence of LDL, which resulted in trafficking of LRP6 to the lysosome, thereby reducing apical sorting of LRP6 and NPC1L1. These results suggest that LRP6 endocytosis proceeds by two routes, depending on the presence of LDL, and that LRP6 controls the intracellular destination of NPC1L1 in hepatocytes.

Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths.

Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.

Atractylenolide I restores HO-1 expression and inhibits Ox-LDL-induced VSMCs proliferation, migration and inflammatory responses in vitro.

Pathogenesis of atherosclerosis is characterized by the proliferation and migration of vascular smooth muscle cells (VSMCs) and inflammatory lesions. The aim of this study is to elucidate the effect of atractylenolide I (AO-I) on smooth muscle cell inflammation, proliferation and migration induced by oxidized modified low density lipoprotein (Ox-LDL). Here, We found that atractylenolide I inhibited Ox-LDL-induced VSMCs proliferation and migration in a dose-dependent manner, and decreased the production of inflammatory cytokines and the expression of monocyte chemoattractant protein-1 (MCP-1) in VSMCs. The study also identified that AO-I prominently inhibited p38-MAPK and NF-κB activation. More importantly, the specific heme oxygenase-1 (HO-1) inhibitor zinc protoporphyrin (ZnPP) IX partially abolished the beneficial effects of atractylenolide I on Ox-LDL-induced VSMCs. Furthermore, atractylenolide I blocked the foam cell formation in macrophages induced by Ox-LDL. In summary, inhibitory roles of AO-I in VSMCs proliferation and migration, lipid peroxidation and subsequent inflammatory responses might contribute to the anti-atherosclerotic property of AO-I.

Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel.

AIMS: To appraise the clinical and genetic evidence that low-density lipoproteins (LDLs) cause atherosclerotic cardiovascular disease (ASCVD). METHODS AND RESULTS: We assessed whether the association between LDL and ASCVD fulfils the criteria for causality by evaluating the totality of evidence from genetic studies, prospective epidemiologic cohort studies, Mendelian randomization studies, and randomized trials of LDL-lowering therapies. In clinical studies, plasma LDL burden is usually estimated by determination of plasma LDL cholesterol level (LDL-C). Rare genetic mutations that cause reduced LDL receptor function lead to markedly higher LDL-C and a dose-dependent increase in the risk of ASCVD, whereas rare variants leading to lower LDL-C are associated with a correspondingly lower risk of ASCVD. Separate meta-analyses of over 200 prospective cohort studies, Mendelian randomization studies, and randomized trials including more than 2 million participants with over 20 million person-years of follow-up and over 150 000 cardiovascular events demonstrate a remarkably consistent dose-dependent log-linear association between the absolute magnitude of exposure of the vasculature to LDL-C and the risk of ASCVD; and this effect appears to increase with increasing duration of exposure to LDL-C. Both the naturally randomized genetic studies and the randomized intervention trials consistently demonstrate that any mechanism of lowering plasma LDL particle concentration should reduce the risk of ASCVD events proportional to the absolute reduction in LDL-C and the cumulative duration of exposure to lower LDL-C, provided that the achieved reduction in LDL-C is concordant with the reduction in LDL particle number and that there are no competing deleterious off-target effects. CONCLUSION: Consistent evidence from numerous and multiple different types of clinical and genetic studies unequivocally establishes that LDL causes ASCVD.

The role of the mLDL-induced activation of the complement system classical pathway and C3 expression stimulation in atherosclerosis.

The role of modified low density lipoprotein in the activation of the classical pathway of the complement system and increasing expression C3 gene in human macrophages is described, role of these processes on the progression of atherosclerotic vascular lesions is considering.

The function of high-density lipoprotein and low-density lipoprotein in the maintenance of mouse ovarian steroid balance.

The membrane proteins, low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SR-BI, gene name Scarb1), are lipoprotein receptors that play central roles in lipoprotein metabolism. Cholesterol bound in high-density lipoprotein (HDL) and LDL is transported into cells mainly by SR-BI and LDLR. The relative contribution of LDL and HDL to the steroidogenic cholesterol pool varies among species and may vary among tissues within one species. To investigate which of these pathways is more important in the supply of cholesterol in mouse ovary, we utilized immunohistochemistry, western blotting, RNAi, and RT-PCR as well as Ldlr-/- mice to explore the uptake of HDL and LDL in the ovary. Our data demonstrate that both SR-BI and LDLR are present in the interstitial cells, thecal cells, and corpora lutea (CLs), and their expression fluctuates with the development of follicles and CLs. The intracellular cholesterol concentration was significantly decreased when Ldlr or Scarb1 was silenced in luteal cells. Furthermore, Ldlr-/- mice had lower progesterone and estrogen levels compared to wild-type mice, and when Ldlr-/- mice were treated with the inhibitor of de novo cholesterol synthesis, lovastatin, serum progesterone, and estrogen concentrations were further reduced. These results demonstrate that both LDLR and SR-BI play important roles in importing cholesterol and that both HDL and LDL are crucial in steroidogenesis in mouse ovaries.

miR-497 accelerates oxidized low-density lipoprotein-induced lipid accumulation in macrophages by repressing the expression of apelin.

microRNAs (miRNAs) play important roles in the pathogenesis of atherosclerosis. A previous study has reported that miR-497 is elevated in advanced atherosclerotic lesions in an apoE-deficient (apoE-/-) mouse model. The purpose of this study is to test whether miR-497 can modulate macrophage foam cell formation, an initiating event in atherosclerosis. We found that miR-497 was upregulated in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). Enforced expression of miR-497 promoted lipid accumulation and decreased cholesterol efflux in oxLDL-exposed THP-1 macrophages. In contrast, downregulation of miR-497 suppressed oxLDL-induced lipid accumulation in THP-1 macrophages. Apelin was identified to be a downstream target of miR-497. Overexpression of miR-497 significantly reduced the expression of apelin in THP-1 macrophages. Interestingly, delivery of a miR-497-resistant variant of apelin significantly inhibited lipid accumulation and enhanced cholesterol efflux in miR-497-overexpressing THP-1 macrophages in response to oxLDL. In addition, miR-497 expression was increased and negatively correlated with apelin protein expression in human atherosclerotic lesions. In conclusion, miR-497 contributes to oxLDL-induced lipid deposition in macrophages largely via targeting of apelin and thus represents a potential therapeutic target for atherosclerosis.

Synaptic Targeting of Kainate Receptors.

When native and recombinant kainate receptors (KARs) are compared, there is a mismatch in several of their functional properties. While both generate currents, synaptic responses mediated by KARs have rarely observed in cultured hippocampal neurons. The recent discovery of auxiliary proteins for KARs, such as Netos, offers an explanation for these discrepancies. We found that the GluK5 KAR subunit and the ancillary proteins, Neto1 and Neto2, are not expressed by hippocampal neurons in culture. Therefore, we used this model to directly test whether these proteins are required for the synaptic localization of KARs. Transfection of GluK4, GluK5, Neto1, or Neto2 into hippocampal neurons was associated with the appearance of synaptic KAR-mediated EPSCs. However, GluK4 or GluK5 alone produced synaptic activity in a significant proportion of cells and with reliable event frequency. While neurons expressing GluK4 or GluK5 subunits displayed synaptic responses with rapid kinetics, the expression of Neto proteins conferred these synaptic responses with their characteristic slow onset and decay rates. These data reveal some requirements for KAR targeting to the synapse, indicating a fundamental role of high affinity KAR subunits in this process.

Anti-inflammatory effect of a novel formulation of coconut inflorescence sap against ox-LDL induced inflammatory responses in human peripheral blood mononuclear cells by modulating TLR-NF-κB signaling pathway.

Oxidized low density lipoprotein (ox LDL) induced inflammatory response was reported to play an important role in the pathogenesis of atherosclerosis. The purpose of this study was to explore the anti-inflammatory effect of a novel formulation of coconut inflorescence sap (CSP); COCOZEN™ against ox-LDL induced inflammatory responses in human peripheral blood mononuclear cells (hPBMCs). The hPBMCs were isolated from healthy human volunteers and cultured in collagen coated plates at 37 °C. The cells were grouped as Group I (Control), Group II (ox-LDL treated) and Group III (ox-LDL + CSP treated). Further analysis of inflammatory markers, reactive oxygen species, mRNA and protein expression levels indicated increased expressions of TLR-4, TNF-α, IL-6 and VCAM-1 in ox-LDL treated group along with the nuclear translocation of NF-κB. Other inflammatory markers such as LOX, PGE2, NO, total COX and lipid peroxidation level were also found to be significantly (p < .05) increased upon Ox-LDL treatment. The treatment with CSP on the other hand was found to down regulate and reverse the ox-LDL-induced alterations indicating its potential anti-inflammatory effect on hPBMCs via TLR-NF-κB signaling pathway.

Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation.

Endothelial biomechanics is emerging as a key factor in endothelial function. Here, we address the mechanisms of endothelial stiffening induced by oxidized LDL (oxLDL) and investigate the role of oxLDL in lumen formation. We show that oxLDL-induced endothelial stiffening is mediated by CD36-dependent activation of RhoA and its downstream target, Rho kinase (ROCK), via inhibition of myosin light-chain phosphatase (MLCP) and myosin light-chain (MLC)2 phosphorylation. The LC-MS/MS analysis identifies 7-ketocholesterol (7KC) as the major oxysterol in oxLDL. Similarly to oxLDL, 7KC induces RhoA activation, MLCP inhibition, and MLC2 phosphorylation resulting in endothelial stiffening. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. An increase in lumen formation was also observed in response to pre-exposing the cells to 7KC, an oxysterol that induces endothelial stiffening, but not to 5α,6α epoxide that does not affect endothelial stiffness. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade, and reversed oxLDL-induced lumen formation. In summary, we show that oxLDL-induced endothelial stiffening is mediated by the CD36/RhoA/ROCK/MLCP/MLC2 pathway and is associated with increased endothelial angiogenic activity.

Electronegative low density lipoprotein induces renal apoptosis and fibrosis: STRA6 signaling involved.

Dyslipidemia has been proven to capably develop and aggravate chronic kidney disease. We also report that electronegative LDL (L5) is the most atherogenic LDL. On the other hand, retinoic acid (RA) and RA receptor (RAR) agonist are reported to be beneficial in some kidney diseases. "Stimulated by retinoic acid 6" (STRA6), one retinol-binding protein 4 receptor, was recently identified to regulate retinoid homeostasis. Here, we observed that L5 suppressed STRA6 cascades [STRA6, cellular retinol-binding protein 1 (CRBP1), RARs, retinoid X receptor α, and retinol, RA], but L5 simultaneously induced apoptosis and fibrosis (TGFß1, Smad2, collagen 1, hydroxyproline, and trichrome) in kidneys of L5-injected mice and L5-treated renal tubular cells. These L5-induced changes of STRA6 cascades, renal apoptosis, and fibrosis were reversed in kidneys of LOX1(-/-) mice. LOX1 RNA silencing and inhibitor of c-Jun N-terminal kinase and p38MAPK rescued the suppression of STRA6 cascades and apoptosis and fibrosis in L5-treated renal tubular cells. Furthermore, crbp1 gene transfection reversed downregulation of STRA6 cascades, apoptosis, and fibrosis in L5-treated renal tubular cells. For mimicking STRA6 deficiency, efficient silencing of STRA6 RNA was performed and was found to repress STRA6 cascades and caused apoptosis and fibrosis in L1-treated renal tubular cells. In summary, this study reveals that electronegative L5 can cause kidney apoptosis and fibrosis via the suppression of STRA6 cascades, and implicates that STRA6 signaling may be involved in dyslipidemia-mediated kidney disease.

VNN1 promotes atherosclerosis progression in apoE-/- mice fed a high-fat/high-cholesterol diet.

Accumulated evidence shows that vanin-1 (VNN1) plays a key part in glucose metabolism. We explored the effect of VNN1 on cholesterol metabolism, inflammation, apoptosis in vitro, and progression of atherosclerotic plaques in apoE(-/-) mice. Oxidized LDL (Ox-LDL) significantly induced VNN1 expression through an ERK1/2/cyclooxygenase-2/PPARα signaling pathway. VNN1 significantly increased cellular cholesterol content and decreased apoAI and HDL-cholesterol (HDL-C)-mediated efflux by 25.16% and 23.13%, respectively, in THP-1 macrophage-derived foam cells (P < 0.05). In addition, VNN1 attenuated Ox-LDL-induced apoptosis through upregulation of expression of p53 by 59.15% and downregulation of expression of B-cell lymphoma-2 127.13% in THP-1 macrophage (P < 0.05). In vivo, apoE(-/-) mice were divided randomly into two groups and transduced with lentivirus (LV)-Mock or LV-VNN1 for 12 weeks. VNN1-treated mice showed increased liver lipid content and plasma levels of TG (124.48%), LDL-cholesterol (119.64%), TNF-α (148.74%), interleukin (IL)-1ß (131.81%), and IL-6 (156.51%), whereas plasma levels of HDL-C (25.75%) were decreased significantly (P < 0.05). Consistent with these data, development of atherosclerotic lesions was increased significantly upon infection of apoE(-/-) mice with LV-VNN1. These observations suggest that VNN1 may be a promising therapeutic candidate against atherosclerosis.

K Domain CR9 of Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1) Is Critical for Aggregated LDL-induced Foam Cell Formation from Human Vascular Smooth Muscle Cells.

Low density lipoprotein receptor-related protein (LRP1) mediates the internalization of aggregated LDL (AgLDL), which in turn increases the expression of LRP1 in human vascular smooth muscle cells (hVSMCs). This positive feedback mechanism is thus highly efficient to promote the formation of hVSMC foam cells, a crucial vascular component determining the susceptibility of atherosclerotic plaque to rupture. Here we have determined the LRP1 domains involved in AgLDL recognition with the aim of specifically blocking AgLDL internalization in hVSMCs. The capacity of fluorescently labeled AgLDL to bind to functional LRP1 clusters was tested in a receptor-ligand fluorometric assay made by immobilizing soluble LRP1 "minireceptors" (sLRP1-II, sLRP1-III, and sLRP1-IV) recombinantly expressed in CHO cells. This assay showed that AgLDL binds to cluster II. We predicted three well exposed and potentially immunogenic peptides in the CR7-CR9 domains of this cluster (termed P1 (Cys(1051)-Glu(1066)), P2 (Asp(1090)-Cys(1104)), and P3 (Gly(1127)-Cys(1140))). AgLDL, but not native LDL, bound specifically and tightly to P3-coated wells. Rabbit polyclonal antibodies raised against P3 prevented AgLDL uptake by hVSMCs and were almost twice as effective as anti-P1 and anti-P2 Abs in reducing intracellular cholesteryl ester accumulation. Moreover, anti-P3 Abs efficiently prevented AgLDL-induced LRP1 up-regulation and counteracted the down-regulatory effect of AgLDL on hVSMC migration. In conclusion, domain CR9 appears to be critical for LRP1-mediated AgLDL binding and internalization in hVSMCs. Our results open new avenues for an innovative anti-VSMC foam cell-based strategy for the treatment of vascular lipid deposition in atherosclerosis.

[Correlations between lipid ratio/oxidative stress status in COPD patients and pulmonary hypertension as well as prognosis].

OBJECTIVE: To investigate the correlations between lipid ratio/oxidative stress status in chronic obstructive pulmonary disease (COPD) patients and pulmonary hypertension as well as the prognosis.
 Methods: A total of 120 patients with COPD were randomly selected and served as the COPD group and 30 healthy persons were selected as the control group. The ratios of low density lipoprotein (LDL)/high-density lipoprotein (HDL), triglyceride (TG)/HDL and total cholesterol (TC)/HDL were measured. The superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) level in the control group and COPD patients were detected. Pulmonary hypertension incidence and 3-year survival rate for COPD patients were statistically analyzed. Spearman rank correlation method was used to analyze relationship between lipid ratio /oxidative stress status and pulmonary hypertension.
 Results: Compared with control group, the ratios of LDL/HDL, TG/HDL and TC/HDL, and the serum MDA level in the COPD group were increased, while the serum SOD and T-AOC level in the COPD group were decreased; compared with stable period, lipid ratios and MDA levels in the acute period were elevated, while serum SOD and T-AOC levels were reduced (P<0.05). Pulmonary hypertension incidence and 3-year survival rates in the COPD group were 56.67% and 81.67% respectively; the lipid ratios and serum MDA levels in COPD patients with pulmonary hypertension were elevated compared with that in COPD patients without pulmonary hypertension; the serum SOD and T-AOC levels in COPD patients with pulmonary hypertension were reduced compared with that in patients without pulmonary hypertension (P<0.05). Spearman rank correlation analysis showed that ratios of LDL/HDL, TG/HDL and TC/HDL, and the serum MDA levels in COPD patients were positively correlated with 3-years pulmonary hypertension incidence (r=0.752, 0.748, 0.752, 0.748; P<0.05), and negatively correlated with 3-years survival rate (r=-0.722, -0.751, -0.736, -0.748; P<0.05); serum SOD and T-AOC levels in COPD patients were negatively correlated with 3-years pulmonary hypertension (r=-0.711, -0.734; P<0.05), and positively correlated with 3-year survival rate (r=0.726, 0.733; P<0.05). 
 Conclusion: Blood lipid ratio and oxidative stress levels in COPD patients are elevated while antioxidant abilities were attenuated. The lipid ratio and oxidative stress status in COPD patients is closely related to the prognosis of pulmonary hypertension. Therefore, blood lipid ratio and oxidative stress status may be used in evaluation of pulmonary hypertension and prognosis for COPD patients.

Cancer stem cell-specific scavenger receptor CD36 drives glioblastoma progression.

Glioblastoma (GBM) contains a self-renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self-renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient-derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self-renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self-renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non-CSCs, increased with exposure to oxidized low-density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages.

Carbamylated low-density lipoprotein induces endothelial dysfunction.

AIMS: Cardiovascular events remain the leading cause of death in Western world. Atherosclerosis is the most common underlying complication driven by low-density lipoproteins (LDL) disturbing vascular integrity. Carbamylation of lysine residues, occurring primarily in the presence of chronic kidney disease (CKD), may affect functional properties of lipoproteins; however, its effect on endothelial function is unknown. METHODS AND RESULTS: Low-density lipoprotein from healthy donors was isolated and carbamylated. Vascular reactivity after treatment with native LDL (nLDL) or carbamylated LDL (cLDL) was examined in organ chambers for isometric tension recording using aortic rings of wild-type or lectin-like-oxidized LDL receptor-1 (LOX-1) transgenic mice. Reactive oxygen species (ROS) and nitric oxide (NO) production were determined using electron spin resonance spectroscopy. The effect of LDL-carbamyl-lysine levels on cardiovascular outcomes was determined in patients with CKD during a median follow-up of 4.7 years. Carbamylated LDL impaired endothelium-dependent relaxation to acetylcholine or calcium-ionophore A23187, but not endothelium-independent relaxation to sodium nitroprusside. In contrast, nLDL had no effect. Carbamylated LDL enhanced aortic ROS production by activating NADPH-oxidase. Carbamylated LDL stimulated endothelial NO synthase (eNOS) uncoupling at least partially by promoting S-glutathionylation of eNOS. Carbamylated LDL-induced endothelial dysfunction was enhanced in LOX-1 transgenic mice. In patients with CKD, LDL-carbamyl-lysine levels were significant predictors for cardiovascular events and all-cause mortality. CONCLUSIONS: Carbamylation of LDL induces endothelial dysfunction via LOX-1 activation and increased ROS production leading to eNOS uncoupling. This indicates a novel mechanism in the pathogenesis of atherosclerotic disease which may be pathogenic and prognostic in patients with CKD and high plasma levels of cLDL.

A lincRNA-DYNLRB2-2/GPR119/GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis.

Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macrophages and atherosclerotic plaque progression in apoE(-/-) mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. In vivo, apoE(-/-) mice were randomly divided into two groups and infected with lentivirus (LV)-Mock or LV-GPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1ß, IL-6, and TNF-α levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibited by infection of apoE(-/-) mice with LV-GPR119. Our findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atherosclerosis in apoE(-/-) mice. These suggest that GPR119 may be a promising candidate as a therapeutic agent.