Transient receptor potential channel TRPM4 favors oxidized low-density lipoprotein-induced coronary endothelial cell dysfunction via a mechanism involving ferroptosis.

Accelerating the repair of damaged endothelium can effectively inhibit the progression of atherosclerosis (AS). Transient receptor potential channel TRPM4 is a non-selective cation channel activated by internal Ca2+, which is expressed in endothelial cells. This study aimed to reveal the potential role of TRPM4 in AS along with the mechanism. Human coronary artery endothelial cells (HCAECs) induced by ox-LDL was regarded as an in vitro model. The impacts of TRPM4 knockdown on cellular inflammation response, oxidative stress, normal endothelial function and lipid peroxidation were evaluated. Given that ferroptosis promotes AS progression, the effects of TRPM4 on intracellular iron ions and ferroptosis-related proteins was determined. Afterwards, HCAECs were treated with ferroptosis inducer erastin, and the influence of ferroptosis in the cellular model was revealed. TRPM4 was elevated in response to ox-LDL treatment in HCAECs. TRPM4 knockdown reduced the inflammation response, oxidative stress and lipid peroxidation caused by ox-LDL, and maintained the normal function of HCAECs. Erastin treatment destroyed the impacts of TRPM4 knockdown that are beneficial for cells to resist ox-LDL, showing the enhancement of the above adverse factors. Together, this study found that TRPM4 knockdown reduced ox-LDL-induced inflammation, oxidative stress, and dysfunction in HCAECs, possibly via a mechanism involving Fe2+ and ferroptosis-related proteins.

Placenta-Derived Extracellular Vesicles From Preeclamptic Pregnancies Impair Vascular Endothelial Function via Lectin-Like Oxidized LDL Receptor-1.

BACKGROUND: Preeclampsia is a complex syndrome that includes maternal vascular dysfunction. Syncytiotrophoblast-derived extracellular vesicles from preeclampsia placentas (preeclampsia-STBEVs) were shown to induce endothelial dysfunction, but an endothelial transmembrane mediator is still unexplored. The LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is a transmembrane scavenger receptor that can cause endothelial dysfunction, and its expression is increased in the endothelium of preeclampsia women. In this study, we hypothesized that LOX-1 mediates the effects of preeclampsia-STBEVs on endothelial function. METHODS: Preeclampsia-STBEVs were collected by perfusion of placentas from women with preeclampsia and in vitro and ex vivo endothelial cell function were assessed. RESULTS: In human umbilical vein endothelial cells, inhibition of LOX-1 with LOX-1 blocking antibody (TS20) reduced the uptake of preeclampsia-STBEVs (61.3±8.8%). TS20 prevented the activation of ERK (extracellular signal-regulated kinase, a kinase downstream of LOX-1) and reduced the activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells; 21.1±8.0%) and nitrative stress (23.2±10.3%) that was induced by preeclampsia-STBEVs. Vascular function was assessed by wire myography in isolated mesenteric arteries from pregnant rats that were incubated overnight with preeclampsia-STBEVs±TS20. TS20 prevented endothelium-dependent vasodilation impairment induced by preeclampsia-STBEVs. Nitric oxide contribution to the relaxation was reduced by preeclampsia-STBEVs, which was prevented by TS20. Superoxide dismutase or apocynin, an inhibitor of NOX (nicotinamide adenine dinucleotide phosphate oxidase), restored the impaired endothelium-dependent vasodilation in arteries exposed to preeclampsia-STBEVs. CONCLUSIONS: Taken together, our findings demonstrate that LOX-1 mediates the endothelial dysfunction induced by preeclampsia-STBEVs. Our study further expands on the mechanisms that may lead to adverse outcomes in preeclampsia and proposes LOX-1 as a potential target for future interventions.

Oxidized LDL receptors: a recent update.

PURPOSE OF REVIEW: LDL in its oxidized form, or 'oxLDL', is now generally acknowledged to be highly proatherogenic and to play a significant role in atherosclerotic plaque formation. Therefore, there has been increasing interest in understanding the significance of oxLDL and its receptors in different phases of atherosclerosis, leading to the accumulation of additional data at the cellular, structural, and physiological levels. This review focuses on the most recent discoveries about these receptors and how they influence lipid absorption, metabolism, and inflammation in various cell types. RECENT FINDINGS: Two crystal structures of lectin-like oxLDL receptor-1 (LOX-1), one with a small molecule inhibitor and the other with a monoclonal antibody have been published. We recently demonstrated that the 'surface site' of LOX1, adjacent to the positively charged 'basic spine region' that facilitates oxLDL binding, is a targetable site for drug development. Further, recent human studies showed that soluble LOX-1 holds potential as a biomarker for cardiovascular disease diagnosis, prognosis, and assessing the efficacy of therapy. SUMMARY: Receptor-mediated oxLDL uptake results in cellular dysfunction of various cell types involved in atherogenesis and plaque development. The current advancements clearly demonstrate that targeting oxLDL-LOX-1 axis may lead to development of future therapeutics for the treatment of atherosclerotic cardiovascular and cerebrovascular diseases.

Oxidized LDL but not angiotensin II induces cardiomyocyte hypertrophic responses through the interaction between LOX-1 and AT1 receptors.

It is well known that lectin-like oxidized low-density lipoprotein (ox-LDL) and its receptor LOX-1, angiotensin II (AngII) and its type 1 receptor (AT1-R) play an important role in the development of cardiac hypertrophy. However, the molecular mechanism is not clear. In this study, we found that ox-LDL-induced cardiac hypertrophy was suppressed by inhibition of LOX-1 or AT1-R but not by AngII inhibition. These results suggest that the receptors LOX-1 and AT1-R, rather than AngII, play a key role in the role of ox-LDL. The same results were obtained in mice lacking endogenous AngII and their isolated cardiomyocytes. Ox-LDL but not AngII could induce the binding of LOX-1 and AT1-R; inhibition of LOX-1 or AT1-R but not AngII could abolish the binding of these two receptors. Overexpression of wild type LOX-1 with AT1-R enhanced ox-LDL-induced binding of two receptors and phosphorylation of ERKs, however, transfection of LOX-1 dominant negative mutant (lys266ala / lys267ala) or an AT1-R mutant (glu257ala) not only reduced the binding of two receptors but also inhibited the ERKs phosphorylation. Phosphorylation of ERKs induced by ox-LDL in LOX-1 and AT1-R-overexpression cells was abrogated by an inhibitor of Gq protein rather than Jak2, Rac1 or RhoA. Genetically, an AT1-R mutant lacking Gq protein coupling ability inhibited ox-LDL induced ERKs phosphorylation. Furthermore, through bimolecular fluorescence complementation analysis, we confirmed that ox-LDL rather than AngII stimulation induced the direct binding of LOX-1 and AT1-R. We conclude that direct binding of LOX-1 and AT1-R and the activation of downstream Gq protein are important mechanisms of ox-LDL-induced cardiomyocyte hypertrophy.

ABCA1 gene R1587K polymorphism could be associated with metabolic syndrome and increased plasma triglyceride concentration in adults from northern Mexico / El polimorfismo R1587K del gen ABCA1 podría estar asociado con el síndrome metabólico y la concentración elevada de triglicéridos en adultos del norte de México

INTRODUCTION: the ABCA1 protein plays a key role in reverse cholesterol transport, promoting its clearance and high-density lipoprotein (HDL) biogenesis. The R1587K (rs2230808) single-nucleotide polymorphism (SNP) in the ABCA1 gene has been associated with dyslipidemia. OBJECTIVES: to investigate the relationship of R1587K genotypes with cardiovascular (CV) risk, metabolic syndrome (MetS), lipid profile, paraoxonase-1 (PON1) activity, and anti-oxLDL titers. METHODS: we performed a cross-sectional study in 57 northern Mexican adults with no reported diseases. The ABCA1 R1587K SNP was detected by real-time polymerase chain reaction (qPCR) using TaqMan allelic discrimination probes. We evaluated the relationship of R1587K with metabolic syndrome and clinical parameters including lipid profile, glucose and insulin, PON1 activity and concentration, anti-oxLDL antibodies, anthropometry and body-composition parameters, and the atherogenic index of plasma calculation. RESULTS: our results show higher triglyceride levels in the RK + KK carriers as compared to RR carriers (p = 0.031). An association between the RK + KK genotype and the presence of MetS (OR = 4.566, 95% CI = 1.386-14.92, p = 0.010) and a tendency towards high CV risk (OR = 3.317, 95% CI = 0.910-8.611, p = 0.069) was observed in comparison to RR carriers; however, there were no differences in HDL-C levels, PON1 activity and concentration, and anti-oxLDL titers among the R1587K genotypes. CONCLUSIONS: in the northern Mexican population, the ABCA1 gene R1587K SNP is present and the RK + KK genotypes are associated with MetS and increased triglyceride concentrations; therefore, it could be a CV risk biomarker. Nevertheless there is a need for further confirmation in longitudinal studies

INTRODUCCIÓN: la proteína ABCA1 juega un papel principal en el transporte reverso del colesterol, promoviendo su eliminación y la biogénesis de HDL. El polimorfismo de un solo nucleótido (SNP) R1587K (rs2230808) del gen ABCA1 se ha asociado con dislipidemias. OBJETIVO: investigar la relación de los genotipos del SNP R1587K con el riesgo cardiovascular (CV), el síndrome metabólico (SM), el perfil de lípidos, la actividad de paraoxonasa 1 (PON1) y los anticuerpos contra las LDL oxidadas (anti-oxLDL). MÉTODOS: se realizó un estudio transversal con 57 adultos del norte de México que reportaron no tener enfermedades diagnosticadas. El SNP R1587K del gen ABCA1 se detectó a través de PCR en tiempo real (qPCR) usando sondas TaqMan para discriminación alélica. Para evaluar la asociación del SNP R1587K con el SM y determinados parámetros clínicos se determinaron el perfil de lípidos, los niveles de glucosa e insulina, la actividad y concentración de PON1, los anticuerpos anti-oxLDL, los parámetros antropométricos y de composición corporal, y el cálculo del índice aterogénico en plasma. RESULTADOS: los resultados mostraron mayores niveles de triglicéridos en los portadores del genotipo RR + KK que en los portadores de RR (p = 0,031). Se observó una asociación entre el genotipo RK + KK y la presencia de SM (OR = 4,566, IC 95% = 1,386-14,92, p = 0,010) y una tendencia hacia un mayor riesgo cardiovascular (OR = 3,317, IC 95% = 0,910-8,611, p = 0,069) al compararlos con los portadores de RR. No se encontraron diferencias en los niveles de HDL-C, la actividad y concentración de PON1 y los anti-oxLDL entre los genotipos R1587K. CONCLUSIONES: el SNP R1587K del gen ABCA1 se encuentra presente en la población del norte de México y el genotipo RK + KK se asocia con el SM y concentraciones elevadas de triglicéridos, por lo que este SNP podría ser un biomarcador de riesgo cardiovascular. Sin embargo, se necesita confirmación a través de estudios longitudinales

Oxidized Low-Density Lipoprotein Receptor in Lymphocytes Prevents Atherosclerosis and Predicts Subclinical Disease.

BACKGROUND: Although the role of Th17 and regulatory T cells in the progression of atherosclerosis has been highlighted in recent years, their molecular mediators remain elusive. We aimed to evaluate the association between the CD69 receptor, a regulator of Th17/regulatory T cell immunity, and atherosclerosis development in animal models and in patients with subclinical disease. METHODS: Low-density lipoprotein receptor-deficient chimeric mice expressing or not expressing CD69 on either myeloid or lymphoid cells were subjected to a high fat diet. In vitro functional assays with human T cells were performed to decipher the mechanism of the observed phenotypes. Expression of CD69 and NR4A nuclear receptors was evaluated by reverse transcription-polymerase chain reaction in 305 male participants of the PESA study (Progression of Early Subclinical Atherosclerosis) with extensive (n=128) or focal (n=55) subclinical atherosclerosis and without disease (n=122). RESULTS: After a high fat diet, mice lacking CD69 on lymphoid cells developed large atheroma plaque along with an increased Th17/regulatory T cell ratio in blood. Oxidized low-density lipoprotein was shown to bind specifically and functionally to CD69 on human T lymphocytes, inhibiting the development of Th17 cells through the activation of NR4A nuclear receptors. Participants of the PESA study with evidence of subclinical atherosclerosis displayed a significant CD69 and NR4A1 mRNA downregulation in peripheral blood leukocytes compared with participants without disease. The expression of CD69 remained associated with the risk of subclinical atherosclerosis in an adjusted multivariable logistic regression model (odds ratio, 0.62; 95% CI, 0.40-0.94; P=0.006) after adjustment for traditional risk factors, the expression of NR4A1, the level of oxidized low-density lipoprotein, and the counts of different leucocyte subsets. CONCLUSIONS: CD69 depletion from the lymphoid compartment promotes a Th17/regulatory T cell imbalance and exacerbates the development of atherosclerosis. CD69 binding to oxidized low-density lipoprotein on T cells induces the expression of anti-inflammatory transcription factors. Data from a cohort of the PESA study with subclinical atherosclerosis indicate that CD69 expression in PBLs inversely correlates with the presence of disease. The expression of CD69 remained an independent predictor of subclinical atherosclerosis after adjustment for traditional risk factors.

The effects of coenzyme Q10 supplementation on gene expression related to insulin, lipid and inflammation in patients with polycystic ovary syndrome.

OBJECTIVE: This research was conducted to assess the effects of coenzyme Q10 (CoQ10) intake on gene expression related to insulin, lipid and inflammation in subjects with polycystic ovary syndrome (PCOS). METHODS: This randomized double-blind, placebo-controlled trial was conducted on 40 subjects diagnosed with PCOS. Subjects were randomly allocated into two groups to intake either 100 mg CoQ10 (n = 20) or placebo (n = 20) per day for 12 weeks. Gene expression related to insulin, lipid and inflammation were quantified in blood samples of PCOS women with RT-PCR method. RESULTS: Results of RT-PCR shown that compared with the placebo, CoQ10 intake downregulated gene expression of oxidized low-density lipoprotein receptor 1 (LDLR) (p < 0.001) and upregulated gene expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) (p = 0.01) in peripheral blood mononuclear cells of subjects with PCOS. In addition, compared to the placebo group, CoQ10 supplementation downregulated gene expression of interleukin-1 (IL-1) (p = 0.03), interleukin-8 (IL-8) (p = 0.001) and tumor necrosis factor alpha (TNF-α) (p < 0.001) in peripheral blood mononuclear cells of subjects with PCOS. CONCLUSIONS: Overall, CoQ10 intake for 12 weeks in PCOS women significantly improved gene expression of LDLR, PPAR-γ, IL-1, IL-8 and TNF-α.

Hospital information system user acceptance factors: User group perspectives.

This study aimed to identify and rank user acceptance factors regarding the implementation of hospital information systems (HIS) based on the views of the following user groups: doctors, nurses, hospital management, and administrative staff (operators). The results should provide guidance for hospital management and HIS developers on meeting user requirements. The study was carried out using both qualitative and quantitative methods. The authors conducted interviews and distributed questionnaires to doctors, nurses, hospital management, and administrative staff in a government-owned Indonesian public hospital. Entropy measurements were used to analyze the questionnaire data. The study identified 15 important HIS user acceptance factors, which were ranked differently by each user group. The results show that non-technological dimensions, such as human and organizational dimensions, influence HIS user acceptance to a greater extent than technological dimensions. More work should be carried out to gain a better understanding of the relationship between user acceptance factors in order to increase the success of HIS implementations.

Recombinant Human Thioredoxin-1 Protects Macrophages from Oxidized Low-Density Lipoprotein-Induced Foam Cell Formation and Cell Apoptosis

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and apoptosis play critical roles in the pathogenesis of atherosclerosis. Thioredoxin-1 (Trx) is an antioxidant that potently protects various cells from oxidative stress-induced cell death. However, the protective effect of Trx on ox-LDL-induced macrophage foam cell formation and apoptosis has not been studied. This study aims to investigate the effect of recombinant human Trx (rhTrx) on ox-LDL-stimulated RAW264.7 macrophages and elucidate the possible mechanisms. RhTrx significantly inhibited ox-LDL-induced cholesterol accumulation and apoptosis in RAW264.7 macrophages. RhTrx also suppressed the ox-LDL-induced overproduction of lectin-like oxidized LDL receptor (LOX-1), Bax and activated caspase-3, but it increased the expression of Bcl-2. In addition, rhTrx markedly inhibited the ox-LDL-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). Furthermore, anisomycin (a p38 MAPK activator) abolished the protective effect of rhTrx on ox-LDL-stimulated RAW264.7 cells, and SB203580 (a p38 MAPK inhibitor) exerted a similar effect as rhTrx. Collectively, these findings indicate that rhTrx suppresses ox-LDL-stimulated foam cell formation and macrophage apoptosis by inhibiting ROS generation, p38 MAPK activation and LOX-1 expression. Therefore, we propose that rhTrx has therapeutic potential in the prevention and treatment of atherosclerosis.

Efectos inflamatorios, oxidativos y aterogénicos del receptor endotelial de la lipoproteína de baja densidad oxidada-1 / Inflammatory, oxidative and atherogenic effects of endothelial lectin-like receptor of oxidized low density lipoprotein-1 / Efeitos inflamatórios, oxidativos e aterogênicos do receptor endotelial da lipoproteína de baixa densidade oxidativa-1

LOX-1 es un receptor endotelial de la familia de las lectinas. Su actividad biológica tiene un fuerte impacto en los fenómenos inflamatorios, oxidativos y aterogénicos endoteliales. Cuando se conoció el receptor de la lipoproteína de baja densidad (RLDL) y su regulación, se afirmó el papel aterogénico del colesterol transportado en esta lipoproteína (C-LDL). Este papel de las lipoproteínas fue la base de la denominación de dislipoproteinemias en reemplazo de dislipemias. En condiciones post-prandiales, las lipoproteínas ricas en triglicéridos, como quilomicrones y lipoproteínas de muy baja densidad (VLDL), son degradadas por la lipoproteína lipasa (LPL) de la pared vascular, produciéndose remanentes de quilomicrones (RQ) y lipoproteínas de densidad intermedia (IDL), respectivamente, que en conjunto se denominan lipoproteínas remanentes (RLPs). Dependiendo del estrés oxidativo las RLPs son oxidables y pueden unirse al LOX-1. También se liberan ácidos grasos que injurian células endoteliales y contribuyen a abrir brechas en el endotelio, que en condiciones fisiológicas es una barrera de células con uniones estrechas. El dominio intracelular de LOX-1 regula el reconocimiento de lipoproteínas de baja densidad oxidadas (LDLOX) y de RLPs. Además, posee un efecto dependiente de los radicales reactivos de oxígeno (ROS). Su dominio transmembrana actúa en el pasaje de LDLOX y monocitos al subendotelio. La inhibición de LOX-1 con anticuerpos específicos impide su unión con LDLOX, restableciendo la barrera entre el lumen vascular y el subendotelio. En cambio, las LDLOX unidas al dominio transmembrana, producen apoptosis de las células endoteliales y suprimen uniones estrechas intercelulares en el endotelio, facilitando la actividad de las moléculas de adhesión leucocitarias que promueven el pasaje al subendotelio de los elementos del lumen, tales como monocitos, plaquetas, LDLOX, RLPs oxidables y lipoproteínas (a) (Lp(a)) semejantes al plasminógeno. Las LDLOX subendoteliales aumentan la movilidad de células musculares lisas. Los monocitos subendoteliales se establecen como residentes, e incorporan LDLOX, convirtiéndose sucesivamente en macrófagos, células espumosas y lesiones aterogénicas. Sin embargo, desde Assmann G y su estudio PROCAM no puede ignorarse el papel de los triglicéridos y colesterol de lipoproteínas de alta densidad (C-HDL) como componentes del cuadro de riesgo en ECV.

LOX-1 is an endothelial receptor belonging to the family of lectins. Its biological activity has a strong impact on inflammatory, oxidative and atherogenic phenomena in endothelium. When Low Density Lipoprotein receptor (RLDL) and its regulation were known, the atherogenic role of the cholesterol transported in LDL (LDL-C) was confirmed. This lipoprotein role in atherosclerosis was the base to use the term dyslipoproteinemia instead of dyslipidemia. In post-prandial conditions, triglyceride-rich lipoproteins like chylomicrons and very low-density lipoproteins (VLDL), are degraded by lipoprotein lipase (LPL) on the vascular wall, with the resultant formation of chylomicron remnants (CR) and intermediate density lipoproteins (IDL) respectively, which as a whole are called remnant lipoproteins (RLPs). Depending on oxidative stress, RLPs are oxidized and then they can bind the LOX-1. In this process, fatty acids are also released, injuring endothelial cells and contributing to open gaps in endothelium, which under physiological conditions, is a barrier of cells with tight junctions. The intracellular domain of LOX-1 regulates the recognition of oxidized LDL (oxLDL) and RLPs, and its effect depends on reactive oxygen species (ROS). LOX-1 transmembrane domain acts in the passage of oxLDL and monocytes to the sub-endothelium. Inhibition of LOX-1 by specific antibodies prevents its binding with OxLDL, restoring the barrier between the vascular lumen and sub-endothelium. By contrast, the oxLDL, attached to the transmembrane domain, produce apoptosis of endothelial cells and the suppression of narrow intercellular junctions in the endothelium. Thus, enabling the activity of leucocyte adhesion molecules that promote the transfer to subendothelial elements lumen of monocytes, platelets, oxLDL, oxidized RLPs and lipoprotein (a) (Lp (a)), similar to plasminogen such as. Sub-endothelial OxLDL increase the mobility of smooth muscle cells. Sub-endothelial monocytes establish as resident, up-take oxLDL and successively become into macrophages, foam cells and atherosclerotic lesions. However, since Assman’s PROCAM study, the role of triglycerides and High Density Lipoprotein-cholesterol (HDL-C), as components of cardiovascular risk, cannot be ignored.

LOX-1 é um receptor endotelial da família das lectinas. Sua atividade biológica tem um importante impacto nos fenômenos inflamatórios, oxidativos e aterogênicos endoteliais. Quando foi conhecido o receptor da lipoproteína de baixa densidade (RLDL) e sua regulação, afirmou-se o papel aterogênico do colesterol transportado nesta lipoproteína (C-LDL). Este papel das lipoproteínas foi a base da denominação de dislipoproteinemias em substituição de dislipidemias. Em condições pós-prandiais, as lipoproteínas ricas em triglicérides como quilomícrons e Lipoproteínas de muito baixa densidade (VLDL) são degradadas pela lipoproteína lipase (LPL) da parede vascular, produzindo remanescentes de quilomícrons (RQ) e lipoproteínas de densidade intermediária (IDL) respectivamente, que em conjunto são chamadas lipoproteínas remanescentes (RLPs). Dependendo do estresse oxidativo, as RLPs são oxidáveis e podem se ligar ao LOX-1. Também são liberados ácidos graxos que injuriam células endoteliais e contribuem na abertura de fendas no endotélio, que em condições fisiológicas é uma barreira de células com uniões estreitas. O domínio intracelular de LOX-1 regula o reconhecimento de lipoproteínas de baixa densidade oxidativa (LDLOX) e de RLPs. Também possui um efeito dependente dos radicais reativos de oxigênio (ROS). Seu domínio transmembrana atua na passagem de LDLOX e monócitos para o subendotélio. A inibição de LOX-1 com anticorpos específicos impede sua união com LDLOX restabelecendo a barreira entre o lúmem vascular e o subendotélio. Entretanto, as LDLOX ligadas ao domínio transmembrana produzem apoptose das células endoteliais e suprimem estreitas junções intercelulares no endotélio, facilitando a atividade das moléculas de adesão leucocitária que promovem a passagem para o subendotélio de elementos do lúmem, tais como monócitos, plaquetas, LDLOX, RLPs oxidáveis e lipoproteínas (a) [Lp(a)] semelhantes ao plasminogênio. As LDLOX subendoteliais aumentam a mobilidade das células musculares lisas. Os monócitos subendoteliais se estabelecem como residentes, e incorporam LDLOX, virando sucessivamente em macrófagos, células espumosas e lesões aterogênicas. No entanto, desde Assman G e seu estudo PROCAM, não pode se ignorar o papel dos triglicérides e do colesterol de lipoproteínas de alta densidade (C-HDL) como componentes do evento de risco em ECV.

OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL.

The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYK's known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (∼9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads.

Vascular Proteomics Reveal Novel Proteins Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Regulating Proliferation and Inflammatory Response.

Neointimal hyperplasia of vascular smooth muscle cells (VSMC) plays a critical role in atherosclerotic plaque formation and in-stent restenosis, but the underlying mechanisms are still incompletely understood. We performed a proteomics study to identify novel signaling molecules organizing the VSMC hyperplasia. The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury. The combination of cellular function assays and a protein network analysis further demonstrated that 27 out of 44 proteins constitute key signaling networks orchestrating the phenotypic change of VSMC from contractile to epithelial-like synthetic. Among the list of proteins, the in vivo validation specifically revealed that six proteins (Rab15, ITR, OLR1, PDHß, PTPε) are positive regulators for VSMC hyperplasia. In particular, the OLR1 played dual roles in the VSMC hyperplasia by directly mediating oxidized LDL-induced monocyte adhesion via NF-κB activation and by assisting the PDGF-induced proliferation/migration. Importantly, OLR1 and PDGFRß were associated in close proximity in the plasma membrane. Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

Molecular hydrogen stabilizes atherosclerotic plaque in low-density lipoprotein receptor-knockout mice.

Hydrogen (H(2)) attenuates the development of atherosclerosis in mouse models. We aimed to examine the effects of H(2) on atherosclerotic plaque stability. Low-density lipoprotein receptor-knockout (LDLR(-/-)) mice fed an atherogenic diet were dosed daily with H(2) and/or simvastatin. In vitro studies were carried out in an oxidized-LDL (ox-LDL)-stimulated macrophage-derived foam cell model treated with or without H(2). H(2) or simvastatin significantly enhanced plaque stability by increasing levels of collagen, as well as reducing macrophage and lipid levels in plaques. The decreased numbers of dendritic cells and increased numbers of regulatory T cells in plaques further supported the stabilizing effect of H(2) or simvastatin. Moreover, H(2) treatment decreased serum ox-LDL level and apoptosis in plaques with concomitant inhibition of endoplasmic reticulum stress (ERS) and reduction of reactive oxygen species (ROS) accumulation in the aorta. In vitro, like the ERS inhibitor 4-phenylbutyric acid, H(2) inhibited ox-LDL- or tunicamycin (an ERS inducer)-induced ERS response and cell apoptosis. In addition, like the ROS scavenger N-acetylcysteine, H(2) inhibited ox-LDL- or Cu(2+) (an ROS inducer)-induced reduction in cell viability and increase in cellular ROS. Also, H(2) increased Nrf2 (NF-E2-related factor-2, an important factor in antioxidant signaling) activation and Nrf2 small interfering RNA abolished the protective effect of H(2) on ox-LDL-induced cellular ROS production. The inhibitory effects of H(2) on the apoptosis of macrophage-derived foam cells, which take effect by suppressing the activation of the ERS pathway and by activating the Nrf2 antioxidant pathway, might lead to an improvement in atherosclerotic plaque stability.

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.

Increased LDL electronegativity in chronic kidney disease disrupts calcium homeostasis resulting in cardiac dysfunction.

Chronic kidney disease (CKD), an independent risk factor for cardiovascular disease, is associated with abnormal lipoprotein metabolism. We examined whether electronegative low-density lipoprotein (LDL) is mechanistically linked to cardiac dysfunction in patients with early CKD. We compared echocardiographic parameters between patients with stage 2 CKD (n = 88) and normal controls (n = 89) and found that impaired relaxation was more common in CKD patients. Reduction in estimated glomerular filtration rate was an independent predictor of left ventricular relaxation dysfunction. We then examined cardiac function in a rat model of early CKD induced by unilateral nephrectomy (UNx) by analyzing pressure-volume loop data. The time constant of isovolumic pressure decay was longer and the maximal velocity of pressure fall was slower in UNx rats than in controls. When we investigated the mechanisms underlying relaxation dysfunction, we found that LDL from CKD patients and UNx rats was more electronegative than LDL from their respective controls and that LDL from UNx rats induced intracellular calcium overload in H9c2 cardiomyocytes in vitro. Furthermore, chronic administration of electronegative LDL, which signals through lectin-like oxidized LDL receptor-1 (LOX-1), induced relaxation dysfunction in wild-type but not LOX-1(-/-) mice. In in vitro and in vivo experiments, impaired cardiac relaxation was associated with increased calcium transient resulting from nitric oxide (NO)-dependent nitrosylation of SERCA2a due to increases in inducible NO synthase expression and endothelial NO synthase uncoupling. In conclusion, LDL becomes more electronegative in early CKD. This change disrupts SERCA2a-regulated calcium homeostasis, which may be the mechanism underlying cardiorenal syndrome.

Diet-induced increase in plasma oxidized LDL promotes early fibrosis in a renal porcine auto-transplantation model.

BACKGROUND: In kidney transplantation, the prevalence of hypercholesterolemia as a co-morbidity factor known to affect graft function, is rising due to the increased number of older donors in response to organ shortage as well as to the hyperlipidemic effects of immunosuppressors in recipient. This study aimed to characterize the effects of hypercholesterolemia on renal graft outcome, investigating the role of oxidized low-density lipoprotein (OxLDL). METHODS: In vivo, we used a porcine preclinical model of renal auto-transplantation modulated by two experimental diets: a normal (n = 6) or a hyperlipidemic diet (n = 5) maintained during the 3 month follow-up after the surgical procedure. Kidney function and OxLDL levels were monitored as well as fibrosis, LOX-1 and TGF beta signaling pathways. In vitro, we used human artery endothelial cells subjected to OxLDL to investigate the TGF beta profibrotic pathway and the role of the scavenger receptor LOX-1. RESULTS: Hyperlipidemic diet-induced increase in plasma OxLDL levels at the time of surgery correlated with an increase in proteinuria 3 months after transplantation, associated with an early graft fibrosis combined with an activation of renal TGF beta signaling. These data suggest a direct involvement of OxLDL in the hyperlipidemic diet-induced activation of the pro-fibrotic TGF beta pathway which seems to be activated by LOX-1 signaling. These results were supported by studies with endothelial cells incubated in culture medium containing OxLDL promoting TGF beta expression inhibited by LOX-1 antibody. CONCLUSIONS: These results implicate OxLDL in the hyperlipidemic diet-promoted fibrosis in transplanted kidneys, suggesting LOX-1 as a potential therapeutic target and reinforce the need to control cholesterol levels in kidney transplant recipients.

[Relationship of ox-LDL/LOX-1 and vascular endothelial dysfunction of diet-induced obese immature rats and nicotinic acid's intervention outcomes].

OBJECTIVE: To explore the effects of nicotinic acid intervention on vascular endothelial dysfunction mediated by oxidized low density lipoprotein (ox-LDL)/lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) in diet-induced obese immature rats and its possible mechanism through detecting the expression levels of ox-LDL and LOX-1 in abdominal aorta. METHODS: A model of diet-induced obese immature rats was established by high-fat diet. And 30 immature rats were divided randomly and equally into control (n = 10), high-fat (n = 10) and drug control (n = 10) groups. At the end of 12 weeks, the levels of serum total cholesterol (TC), triglyceride (TG), LDL and high-density lipoprotein (HDL) were examined. The levels of ox-LDL, soluble intercellular adhesion molecule-1 (sICAM-1), endothelin and nitric oxide (NO) were detected. The gene and protein expressions of LOX-1 and ICAM-1 in abdominal aorta were detected. And the location protein expressions of LOX-1 and ICAM-1 were examined. RESULTS: High-fat diet induced hyperlipidemia and obesity in immature rats. The serum levels of TG, TC, LDL, ox-LDL and endothelin in high-fat and drug control groups were all higher than control group ((0.98 ± 0.12) and (0.69 ± 0.06) vs (0.49 ± 0.06) mmol/L, (2.11 ± 0.16) and (1.62 ± 0.12) vs (1.30 ± 0.12) mmol/L, (0.71 ± 0.04) and (0.50 ± 0.03) vs (0.30 ± 0.04) mmol/L, (44.2 ± 5.1) and (33.7 ± 2.1) vs (26.6 ± 2.9) µg/L, (187 ± 10) and (157 ± 6) vs (118 ± 7) pg/ml). The indices in high-fat group were higher than those in drug control group (all P < 0.01) . The levels of HDL and NO in high-fat and drug control groups were lower than those in control group (all P < 0.01); the levels of HDL and NO in high-fat group lower than those in drug control group (all P < 0.01). And the levels of LOX-1, ICAM-1 protein and mRNA in high-fat group were higher than those in drug control and control groups (all P < 0.01).ox-LDL was correlated positively with LOX-1, TC, TG, LDL, endothelin and ICAM-1 (r = 0.918, 0.867, 0.857, 0.834, 0.869, 0.644, all P < 0.01) , but negatively with NO and HDL (r = -0.823, -0.872, P < 0.01) . CONCLUSION: Early treatment of nicotinic acid can protect endothelial function through inducing therapeutic effects on hyperlipidemia and antioxidation and down-regulating the expression level of ox-LDL/LOX-1 in vascular endothelium.

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.

Inhibition of LDL-oxidation and antioxidant properties related to polyphenol content of hydrophilic fractions from seaweed Halimeda Incrassata (Ellis) Lamouroux

LDL oxidation and oxidative stress are closely related to atherosclerosis. Therefore, natural antioxidants have been studied as promising candidates. In the present study, the LDL oxidation inhibition activity of bioactive compounds from Halimeda incrassata seaweed. associated to antioxidant capacity, was evaluated in vitro. Experimental work was conducted with lyophilized aqueous extract and phenolic-rich fractions of the seaweed and their effect on LDL oxidation was evaluated using heparin-precipitated LDL (hep-LDL) with exposure to Cu2+ ions and AAPH as the free radical generator. H. incrassata had a protective effect for hep-LDL in both systems and the presence of phenolic compounds contributed to the activity where phenolic-rich fractions showed significant capacity for inhibition of oxidation mediated by Cu2+ ions. The observed effect could be related to the antioxidant potential of polar fractions evidenced by reducing activity and DPPH• radical scavenging. The results obtained in vitro further support the antioxidant and LDL oxidation inhibition properties of H. incrassata and further knowledge toward future phytotherapeutic application of the seaweed.

A oxidação da LDL e o estresse oxidativo estão intimamente relacionados com a aterosclerose. Por isso, os antioxidantes naturais têm sido estudados como candidatos promissores. No presente trabalho foi avaliada in vitro a capacidade de inibição da oxidação da LDL pelos compostos bioativos da alga Halimeda incrassata em associação à capacidade antioxidante. O trabalho experimental foi conduzido com extratos polares (extrato aquoso liofilizado e frações ricas em fenólicos) e seu efeito na oxidação da LDL foi avaliado usando LDL precipitada com heparina (hep-LDL), oxidada com íons de Cu2+ e AAPH, como geradores de radicais livres. A H. incrassata apresentou efeito protetor para hep-LDL em ambos sistemas e a presença de compostos fenólicos contribuiu para a atividade em que as frações ricas em fenólicos demonstram capacidade significativa em inibir a oxidação mediada pelos íons de Cu2+. O efeito observado deve estar relacionado com o potencial antioxidante das frações polares medido pela atividade redutora e varredura do radical DPPH. Os resultados obtidos demonstram as propriedades antioxidantes e de inibição da oxidação da LDL da H. incrassata e podem contribuir para as evidências de futuras aplicações fitoterapêuticas desta alga.

Chlamydia pneumoniae binds to the lectin-like oxidized LDL receptor for infection of endothelial cells.

The association of Chlamydia pneumoniae and atherosclerosis has been well documented. Recently, it has been demonstrated that C. pneumoniae up-regulates expression of the lectin-like ox-LDL receptor (LOX-1) in endothelial cells. Many of the pro-atherogenic effects of ox-LDL occur through its activation and uptake by LOX-1. This class E scavenger receptor contains a carbohydrate-recognition domain common to the C type lectin family. Previously, we have demonstrated that the major outer membrane protein of the chlamydiae is glycosylated and glycan removal abrogates infectivity of C. pneumoniae for endothelial cells. In this study, we investigated whether C. pneumoniae binds to LOX-1. The results show that 1) infection of endothelial cells by C. pneumoniae is inhibited by ligands that bind to the LOX-1 receptor, but not by ligands binding to other scavenger receptors; 2) anti-LOX-1 antibody inhibits C. pneumoniae infectivity, while antibodies against other scavenger receptors do not; 3) anti-LOX-1 antibody inhibits attachment of C. pneumoniae to endothelial cells; and 4) C. pneumoniae co-localizes with LOX-1. These effects were not observed for Chlamydia trachomatis. In conclusion, C. pneumoniae binds to the LOX-1 receptor, which is known to promote atherosclerosis.