Malondialdehyde-Acetaldehyde Modified (MAA) Proteins Differentially Effect the Inflammatory Response in Macrophage, Endothelial Cells and Animal Models of Cardiovascular Disease.

Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis. Currently, the mechanism(s) by which inflammation contributes to this disease are not entirely understood. Inflammation is known to induce oxidative stress, which can lead to lipid peroxidation. Lipid peroxidation can result in the production of reactive by-products that can oxidatively modify macromolecules including DNA, proteins, and lipoproteins. A major reactive by-product of lipid peroxidation is malondialdehyde (MDA). MDA can subsequently break down to form acetaldehyde (AA). These two aldehydes can covalently interact with the epsilon (ε)-amino group of lysines within proteins and lipoproteins leading to the formation of extremely stable, highly immunogenic malondialdehyde/acetaldehyde adducts (MAA-adducts). The aim of this study was to investigate the inflammatory response to MAA-modified human serum albumin (HSA-MAA) and low-density lipoprotein (LDL-MAA). We found that animals injected with LDL-MAA generate antibodies specific to MAA-adducts. The level of anti-MAA antibodies were further increased in an animal model of atherosclerosis fed a Western diet. An animal model that combined both high fat diet and immunization of MAA-modified protein resulted in a dramatic increase in antibodies to MAA-adducts and vascular fat accumulation compared with controls. In vitro exposure of endothelial cells and macrophages to MAA-modified proteins resulted in increased fat accumulation as well as increased expression of adhesion molecules and pro-inflammatory cytokines. The expression of cytokines varied between the different cell lines and was unique to the individual modified proteins. The results of these studies demonstrate that different MAA-modified proteins elicit unique responses in different cell types. Additionally, the presence of MAA-modified proteins appears to modulate cellular metabolism leading to increased accumulation of triglycerides and further progression of the inflammatory response.

Auto-Antibody Production During Experimental Atherosclerosis in ApoE-/- Mice.

Current models stipulate that B cells and antibodies function during atherosclerosis in two distinct ways based on antibody isotype, where IgM is protective and IgG is inflammatory. To examine this model, we generated ApoE-/- Aid-/- mice, which are unable to produce IgG antibodies due to the absence of activation-induced deaminase (AID) but maintain high plasma cholesterol due to the absence of apolipoprotein E (APOE). We saw a dramatic decrease in plaque formation in ApoE-/- Aid-/- mice compared to ApoE-/- mice. Rigorous analysis of serum antibodies revealed both ApoE-/- and ApoE-/- Aid-/- mice had substantially elevated titers of IgM antibodies compared to C57BL/6J controls, suggesting a more complex dynamic than previously described. Analysis of antigen specificity demonstrated that ApoE-/- Aid-/- mice had elevated titers of antibodies specific to malondialdehyde-oxidized low density lipoprotein (MDA-oxLDL), which has been shown to block macrophage recruitment into plaques. Conversely, ApoE-/- mice showed low levels of MDA-oxLDL specificity, but had antibodies specific to numerous self-proteins. We provide evidence for a hierarchical order of antibody specificity, where elevated levels of MDA-oxLDL specific IgM antibodies inhibit plaque formation. If the level of MDA-oxLDL specific IgM is insufficient, self-reactive IgM and IgG antibodies are generated against debris within the arterial plaque, resulting in increased inflammation and further plaque expansion.

Genetic Ablation of Transmembrane Prolyl 4-Hydroxylase Reduces Atherosclerotic Plaques in Mice.

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Compensatory IgM to the Rescue: Patients with Selective IgA Deficiency Have Increased Natural IgM Antibodies to MAA-LDL and No Changes in Oral Microbiota.

IgA is the most abundant Ab in the human body. However, most patients with selective IgA deficiency (SIgAD) are asymptomatic. IgM, and to lesser extent IgG Abs, are generally presumed to compensate for the lack of IgA in SIgAD by multiplying and adopting functions of IgA. We used data from the Northern Finland Birth Cohort 1966 to investigate whether SIgAD patients have differences in levels of natural Abs to oxidized epitopes compared with 20 randomly selected healthy controls. First, we screened the saliva and serum samples from the Northern Finland Birth Cohort 1966 cohort (n = 1610) for IgA concentration. We detected five IgA-deficient subjects, yielding a prevalence of 0.3%, which is consistent with the general prevalence of 0.25% in the Finnish population. To detect natural Abs, we used malondialdehyde acetaldehyde-low-density lipoprotein (MAA-LDL), an Ag known to bind natural Abs. In this study, we show that natural secretory IgM and IgG Abs to MAA-DL were significantly increased in subjects with SIgAD. Given that secretory IgA is an important part of mucosal immune defense and that, in the gut microbiota, dysbiosis with SIgAD patients has been observed, we characterized the oral bacterial microbiota of the subjects with and without SIgAD using high-throughput 16S rRNA gene sequencing. We found no significant alterations in diversity and composition of the oral microbiota in subjects with SIgAD. Our data suggest that increased levels of secretory natural Abs in patients with SIgAD could be a compensatory mechanism, providing alternative first-line defense against infections and adjusting mucosal milieu to maintain a healthy oral microbiota.

Extracellular-vesicle containing miRNA-503-5p released by macrophages contributes to atherosclerosis.

Endothelial dysfunction, and the differentiation of smooth muscle cells (SMCs) into proliferative, secretory phenotypes, are two major pathophysiological processes in atherosclerosis. SMCs have the potential to recruit macrophages in atherosclerotic plaques, in which macrophages drive inflammatory responses. In this study, we found that microRNA-503-5p (miR-503-5p) was enriched in either extracellular vesicles (EVs), secreted by oxidized low-density lipoprotein-treated macrophages, or the EVs from peripheral blood mononuclear cells of atherosclerosis patients. miR-503-5p was transferred intercellularly from macrophages to the co-cultured human coronary artery endothelial cells (HCAECs) and HCASMCs via EVs, thus reducing the proliferative and angiogenic abilities of HCAECs and accelerating the proliferative and migrating abilities of HCASMCs. Smad family members 1, 2 and 7 were negatively regulated by miR-503-5p in HCAECs and HCASMCs. miR-503-5p was verified as an enhancer of inflammatory cytokines and adhesion molecules released by macrophages, in part via the down-regulation of smad family members 1, 2 and 7. The inhibition of miR-503-5p by lentivirus reduced atherosclerotic lesion formations in the aorta of atherosclerotic mice. Our work demonstrated a miR-503-5p- and EV-mediated mechanism for macrophage communication with HCAECs and HCASMCs in atherosclerosis. miR-503-5p is pro-atherosclerotic stimuli that may be a therapeutic target for atherosclerosis treatment.

The neurorepellent, Slit2, prevents macrophage lipid loading by inhibiting CD36-dependent binding and internalization of oxidized low-density lipoprotein.

Atherosclerosis is characterized by retention of modified lipoproteins, especially oxidized low density lipoprotein (oxLDL) within the sub-endothelial space of affected blood vessels. Recruited monocyte-derived and tissue-resident macrophages subsequently ingest oxLDL by binding and internalizing oxLDL via scavenger receptors, particularly CD36. The secreted neurorepellent, Slit2, acting through its transmembrane receptor, Roundabout-1 (Robo-1), was previously shown to inhibit recruitment of monocytes into nascent atherosclerotic lesions. The effects of Slit2 on oxLDL uptake by macrophages have not been explored. We report here that Slit2 inhibits uptake of oxLDL by human and murine macrophages, and the resulting formation of foam cells, in a Rac1-dependent and CD36-dependent manner. Exposure of macrophages to Slit2 prevented binding of oxLDL to the surface of cells. Using super-resolution microscopy, we observed that exposure of macrophages to Slit2 induced profound cytoskeletal remodeling with formation of a thick ring of cortical actin within which clusters of CD36 could not aggregate, thereby attenuating binding of oxLDL to the surface of cells. By inhibiting recruitment of monocytes into early atherosclerotic lesions, and the subsequent binding and internalization of oxLDL by macrophages, Slit2 could represent a potent new tool to combat individual steps that collectively result in progression of atherosclerosis.

Reduced SULT2B1b expression alleviates ox-LDL-induced inflammation by upregulating miR-148-3P via inhibiting the IKKß/NF-κB pathway in macrophages.

Atherosclerosis is a lipid-driven chronic inflammatory disease in which lipid-laden macrophage foam cells lead to inflamed lesions in arteries. Previous studies have proven that sulfotransferase 2B1b (SULT2B1b) has several roles in the regulation of lipid metabolism and the inflammatory response. However, little is known about the functions of SULT2B1b in ox-LDL-induced inflammation in macrophages. In this study, after treatment with either ox-LDL alone or combined with transfection of siRNAs targeting SULT2B1b, IL-6, TNF-α, NF-κB, IKKß and IκB mRNA and protein expression were determined in Raw264.7 cells by real-time PCR and Western blot, respectively. The proliferative capacity was determined by EdU staining and Cell Counting Kit-8. Our data demonstrated that SULT2B1b knockdown could reduce phosphorylated NF-κB levels and downregulate IKKß protein levels. Additionally, IκB levels were increased and the proliferation of ox-LDL stimulated cells was inhibited after SULT2B1b silencing. Downregulation of SULT2B1b expression was found to upregulate miR-148a-3p expression by microarray assay, while IKKß was a miR-148a-3p target gene. Our study suggests that SULT2B1b knockdown could promote miR148a-3p expression and inhibit activation of the IKKß/NF-κB signalling pathway, which suppressed the inflammatory response in macrophages. Therefore, targeting the SULT2B1b gene might be potentially beneficial for atherosclerosis prevention by decreasing the inflammatory response.

Pyroptosis is a critical immune-inflammatory response involved in atherosclerosis.

Pyroptosis is a form of programmed cell death activated by various stimuli and is characterized by inflammasome assembly, membrane pore formation, and the secretion of inflammatory cytokines (IL-1ß and IL-18). Atherosclerosis-related risk factors, including oxidized low-density lipoprotein (ox-LDL) and cholesterol crystals, have been shown to promote pyroptosis through several mechanisms that involve ion flux, ROS, endoplasmic reticulum stress, mitochondrial dysfunction, lysosomal rupture, Golgi function, autophagy, noncoding RNAs, post-translational modifications, and the expression of related molecules. Pyroptosis of endothelial cells, macrophages, and smooth muscle cells in the vascular wall can induce plaque instability and accelerate atherosclerosis progression. In this review, we focus on the pathogenesis, influence, and therapy of pyroptosis in atherosclerosis and provide novel ideas for suppressing pyroptosis and the progression of atherosclerosis.

Discrimination between minimally modified LDL and fully oxidized LDL using monoclonal antibodies.

Low density lipoprotein (LDL) can be oxidized in a stepwise process that leads to the production of minimally modified low density lipoprotein (mm-LDL), in which only the lipid component is oxidized, and then of fully oxidized LDL (oxLDL), in which both the lipids and the protein are oxidized. The thiobarbituric acid-reactive substances (TBARS) assay is a recognized method for determination of oxidized LDL, however this method is unable to distinguish between mm-LDL and oxLDL. In this study, seven specific monoclonal antibodies (mAbs) against human LDL were generated and selectively bound to the apolipoprotein B-100 (apoB-100) component of LDL. Oxidized LDL was produced by incubation of human LDL with 10 µM CuSO4 for various times. The TBARS assay revealed that the optimal incubation time to achieve maximal lipid oxidation was 9 h. Indirect ELISA using the newly generated mAbs was implemented to differentiate between mm-LDL and oxLDL and it was found that binding of the mAbs to oxLDL was significantly decreased after 48 h of incubation, reflecting the oxidative modification of apoB-100. Our results suggest that the optimal times for incubation of LDL with CuSO4 for generation of mm-LDL and oxLDL were 9 h and 48 h, respectively.

Antibodies to malondialdehyde-acetaldehyde modified low-density lipoprotein in patients with newly diagnosed inflammatory joint disease.

Objective:To assess antibodies to malondialdehyde-acetaldehyde-modified low-density lipoprotein (MAA-LDL) in patients with newly diagnosed inflammatory joint disease.Method: Patients with rheumatoid arthritis (RA), spondyloarthritis (SpA), and undifferentiated arthritis (UA), participating in the Northern Savo 2010 Study, were evaluated for metabolic syndrome (MetS), metabolic and inflammatory markers, antibodies to MAA-LDL, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis.Results: Among 135 newly diagnosed untreated patients, of whom 53 (39%) were diagnosed to have RA, 44 (33%) SpA, and 38 (28%) UA, 49%, 30%, and 47%, respectively, had MetS. After adjusting for age and gender, anti-MAA-LDL immunoglobulin (Ig)A (p = 0.009), IgG (p = 0.031), and IgM (p = 0.001) levels differed between the diagnostic categories, but not in patients with MetS present or absent. All antibody classes to MAA-LDL correlated with erythrocyte sedimentation rate (ESR), and IgA and IgG antibodies with high-sensitivity C-reactive protein (hs-CRP). IgA antibodies to MAA-LDL correlated with rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPAs), fasting plasma glucose, IgA antibodies to A. actinomycetemcomitans, and in IgA and IgG antibodies to P. gingivalis.Conclusion: Among various arthritis groups, antibodies to MAA-LDL were most common in RA. Antibodies to modified lipoproteins were associated with inflammation measured by ESR and hs-CRP. IgA antibodies to MAA-LDL correlated with age, antibodies to periodontal bacteria, RF, ACPA, and fasting glucose. Associations between antibodies to MAA-LDL and antibodies to periodontal bacteria, RA-associated antibodies, inflammatory parameters, and plasma glucose already reflect cardiovascular burden in inflammatory joint diseases at diagnosis.

SERUM LEVELS OF ANTIBODIES AGAINST OXIDATION-SPECIFIC EPITOPES ARE DECREASED IN PATIENTS WITH RETINAL VEIN OCCLUSION.

PURPOSE: Oxidative stress and inflammation have been implicated in the development of retinal vein occlusion (RVO). Oxidation-specific epitopes (OSEs) represent products of oxidative stress that can trigger vascular inflammation and thrombosis. Natural occurring antibodies have been shown to bind oxidation-specific epitopes thereby inhibiting their inflammatory potential and promoting their removal. METHODS: This prospective cross-sectional study included 270 patients with RVO and 81 in-hospital control patients. We measured three types of serum levels of oxidation-specific epitope-specific immunoglobulin M and immunoglobulin G antibodies (anti-copper-oxidized LDL [CuOx-LDL], antiphosphocholine [PC], anti-malondialdehyde-modified LDL [MDA-LDL]). History of arterial hypertension, hyperlipidemia, myocardial infarction, diabetes mellitus, stroke, smoking status, and several laboratory parameters were determined to control for potential confounders. RESULTS: Compared with controls, patients with RVO had significantly lower levels of immunoglobulin M and immunoglobulin G antibodies against CuOx-LDL and PC, and significantly lower levels of immunoglobulin G but not immunoglobulin M antibodies against MDA-LDL. The association between RVO patients and lower levels of these antibodies prevailed upon multivariable adjustment. CONCLUSION: These prospective data show that antibodies against oxidation-specific epitope are lower in patients with RVO compared with control patients and support the concept that oxidative stress and inflammation play key roles in the development and subsequent complications in RVO.

New insights into immunomodulation via overexpressing lipoic acid synthase as a therapeutic potential to reduce atherosclerosis.

Atherosclerosis is a systemic chronic inflammatory disease. Many antioxidants including alpha-lipoic acid (LA), a product of lipoic acid synthase (Lias), have proven to be effective for treatment of this disease. However, the question remains whether LA regulates the immune response as a protective mechanism against atherosclerosis. We initially investigated whether enhanced endogenous antioxidant can retard the development of atherosclerosis via immunomodulation. To explore the impact of enhanced endogenous antioxidant on the retardation of atherosclerosis via immune regulation, our laboratory has recently created a double mutant mouse model, using apolipoprotein E-deficient (Apoe-/-) mice crossbred with mice overexpressing lipoic acid synthase gene (LiasH/H), designated as LiasH/HApoe-/- mice. Their littermates, Lias+/+Apoe-/- mice, served as a control. Distinct redox environments between the two strains of mice have been established and they can be used to facilitate identification of antioxidant targets in the immune response. At 6 months of age, LiasH/HApoe-/- mice had profoundly decreased atherosclerotic lesion size in the aortic sinus compared to their Lias+/+Apoe-/- littermates, accompanied by significantly enhanced numbers of regulatory T cells (Tregs) and anti-oxidized LDL autoantibody in the vascular system, and reduced T cell infiltrates in aortic walls. Our results represent a novel exploration into an environment with increased endogenous antioxidant and its ability to alleviate atherosclerosis, likely through regulation of the immune response. These outcomes shed light on a new therapeutic strategy using antioxidants to lessen atherosclerosis.

Treatment with a Toll-like Receptor 7 ligand evokes protective immunity against atherosclerosis in hypercholesterolaemic mice.

BACKGROUND: The interplay between innate and adaptive immunity is central in life-threatening clinical complications of atherosclerosis such as myocardial infarction and stroke. The specific mechanisms involved and their protective versus detrimental effects in the disease process remain poorly understood. We have previously shown that higher levels of Toll-like receptor 7 (TLR7) expression in human atherosclerotic lesions are correlated with better patient outcome. OBJECTIVE: In this study, we explored whether TLR7 activation can ameliorate disease in experimental atherosclerosis in mice. METHODS: Apolipoprotein E deficient mice (Apoe-/- ) with established disease were injected for five weeks intraperitoneally with the TLR7 ligand R848. Local effects were evaluated by characterization of the lesion. Systemic effects of the treatment were investigated by immune composition analysis in the spleen and plasma measurements. RESULTS: The in vivo treatment arrested lesion progression in the aorta. We also detected expansion of marginal zone B cells and Treg in the spleen together with increased plasma IgM antibodies against oxidized low-density lipoprotein (oxLDL) and reduced plasma cholesterol levels. These changes were accompanied by increased accumulation of IgM antibodies, decreased necrosis and fewer apoptotic cells in atherosclerotic lesions. CONCLUSIONS: Our findings show that TLR7 stimulation could ameliorate atherosclerotic lesion burden and reduce plasma cholesterol in Apoe-/- mice. TLR7 stimulation was associated with an atheroprotective B-cell and Treg response, which may have systemic and local effects within lesions that could prevent arterial lipid accumulation and inflammation.

Humoral immune response to heat shock protein 60 of Aggregatibacter actinomycetemcomitans and cross-reactivity with malondialdehyde acetaldehyde-modified LDL.

Atherosclerosis is a chronic inflammatory disease and major cause of mortality worldwide. One of the crucial steps for atherosclerotic plaque development is oxidation of low-density lipoprotein (LDL). Through the oxidation, highly immunogenic epitopes are created and the immune system is activated. Association between atherosclerosis and periodontal diseases is well documented, and one of the main oral pathogens common in periodontitis is Aggregatibacter actinomycetemcomitans (Aa). Heat shock protein 60 (HSP60) is an important virulence factor for Aa bacteria and a strong activator of the immune system. Cross-reactivity of HSP60 and oxidized LDL (OxLDL) antibodies could be a potential mechanism in the progression of atherosclerosis and one possible link between atherosclerosis and periodontitis. Human plasma samples from neonates and mothers were analyzed to determine if antibody titer to Aa-HSP60 protein is already present in newborns. Further objectives were to characterize antibody response in Aa-HSP60 immunized mice and to determine possible antibody cross-reaction with oxidized LDL. We demonstrated that newborns already have IgM antibody levels to Aa-HSP60. We also showed that in mice, Aa-HSP60 immunization provoked IgG and IgM antibody response not only to Aa-HSP60 but also to malondialdehyde acetaldehyde-modified LDL (MAA-LDL). Competition assay revealed that the antibodies were specific to Aa-HSP60 and cross-reacted with MAA-LDL. Our results suggest a possibility of molecular mimicry between Aa-HSP60 and MAA-LDL, making it intriguing to speculate on the role of HSP60 protein in atherosclerosis that manifests at young age.

Circular RNA circ-RELL1 regulates inflammatory response by miR-6873-3p/MyD88/NF-κB axis in endothelial cells.

Endothelial inflammation is an important contributor to the pathology of atherosclerotic cardiovascular disease (ASCVD). Circular RNAs (circRNAs) function and role in endothelium inflammation still unknown. In our present study, we firstly identified that circ-RELL1 plays a proinflammatory role in ox-LDL-induced HUVECs through high-throughput circRNA microarray assays. Knockdown circ-RELL1 can reduce the expression of ICAM1 and VCAM1 in ox-LDL induced endothelium inflammation. Mechanistically, circ-RELL1 directly bound to miR-6873-3p in cytoplasm. Subsequently miR-6873-3p reduced MyD88 (myeloid differentiation primary response 88) protein expression and alleviated MyD88 medicated NF-κB activation. Furthermore, circ-RELL1 can abolish the inhibition of inflammation response by miR-6873-3p. Our findings illustrate a novel regulatory pathway that circ-RELL1 modulate inflammatory response by miR-6873-3p/MyD88/NF-κB axis in ox-LDL induced endothelial cells, which provides a potential therapeutic candidate for endothelium inflammation in atherosclerotic cardiovascular disease.

The neoepitopes on methylglyoxal (MG) glycated LDL create autoimmune response; autoimmunity detection in T2DM patients with varying disease duration.

AIMS: Non-enzymatic reaction of biomolecules leads to the formation of advanced glycation end products (AGEs). AGEs plays significant role in the pathophysiology of type 2 diabetes mellitus. Methylglyoxal (MG) is a highly reactive carbonyl compound which causes formation of early (ketoamines), intermediate (dicarbonyls) and advanced glycation end products (AGEs). Glycation also results in the generation of free radicals causing structural perturbations which leads to the generation of neoantigenic epitopes on LDL molecules. The aim of the present study was to investigate whether the modification of LDL results in auto-antibodies generation in type 2 diabetes patients'. METHODS: The binding affinity of circulating autoantibodies in patients against native and MG modified LDL were assessed as compared with healthy and age-matched controls (n = 50) and T2DM patients with disease duration (DD) 5-15 yrs (n = 80) and DD > 15 yrs (n = 50) were examined by direct binding ELISA. KEYFINDINGS: The high affinity binding were observed in 50% of T2DM with DD 5-15 and 62% of T2DM with DD > 15 of patient's sera antibodies to MG-LDL antigen, in comparison to its native analog (P < 0.05). NHS sera showed negligible binding with both native and glycated LDL. Competitive inhibition ELISA results exhibit greater affinity sera IgG than the direct binding ELISA results. The increase in glycation intermediate and ends product were also observed in T2DM patient's sera and NHS sera. SIGNIFICANCE: There might be the generation of neoantigenic epitopes on LDL macromoleucle which results in generation of antibodies in T2DM. The prevalence of antibodies was dependent on disease duration.

Increased macrophage activation mediated by caspase recruitment domain 6 knockdown through negatively targeting AMPK.

Caspase recruitment domain 6 (CARD6) was initially implicated in the immune system and oncogenesis, which has also been emerged to play an important role in cardio-metabolic diseases. Nevertheless, the potential role of CARD6 on macrophage activation remains unknown. In the present study, we observed a decreased CARD6 expression in bone marrow derived macrophages (BMDMs) and mouse peritoneal macrophages (MPMs) isolated from ApoE deficiency mice and administrated with OX-LDL, which were tested by RT-PCR and western bolt analysis. Moreover, the immunofluorescence co-staining revealed that a weaker immunoreactivity of CARD6 was found and primary located in cytoplasm of macrophages induced by OX-LDL. Phenotypically, loss-of-function of CARD6 dramatically increased pro-inflammatory M1 macrophage but decreased resolving M2 macrophage markers expression. Additionally, CARD6 knockdown significantly promoted cholesterol uptake but attenuated cholesterol efflux, which lead to increased foam cell formation. Mechanistically, a downregulated AMP-activated protein kinase (AMPK) expression was required for the promoted effect of CARD6 knockdown on macrophage activation. Taken together, these results suggest that CARD6 protects against macrophage activation partially through activation of AMPK-dependent mechanism.

Poria cocos polysaccharides attenuated ox-LDL-induced inflammation and oxidative stress via ERK activated Nrf2/HO-1 signaling pathway and inhibited foam cell formation in VSMCs.

Oxidative stress, inflammation, and foam cell formation in vascular smooth muscle cells (VSMCs) are considered to play crucial roles in the pathogenesis of atherosclerosis. Poria cocos polysaccharides (PCP) has been shown to possess anti-inflammatory, antitumor and anti-oxidative properties. In this study we explored the effects of PCP on ox-LDL-induced inflammation, oxidative stress and foam cell formation in VSMCs. PCP significantly attenuated ox-LDL-induced oxidative stress, as evidenced by the decreased reactive oxygen species (ROS) and MDA levels, and the increased SOD activity in VSMCs. PCP suppressed the induction effect of ox-LDL on inflammatory cytokines and inflammatory mediators. PCP also substantially inhibited VSMCs foam cell formation and intracellular lipids accumulation. Mechanistically, PCP suppressed ox-LDL-induced up-regulation of LOX-1, which is responsible for ox-LDL uptake. Western blotting suggested that PCP activated ERK1/2 signaling pathway, increased Nrf2 translocated from cytoplasm to nucleus and heme oxygenase-1 (HO-1) expression. Up-regulation of PCP on Nrf2/HO-1 signaling was reversed by pretreatment with ERK inhibitor PD98059, indicating the involvement of ERK in PCP activation of Nrf2/HO-1 signaling. In conclusion, these results demonstrated that PCP exerted its protection against oxidative stress and inflammation via the ERK/Nrf2/HO-1 signaling pathway and that PCP may be a promising candidate for the therapy of atherosclerosis.

Atherosclerosis: Beyond the lipid storage hypothesis. The role of autoimmunity.

Atherosclerosis has long been considered as a lipid storage disease. Recent data suggest that autoimmune mechanisms seem to be involved in the pathophysiology of atherosclerosis. The presence of activated endothelial vascular cells, neutrophils, macrophages, T and to a lesser extent B cells in atherosclerotic plaques, together with the proinflammatory cytokine burden suggest mobilization of both innate and adaptive immune pathways in atherosclerosis pathobiology. The development of antibodies to oxidized low-density lipoprotein (ox-LDL), the experimental induction of atherosclerosis either via the transfer of T cells or immunization with autoantigens such as ß2 glycoprotein Ι (ß2-GPI) and heat shock proteins (HSP) further support the autoimmune nature of atherosclerosis. However, classical immunosuppressive and immune-modulatory drugs, successfully used in the therapy of autoimmune rheumatic diseases have shown limited benefits so far in the treatment of atherosclerosis.