Experimental Atherogenesis Research Articles

Effect of Aspirin on Experimental Atherosclerosis in Rabbits Submitted to Hypercholesterolemic Diet

Aspirin, besides its antithrombotic activity, has also been quoted for protective effect reducing new coronary lesions. Objective: Considering the growing interest in the study of drugs that prevent the progression of atherosclerotic lesion, the objective of this study was to determine the effect of aspirin in experimental atherogenesis induced in rabbits fed with cholesterol rich diets. Method: Thirty adult, New Zealand white male rabbits, with 3.4 kg of body weight were submitted to a 1.5% cholesterol-rich-diet for 9 weeks. Aspirin was triturated, mixed with chloroform and incorporated to the normal chow. The drug was given to the rabbits every day in portions of 20 g of chow with 100 mg of aspirin. The rabbits were divided in 3 groups as follows: A) Cholesterol-rich-diet (n=10); B) Cholesterol-rich-diet plus aspirin (n=10) and C) Normal chow (n=10). Blood samples were collected before starting the diet, at 5 weeks and at sacrifice (9 weeks) for determination of total cholesterol, triglycerides, enzymes and hematological tests. After sacrifice, staining of the aorta was done by Sudan III for visualization of sudanophillic plaques. The percentual of aorta couvered with lipidic deposits were determined by computerized planimetry. Results: Total cholesterol (mg/dl) was per group: A- TO=52, T5=424 and T9=1.483; B- TO=32, T5=755 and T9=1.436; C- TO=41, T5=22 and T9=27. Planimetry data did not differ among groups A (23.3%) and B (27.3%). Scanning microscopy – the interpretation of the document images in three groups showed insignificant platelet deposition in all aortic segments both in groups B and C. Conclusion: This experiment enhances the theoretic basis for the protective effect of aspirin as an antiaggregant factor in the experimental aortic atherosclerotic lesion.

Mechanisms of endothelial cell activation by endocannabinoid 2-arachidonoylglycerol

Abstract Background Endothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to restore vascular function and to prevent stent thrombosis. The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Earlier studies have demonstrated the relevance of this endocannabinoid in human pathophysiology during coronary artery disease and in murine experimental atherogenesis. However, evidence on the impact of 2-AG on endothelial cell function remains scarce. Methods Endothelial repair was studied in two treatment groups of wildtype mice following electrical denudation of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO. The residual endothelial gap at five days was visualized by Evan's blue staining in either group. In vitro, the effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability was assessed by an XTT-based assay. Endothelial activation was studied by an adhesion assay of THP-1 monocytes to 2-AG-preconditioned HCAEC. Activation of HCAEC adhesion molecules was characterized by flow cytometry. Results Elevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery, resulting in a residual denudation at 5 days of 2291±286 μm vs. 1505±223 μm (n=18–19; p<0.05). In vitro, 2-AG significantly reduced viability of HCAEC at 24 hours (0.31±0.10 vs. 1.00±0.08; n=3; p<0.01). Finally, 2-AG promoted HCAEC activation resulting in a significant increase in THP-1 monocyte adhesion to HCAEC following pre-treatment of HCAEC with 2-AG (0.17±0.03 THP-1 cells per HCAEC vs. 0.07±0.01 THP-1 cells per HCAEC; n=3; p<0.05). Adhesion molecules E-selectin, ICAM-1 and VCAM-1, that are known to be regulated by 2-AG in the venous endothelium, remained unchanged in arterial endothelial cells. Besides, HCAEC migration, ROS-production, expression of NADPH oxidases and secretion of inflammatory cytokines were unaffected by 2-AG. Conclusion Elevated 2-AG levels hamper endothelial repair and impair HCAEC proliferation while facilitating adhesion of monocytes. Intriguingly, the underlying mechanisms in the arterial vascular bed appear distinct from venous endothelium. Given that 2-AG is elevated during coronary artery disease in humans, 2-AG might impair reendothelialization after angioplasty and thus impact on clinical outcomes. Funding Acknowledgement Type of funding source: None

The Heat Shock Protein 27 Immune Complex Enhances Exosomal Cholesterol Efflux.

Previously, we demonstrated that Heat Shock Protein 27 (HSP27) reduces the inflammatory stages of experimental atherogenesis, is released by macrophage (MΦ) exosomes and lowers cholesterol levels in atherosclerotic plaques. Recently, we discovered that natural autoantibodies directed against HSP27 enhance its signaling effects, as HSP27 immune complexes (IC) interact at the cell membrane to modulate signaling. We now seek to evaluate the potential role of the HSP27 IC on MΦ exosomal release and cholesterol export. First, in human blood samples, we show that healthy control subjects have 86% more exosomes compared to patients with coronary artery disease (p < 0.0001). Treating human THP-1 MΦ with rHSP27 plus a validated anti-HPS27 IgG antibody increased the abundance of exosomes in the culture media (+98%; p < 0.0001) as well as expression of Flotillin-2, a marker reflective of exosomal release. Exosome cholesterol efflux was independent of Apo-A1. THP-1 MΦ loaded with NBD-labeled cholesterol and treated with the HSP27 IC showed a 22% increase in extracellular vesicles labeled with NBD and a 95% increase in mean fluorescent intensity. In conclusion, exosomal abundance and secretion of cholesterol content increases in response to HSP27 IC treatment, which may represent an important therapeutic option for diseases characterized by cholesterol accumulation.

The endocannabinoid 2-arachidonoylglycerol inhibits endothelial function and repair

BackgroundEndothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to prevent stent failure. Previous studies have highlighted the role of 2-arachidonoylglycerol (2-AG) in murine experimental atherogenesis and in human coronary artery disease. However, the impact of 2-AG on endothelial repair and leukocyte–endothelial cell adhesion is still unknown. MethodsEndothelial repair was studied in two treatment groups of wildtype mice following electrical injury of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO (vehicle). The effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability, leukocyte–endothelial cell adhesion, surface expression of adhesion molecules, and expression of endothelial NO synthase (NOS3) was studied in vitro. ResultsElevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery. In vitro, 2-AG significantly reduced viability of HCAEC. Additionally, 2-AG promoted adhesion of THP-1 monocytes to HCAEC following pre-treatment of the HCAEC with 2-AG. Adhesion molecules (E-selectin, ICAM-1 and VCAM-1) remained unchanged in arterial endothelial cells, whereas 2-AG suppressed the expression of NOS3 in HCAEC. Conclusion and translational aspectElevated 2-AG levels hamper endothelial repair and HCAEC proliferation, while simultaneously facilitating leukocyte–endothelial cell adhesion. Given that 2-AG is elevated in patients with coronary artery disease and non-ST-segment elevation myocardial infarction, 2-AG might decrease reendothelialization after angioplasty and thus impact the clinical outcomes.

Biophysical analyses and functional implications of the interaction between Heat Shock Protein 27 and antibodies to HSP27

Heat Shock Protein 27 (HSP27) is a small molecular chaperone that reduces the development of atherosclerosis by lowering plasma cholesterol levels as well as inflammation. Human studies show an inverse correlation between atherosclerotic burden and HSP27 expression, and are supported by murine models in which augmenting HSP27 levels curbs experimental atherogenesis. Natural HSP27 auto-antibodies (AAb) are found in human plasma, however their role in modulating the athero-protective effects of HSP27 is unknown. The purpose of this study is to characterize the biophysical interaction between human recombinant HSP27 and AAb. A validated polyclonal anti-HSP27 IgG antibody (PAb) was used to mimic natural AAb. Homology modeling and secondary structure prediction tools facilitated the design of HSP27 truncation and phosphorylation mutants. Secondary structural changes were identified using Circular Dichroism (CD) and Dynamic Light Scattering (DLS). Similar to prior structural investigations of HSP27, there was a predominance of α-helical content in the N-terminal truncation and dephosphorylation (“AA”) mutants. The α-crystallin domain (ACD) predominantly consists of β-strands, with the addition of the N-terminal increasing helical content and the C-terminal maintaining β structure. With increasing ratios of PAb to HSP27 β structure abundance and particle size increased, with a similar trend observed with the N-terminus, C-terminus and ACD peptides but an opposite trend with the phosphorylation peptides. Taken together, these studies provide insights into the interaction of HSP27 and its AAb that ultimately may aid in optimizing the design of HSP27 peptidomimetics with anti-atherogenic potential.

Stage-dependent differential effects of interleukin-1 isoforms on experimental atherosclerosis.

Targeting interleukin-1 (IL-1) represents a novel therapeutic approach to atherosclerosis. CANTOS demonstrated the benefits of IL-1β neutralization in patients post-myocardial infarction with residual inflammatory risk. Yet, some mouse data have shown a prominent role of IL-1α rather than IL-1β in atherosclerosis, or even a deleterious effect of IL-1 on outward arterial remodelling in atherosclerosis-susceptible mice. To shed light on these disparate results, this study investigated the effect of neutralizing IL-1α or/and IL-1β isoforms starting either early in atherogenesis or later in ApoE-/- mice with established atheroma. The neutralization of IL-1α or of both IL-1 isoforms impaired outward remodelling during early atherogenesis as assessed by micro-computed tomographic and histologic assessment. In contrast, the neutralization of IL-1β did not impair outward remodelling either during early atherogenesis or in mice with established lesions. Interleukin-1β inhibition promoted a slant of blood monocytes towards a less inflammatory state during atherogenesis, reduced the size of established atheromata, and increased plasma levels of IL-10 without limiting outward remodelling of brachiocephalic arteries. This study established a pivotal role for IL-1α in the remodelling of arteries during early experimental atherogenesis, whereas IL-1β drives inflammation during atherogenesis and the evolution of advanced atheroma in mice.

Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.

Previously, we reported that elevated serum levels of heat shock protein 27 (HSP27) are predictive of a lower risk of having a heart attack, stroke, or death from cardiovascular disease. Moreover, augmenting HSP27 (or the murine ortholog, HSP25) attenuated experimental atherogenesis, reduced inflammation, and lowered cholesterol levels. Recently, we noted that HSP27 activates NF-κB via TLR-4, resulting in attenuation of plaque inflammation; however, the precise anti-atherosclerosis mechanisms mediated by extracellular HSP27 are incompletely understood. Our purpose in this study was to investigate the existence of HSP27 in extracellular vesicles (EVs) and whether HSP27 elicited atheroprotective effects on target cells. Here, we provide evidence that HSP27 localizes to EVs derived from THP-1 cells using transmission electron microscopy (TEM) and immunogold labeling, Western blotting, ELISA, and fluorescence-activated cell sorting. TEM imaging indicated that HSP27 is found at the exosomal membrane. Multiple reactor monitor-mass spectrometric analysis of large vesicles, which included microparticles and exosomes, isolated from human plasma, also led to detection of HSP27 using the unique signature peptide, R.LFDQAFGLPR.L. Studies using THP-1 and human embryonic kidney cells show that HSP27-laden exosomes significantly stimulated NF-κB activation ( P < 0.001) and release of IL-10 ( P < 0.0001), suggesting that HSP27 may be important exosomal cargo with beneficial anti-inflammatory effects.-Shi, C., Ulke-Lemée, A., Deng, J., Batulan, Z., O'Brien, E. R. Characterization of heat shock protein 27 in extracellular vesicles: a potential anti-inflammatory therapy.

Roles of PAD4 and netosis in experimental atherosclerosis and arterial injury: Implications for superficial erosion

Aim: Neutrophils likely contribute to the thrombotic complications of human atheromata. In particular, neutrophil extracellular traps (NETs) could exacerbate local inflammation and amplify and propagate arterial intimal injury and thrombosis. Peptidyl arginine deiminase 4 (PAD4) participates in NET formation, but understanding of this enzyme’s role in atherothrombosis remains scant. This study tested the hypothesis that PAD4 and NETs influence experimental atherogenesis and in processes implicated in superficial erosion, a form of plaque complication we previously associated with NETs.

HSP27 immunization attenuates atherogenesis by markedly reducing plasma PCSK9 and cholesterol levels

Aim: Augmentation of Heat Shock Protein 27 (HSP27) serum levels attenuates experimental atherogenesis in part by reducing cholesterol levels and inflammation. Higher serum HSP27 levels are associated with reduced 5-year risk of cardiovascular events. Recently, we documented the presence of natural antibodies (AABs) to HSP27.

Roles of PAD4 and NETosis in Experimental Atherosclerosis and Arterial Injury: Implications for Superficial Erosion.

Neutrophils likely contribute to the thrombotic complications of human atheromata. In particular, neutrophil extracellular traps (NETs) could exacerbate local inflammation and amplify and propagate arterial intimal injury and thrombosis. PAD4 (peptidyl arginine deiminase 4) participates in NET formation, but an understanding of this enzyme's role in atherothrombosis remains scant. This study tested the hypothesis that PAD4 and NETs influence experimental atherogenesis and in processes implicated in superficial erosion, a form of plaque complication we previously associated with NETs. Bone marrow chimeric Ldlr deficient mice reconstituted with either wild-type or PAD4-deficient cells underwent studies that assessed atheroma formation or procedures designed to probe mechanisms related to superficial erosion. PAD4 deficiency neither retarded fatty streak formation nor reduced plaque size or inflammation in bone marrow chimeric mice that consumed an atherogenic diet. In contrast, either a PAD4 deficiency in bone marrow-derived cells or administration of DNaseI to disrupt NETs decreased the extent of arterial intimal injury in mice with arterial lesions tailored to recapitulate characteristics of human atheroma complicated by erosion. These results indicate that PAD4 from bone marrow-derived cells and NETs do not influence chronic experimental atherogenesis, but participate causally in acute thrombotic complications of intimal lesions that recapitulate features of superficial erosion.

Rac2 Modulates Atherosclerotic Calcification by Regulating Macrophage Interleukin-1β Production.

The calcium composition of atherosclerotic plaque is thought to be associated with increased risk for cardiovascular events, but whether plaque calcium itself is predictive of worsening clinical outcomes remains highly controversial. Inflammation is likely a key mediator of vascular calcification, but immune signaling mechanisms that promote this process are minimally understood. Here, we identify Rac2 as a major inflammatory regulator of signaling that directs plaque osteogenesis. In experimental atherogenesis, Rac2 prevented progressive calcification through its suppression of Rac1-dependent macrophage interleukin-1β (IL-1β) expression, which in turn is a key driver of vascular smooth muscle cell calcium deposition by its ability to promote osteogenic transcriptional programs. Calcified coronary arteries from patients revealed decreased Rac2 expression but increased IL-1β expression, and high coronary calcium burden in patients with coronary artery disease was associated with significantly increased serum IL-1β levels. Moreover, we found that elevated IL-1β was an independent predictor of cardiovascular death in those subjects with high coronary calcium burden. Overall, these studies identify a novel Rac2-mediated regulation of macrophage IL-1β expression, which has the potential to serve as a powerful biomarker and therapeutic target for atherosclerosis.

Silencing of CD40 in vivo reduces progression of experimental atherogenesis through an NF-κB/miR-125b axis and reveals new potential mediators in the pathogenesis of atherosclerosis

Background and aimsCD40/CD40L signaling exerts a critical role in the development of atherosclerosis, and microRNAs (miRNAs) are key regulators in vascular inflammation and plaque formation. In this work, we investigated mRNA/miRNA expression during progression of atherosclerotic lesions through CD40 silencing. Methods: We silenced CD40 with a specific siRNA in ApoE−/− mice and compared expression of mRNA/miRNA in ascending aorta with scrambled treated mice. ResultssiRNA-CD40 treated mice significantly reduced the extension and severity of atherosclerotic lesions, as well as the number of F4/80+, galectin-3+ macrophages and NF-κB+ cells in the intima. Genome-wide mRNA/miRNA profiling allowed the identification of transcripts, which were significantly upregulated during atherosclerosis; among them, miR-125b and miR-30a, Xpr1, a regulator of macrophage differentiation, Taf3, a core transcription factor and the NF-κB activator Ikkβ, whereas, the NF-κB inhibitor Ikbα was downregulated during disease progression. All those changes were reversed upon CD40 silencing. Interestingly, TAF3, XPR1 and miR-125b were also overexpressed in human atherosclerotic plaques. Murine Taf3 and Xpr1 were detected in the perivascular adipose tissue (PVAT), and Taf3 also in intimal foam cells. Finally, expression of miR-125b was regulated by the CD40-NF-κB signaling axis in RAW264.7 macrophages. ConclusionsCD40 silencing with a specific siRNA ameliorates progression of experimental atherosclerosis in ApoE−/− mice, and evidences a role for NF-κB, Taf3, Xpr1, and miR-125b in the pathogenesis of atherosclerosis.

Anti-atherogenic effect of Humulus japonicus in apolipoprotein E-deficient mice.

Humulus japonicus (HJ) is used as a traditional medicine in Korea owing to its multiple properties including anti-mycobacterial, antioxidant and antihypertensive effects. The present study aimed to examine the anti-inflammatory and anti-atherogenic effects of a methanol extract of HJ. In lipopolysaccharide-stimulated RAW 264.7 cells, HJ significantly suppressed the mRNA expression and secretion of pro-inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1β and IL-6)], and the release of inflammatory mediators such as nitrite and prostaglandin E2, together with a concomitant decrease in the mRNA levels of inducible nitric oxide synthase and cyclooxygenase-2. To examine whether HJ is capable of inhibiting experimental atherogenesis in an animal model, we randomly divided apolipoprotein E-deficient (apoE−/−) mice into three groups: mice fed an atherogenic diet plus vehicle (0.5% carboxymethyl cellulose) as the control vehicle group, and mice fed an atherogenic diet plus either 100 (HJ100) or 500 mg/kg (HJ500) of HJ as the experimental groups. After 12 weeks of HJ administration, lipid accumulation and the formation of atherosclerotic lesions in the aorta (en face) and the aortic sinus markedly decreased in the HJ500 group compared with the corresponding values in the vehicle control group. Moreover, monocyte and macrophage infiltration in the aortic sinus was markedly reduced in the HJ500 group. Reverse transcription-quantitative polymerase chain reaction analysis of the whole aorta showed that the mRNA levels of intercellular adhesion molecule-1, monocyte chemoattractant protein-1, CD68 and IL-18 were significantly decreased in the HJ500 group. Collectively, these findings suggest that HJ may suppress atherosclerosis by inhibiting lipid accumulation and the expression of pro-atherogenic factors, and it may be effective at preventing the development of atherosclerosis.

Neutrophil-derived myeloperoxidase promotes atherogenesis and neointima formation in mice

BackgroundMyeloperoxidase (MPO), expressed mainly in neutrophils, is an enzyme linked to inflammation and oxidative stress. MPO is an independent prognostic marker in healthy individuals as well as in patients with coronary artery disease. In this present study we analyze the role of MPO in experimental atherogenesis and neointima formation after vascular injury in mice. Methods and results6–8weeks old apolipoprotein E-deficient (ApoE−/−) mice were fed a high-cholesterol diet for 8weeks with concomitant treatment with two different doses (10μg/mgbw vs. 20μg/mgbw) of 4-ABAH (MPO inhibitor). Application at lower dosage did not affect oxidative stress, endothelial function and atherosclerotic plaque development. 4-ABAH in higher dosage decreased inflammatory markers and vascular oxidative stress, consecutively improved endothelial function and reduced significantly atherosclerotic plaque development. To assess the role of circulating intracellular MPO, irradiated ApoE−/− mice were repopulated with bone marrow-derived cells from MPO−/− mice and were fed a high-cholesterol diet for 8weeks. This MPO deficiency resulted in alleviated inflammation, reduced oxidative stress and improved endothelial function with a significant impact on plaque formation. To understand the possible role of MPO in vascular remodeling, we tested its effects on neointima formation following vascular injury in mice. MPO inhibition by 4-ABAH reduced significantly neointima formation. It was significantly reduced in MPO deficient mice, whereas transfer of spleen-derived neutrophils from WT mice enhanced it. ConclusionOur data suggests a central role of MPO in the pathogenesis of atherogenesis and prefers pharmacological MPO inhibition as a therapeutic strategy for prevention and therapy of atherosclerosis and restenosis.

Abstract 17627: Is the Post-menopausal Rise in PCSK9 Levels Due to Loss of the Estrogen Responsive Anti-atherogenic Protein, Heat Shock Protein 27? Insights Into a Potential New PCSK9 Lowering Therapy

Introduction: Previously we reported that elevated serum levels of Heat Shock Protein 27 represent a predictive biomarker of reduced cardiovascular clinical events. Moreover, attenuation of experimental atherogenesis, characterized by reduced cholesterol levels in both the artery wall and serum, can be achieved by augmenting serum HSP27 levels (e.g., with recombinant HSP27). PCSK9 plays a central role in regulating the abundance of hepatic LDL-receptors, and hence serum and plaque cholesterol levels. Interestingly, PCSK9 levels rise with menopause (Dallas Heart Study) and we previously published that HSP27 levels are markedly attenuated post-ovariectomy. Hence, we hypothesize that HSP27 augments hepatic cholesterol uptake via reductions in PCSK9 levels. Methods / Results: 1. In vitro studies: HepG2 (liver) cells were maintained in culture for 24 hours with or without rHSP27. The hepatic levels of apoB100 did not change with rHSP27 treatment. ApoB100 containing lipoproteins were reduced in the cell culture media with rHSP27 treatment (Western Blot: p=0.008; ELISA: p=0.0025). rHSP27 treatment reduced hepatic PCSK9 protein levels (Western Blot: p=0.019), increased LDL receptor protein levels (Western Blot: p=0.001) and increased (fluorescent) LDL uptake (p=0.022). Levels of hepatic SREBP2 and HMG-CoA reductase proteins were unaltered with rHSP27 treatment. 2. In vivo studies: Female ApoE-/- mice fed high fat diet and injected weekly with rHSP27 or PBS for 8 weeks. Serum total cholesterol and plasma PCSK9 levels were ~20-40% lower in the rHSP27 treated vs. control mice. Mice treated with rHSP27 showed reductions in aortic lesion area of ~40% for en face and ~35% for cross-sectional sinus tissue sections. Conclusions: These findings suggest that HSP27, an endogenous estrogen responsive protein, can lower extracellular cholesterol levels by reducing PCSK9 expression. Hence there is promise for the development of HSP27 “replacement” therapy for the prevention and modification of atherosclerosis via reductions in PCSK9 expression. Current clinical studies examining the relationship between HSP27 and PCSK9 levels in women undergoing prophylactic ovariectomy for BRAC1/2 (i.e., the “Angelina Effect”) are ongoing.

Abstract 17586: HSP27 Mediated Atheroprotection Requires GM-CSF Induction: Potential Role in Reverse Cholesterol Transport? (Best of Basic Science Abstract)

Introduction: Previously, we demonstrated that Heat Shock Protein 27 (HSP27) reduces experimental atherogenesis and patients with elevated serum HSP27 levels are relatively protected against future CV events. We previously reported that extracellular HSP27 activates the NF-kB pathway and increases the expression of GM-CSF. While the potential atheroprotective role of GM-CSF remains controversial, clinical reports highlight its effect on reducing serum cholesterol levels. We now hypothesize that GM-CSF plays an important role in HSP27-mediated atheroprotection, potentially by up-regulating reverse cholesterol transport (RCT). Methods / Results: 1. NF-KB signaling in vitro: Treatment of macrophages with recombinant HSP27 (rHSP27) activated NF-kB 15-fold via TLR-4 (NF-kB reporter gene assay; controls: truncated rHSP27 peptide and TLR-4 deficient cells; p&lt;0.05). While rHSP27 substantially induced GM-CSF expression and secretion (300- and 400-fold respectively; p&lt;0.05) both processes were blocked by NF-kB inhibitors. 2. In vivo role of GM-CSF induction in HSP27-mediated atheroprotection: Aortic atherogenesis in apoE null treated with rHSP27 was reduced by an expected 40%, while ApoE null / GM-CSF null did not respond to rHSP27 treatment. Moreover, apoE null / GM-CSF null macrophages expressed ~33% lower levels of the reverse cholesterol transporters, ABC-A1 and ABC-G1. 3. rHSP27 &amp; Reverse Cholesterol Transport: THP-1 macrophages pre-treated with oxLDL and incubated with rHSP27 for 24h showed enhanced mRNA and protein expression of ABCA1 and ABCG1 (p&lt;0.01). In a RCT assay, THP-1 macrophages labeled with NBD-cholesterol and treated with rHSP27 for 24h before being incubated with apoA1 or HDL showed increased cholesterol efflux compared to controls (p&lt;0.0001 for both). Conclusion: HSP27 up regulates expression of GM-CSF that appears to play an important role in RCT-mediated atheroprotection.

Abstract 17326: Clues to Attenuating Plaque Inflammation: Heat Shock Protein-27 Augments Macrophage Histone Deacetylase-6 Activity and Cytoskeletal Migratory Capacity

Introduction: We recently demonstrated that elevated serum levels of Heat Shock Protein 27 (HSP27) are associated with reduced 5-year CV events and that extracellular HSP27 attenuates experimental atherogenesis and lesion progression. Indeed, HSP27 therapy reduces plaque macrophage and cholesterol content and enhances histological features associated with resilience to plaque rupture. Cell motility and adhesion involves dynamic microtubule acetylation/deacetylation, a process regulated by the enzyme histone deacetylase 6 (HDAC6), a major cytoplasmic α-tubulin deacetylase. We hypothesize that HSP27 enhances HDAC6 activity to promote favorable plaque remodeling by enhancing macrophage migration. Methods / Results: 1. The HDAC6 activity of THP-1 macrophages treated with recombinant HSP27 was assessed using a fluorescent plate reader ELISA. In a dose-dependent fashion, rHSP27 treatment resulted in a 100% increase in HDAC-6 activity compared to the control and was independent of HSP27 phosphorylation. Moreover, HDAC-6 activity was enhanced 100% in peritoneal macrophages harvested from rHsp27-treated ApoE-/- vs. control mice. With the addition of cycloheximide HDAC-6 activity was not prolonged, suggesting that HSP27 did not have an effect on the enzymatic half-life. 2. When HSP27 expression was knocked down in THP-1 cells transfected with a lentivirus carrying specific shRNA-HSP27 vector there was a reduction in HDAC6 activity and an increase in tubulin acetylation - and both of these effects were rescued with the addition of rHSP27. 3. Using a Boyden chamber cell migration assay, we found that rHSP27 treatment increased migration of THP-1 cells without and with ox-LDL loading by ~2- and ~1.5-fold; respectively - an effect that was blocked by either Tubacin (a specific inhibitor of HDAC6) or HSP27 knockdown. Conclusion: Extracellular HSP27 is capable of up-regulating HDAC6 activity in macrophages, an effect associated with enhanced macrophage migratory capacity. These data suggest that HSP27 may be important for potentially improving macrophage egress from plaques thereby resulting in reduced cholesterol content and more stable lesions - factors that may explain the proven predictive biomarker value of elevated serum HSP27 levels.

Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter.

Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms1,2. Interruption of IL18 action reduces atherosclerosis in mice3,4. This study shows that the absence of IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E-deficient (Apoe−/−) mice, nor does it affect IL18 cell surface binding or signaling. IL18 antibody-mediated immunoprecipitation identified an interaction between IL18 and Na-Cl co-transporter (NCC), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney5. Yet, we find NCC expression and colocalization with IL18r in atherosclerotic lesions and both molecules form a complex. IL18 also binds to the cell surface and induces cell signaling and down-stream cytokine expression in NCC-transfected COS-7 cells that do not express IL18r. In Apoe−/− mice, combined deficiency of IL18r and NCC, but not single deficiency, protects mice from atherosclerosis. Peritoneal macrophages from Apoe−/− mice or those lacking IL18r or NCC respond to IL18 binding or IL18 induction of cell signaling and cytokine and chemokine production, but those with combined deficiency of IL18r and NCC do not. This study identifies NCC as an IL18-binding protein that coordinates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

Inflammation blood and tissue factors of plaque growth in an experimental model evidenced by a systems approach.

Purpose: The multifactorial pathogenesis of coronary atherosclerotic lesion formation has been investigated in a swine model of high cholesterol diet induced atherogenesis and data processed by a systems approach.Methods: Farm pigs were fed on standard or high cholesterol diet of 8 and 16 weeks duration. Plasma assessment of total cholesterol, HDL, LDL, and ELISA of some cytokines and ICAM-1 were performed on baseline and end-diet samples. Segments of the right coronary artery were incubated for 24 h in serum-free medium to collect secreted proteins and their expression analyzed by mass spectrometry. Data of plasma and tissue factors were processed by a statistical systems inference approach: both histologic parameters of coronary intimal thickness (IT) and of lesion area (LA) were chosen as dependent variables (coronary atherosclerotic burden).Results: Relations among plasma adhesion molecules, cytokines, lipoproteins, tissue proteins and histology indexes were integrated in a model regression scheme. Bayesian model averaging (BMA) variable selection was chosen as a method to identify relevant factors associated to atherosclerotic burden: TNFα was identified as an associated plasma marker, oxLDL and HDL as relevant lipoproteins; macrophage function related antioxidant Catalase enzyme, lysosome associated Cathepsin D, S100-A10, and Transforming growth factor-beta-induced protein ig-h3 were identified and selected as associated to atherogenesis outcome.Conclusions: The results of this systems approach are consistent with the hypothesis that, in high cholesterol diet-induced experimental atherogenesis, the interaction between plasma cytokines, lipoproteins and artery-specific proteins, influences lesion initiation and growth. In particular, some macrophage function related proteins are found significantly and positively associated to atherosclerotic burden, suggesting a novel molecular framework into the atherogenesis-inflammatory disorder.

Apolipoprotein A-I at the Interface of Vascular Inflammation and Arthritis

Decreased high-density lipoprotein cholesterol (HDL-C) levels are associated with an increased risk of atherosclerosis and its atherothrombotic complications. This has spawned numerous randomized human atherosclerosis trials of measures to indirectly or directly elevate HDL to directly test the HDL hypothesis.1,2 HDL functions, beyond reverse cholesterol transport, include antithrombotic effects, stimulation of endothelial nitric oxide release, and shuttling of microRNAs. Moreover, HDL and its principal apolipoprotein A-I (apoA-I) suppress vascular inflammatory responses in experimental atherogenesis and arterial injury,3,4 though such effects seem modulated by genetic background.5 Significantly, primary extravascular inflammatory processes promote quantitative and qualitative modulation of HDL and apoA-I. This is exemplified by 3 conditions (rheumatoid arthritis [RA], systemic lupus erythematosus [SLE], and gout) in which there is coexisting inflammatory arthritis and increased susceptibility to atherosclerosis, but with differences in mechanisms affecting HDL levels and properties of plasma HDL (see Figure). Figure. Schematic model of putative interfaces of high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) with 3 specific human arthritic diseases. The model summarizes past literature and incorporates new findings from Wu et al,16 in which HDL and apoA-I limit the models of chronic inflammatory arthritis stimulated by anticollagen antibodies or by exogenous toll-like receptor (TLR) 2 ligand signaling. Systemic lupus erythematosus (SLE) and gout are prominent among additional interfaces between altered HDL and apoA-I with arthritic diseases, as discussed in the text. ABCA1 indicates ATP-binding cassette transporter A1; NFκB, nuclear factor-κB; RA, rheumatoid arthritis; and TNF-α, tumor necrosis facor-α. …