Fatty Streak Formation Research Articles

Endothelial Cell-Specific Deletion of P2Y2 Receptor Promotes Plaque Stability in Atherosclerosis-Susceptible ApoE-Null Mice.

Nucleotide P2Y2 receptor (P2Y2R) contributes to vascular inflammation by increasing vascular cell adhesion molecule-1 expression in endothelial cells (EC), and global P2Y2R deficiency prevents fatty streak formation in apolipoprotein E null (ApoE-/-) mice. Because P2Y2R is ubiquitously expressed in vascular cells, we investigated the contribution of endothelial P2Y2R in the pathogenesis of atherosclerosis. EC-specific P2Y2R-deficient mice were generated by breeding VEcadherin5-Cre mice with the P2Y2R floxed mice. Endothelial P2Y2R deficiency reduced endothelial nitric oxide synthase activity and significantly altered ATP- and UTP (uridine 5'-triphosphate)-induced vasorelaxation without affecting vasodilatory responses to acetylcholine. Telemetric blood pressure and echocardiography measurements indicated that EC-specific P2Y2R-deficient mice did not develop hypertension. We investigated the role of endothelial P2Y2R in the development of atherosclerotic lesions by crossing the EC-specific P2Y2R knockout mice onto an ApoE-/- background and evaluated lesion development after feeding a standard chow diet for 25 weeks. Histopathologic examination demonstrated reduced atherosclerotic lesions in the aortic sinus and entire aorta, decreased macrophage infiltration, and increased smooth muscle cell and collagen content, leading to the formation of a subendothelial fibrous cap in EC-specific P2Y2R-deficient ApoE-/- mice. Expression and proteolytic activity of matrix metalloproteinase-2 was significantly reduced in atherosclerotic lesions from EC-specific P2Y2R-deficient ApoE-/- mice. Furthermore, EC-specific P2Y2R deficiency inhibited nitric oxide production, leading to significant increase in smooth muscle cell migration out of aortic explants. EC-specific P2Y2R deficiency reduces atherosclerotic burden and promotes plaque stability in ApoE-/- mice through impaired macrophage infiltration acting together with reduced matrix metalloproteinase-2 activity and increased smooth muscle cell migration.

In This Issue

HomeCirculation ResearchVol. 119, No. 6In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published2 Sep 2016https://doi.org/10.1161/RES.0000000000000120Circulation Research. 2016;119:697Ogg1 Is Atheroprotective (p e76)Download figureDownload PowerPointDNA repair enzyme OGG1 is protective against atherosclerosis, report Tumurkhuu et al.Inflammation is a major factor in the progression of atherosclerosis. It is mediated by a progressive influx of monocytes and macrophages into the injured vessel wall, leading to the formation of fatty streaks that develop into arterial lesions. Inflammation within such plaques is further sustained by abundant mitochondrial DNA damage—caused by reactive oxygen species—that further exacerbate pathology. It has been previously reported that mice lacking a key mitochondrial DNA repair enzyme—oxyguanine glycosylase 1 (OGG1)—have larger fatty streaks in their arteries. And now, Tumurkhuu and colleagues report that genetic deletion of OGG1 increases mitochondrial DNA damage and inflammatory cytokines in the plaque, which results in the formation of inflammasomes in macrophages and larger arterial plaques in atherosclerosis-prone mice. They also found that deficiency of a key inflammasome component, NLRP3, counteracted the effects of OGG1 loss and that OGG1 expression tended to drop as atherosclerosis pathology progressed. In mouse and human cells, OGG1 was suppressed by the proatherogenic microRNA miR-33. The levels of OGG1 were reduced in human coronary and carotid artery plaques compared to normal vessels. Together, these results indicate that boosting OGG1, or mitochondrial DNA repair, in plaques could be a putative therapeutic strategy to slow down the progression of atherosclerotic lesions.LNK Deficiency Promotes Atherosclerosis and Thrombosis (p e91)Download figureDownload PowerPointWang et al discover that a lack of LNK promotes atherosclerosis and thrombosis.Genome-wide association studies (GWAS) have found that a single nucleotide polymorphism (SNP) at the lymphocyte specific adaptor protein (LNK) gene locus is linked with an increase in the risk of coronary heart disease (CHD). The same SNP has also been linked to hypertension, autoimmunity, and increased platelet counts. However, it remains unclear how this genetic variant increases CHD risk. In mice, LNK suppresses thrombopoietin signaling, which in turn suppresses proliferation of hematopoietic stem cells (HSCs). Consistent with these findings, Wang and colleagues now show that, in humans, the LNK risk allele is associated with increased production of HSCs and platelet-producing cells, suggesting that the risk-variant protein has reduced functionality. Using LNK-deficient mice as a model for humans carrying the LNK risk allele, the team showed that reduced LNK function not only boosted leukocyte and platelet production, but it also increased platelet activation. In combination with hyperlipidemia, these effects exacerbated both atherosclerosis and thrombosis. Together, the results indicate that carriers of the LNK risk allele produce more blood cells and activated platelets, which heightens the threat of thrombosis and atherosclerosis. Such individuals presenting with high cholesterol might thus benefit from early and more aggressive treatment.miR-184 and Adipogenesis (p 731)Download figureDownload PowerPointGurha et al suggest dysregulation of microRNA miR-184 contributes to arrhythmogenic cardiomyopathy.Arrhythmogenic cardiomyopathy (AC) is a hereditary condition characterized by the gradual replacement of ventricular cardiomyocytes with fibro-adipocytes causing arrhythmia (particularly of the right ventricle), heart failure, and, possibly, sudden cardiac death. Proteins of the intercalated discs—cell-to-cell adhesion structures in the heart—especially plakophilin2 (PKP2) are commonly mutated in AC, but how these mutations lead to pathology is unclear. To investigate the molecular mechanisms of AC, Gurha and colleagues searched for microRNAs (miRs) that were dysregulated in PKP2-deficient cardiac cells. They found that of the 59 differentially expressed miRs, miR-184 was the most dramatically reduced in this model as well as in two independent mouse models of AC. Suppression of miR-184, which was caused by hypermethylation of the gene locus, led in turn to upregulation of genes involved in, among other things, adipogenesis. This induction of adipogenic genes, together with the observation that the cells tended to accumulate fat droplets, could explain why cardiomyocytes adopt a fibro-adipocytic phenotype in AC. Overexpression of miR-184 in the cells had the opposite effects: reducing fat droplet accumulation and reducing adipogenic gene expression. These results, identifying miR-184 as a novel player in AC, deserve further study. Previous Back to top Next FiguresReferencesRelatedDetails September 2, 2016Vol 119, Issue 6 Advertisement Article InformationMetrics © 2016 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000120 Originally publishedSeptember 2, 2016 PDF download Advertisement

Perilla Oil Reduces Fatty Streak Formation at Aortic Sinus via Attenuation of Plasma Lipids and Regulation of Nitric Oxide Synthase in ApoE KO Mice.

Consumption of n-3 polyunsaturated fatty acids (PUFA) is associated with a reduced incidence of atherosclerosis. Perilla oil (PO) is a vegetable oil rich in α-linolenic acid (ALA), an n-3 PUFA. In this study, antiatherogenic effects and related mechanisms of PO were investigated in atherosclerotic mice. Apolipoprotein E knockout (ApoE KO) mice (male, n=27) were fed high-cholesterol and high-fat diets containing 10% w/w lard (LD), PO, or sunflower oil (SO) for 10weeks. Plasma triglyceride, total cholesterol, and low-density lipoprotein cholesterol concentrations reduced in the PO and SO groups compared to the concentrations in the LD group (P<0.05). The PO group showed reduced fatty streak lesion size at the aortic sinus (P<0.05) compared to the sizes in the LD and SO groups. A morphometric analysis showed enhancement of endothelial nitric oxide synthase expression and reduction of inducible nitric oxide synthase expression in the PO group compared to that in the LD group (P<0.05). Furthermore, aortic protein expression of intercellular cell adhesion molecule 1 and vascular cell adhesion molecule 1 was diminished in the PO group compared to that in the LD and SO groups (P<0.05). These findings suggested that PO inhibited the development of aortic atherosclerosis by improving the plasma lipid profile, regulating nitric oxide synthase, and suppressing the vascular inflammatory response in the aorta of ApoE KO mice.

Deletion of P2Y2 receptor reveals a role for lymphotoxin-α in fatty streak formation

BackgroundLymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE−/−) deficient mice. Methods and resultsEn face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE−/− mice, ApoE−/− mice lacking the P2Y2R gene (ApoE−/−/P2Y2R−/−) did not develop fatty streak lesions when fed a standard chow diet for 15weeks. Systemic and CD4+ T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE−/−/P2Y2R−/−mice. Anti-LTα preventive treatment was initiated in ApoE−/−mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE−/−mice exhibited atherosclerotic lesions at any developmental stage. SignificanceApoE−/− mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.

Experimental Animal Models Evaluating the Causal Role of Lipoprotein(a) in Atherosclerosis and Aortic Stenosis.

Lipoprotein(a) [Lp(a)], comprised of apolipoprotein(a) [apo(a)] and a low-density lipoprotein-like particle, is a genetically determined, causal risk factor for cardiovascular disease and calcific aortic valve stenosis. Lp(a) is the major plasma lipoprotein carrier of oxidized phospholipids, is pro-inflammatory, inhibits plasminogen activation, and promotes smooth muscle cell proliferation, as defined mostly through in vitro studies. Although Lp(a) is not expressed in commonly studied laboratory animals, mouse and rabbit models transgenic for Lp(a) and apo(a) have been developed to address their pathogenicity in vivo. These models have provided significant insights into the pathophysiology of Lp(a), particularly in understanding the mechanisms of Lp(a) in mediating atherosclerosis. Studies in Lp(a)-transgenic mouse models have demonstrated that apo(a) is retained in atheromas and suggest that it promotes fatty streak formation. Furthermore, rabbit models have shown that Lp(a) promotes atherosclerosis and vascular calcification. However, many of these models have limitations. Mouse models need to be transgenic for both apo(a) and human apolipoprotein B-100 since apo(a) does not covalently associated with mouse apoB to form Lp(a). In established mouse and rabbit models of atherosclerosis, Lp(a) levels are low, generally < 20 mg/dL, which is considered to be within the normal range in humans. Furthermore, only one apo(a) isoform can be expressed in a given model whereas over 40 isoforms exist in humans. Mouse models should also ideally be studied in an LDL receptor negative background for atherosclerosis studies, as mice don't develop sufficiently elevated plasma cholesterol to study atherosclerosis in detail. With recent data that cardiovascular disease and calcific aortic valve stenosis is causally mediated by the LPA gene, development of optimized Lp(a)-transgenic animal models will provide an opportunity to further understand the mechanistic role of Lp(a) in atherosclerosis and aortic stenosis and provide a platform to test novel therapies for cardiovascular disease.

Atherosclerotic cardiovascular disease: a review of initiators and protective factors

Atherosclerotic cardiovascular disease (CVD) is a collective term comprising of a group of disorders of the heart and blood vessels. These diseases are the largest cause of morbidity and premature death worldwide. Coronary heart disease and cerebrovascular disease (stroke) are the most frequently occurring diseases. The two major initiators involved in the development of atherosclerotic CVD are vascular production of reactive oxygen species (ROS) and lipid oxidation. In atherosclerosis development, ROS is associated with rapid loss of anti-inflammatory and anti-atherogenic activities of the endothelium-derived nitric oxide (NO(·)) resulting in endothelial dysfunction. In part involving activation of the transcription factor NF-κB, ROS have been involved in signaling cascades leading to vascular pro-inflammatory and pro-thrombotic gene expression. ROS is also a potent activator of matrix metalloproteinases (MMPs), which indicate plaque destabilization and rupture. The second initiator involved in atherosclerotic CVD is the oxidation of low-density lipoproteins (LDL). Oxidation of LDL in vessel wall leads to an inflammatory cascade that activates atherogenic pathway leading to foam cell formation. The accumulation of foam cells leads to fatty streak formation, which is the earliest visible atherosclerotic lesion. In contrast, the cardiac sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) and hepatic apolipoprotein E (apoE) expression can improve cardiovascular function. SERCA2a regulates the cardiac contractile function by lowering cytoplasmic calcium levels during relaxation, and affecting NO(·) action in vascular cells, while apoE is a critical ligand in the plasma clearance of triglyceride- and cholesterol-rich lipoproteins.

Study of pomegranate (Punica granatum L.) peel extract containing anthocyanins on fatty streak formation in the renal arteries in hypercholesterolemic rabbits.

Background:The influence of the supplementation of pomegranate peel extract containing anthocyanins on atherosclerotic plaque formation induced by hypercholesterolemia was investigated in renal arteries in rabbits.Materials and Methods:After the determination of polyphenol and anthocyanin's content of P. granatum peel hydroalcoholic extract, 30 male rabbits were randomly divided into three groups. They were fed basic diet, hypercholesterolemic diet and hypercholesterolemic diet along with P. granatum peel extract (polyphenolic content for each rabbit 1 g/kg diet) for 2 month. Blood samples were collected at the begging, middle and end of the study in order to measure lipid concentration and oxidative and antioxidative status variables, and renal arteries were taken for the assessment of atherosclerotic plaques at the end of the study.Results:The results reveal that P. granatum peel extract significantly increases serum antioxidant capacity in the extract recipient group in comparison with hypercholesterolemic control (P < 0.05). No significant differences are observed in total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, very low-density lipoprotein and in mean size of accumulated fatty streaks in renal arteries in the extract treatment group in comparison with hypercholesterolemic control (P > 0.05).Conclusion:The results of this study indicate that consumption of pomegranate peel extract containing anthocyanins (polyphenol content 1 g/kg diet) despite of a significant increase in serum antioxidant capacity cannot protect the kidneys from hypercholesterolemia-induced damages during the treatment period.

Increased diet-induced fatty streak formation in female mice with deficiency of liver-derived insulin-like growth factor-I.

The role of endocrine IGF-I for atherosclerosis is unclear. We determined the importance of circulating, liver-derived IGF-I for fatty streak formation in mice. Mice with adult, liver-specific IGF-I inactivation (LI-IGF-I−/− mice, serum IGF-I reduced by approximately 80 %) and control mice received an atherogenic (modified Paigen) diet between 6 and 12 months of age. At study end, Oil Red O staining of aortic root cryosections showed increased fatty streak area and lipid deposition in female but not in male LI-IGF-I−/− mice compared to controls. Mac-2 staining of aortic root and measurements of CD68 mRNA level in femoral artery revealed increased macrophage accumulation in proportion to the increased fatty streak area in female LI-IGF-I−/− mice. Moreover, female LI-IGF-I−/− mice displayed increased serum cholesterol and interleukin-6 as well as increased vascular cell-adhesion molecule 1 (VCAM1) mRNA levels in the femoral artery and elevated VCAM1 protein expression in the aortic root. Thus, increased diet-induced fatty streak formation in female LI-IGF-I−/− mice was associated with increased serum cholesterol and signs of systemic inflammation, endothelial activation, lipid deposition, and macrophage infiltration in the vascular wall.

Abstract 18300: Association Between a Genetic Risk Score for Clinical CAD and Early Stage Lesions in the Coronary and Aorta Provides Insights Into the Pathophysiology of Atherosclerosis

Autopsy studies suggest that the fatty streak is the earliest identifiable lesion of atherosclerosis. However, not all fatty streaks progress to more advanced lesions of atherosclerosis. Furthermore, the correlation between the extent of fatty streaks in younger persons and the extent of more advanced lesions in older persons as well as the community rates of CAD is substantially lower for the aorta compared to the coronaries. These observations suggest that the pathophysiologic processes leading to the formation of fatty streaks and raised atherosclerotic lesions may differ between the two vascular beds. To test this hypothesis, we analyzed genetic data from 564 white participants of the Pathobiological Determinants of Atherosclerosis in Youth study between the age of 15 and 34 at the time of accidental death. We ranked subjects by the percent surface area of involvement of fatty streaks and raised lesions in their right coronary artery, thoracic aorta, and abdominal aorta, respectively, on post-mortem examination. We then defined cases within each lesion type and artery location as subjects with a sex specific ranking that was in the top quartile. We found a genetic risk score (GRS) composed of high-risk alleles at 32 loci for clinical CAD operating independent of traditional risk factors to be robustly associated with the probability of being a case for both fatty streaks and raised atherosclerotic lesions in the right coronary artery (Table). In contrast, the same GRS exhibited no association with case status for fatty streaks in the aorta and only a weak trend for association with case status for raised lesions in the aorta. Our findings support the notion that not all fatty streaks are equivalent in their predisposition to atherosclerosis and suggest that some atherosclerosis susceptibility loci uncovered through GWAS studies of clinical CAD may only be active in the coronary tree.

Scavenger receptors and non-coding RNAs: relevance in atherogenesis.

Scavenger receptors (SRs), which recognize modified low-density lipoprotein (LDL) by oxidation or acetylation, are a group of receptors on plasma membrane of macrophages and other cell types. These receptors by facilitating modified LDL uptake are a primary step in the intracellular accumulation of modified LDL and formation of fatty streak. Non-coding RNAs (ncRNAs) are a group of functional RNA nucleotides that are not translated into protein, and include microRNAs (miRs), snoRNAs, siRNAs, snRNAs, exRNAs, piRNAs, and the long ncRNAs (lncRNAs). Recently, ncRNAs have received much attention due to their effects in a variety of disease states such as atherosclerotic cardiovascular disease and cancers. A host of ncRNAs, such as miRs and lncRNAs, have been found to be involved in the regulation of SRs and the inflammatory cascade and subsequently atherosclerosis. Here, we review this important area to create interest in this growing field among researchers and clinicians alike.

Angiopoietin-2 blocking antibodies reduce early atherosclerotic plaque development in mice.

ObjectiveAngiopoietin-2 (Ang-2) blocking agents are currently undergoing clinical trials for use in cancer treatment. Ang-2 has also been associated with rupture-prone atherosclerotic plaques in humans, suggesting a role for Ang-2 in plaque stability. Despite the availability of Ang-2 blocking agents, their clinical use is still lacking. Our aim was to establish if Ang-2 has a role in atheroma development and in the transition of subclinical to clinically relevant atherosclerosis. We investigated the effect of antibody-mediated Ang-2 blockage on atherogenesis after in a mouse model of atherosclerosis.MethodsHypercholesterolemic (low-density lipoprotein receptor−/− apolipoprotein B100/100) mice were subjected to high-cholesterol diet for eight weeks, one group with and one group without Ang-2 blocking antibody treatment during weeks 4–8.To enhance plaque development, a peri-adventitial collar was placed around the carotid arteries at the start of antibody treatment. Aortic root, carotid arteries and brachiocephalic arteries were analyzed to evaluate the effect of Ang-2 blockage on atherosclerotic plaque size and stable plaque characteristics.ResultsAnti-Ang-2 treatment reduced the size of fatty streaks in the brachiocephalic artery (−72%, p < 0.05). In addition, antibody-mediated Ang-2 blockage reduced plasma triglycerides (−27%, p < 0.05). In contrast, Ang-2 blockage did not have any effect on the size or composition (collagen content, macrophage percentage, adventitial microvessel density) of pre-existing plaques in the aortic root or collar-induced plaques in the carotid artery.ConclusionsAng-2 blockage was beneficial as it decreased fatty streak formation and plasma triglyceride levels, but had no adverse effect on pre-existing atherosclerosis in hypercholesterolemic mice.

Abstract 626: Vascular Endothelial HSP60 and Von Willebrand Factor Induction Are Both Involved in Promoting Fatty-Streak Formation in ApoE Null Mice

Objectives: This study was designed to determine the murine atherosclerosis in ApoE null mice with inducible expression of human heat shock protein 60 (hHSP60) in vascular endothelium. Background: Autoimmunity to HSP60 may be involved in eliciting early atherosclerotic lesions. Most classical risk factors of atherosclerosis were previously shown in cell culture models to induce HSP60 expression in vascular ECs and in vivo particularly at regions predilected to lesions. However, it has not been demonstrated directly in an animal model whether HSP60 induction in ECs are capable of influencing lesion formation. Methods: We developed ApoE -/- ::Cdh5-CreER T2 ::G-Lox-HSP60 triple Tg mouse model with tamoxifen-induced hHSP60 expression in ECs. Eight week-old triple TG mice were fed by tamoxifen citrate-contained chow for 2 weeks, followed by high fat chow (HF) for additional 4 and 8 weeks before sacrificed for oil red staining for fatty streaks and IHC staining. Results: In tamoxifen-fed triple Tg mouse, vascular EC expression of hHSP60 increased fatty-streak formation and increased the severity of lesions in comparison with uninduced triple Tg or wildtype ApoE mouse. In triple Tg mouse, we found tamoxifen strongly induced HSP60 expression in ECs of the lesions and to a lesser degree in ECs of veins and other arterial regions not predilected to lesions. Concomitant VWF induction at ECs and subendothelial regions was observed at areas with increased HSP60 expression. Conclusions: hHSP60 induction at vascular ECs accelerates fatty-steak formation in ApoE null mice that may involve increasing local VWF expression.

Abstract 15768: Mitochondria-Targeted DNA Repair Glycosylase Ogg1 Suppresses Early Stage of Atherogenesis in Ogg1 Deficient Mice

Introduction: Reactive oxygen species (ROS) play a key role in the development of atherosclerosis. Mitochondria are a main source of endogenous ROS in the cell. Mitochondrial DNA (mtDNA) is sensitive to oxidation and our previous results from cultured cell and intact animal models suggest that increasing mtDNA repair prevents both oxidative mtDNA damage and associated cytotoxicity and cellular dysfunction. Involvement of oxidative mtDNA damage in disorders characterized by chronic oxidative stress has been less thoroughly studied. Hypothesis: In the present study we tested the hypothesis that transgenic modulation of Ogg1, a DNA glycosylase mediating the first step in the base excision repair of oxidative mtDNA damage, coordinately regulates atherogenesis in mice fed a high fat diet. Methods: Wild type (WT) mice, Ogg1 knock-out (KO) mice and KO mice transgenically overexpressing mitochondria-targeted Ogg1 (KO-Tg) were fed pro-atherogenic Western type diet for 14 weeks and analyzed for mtDNA damage and signs of atherogenesis. Results: KO mice fed a high fat diet had increased oxidative mtDNA damage in cardiac tissue, whereas KO-Tg animals did not differ from WT mice (WT: 0.18 ± 0.04; KO: 0.35 ± 0.04; KO-Tg: 0.15 ± 0.01 lesions per 10 4 bp; n=3, P&lt;0.05; quantitative Southern blot analysis). We did not observe significant atherosclerotic plaque formation in the aortic valve of animals from any group; however appearance of fatty streaks, indicative of early plaque development, was more evident in KO mice (WT: 179 ± 20; KO: 384 ± 59 pixels, n=4, P&lt;0.05; immunohistochemistry for Fc receptor - general marker of inflammatory cells). This effect was completely blocked in KO-Tg mice. We found increased number of apoptotic cells in the aortic valve of KO, but not KO-Tg mice (WT: 2.00 ± 0.50; KO: 4.25 ± 0.48; KO-Tg: 2.14 ± 0.85 apoptotic cells, n=4, P&lt;0.05; TUNEL assay). Conclusion: Our data demonstrate that Ogg1 deficiency in mice fed a high fat diet leads to increased oxidative mtDNA damage, appearance of fatty streaks and cell apoptosis. In contrast, enhancement of mtDNA repair with mitochondria-targeted Ogg1 reduces fatty streaks formation and apoptosis induced by a high fat diet. These results suggest mtDNA damage and repair could be important targets for atheroprotection.

Cholesterol crystal-induced endothelial cell activation is complement-dependent and mediated by TNF

Cholesterol crystals are known to be a hallmark of atherosclerosis with recent studies demonstrating deposition of these crystals in early fatty streak formation as well as penetrating the intima following plaque rupture. Inflammation has also become a central focus in atheroma development and endothelial cell activation is recognized as necessary for the recruitment of inflammatory cells to the plaque. However, the extent to which cholesterol crystals can induce inflammation and activate endothelial cells is not known. To investigate this, we developed a novel model activating human umbilical vein endothelial cells using lepirudin anticoagulated human whole blood. We found that cholesterol crystals caused a marked and dose-dependent increase in the adhesion molecules E-selectin and ICAM-1 on the surface of the endothelial cells after incubation with whole blood. There was no activation of the cells when the crystals were incubated in medium alone, or in human serum, despite substantial crystal-induced complement activation in serum. Complement inhibitors at the C3 and C5 levels reduced the whole blood induced endothelial cell activation by up to 89% (p<0.05) and abolished TNF release (p<0.01). Finally, the TNF inhibitor infliximab reduced endothelial activation to background levels (p<0.05). In conclusion, these data demonstrate that endothelial activation by cholesterol crystals is mediated by complement-dependent TNF release, and suggests that complement-inhibition might have a role in alleviating atherosclerosis-induced inflammation.

Curcumin modulation of high fat diet-induced atherosclerosis and steatohepatosis in LDL receptor deficient mice

ObjectiveConsuming curcumin may benefit health by modulating lipid metabolism and suppressing atherogenesis. Fatty acid binding proteins (FABP-4/aP2) and CD36 expression are key factors in lipid accumulation in macrophages and foam cell formation in atherogenesis. Our earlier observations suggest that curcumin's suppression of atherogenesis might be mediated through changes in aP2 and CD36 expression in macrophages. Thus, this study aimed to further elucidate the impact of increasing doses of curcumin on modulation of these molecular mediators on high fat diet-induced atherogenesis, inflammation, and steatohepatosis in Ldlr−/− mice. MethodsLdlr−/− mice were fed low fat (LF) or high fat (HF) diet supplemented with curcumin (500 HF + LC; 1000 HF + MC; 1500 HF + HC mg/kg diet) for 16 wks. Fecal samples were analyzed for total lipid content. Lipids accumulation in THP-1 cells and expression of aP2, CD36 and lipid accumulation in peritoneal macrophages were measured. Fatty streak lesions and expression of IL-6 and MCP-1 in descending aortas were quantified. Aortic root was stained for fatty and fibrotic deposits and for the expression of aP2 and VCAM-1. Total free fatty acids, insulin, glucose, triglycerides, and cholesterol as well as several inflammatory cytokines were measured in plasma. The liver's total lipids, cholesterol, triglycerides, and HDL content were measured, and the presence of fat droplets, peri-portal fibrosis and glycogen was examined histologically. ResultsCurcumin dose-dependently reduced uptake of oxLDL in THP-1 cells. Curcumin also reduced body weight gain and body fat without affecting fat distribution. During early intervention, curcumin decreased fecal fat, but at later stages, it increased fat excretion. Curcumin at medium doses of 500–1000 mg/kg diet was effective at reducing fatty streak formation and suppressing aortic expression of IL-6 in the descending aorta and blood levels of several inflammatory cytokines, but at a higher dose (HF + HC, 1500 mg/kg diet), it had adverse effects on some of these parameters. This U-shape like trend was also present when aortic root sections were examined histologically. However, at a high dose, curcumin suppressed development of steatohepatosis, reduced fibrotic tissue, and preserved glycogen levels in liver. ConclusionCurcumin through a series of complex mechanisms, alleviated the adverse effects of high fat diet on weight gain, fatty liver development, dyslipidemia, expression of inflammatory cytokines and atherosclerosis in Ldlr−/− mouse model of human atherosclerosis. One of the mechanisms by which low dose curcumin modulates atherogenesis is through suppression of aP2 and CD36 expression in macrophages, which are the key players in atherogenesis. Overall, these effects of curcumin are dose-dependent; specifically, a medium dose of curcumin in HF diet appears to be more effective than a higher dose of curcumin.

T-Cell Immunoglobulin and Mucin Domain 3 Acts as a Negative Regulator of Atherosclerosis

Atherosclerosis is a chronic autoimmune-like disease in which lipids and fibrous elements accumulate in the arterial blood vessels. T cells are present within atherosclerotic plaques, and their activation is partially dependent on costimulatory signals, which can either provide positive or negative signals that promote T-cell activation or limit T-cell responses, respectively. T-cell immunoglobulin and mucin domain 3 (Tim-3) is a coinhibitory type 1 transmembrane protein that affects the function of several immune cells involved in atherosclerosis, such as monocytes, macrophages, effector T cells, and regulatory T cells. In the present study, we determined the role of Tim-3 in the development of atherosclerosis. Western-type diet-fed low-density lipoprotein receptor-deficient (LDLr(-/-)) mice were treated with an anti-Tim-3 antibody for 3 and 8 weeks. Anti-Tim-3 administration increased fatty streak formation with 66% and increased atherosclerotic plaque formation after 8 weeks with 35% in the aortic root and with 50% in the aortic arch. Furthermore, blockade of Tim-3 signaling increased percentages of circulating monocytes with 33% and lesional macrophages with 20%. In addition, anti-Tim-3 administration increased CD4(+) T cells with 17%, enhanced their activation status, and reduced percentages of regulatory T cells with 18% and regulatory B cells with 37%. It is known that Tim-3 acts as a negative regulator of both innate and adaptive immune responses, and in the present study, we show that anti-Tim-3 treatment augments lesion development, accompanied by an increase in the number of monocytes/macrophages and CD4(+) T cells and by decreased regulatory T cells and regulatory B cells.

How Do Elevated Triglycerides and Low HDL-Cholesterol Affect Inflammation and Atherothrombosis?

This review article summarizes recent research into the mechanisms as to how elevated levels of triglyceride (TG) and low levels of high- density- lipoprotein cholesterol (HDL-C) contribute to inflammation and atherosclerosis. Evidence supports the role of TG-rich lipoproteins in signaling mechanisms via apolipoproteins C-III and free fatty acids leading to activation of NFKβ, VCAM-1 and other inflammatory mediators which lead to fatty streak formation and advanced atherosclerosis. Moreover, the cholesterol content in TG-rich lipoproteins has been shown to predict CAD risk better than LDL-C. In addition to reverse cholesterol transport, HDL has many other cardioprotective effects which include regulating immune function. The "functionality" of HDL appears more important than the level of HDL-C. Insulin resistance and central obesity underlie the pathophysiology of elevated TG and low HDL-C in metabolic syndrome and type 2 diabetes. Lifestyle recommendations including exercise and weight loss remain first line therapy in ameliorating insulin resistance and the adverse signaling processes from elevated levels of TG-rich lipoproteins and low HDL-C.

Abstract 463: TLT-1 Deficiency Affects Hypercholesterolemia and Progression of the Atherosclerotic Plaque in Apoe Null Mice

Platelet activation at sites of inflammation triggers the secretion of molecules that induce the transition of atherosclerosis from fatty streak to an acute disease, featuring an increased vulnerability of the atherosclerotic lesion that results in plaque rupture and thrombosis. TLT-1 (Triggering Receptor Expressed in Myeloid cells (TREM)-like transcript-1) is a molecule exclusively found in the α-granules of megakarocytes and platelets and has a demonstrated effect in inflammatory responses. Upon platelet activation, TLT-1 is moved to the platelet surface, while its soluble form, s-TLT-1, is secreted and detected in serum. Studies using the C57Bl/6 treml1 - /- mouse demonstrated a predisposition to hemorrhage after an acute inflammatory challenge suggesting that TLT-1 may be a key regulatory molecule in the interface between hemostatic and inflammatory mechanisms. Because we have found that sTLT-1 levels are significantly elevated in apoE mice when compared to wild type, we hypothesized that TLT-1 may be playing an important role in the progression of atherosclerosis. To address this possibility, we generated apoE - /- / treml1 - /- double knockout mice [DN]. Assessment of lesions after 4 weeks high-fat diet (HFD) demonstrated that at early stages, TLT-1 deficiency accelerates fatty streak formation. After 20 weeks on HFD, lesions in both apoE - /- and [DN] mice progressed to an advance fibrous plaque stage. Although their lesion sizes were not substantially different, lesion compositions were. The mechanistic basis of these differences appears to be that the [DN] mice have significantly higher cholesterol levels when compared to apoE - /- mice. The increased cholesterol levels extend to the treml1 -/- mouse when compared to wild type mice at 4 weeks on HFD, this difference, however, gradually subsides as wild type mice cholesterol levels increase over 20 weeks. Interestingly, cholesterol levels in 50 week old mice on chow diet revealed minimal differences between test and control mice suggesting the higher cholesterol levels are related to increased dietary intake. Our work defines a surprising role for TLT-1 in the regulation of serum cholesterol levels during atherogenesis.

Arginase inhibition by piceatannol-3′-O-β-D-glucopyranoside improves endothelial dysfunction via activation of endothelial nitric oxide synthase in ApoE-null mice fed a high-cholesterol diet

Elevated plasma cholesterol is a hallmark of numerous cardiovascular diseases that are closely linked to endothelial dysfunction indicating decreased nitric oxide (NO) production in the endothelium. It has been previously demonstrated that piceatannol-3'-O-β-D-glucopyranoside (PG) inhibits arginase activity and reciprocally regulates NO production. Here, we aimed to ascertain whether PG ameliorates vascular function in wild-type (WT) and atherogenic model mice [apolipoprotein E-null mice (ApoE-/-)] and to investigate the possible underlying mechanism. Preincubation of aortic vessels from WT mice fed a normal diet (ND) with PG attenuated vasoconstriction response to U46619 and phenylephrine (PE), while the vasorelaxant response to acetylcholine (Ach) was markedly enhanced in an endothelium-dependent manner. However, the endothelium-independent NO donor, sodium nitroprusside (SNP), did not change vessel reactivity. In thoracic aorta from ApoE-/- mice, a high-cholesterol diet (HCD) induced an increase in arginase activity, a decrease in NO release and an increase in reactive oxygen species generation that was reversed by treatment with PG. The effect of PG was associated with enhanced stability of the eNOS dimer and was not dependent on the expression levels of arginase II and eNOS proteins, although eNOS expression was increased in ApoE-/- mice fed an HCD. Furthermore, PG treatment attenuated the PE-dependent contractile response, and significantly improved the Ach-dependent vasorelaxation response in aortic rings from ApoE-/- mice fed an HCD. On the other hand, PG incubation neither altered the contractile response to a high K+ solution nor the relaxation response to SNP. When analyzing the L-arginine content using high-performance liquid chromatography, PG incubation increased the intracellular L-arginine concentration. PG administration in the drinking water significantly reduced fatty streak formation in ApoE-/- mice fed an HCD. These data indicate that PG improves the pathophysiology of cholesterol-mediated endothelial dysfunction. Therefore, we conclude that the development of PG as a novel effective therapy for preventing atherosclerotic diseases is warranted.

The Last Contribution of Hemophiliacs to Blood Coagulation: Atherosclerosis Is Not Prevented by Hypocoagulability

“In principio omnes errant hemophiliacs”. In the past, all bleeding patients were considered as hemophiliacs. Quick demonstrated in the mid thirties that hemophiliacs had a normal prothrombin time (P.T.). This allowed the first distinction of “bleeding patients” in two groups, some with a normal P.T. and some with a prolonged P.T. [1]. Only in 1947, “hemophilia” could be suspected to comprise two different diseases, when Pawlowski in Argentina showed the correction of the defect after mixing the plasmas of two patients known to have hemophilia, thereby suggesting the existence of two types of defect [2]. This was independently confirmed later on by Aggeler et al in the U.S.A. and in England by the Oxford Group in 1952 [3, 4]. Since that time the contribution of hemophilic patients to the understanding of blood coagulation was astonishing and could be summarized along the following lines, without the presumption to be complete: 1). Cryoprecipitates corrected Hemophilia A but not, or very little the hemophilia B defect [5]. 2). Immunological studies on hemophilia A and B have opened new horizons on VWD and demonstrated the existence of hemophilia variants with cross reacting material (CRM) [6, 7]. These studies were then instrumental in the classification of other clotting defects, especially, FX and F VII defects [8]. 3). A hemophilia B variant, hemophilia BM, showed a prolonged prothrombin time when a reagent containing ox-brain thomboplastin was used in the assay system [9]. This opened new horizons in prothrombin complex diseases with regard to their reactivity towards tissue thromploplastins of different origin [10]. 4). Another peculiar contribution was the observation that, in a special type of hemophilia B (Hemophilia B Leyden) there was a variable correction of the defect as the patients aged [11]. This cast light on the androgen-dependent maturation of FIX at the hepatocyte level. 5). Hemophilia patients payed heavily for our delays and faults in the developing of safe concentrates. The pain, sacrifice and the size of the cemetery of hemophiliacs has still to be properly acknowledged and described. 6). Synoviectomy carried out in hemophiliacs in an attempt to impede or delay the occurrence of the arthropathy has considerably spurred the use of this procedure in several orthopedic diseases [12]. 7). The development in some patients of antibodies after replacement therapy has opened important lines of investigation in the genetic control of immunological mechanisms. 8). The widespread treatment of HIV and hepatitis B and C in hemophilia patients has supplied valuable information for the management of these infections even in non-bleeding subjects. 9). The role of liver transplantation in the management of hemophilia is still disputed. However some success has been obtained even by the live donor transplant [13-15]. 10). The status of genetic therapy of hemophilia is still open and very controversial but hemophiliacs have given a contribution even to this field [16, 17]. They represent, at least on theoretical grounds, ideal subjects for gene therapy [16]. Now, hemophiliacs supply us with a new important information. They have shown that atherosclerosis’ may occur in them as in normal persons [18, 19]. Old studies had suggested that the clotting defect could exercise a protection against atherosclerosis. They were wrong. Now it is known that this is not the case. A new medical field has been opened. How to treat hemophilia patients with atherosclerosis and its complications, notably acute coronary syndromes. So, after so many contributions, another major contribution has emerged. Thanks to the improved overall care and to the safety of replacement therapy, the hemophilia population has aged and with age , age related problems have appeared [19, 20]. The demonstration that atherosclerosis may occur in patients with hemophilia and with other congenital coagulation disorders is an event of great importance in blood coagulation. It indicates that hypocoagulability does not prevent the aging of the arteries, namely the formation of fatty streaks or plaques in the intima of the arteries. The focus on the pathogenesis of atherosclerosis must rest therefore on lipids (mainly cholesterol) and inflammation. Congenital hypocoagulability may still protect from atherothrombosis which refers to the formation of a thrombus on fissured on ulcerated plaques. However an arterial occlusion may occur without the formation of a thrombus. This may explain at least part of the myocardial infarctions (M.I.) seen in patients with Hemophilia and other clotting disorders. Autopsy and coronographic data indicate that atherosclerotic streaks and plaques are always present, sometimes complicated by the formation of a thrombus [21, 22]. Manuscript accepted for publication May 27, 2013