Early Stages Of Atherogenesis Research Articles

Cardioprotection by Phytochemicals via Antiplatelet Effects and Metabolism Modulations.

The multi-factorial aetiology is the characteristic element of cardiac disorders. Further scientific research had recognized for a long time that platelet function is related to the risk of developing atherosclerosis. Activated blood platelets play a central role in this chronic inflammatory condition as they contribute to plaque formation within blood vessels in the early stages of atherogenesis. The present review article summarizes the cardioprotective role played by Phytochemicals via antiplatelet effects. Also, various metabolic modifications have been included that have protective effect during cardiac pathology.

Involvement of Mitochondrial (mt) DNA Oxidative Damage and Circulating Fragments in Early Stages of Atherogenesis

Mitochondrial dysfunction underlies many processes in the development of atherosclerosis, including generation of reactive oxygen species, apoptosis, and inflammation. Oxidative mtDNA damage is a proximate cause of mitochondrial dysfunction and there is growing evidence that it can directly promote atherosclerosis. The mechanism of this action remains unclear, but may include initiation of apoptosis and inflammation by mtDNA oxidation. Previously, we have demonstrated protective role of mitochondria‐targeted DNA glycosylase Ogg1 against oxidative stress and apoptosis in cell culture and against vascular injury in animal models. In the present study we tested the hypothesis that transgenic modulation of Ogg1 coordinately regulates atherogenesis in mice fed a high fat diet. Wild type (WT) mice, Ogg1 knock‐out (KO) mice and KO mice transgenically overexpressing mitochondria‐targeted Ogg1 (KO‐Tg) were fed Western diet and analyzed for mtDNA damage and signs of atherogenesis. When compared to WT animals, KO mice displayed significantly higher mtDNA oxidative damage in cardiac tissue, elevated cell apoptosis and increased size of fatty streaks in aortic valve. These effects were eliminated in KO‐Tg mice. Interestingly, mtDNA oxidative damage correlated with the abundance of plasma pro‐inflammatory mtDNA fragments. These results indicate that enhanced mtDNA repair suppresses apoptosis and atherogenesis, suggesting that mtDNA and its integrity could be an important target for atheroprotection. Supported by NIH.

Activation of an Innate Immune Receptor, Nod1, Accelerates Atherogenesis in Apoe−/− Mice

Atherosclerosis is essentially a vascular inflammatory process in the presence of an excess amount of lipid. We have recently reported that oral administration of a nucleotide-binding oligomerization domain (Nod)-1 ligand, FK565, induced vascular inflammation in vivo. No studies, however, have proven the association between Nod1 and atherosclerosis in vivo. To investigate a potential role of NOD1 in atherogenesis, we orally administered FK565 to apolipoprotein E knockout (Apoe(-/-)) mice for 4 wk intermittently and performed quantification of atherosclerotic lesions in aortic roots and aortas, immunohistochemical analyses, and microarray-based gene expression profiling of aortic roots. FK565 administration accelerated the development of atherosclerosis in Apoe(-/-) mice, and the effect was dependent on Nod1 in non-bone marrow origin cells by bone marrow transplantation experiments. Immunohistochemical studies revealed the increases in the accumulation of macrophages and CD3 T cells within the plaques in aortic roots. Gene expression analyses of aortic roots demonstrated a marked upregulation of the Ccl5 gene during early stage of atherogenesis, and the treatment with Ccl5 antagonist significantly inhibited the acceleration of atherosclerosis in FK565-administered Apoe(-/-) mice. Additionally, as compared with Apoe(-/-) mice, Apoe and Nod1 double-knockout mice showed reduced development of atherosclerotic lesions from the early stage as well as their delayed progression and a significant reduction in Ccl5 mRNA levels at 9 wk of age. Data in the present study show that the Nod1 signaling pathway in non-bone marrow-derived cells contributes to the development of atherosclerosis.

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<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<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<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.

Interleukin 10-coated nanoparticle systems compared for molecular imaging of atherosclerotic lesions

Atherosclerosis (AS) is one of the leading causes of mortality in high-income countries. Early diagnosis of vulnerable atherosclerotic lesions is one of the biggest challenges currently facing cardiovascular medicine. The present study focuses on developing targeted nanoparticles (NPs) in order to improve the detection of vulnerable atherosclerotic-plaques. Various biomarkers involved in the pathogenesis of atherosclerotic-plaques have been identified and one of these promising candidates for diagnostic targeting is interleukin 10 (IL10). IL10 has been shown to be a key anti-inflammatory responding cytokine in the early stages of atherogenesis, and has already been used for therapeutic interventions in humans and mice. IL10, the targeting sequence, was coupled to two different types of NPs: protamine-oligonucleotide NPs (proticles) and sterically stabilized liposomes in order to address the question of whether the recognition and detection of atherosclerotic-lesions is primarily determined by the targeting sequence itself, or whether it depends on the NP carrier system to which the biomarker is coupled. Each IL10-targeted NP was assessed based on its sensitivity and selectivity toward characterizing atherosclerotic-plaque lesions using an apolipoprotein E-deficient mouse as the model of atherosclerosis. Aortas from apolipoprotein E-deficient mice fed a high fat diet, were stained with either fluorescence-labeled IL10 or IL10-coupled NPs. Ex vivo imaging was performed using confocal laser-scanning microscopy. We found that IL10-targeted proticles generated a stronger signal by accumulating at the surface of atherosclerotic-plaques, while IL10-targeted, sterically stabilized liposomes showed a staining pattern deeper in the plaque compared to the fluorescence-labeled IL10 alone. Our results point to a promising route for enhanced in vivo imaging using IL10-targeted NPs. NPs allow a higher payload of signal emitting molecules to be delivered to the atherosclerotic-plaques, thus improving signal detection. Importantly, this allows for the opportunity to visualize different areas within the plaque scenario, depending on the nature of the applied nanocarrier.

Abstract 653: Endothelial ASC Modulates Early Atherosclerosis by Limiting Monocyte Recruitment

The inflammasome consists of an innate immune receptor, the adaptor molecule ASC, and the effector Caspase-1 that is responsible for processing signature cytokines such as IL-1beta. The exact role of the inflammasome in atherosclerosis is not clear since both pathogenic and dispensable roles have been reported. Notably, ASC has functions that may impact atherosclerosis but are unrelated to the inflammasome, for example the induction of apoptosis and the regulation of pro-inflammatory transcription factors. The various roles of ASC (i.e. IL-1beta processing, cell death, NF-kappaB and AP-1 regulation) may modulate the progression of atherosclerosis depending on lesion stage and explain the divergent findings highlighted above. To examine the role of ASC in lesion progression, we bred ASC -deficient mice onto the Ldlr -/- background and initiated a cholesterol-rich diet (CRD). In the early stages of atherogenesis (2 weeks of CRD), ASC -/- Ldlr -/- mice had a significantly increased lesion surface area, leukocyte content, and rate of Ly6C hi monocyte recruitment in the ascending aorta. Bone marrow transplant experiments suggested that ASC deficiency in the vessel wall was responsible for this phenotype. Knockdown of ASC by siRNA in cultured human umbilical vein endothelial cells lead to increased TNF-alpha-induced expression of VCAM1 and monocyte adhesion in parallel-plate flow chamber assays. Monocyte adhesion was not affected by silencing Caspase-1, suggesting that the effect of ASC was independent of the inflammasome. Preliminary findings implicate MAPK family members in ASC-mediated regulation of endothelial adhesion molecules. At 16 weeks of a CRD ASC -/- Ldlr -/- mice had a significantly decreased lesion size compared with controls. Lesions from ASC -/- Ldlr -/- mice had a smaller necrotic core area accompanied by decreased numbers of apoptotic leukocytes as determined by active Caspase-3 and TUNEL positivity. These data are in accordance with the pro-atherogenic role of ASC in later stages of atherosclerosis. However our findings demonstrate a novel protective role for ASC in the early stage, which represents a non-inflammasome function via modulation of endothelial-associated adhesion molecules.

Coronary endothelial dysfunction induced by Nlrp3 inflammasome activation during hypercholesterolemia: beyond inflammation (851.13)

Recent studies have shown that Nlrp3 inflammasomes function as a major intracellular molecular machinery to switch on the inflammatory response upon sensing danger signals such as elevated plasma levels of cholesterol during hypercholesterolemia (HC). The present study tested the hypothesis that endothelial Nlrp3 inflammasome activation has uncanonical direct actions on endothelial cells (ECs) producing endothelial dysfunction prior to or during inflammation, which contribute to vascular injury at early stages of atherogenesis. Endothelial dysfunction was assessed by evaluating endothelium‐dependent vasodilation (EDVD) in isolated, perfused and pressurized coronary arteries in response to bradykinin and acetylcholine. HC induced by poloxamer 407 impaired EDVD in Nlrp3+/+ mice, which was markedly ameliorated in Nlrp3‐/‐ mice. Pretreatment of coronary arteries with inflammasome products showed that IL‐1β or HMGB1 impaired EDVD in normal Nlrp3+/+ mice suggesting a detrimental effect of IL‐1β or HMGB1 on the endothelium. In contrast, HC‐induced impairment of EDVD in Nlrp3+/+ mice was attenuated when these mice were treated with caspase‐1 inhibitor WEHD or HMGB1 inhibitor glycyrrhizin, but not with IL‐1β receptor antagonist. Confocal microscopic analysis demonstrated that in Nlrp3+/+ mice, HC increased caspase‐1 activity and HMGB1 expression in coronary arterial endothelium, but did not alter the levels of IL‐1β or Mϕ marker F4/80. In cultured ECs, cholesterol crystals (CCs) activated Nlrp3 inflammasomes and increased caspase‐1 activity. Further, CCs impaired EDVD in coronary arteries from Nlrp3+/+ mice, but not in Nlrp3‐/‐ mice. Taken together, these results suggest that activation of endothelial Nlrp3 inflammasomes directly impair endothelial function beyond its canonical inflammatory effects such as Mϕ recruitment and T‐cell infiltration. This non‐inflammatory action of Nlrp3 inflammasome activation may initiate or exacerbate vascular injury during HC.Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316

Compromised bioavailable IGF-1 of black men relates favourably to ambulatory blood pressure: The SABPA study

ObjectivesInsulin-like growth factor-1 (IGF-1) has potent endothelial-protective, anti-platelet and anti-thrombotic activities, and also exerts mitogenic and proliferatory actions on vascular smooth muscle cells. Conflicting reports exist regarding the role of IGF-1 in vascular protection and atherogenesis. We therefore investigated the relationships of ambulatory blood pressure (BP) and carotid intima-media thickness (cIMT) with a range of components of the IGF-1 axis in a bi-ethnic population. MethodsWe included black (N = 86) and white (N = 101) men and measured growth hormone, total IGF-1, insulin-like growth factor binding protein-3 (IGFBP-3), and pregnancy-associated plasma protein-A (PAPP-A) levels. ResultsAmbulatory BP was almost 10 mmHg higher in black men (137/88 mmHg versus 128/80 mmHg; both p < 0.001), accompanied by an adverse profile of the IGF-axis for all measured components (all p < 0.01), including reduced bioavailable IGF-1 (IGF-1/IGFBP-3; p = 0.006) and tissue IGF-1 accessibility index as represented by IGF-1.PAPP-A/IGFBP-3 (p < 0.001). Single, partial and multiple regression analyses confirmed an independent inverse association between ambulatory systolic BP and bioavailable IGF-1 in black men (R2 = 0.24; β = −0.22; p = 0.035). cIMT was similar in the ethnic groups (p = 0.34), and was negatively associated with bioavailable IGF-1 in white men (R2 = 0.42; β = −0.17; p = 0.039) prior to adjustment for γ-glutamyl transferase (R2 = 0.45; β = −0.10; p = 0.25). ConclusionAmbulatory systolic BP is inversely related to bioavailable IGF-1 in black men who displayed low IGF-1 concentrations. An inverse relation was found between cIMT and IGF-1 in white men, which disappeared after correction for γ-glutamyl transferase – opposing reports of a detrimental role of IGF-1 in the early stages of atherogenesis.

Chitotriosidase as a possible marker of clinically evidenced atherosclerosis in dyslipidemic children

A correlation has been clearly shown between inflammation markers and subclinical atherosclerosis markers in the early stages of atherogenesis in subjects with familial hypercholesterolemia (FH). The aim of this study was to investigate potential inflammation markers in the diagnosis of atherosclerosis in children with FH. A total of 48 dyslipidemic children and 24 healthy age-matched control subjects were taken into study. Inflammation and macrophage activation markers (hsCRP, myeloperoxidase, chitotriosidase, YKL-40, TNF-α, IL-6, IL-18, MMP-1 and MMP-9) and lipid parameters of all patients were measured. Carotid intima-media thickness (cIMT) and flow-mediated dilation (FMD) levels were determined. Our data suggested that clinically evidenced (by cIMT and FMD levels) atherosclerosis starts in the early ages in hypercholesterolemic children. Higher cholesterol levels strongly correlated with macrophage activation markers (ChT, YKL-40 and myeloperoxidase). ChT and YKL-40 seem to be the more predictable markers of atherosclerosis even in early ages (<6 years old) than other classical inflammation markers such as hs-CRP, IL-6 and TNF-α.

NMR Spectroscopy of Macrophages Loaded with Native, Oxidized or Enzymatically Degraded Lipoproteins

Oxidized and enzymatically modified low-density lipoproteins (oxLDL and eLDL) play a key role in early stages of atherogenesis. Their uptake by recruited macrophages leads to endolysosomal phospholipidosis or foam cell formation, respectively, each of which is preceded by highly differential lipid restructuring processes. We applied 1H-NMR spectroscopy (NMRS) to elucidate these structural rearrangements both in consequence of lipoprotein modifications and following phagocytosis. Being specifically sensitive to the mobile lipid subset, NMRS of oxLDL and eLDL revealed a partial and total immobilization of lipids, respectively. NMRS of intact macrophages showed a sixfold increase in mobile lipids in case of loading with eLDL but no significant changes for oxLDL or native LDL. This finding reflected the disparate lipid storage in lipid droplets and in multilamellar endolysosomal clusters when loaded with either eLDL or oxLDL, respectively. Moreover, a significant shift of the degree of saturation towards mainly polyunsaturated fatty acid chains was found for the mobile lipid pool in eLDL-loaded macrophages. Additional analyses of lipid extracts by NMRS and mass spectrometry (MS) reflected these changes in lipid content and in fatty acid composition only partially. In summary, in-cell NMRS represents a unique lipidomics tool to investigate structural changes within the mobile lipid pool following atherogenic triggers that can be not detected by the analysis of lipid extracts by MS or NMRS.

Mitochondrial DNA damage promotes atherosclerosis and is associated with vulnerable plaque

Mitochondrial DNA (mtDNA) damage is associated with atherosclerotic disease in man. However, when mtDNA damage occurs, whether it promotes atherogenesis and whether the damage is associated with plaque volume or vulnerability are unknown.To assess the role of mtDNA defects in atherosclerosis, we first performed a time-course study in apolipoprotein E deficient (ApoE−/−) mice. MtDNA damage was present at the earliest stages of atherogenesis, before histological evidence of disease, with mitochondrial dysfunction occurring in advanced disease. We then studied ApoE−/− mice that were doubly deficient for a proof reading deficiency of mitochondrial DNA polymerase (PolG−/−ApoE−/− mice). PolG−/−ApoE−/− mice had increased plaque burden and hypercholesterolaemia, despite a marked reduction in adiposity and no increase of reactive oxygen species. PolG−/−ApoE−/− mice had increased aortic mtDNA damage and decreased expression and respiration of complexes that have mtDNA-encoded subunits. PolG−/−ApoE−/− smooth muscle cells showed reduced ATP content, impaired proliferation, and increased apoptosis.To determine whether MtDNA damage correlates with human disease we studied 1096 plaques in 170 patients who had undergone three-vessel virtual histology intravascular ultrasound of their coronary arteries at Papworth Hospital. mtDNA damage correlated strongly with the number of vulnerable lesions but not plaque volume.Our results indicate that mtDNA damage occurs early in atherosclerosis and leads to respiratory dysfunction without increased oxidative stress. mtDNA damage causes impaired bioenergetics, changes cell proliferation and apoptosis, and promotes hypercholesterolaemia and atherosclerosis. mtDNA damage may also be a novel marker for unstable atherosclerosis in man. FundingBritish Heart Foundation.

Abstract TMP112: F11 Receptor Antagonists As Therapeutic Agents For Atherosclerosis And Thrombosis

Rationale and Objective: The F11 Receptor (F11R; aka Junctional Adhesion Molecule-A, JAM-A) is a cell adhesion protein present on the surface of circulating platelets and within tight junctions of endothelial cells (EC). We reported evidence for the critical role of F11R in the adhesion of platelets to cytokine-inflamed endothelium. Because platelet adhesion to an inflamed endothelium is one of the early steps of plaque formation in non-denuded blood vessels, we concluded that exposure of de novo synthesized F11R molecules on the luminal surface of EC is a crucial step in early stages of atherogenesis, leading to atherosclerosis, heart attacks and strokes. Utilizing recombinant F11R protein and F11R peptides, we have demonstrated that the expression of F11R on the luminal surface of inflamed EC initiates platelet adhesion to the inflamed EC thru binding of F11R molecules and this binding is inhibited by F11R antagonists. The present report provides new in-vivo evidence for the critical role of F11R antagonists in the formation of atherosclerotic plaques in apoE -/- mice. Methods and Results: The effect of F11R peptide on the formation of atherosclerotic plaques was determined in apoE -/- mice from 12 to 20 weeks of age. Fourteen apoE -/- mice were obtained from the Jackson Laboratory and fed a Western-type diet. Seven mice were injected with an F11R peptide (4.3 mg/100μl) every day for either a 3-, 4- or a 5- month period. Control animals were injected with diluent for the same period of time. The aortae and hearts were dissected and the aortic arch was photographed. An aortic lesion en face assay and morphometric lesion analysis was performed. Mice treated with the F11R peptide demonstrated healthy appearance with significantly decreased atherosclerotic lesion size as compared to unhealthy, control animals with large lesion sizes. Conclusions: F11R peptide inhibits the development of plaque formation in Apo E -/- mice, an animal model of atherosclerosis. The results of combined in vitro and in vivo studies provide information that development of small peptide analogs or peptidiomimetics which antagonize the pathological activity of F11R can be used as therapeutic agents for the prevention of atherosclerosis and thrombosis leading to heart attacks and strokes.

Serum Interleukin-6 level in children with type 1 diabetes mellitus

pancreatic islets in which a process of programmed cell death (apoptosis) is elicited in the β-cells by interaction of activated T-cells and proinflammatory cytokines in the immune infiltrate. Interleukin-6 (IL-6) is a pleiotropic cytokine with a key impact on both immunoregulation and nonimmune events in many cell types .Objective: to assess the level of serum IL-6 as an inflammatory marker in type 1 diabetic children, with correlation to FBG and HbA1c.Subjects and methods: 45 type 1 diabetic child (20 males and 25 females), mean age 10.9± 3.4 years who attended the National Diabetic Center, Al-Mustansiria university were included in this study. 45 apparently healthy controls matched for age and sex were participated in this study .Fasting venous blood samples were collected from all the subjects. The serum was used for analyzing HbA1c, Fasting Blood Glucose (FBG) and IL-6, HbA1c was estimated by high performance liquid chromatography ,Serum glucose level was determined enzymatically and serum IL-6 was measured by enzyme linked immune sorbent assay.Analysis of data was performed using the statistically package for social science (SPSS) version 17.0.Results: Mean serum IL-6 level in type 1 diabetic children were significantly higher compared to the healthy controls (30.9 Pg/ml ±10.85versus 10.57 Pg/ml±1.98 (P ≤0.0001), and positive strong correlation was found between serum IL-6 and FBS, HbA1C ,BMI.Conclusion: There is a low-level of chronic inflammatory state in type 1 diabetic children reflected by the level of serum IL-6, that may play a key role in the early stages of atherogenesis and the development of microvascular complications.

Oxidized LDL level is related to gene expression of tumour necrosis factor super family members in children and young adults with familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) is associated with increased risk of premature atherosclerosis. Inflammation is a key event in atherogenesis, and we have previously reported an inflammatory imbalance between tumour necrosis factor (TNF)α and interleukin-10 in children with FH. Based on the potential role of TNF-related molecules in inflammation, we investigated the regulation of other members of the TNF superfamily (TNFSF)/TNF receptor superfamily (TNFRSF) in children and young adults with FH and matched healthy controls. Expression of TNFSF/TNFRSF genes in peripheral blood mononuclear cells (PBMCs) was quantified in children and young adults with FH prior to (n = 42) and after statin treatment (n = 10) and in controls (n = 25) by quantitative real-time polymerase chain reaction. First we found that, compared with controls, the mRNA levels of OX40L, BAFFR and TRAILR1 were significantly higher, whereas TRAIL and TRAILR3 were significantly lower in children and young adults with FH. Secondly, levels of oxidized low-density lipoprotein (oxLDL) were significantly raised in the FH group, and correlated with the expression of OX40L, BAFFR and TRAILR1. Thirdly, oxLDL increased mRNA levels of BAFFR, TRAILR1 and TRAILR4 in PBMCs ex vivo from individuals with FH. Fourthly, OX40, acting through OX40L, enhanced the oxLDL-induced expression of matrix metalloproteinase-9 in THP-1 monocytes in vitro. Finally, after statin treatment in children with FH (n = 10), mRNA levels of OX40L and TRAILR1 decreased, whereas levels BAFF, TRAIL and TRAILR3 increased. Our findings suggest the involvement of some TNFSF/TNFRSF members and oxLDL in the early stages of atherogenesis; this may potentially contribute to the accelerated rate of atherosclerosis observed in individuals with FH.

Effect of everolimus on pre-existing atherosclerosis in LDL-receptor deficient mice

ObjectiveProliferation signal inhibitors/mTOR-inhibitors have been shown to reduce de novo development of hypercholesterolemic atherosclerosis in animal models. However, their effect on pre-existing atherosclerosis has not yet been studied. Methods and resultsFeeding LDL-R-KO mice a high cholesterol diet for 12 weeks resulted in formation of moderate fibroatheroma (induction phase). Sixty mice received either everolimus (1 or 5mg/kg) or no everolimus for further 12 weeks (treatment phase). Everolimus significantly enhanced hypercholesterolemia (plasma cholesterol +45%, p<0.001). Atherosclerosis progressed obstructively in treated and non-treated mice. Everolimus (5mg/kg) tended to reduced progression in aortic root lesions (0.28±0.02 vs. 0.33±0.03mm2, p=ns) and brachiocephalic lesions (0.044±0.006 vs. 0.066±0.012mm2, p=ns) but without significance. Everolimus (5mg/kg) resulted in an arrest of CD68 positive plaque area (p=0.03) and nearly halved CD68 fraction (p=0.05) in aortic root lesions but not in brachiocephalic lesions. Taken together, despite a trend to reduced progression and inflammatory cell content there was less conclusive net effect of everolimus treatment than expected. ConclusionA higher potential of everolimus in the treatment of atherosclerosis might be obscured by its concomitant hypercholesterolemia. Considering stronger effects in previous studies we suggest that everolimus might exert more potent anti-atherogenic properties in earlier stages of atherogenesis than in advanced atherosclerosis.

02 Mitochondrial DNA damage, dysfunction and atherosclerosis

RationaleAtherosclerosis remains the leading cause of death in the western world. Mitochondrial DNA (mtDNA) damage has been associated with the disease, but it is unknown when the damage occurs relative...

Effect of Zinc and Nitric Oxide on Monocyte Adhesion to Endothelial Cells under Shear Stress

This study describes the effect of zinc on monocyte adhesion to endothelial cells under different shear stress regimens, which may trigger atherogenesis. Human umbilical vein endothelial cells were exposed to steady shear stress (15 dynes/cm(2) or 1 dyne/cm(2)) or reversing shear stress (time average 1 dyne/cm(2)) for 24 h. In all shear stress regimes, zinc deficiency enhanced THP-1 cell adhesion, while heparinase III reduced monocyte adhesion following reversing shear stress exposure. Unlike other shear stress regimes, reversing shear stress alone enhanced monocyte adhesion, which may be associated with increased H(2)O(2) and superoxide together with relatively low levels of nitric oxide (NO) production. L-N(G)-Nitroarginine methyl ester (L-NAME) treatment increased monocyte adhesion under 15 dynes/cm(2) and under reversing shear stress. After reversing shear stress, monocyte adhesion dramatically increased with heparinase III treatment followed by a zinc scavenger. Static culture experiments supported the reduction of monocyte adhesion by zinc following endothelial cell cytokine activation. These results suggest that endothelial cell zinc levels are important for the inhibition of monocyte adhesion to endothelial cells, and may be one of the key factors in the early stages of atherogenesis.

MMP-2 and MMP-9 are prominent matrix metalloproteinases during atherosclerosis development in the Ldlr-/-Apob100/100 mouse

Matrix-degrading proteases capable of degrading components of the extracellular matrix may play an important role in development and progression of atherosclerotic lesions. In the present study, we used the Ldlr(-/-)Apob(100/100) mouse model, which has a plasma lipoprotein profile similar to that of humans with atherosclerosis, to study the expression of matrix metalloproteinases (MMPs) during early stages of atherosclerosis development. We analyzed the expression of 11 proteases and three protease inhibitors in 5- to 40-week-old Ldlr(-/-)Apob(100/100) mice. Expression and activity of MMP-2 and MMP-9 was increased in advanced atherosclerotic lesions followed by macrophage infiltration as shown by real-time PCR, gel-based and in situ zymography and immunohistochemistry. Expression of other investigated MMPs did not increase during disease progression. However, the mRNA expression of MMP-8 and MMP-13 was down-regulated, which could explain the relatively high amount of collagen observed in the vessels in this model. In conclusion, low proteolytic expression at early stages of atherogenesis and a limited repertoire of proteolytic enzymes were associated with the progression of atherosclerosis in Ldlr(-/-)Apob(100/100) mice. The study suggests that MMP-2 and MMP-9 are the main proteases involved in atherogenesis in this mouse model.

A reversal of Glagov's hypothesis; a preliminary demonstration by cardiac magnetic resonance

According to Glagov's hypothesis, the early stages of atherogenesis (stenosis <40%) are associated with positive remodeling of the outer arterial wall resulting in paradoxical preservation of the luminal area, and as stenosis exceeds 40%, further plaque accumulation results in a decrease in the area of the lumen. This study examines the effects of statins on human carotid atherosclerotic plaque to investigate if a reversal of Glagov's hypothesis occurs with statin therapy.

The changes in the endothelial expression of cell adhesion molecules and iNOS in the vessel wall after the short-term administration of simvastatin in rabbit model of atherosclerosis

Cell adhesion molecules P-selectin, VCAM-1 and ICAM-1 play an important role in the pathogenesis of atherosclerosis. High levels of nitric oxide (NO) produced by inducible NO synthase (iNOS) have been associated with atherosclerotic processes. Simvastatin is an HMG-CoA reductase inhibitor responsible for many clinical benefits. The aim of this study was to detect and quantify changes in endothelial expression of P-selectin, VCAM-1, ICAM-1 and iNOS in the vessel wall after the shortterm administration of simvastatin in a rabbit model of atherosclerosis. Eighteen New Zealand White rabbits were randomly divided into three groups (n=6). In the cholesterol group, rabbits consumed an atherogenic diet (0.4% cholesterol) for eight weeks. In the simvastatin group, rabbits consumed an atherogenic diet for six weeks and then consumed an atherogenic diet supplemented with simvastatin (10 mg kg(-1)) for two weeks. Biochemical analysis showed that administration of simvastatin led to an almost two-fold lowering of the total serum cholesterol, VLDL, LDL and HDL, but not triglycerides, compared with the cholesterol-fed rabbits only. Stereological analysis of the immunohistochemical staining revealed that administration of simvastatin (10 mg kg(-1) daily) in an atherogenic diet decreased the endothelial expression of P-selectin, ICAM-1 and iNOS in both aortic arch and carotid artery compared with the cholesterol fed-rabbits only. We conclude that simvastatin has beneficial effects on endothelial function by decreasing expression of P-selectin, ICAM-1 and iNOS in endothelial cells in the very early stages of atherogenesis.