Increased Lesion Formation Research Articles

Endothelial nitric oxide synthase (eNOS) S1176 phosphorylation status governs atherosclerotic lesion formation.

We have previously demonstrated the in vivo importance of the Akt-eNOS substrate-kinase relationship, as defective postnatal angiogenesis characteristic of global Akt1-null mice is rescued when bred to 'gain-of-function' eNOS S1176D mutant mice. While multiple studies support the vascular protective role of endothelial NO generation, the causal role of Akt1-dependent eNOS S1176 phosphorylation during atherosclerotic plaque formation is not yet clear. We herein bred congenic 'loss-of-function' eNOS S1176A and 'gain-of-function' eNOS S1176D mutant mice to the exacerbated atherogenic Akt1-/-; ApoE-/- double knockout mice to definitively test the importance of Akt-mediated eNOS S1176 phosphorylation during atherogenesis. We find that a single amino acid substitution at the eNOS S1176 phosphorylation site yields divergent effects on atherosclerotic plaque formation, as an eNOS phospho-mimic aspartate (D) substitution at S1176 leads to favorable lipid profiles and decreased indices of atherosclerosis, even when on a proatherogenic Akt1 global deletion background. Conversely, mice harboring an unphosphorylatable mutation to alanine (S1176A) result in increased plasma lipids, increased lesion formation and cellular apoptosis, phenocopying the physiological consequence of eNOS deletion and/or impaired enzyme function. Furthermore, gene expression analyses of whole aortas indicate a combinatorial detriment from NO deficiency and Western Diet challenge, as 'loss-of-function' eNOS S1176A mice on a Western Diet present a unique expression pattern indicative of augmented T-cell activity when compared to eNOS S1176D mice. By using genetic epistasis approaches, we conclusively demonstrate that Akt-mediated eNOS S1176 phosphorylation and subsequent eNOS activation remains to be the most physiologically relevant method of NO production to promote athero-protective effects.

The role of CD45 in the regulation of B cell subset functions in Atherosclerosis

Abstract Atherosclerosis is an inflammatory disease of the large and medium size arteries characterized by deposition of oxidized lipids within the vessel. While specific self-antigens are not well characterized, data show that the immune response is involved in atherogenesis. The role of B cells in atherosclerosis is subset specific. FO B cells are proatherogenic, while B1 and MZ B cells serve as protective. B cell receptor signaling is important in B cell activation, differentiation, and functions, while important costimulatory molecules, like CD45, assist in initiating signal propagation. To date, it is unknown whether B cell subsets respond differently to BCR signaling or if CD45-dependent regulation of BCR signaling is subset specific in homeostatic or atherosclerotic conditions. Taking advantage of transgenic mice that express low levels of CD45(CD45L/L), we examined activation of CD45L/L B cells through Ca2+ flux. We show that BCR-induced B cell activation differs between subsets and CD45-dependent modulation of BCR signaling is subset specific. MZ and B1 have high levels of Ca2+ flux, with MZ B cell activation the most affected by CD45 expression. To test the effects of CD45L/L B in atherosclerosis, we performed adoptive transfer of CD45L/L or WT B cells into B cell-deficient atherosclerotic prone mice. After 23–38 wks of western diet feeding, CD45L/L B cell recipients had increased lesion formation despite an increase of protective MZ B cells. This increase in atherogenesis could be due to MZ CD45L/L B cells having reduced activation, disrupting their protective ability. This suggests that the subset-specific modulation of BCR signaling through CD45 plays a role in the regulation of B cell functions in atherosclerosis. This work was supported by American Heart Association AIREA grant 8AIREA33960546.

Dicer promotes endothelial recovery and limits lesion formation after vascular injury through miR-126-5p

BackgroundThe recovery of endothelial cells (ECs) after vascular injury is mainly mediated by the proliferation of resident ECs, thereby reducing neointima formation. The RNase Dicer processes microRNAs (miRNAs) and regulates EC function by controlling miRNA-mediated regulation of gene expression. This study aimed to investigate the impact of miRNA biogenesis in ECs on endothelial repair during lesion formation after vascular injury. Methods and resultsTo study the effect of Dicer on ECs during neointima formation, conditional deletion of Dicer was induced in Apoe−/− mice (EC-Dicerflox) by tamoxifen injection. Following wire-induced injury to carotid arteries of EC-Dicerflox mice, the EC recovery was impaired and the neointima formation and lesional macrophage accumulation was increased. Moreover, conditional deletion of Dicer in ECs diminished the expression of miR-126-5p in EC-Dicerflox mice. Notably, reconstitution of miR-126-5p in the injured arteries of EC-Dicerflox mice using miR-126-5p mimic, prevented the impaired endothelial recovery and increased lesion formation observed in EC-Dicerflox mice. ConclusionsDeficiency of endothelial Dicer diminished endothelial recovery and promoted neointima formation probably due to impaired miR-126-5p expression. Treatment with miR-126-5p mimics promotes endothelial recovery and thereby limits neointima formation. Thus, miR-126-5p therapy represents a potential approach to improve endothelial recovery and prevent restenosis following vascular injury.

Hypercholesterolemia-Induced Loss of Flow-Induced Vasodilation and Lesion Formation in Apolipoprotein E-Deficient Mice Critically Depend on Inwardly Rectifying K+ Channels.

BackgroundHypercholesterolemia‐induced decreased availability of nitric oxide (NO) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K+ (Kir) channels and that Kir2.1 is an upstream mediator of flow‐induced NO production. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia‐induced inhibition of flow‐induced NO production and flow‐induced vasodilation (FIV). We also tested the role of Kir2.1 in the development of atherosclerotic lesions.Methods and ResultsKir2.1 currents are significantly suppressed in microvascular endothelial cells exposed to acetylated–low‐density lipoprotein or isolated from apolipoprotein E–deficient (Apoe −/−) mice and rescued by cholesterol depletion. Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe −/−mice, Kir2.1 +/− /Apoe −/− exhibit the same blunted FIV and flow‐induced NO response as Apoe −/−or Kir2.1 +/− alone, but while FIV in Apoe −/− mice can be rescued by cholesterol depletion, in Kir2.1 +/− /Apoe −/− mice cholesterol depletion has no effect on FIV. Endothelial‐specific overexpression of Kir2.1 in arteries from Apoe −/− and Kir2.1 +/− /Apoe −/− mice results in full rescue of FIV and NO production in Apoe −/− mice with and without the addition of a high‐fat diet. Conversely, endothelial‐specific expression of dominant‐negative Kir2.1 results in the opposite effect. Kir2.1 +/− /Apoe −/−mice also show increased lesion formation, particularly in the atheroresistant area of descending aorta.ConclusionsWe conclude that hypercholesterolemia‐induced reduction in FIV is largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow‐induced NO production, whereas the stages downstream of flow‐induced Kir2.1 activation appear to be mostly intact. Kir2.1 channels also have an atheroprotective role.

The Prebiotic Inulin Aggravates Accelerated Atherosclerosis in Hypercholesterolemic APOE*3-Leiden Mice.

The prebiotic inulin has proven effective at lowering inflammation and plasma lipid levels. As atherosclerosis is provoked by both inflammation and hyperlipidemia, we aimed to determine the effect of inulin supplementation on atherosclerosis development in hypercholesterolemic APOE*3-Leiden (E3L) mice. Male E3L mice were fed a high-cholesterol (1%) diet, supplemented with or without 10% inulin for 5 weeks. At week 3, a non-constrictive cuff was placed around the right femoral artery to induce accelerated atherosclerosis. At week 5, vascular pathology was determined by lesion thickness, vascular remodeling, and lesion composition. Throughout the study, plasma lipids were measured and in week 5, blood monocyte subtypes were determined using flow cytometry analysis. In contrast to our hypothesis, inulin exacerbated atherosclerosis development, characterized by increased lesion formation and outward vascular remodeling. The lesions showed increased number of macrophages, smooth muscle cells, and collagen content. No effects on blood monocyte composition were found. Inulin significantly increased plasma total cholesterol levels and total cholesterol exposure. In conclusion, inulin aggravated accelerated atherosclerosis development in hypercholesterolemic E3L mice, accompanied by adverse lesion composition and outward remodeling. This process was not accompanied by differences in blood monocyte composition, suggesting that the aggravated atherosclerosis development was driven by increased plasma cholesterol.

Deficiency of secreted IgM antibodies causes increased plasma IgE levels that promote atherosclerosis

Objectives: Deficiency of secreted IgM antibodies (sIgM-/-) in Ldlr-/- mice results in accelerated atherosclerosis. However, the underlying mechanisms have not been elucidated.We aim to identify mechanisms by which secreted IgM deficiency results in increased lesion formation.

Abstract 659: Macrophage Beta3 Integrin Deficiency Promotes Atherosclerosis Development Through ERK-Dependent Signaling

Atherosclerosis is a chronic inflammation of the arterial wall caused by accumulation of cholesterol. This complex process involves endothelial and smooth muscle cells as well as the recruitment and differentiation of circulating monocytes. Integrins are membrane-bound molecules that are involved in signaling between cells and the extracellular matrix, thereby influencing intracellular signaling and cytoskeletal stability. We and others have described the in vivo consequences of germline deletion of the β3 integrin in inflammation, atherosclerosis and cancer. However, the distinct pathophysiological consequences of its tissue-specific deletion in macrophage function in development of atherosclerotic lesions remain mostly elusive. Our aim was to characterize for the first time the phenotype of mice with tissue-specific deletion of β3 integrin in macrophages (LyzMCre) under high fat diet (HFD) condition on atherosclerotic prone backgrounds (ApoE-/- and LDLr-/-), and to elucidate the signaling mechanism involved using in vitro methods. Tissue specific β3 integrin deficiency in macrophages alone is sufficient to cause increased lesion formation in the aorta of mice on HFD on both backgrounds used suggesting an important atheroprotective role played by this integrin. β3 integrin-deficient bone marrow-derived macrophages (BMDM) treated with oxLDL in vitro demonstrated a strong cholesterol uptake and increased foam cell formation. We identified that β3 integrin deficiency in macrophages caused an early activation of Ras followed by ERK phosphorylation, deficient cholesterol efflux and decreased expression of cholesterol transporter (ABCA1) which is a major regulator of cellular cholesterol. Inhibition of the ERK pathway reduced foam cell formation of macrophages at least partially by restoring expression of ABCA1. Taken together, our results show that macrophage β3 integrin is an important signaling molecule for cellular activation. Its deficiency showed important functional consequences in regard to inflammation and atherosclerosis that could be possibly modulated by interfering with downstream signaling.

Virulence determinants of Pseudomonas syringae strains isolated from grasses in the context of a small type III effector repertoire.

BackgroundPseudomonas syringae is pathogenic to a large number of plant species. For host colonization and disease progression, strains of this bacterium utilize an array of type III-secreted effectors and other virulence factors, including small secreted molecules such as syringolin A, a peptide derivative that inhibits the eukaryotic proteasome. In strains colonizing dicotyledonous plants, the compound was demonstrated to suppress the salicylic-acid-dependent defense pathway. Here, we analyze virulence factors of three strains colonizing wheat (Triticum aestivum): P. syringae pathovar syringae (Psy) strains B64 and SM, as well as P. syringae BRIP34876. These strains have a relatively small repertoire of only seven to eleven type III secreted effectors (T3Es) and differ in their capacity to produce syringolin A. The aim of this study was to analyze the contribution of various known virulence factors in the context of a small T3E repertoire.ResultsWe demonstrate that syringolin A production enhances disease symptom development upon direct infiltration of strains into wheat leaves. However, it is not universally required for colonization, as Psy SM, which lacks syringolin biosynthesis genes, reaches cell densities comparable to syringolin A producer P. syringae BRIP34876. Next, we show that despite the small set of T3E-encoding genes, the type III secretion system remains the key pathogenicity determinant in these strains, and that phenotypic effects of deleting T3E-coding genes become apparent only when multiple effectors are removed.ConclusionsWhereas production of syringolin A is not required for successful colonization of wheat leaves by P. syringae strains, its production results in increased lesion formation. Despite the small number of known T3Es encoded by the analyzed strains, the type III secretion system is essential for endophytic growth of these strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0304-5) contains supplementary material, which is available to authorized users.

Abstract 156: Macrophage Low-Density Receptor--Related Protein 1 Deficiency Facilitates Atherosclerosis Regression in Hyperlipidemic Mice

Atherosclerosis regression is characterized by egress of macrophages out of the artery wall. We have previously shown that macrophages lacking low-density receptor related protein 1 (MFLRP1-/-) are pro-inflammatory and lead to increased lesion formation in apoE-/- mice. To study the role of macrophage inflammation during atherosclerosis regression, bone marrow from four different types of mice (wild-type, MFLRP1-/-, apoE-/- and apoE-/-/ MFLRP1-/-) was transplanted into apoE-/- recipient mice who had been fed a Western-type diet for 12 weeks. ApoE-/- recipient mice transplanted with apoE-/- bone marrow were sacrificed 2 weeks post-BMT for determination of baseline aortic atherosclerosis. After 8 weeks on chow diet, cholesterol levels were normalized in mice reconstituted with wild-type (WT) and MFLRP1-/- bone marrow (157± 36 mg/dl and 136 ± 33 mg/dl, respectively), and significantly lowered in mice with apoE-/- (302±33 mg/dl) or apoE-/-/ MFLRP1-/- (294±52 mg/dl) macrophages compared to baseline (387±34 mg/dl). Total atherosclerotic lesion area in the aortic root decreased by 15% in mice receiving WT macrophages, and decreased by an additional 10% in mice transplanted with LRP1 deficient macrophages (p<0.05 compared to WT). Similarly, mice reconstituted with apoE-/-/ MFLRP1-/- bone marrow had 15% (p < 0.01) smaller lesion size than mice receiving apoE-/- marrow. The lesion area positive for CD68 was significantly smaller in MFLRP1-/- mice compared to WT mice, and in apoE-/-/ MFLRP1-/- mice compared to apoE-/- mice. The ratio of necrotic to total lesion area was significantly lowered by WT and LRP1-/- macrophages, and was also reduced in recipients of apoE-/-/MFLRP1-/- compared to apoE-/- bone marrow. Here we demonstrate that absence of LRP1 in macrophages, which is known to cause pro-inflammatory changes, promotes atherosclerosis regression. Our study supports the novel idea that pro-inflammatory macrophages efficiently egress from the plaque in a regressive environment caused by switching from a high-fat to a chow diet. This observation sets the stage for a change in paradigm on how to target inflammation for prevention of atherosclerotic cardiovascular events.

Intermittent hypoxia and hypercapnia induce pulmonary artery atherosclerosis and ventricular dysfunction in low density lipoprotein receptor deficient mice

Patients with obstructive sleep apnea, who experience episodic hypoxia and hypercapnia during sleep, often demonstrate increased inflammation, oxidative stress, and dyslipidemia. We hypothesized that sleep apnea patients would be predisposed to the development of atherosclerosis. To dissect the mechanisms involved, we developed an animal model in mice whereby we expose mice to intermittent hypoxia/hypercapnia (IHH) in normobaric environments. Two- to three-month-old low-density lipoprotein receptor deficient (Ldlr(-/-)) mice were fed a high-fat diet for 8 or 16 wk while being exposed to IHH for either 10 h/day or 24 h/day. Plasma lipid levels, pulmonary artery and aortic atherosclerotic lesions, and cardiac function were then assayed. Surprisingly, atherosclerosis in the aorta of IHH mice was similar compared with controls. However, in IHH mice, atherosclerosis was markedly increased in the trunk and proximal branches of the pulmonary artery of exposed mice; even though plasma cholesterol and triglycerides were lower than in controls. Hemodynamic analysis revealed that right ventricular maximum pressure and isovolumic relaxation constant were significantly increased in IHH exposed mice and left ventricular % fractional shortening was reduced. In conclusion, 1) Intermittent hypoxia/hypercapnia remarkably accelerated atherosclerotic lesions in the pulmonary artery of Ldlr(-/-) mice and 2) increased lesion formation in the pulmonary artery was associated with right and left ventricular dysfunction. These findings raise the possibility that patients with obstructive sleep apnea may be susceptible to atherosclerotic disease in the pulmonary vasculature, an observation that has not been previously recognized.

Atherosclerosis Susceptibility Loci Identified in an Extremely Atherosclerosis‐Resistant Mouse Strain

BackgroundC3H/HeJ (C3H) mice are extremely resistant to atherosclerosis, especially males. To understand the underlying genetic basis, we performed quantitative trait locus (QTL) analysis on a male F2 (the second generation from an intercross between 2 inbred strains) cohort derived from an intercross between C3H and C57BL/6 (B6) apolipoprotein E–deficient (Apoe−/−) mice.Methods and ResultsTwo hundred forty‐six male F2 mice were started on a Western diet at 8 weeks of age and kept on the diet for 5 weeks. Atherosclerotic lesions in the aortic root and fasting plasma lipid levels were measured. One hundred thirty‐four microsatellite markers across the entire genome were genotyped. Four significant QTLs on chromosomes (Chr) 2, 4, 9, and 15 and 4 suggestive loci on Chr1, Chr4, and Chr7 were identified for atherosclerotic lesions. Unexpectedly, the C3H allele was associated with increased lesion formation for 2 of the 4 significant QTLs. Six loci for high‐density lipoprotein (HDL), 6 for non‐HDL cholesterol, and 3 for triglycerides were also identified. The QTL for atherosclerosis on Chr9 replicated Ath29, originally mapped in a female F2 cohort derived from B6 and C3H Apoe−/− mice. This locus coincided with a QTL for HDL, and there was a moderate, but statistically significant, correlation between atherosclerotic lesion sizes and plasma HDL cholesterol levels in F2 mice.ConclusionsThese data indicate that most atherosclerosis susceptibility loci are distinct from those for plasma lipids except for the Chr9 locus, which exerts effect through interactions with HDL.

Hematopoietic Interferon Regulatory Factor 8-Deficiency Accelerates Atherosclerosis in Mice

Inflammatory leukocyte accumulation drives atherosclerosis. Although monocytes/macrophages and polymorphonuclear neutrophilic leukocytes (PMN) contribute to lesion formation, sequelae of myeloproliferative disease remain to be elucidated. We used mice deficient in interferon regulatory factor 8 (IRF8(-/-)) in hematopoietic cells that develop a chronic myelogenous leukemia-like phenotype. Apolipoprotein E-deficient mice reconstituted with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow displayed an exacerbated atherosclerotic lesion formation compared with controls. The chronic myelogenous leukemia-like phenotype in mice with IRF8(-/-) bone marrow, reflected by an expansion of PMN in the circulation, was associated with an increased lesional accumulation and apoptosis of PMN, and enlarged necrotic cores. IRF8(-/-) compared with IRF8(+/+) PMN displayed unaffected reactive oxygen species formation and discharge of PMN granule components. In contrast, accumulating in equal numbers at sites of inflammation, IRF8(-/-) macrophages were defective in efferocytosis, lipid uptake, and interleukin-10 cytokine production. Importantly, depletion of PMN in low-density lipoprotein receptor or apolipoprotein E-deficient mice with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow abrogated increased lesion formation. These findings indicate that a chronic myelogenous leukemia-like phenotype contributes to accelerated atherosclerosis in mice. Among proatherosclerotic effects of other cell types, this, in part, is linked to an expansion of functionally intact PMN.

AtRbohF is a crucial modulator of defence‐associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis

This work investigated the contribution of AtRbohD and AtRbohF to regulating defence-associated metabolism during three types of interaction: (i) incompatible and (ii) compatible interaction with Pseudomonas syringae; and (iii) intracellular oxidative stress in the catalase-deficient cat2 background. In all three cases, loss of function of either gene modulated the response of defence compounds. AtRbohF gene function was necessary for rapid and full induction of salicylic acid (SA) during compatible and incompatible interactions, and for resistance to virulent bacteria. Both artrboh mutations modulated the effects of intracellular ROS in the cat2 background, although the predominant effect was mediated by atrbohF. Loss of this gene function increased lesion formation in cat2 but uncoupled this effect from cat2-triggered induction of SA and camalexin, accumulation of glutathione and disease resistance, all of which were much lower in cat2 artbohF than in cat2. A detailed comparison of GC-TOF-MS profiles produced by the three interactions revealed considerable overlap between cat2 effects and those produced by bacterial infection in the wild-type background. Analysis of the impact of the two atrboh mutations on these profiles provided further evidence that AtRbohF interacts closely with intracellular oxidative stress to tune dynamic metabolic responses during infection. Thus, AtRbohF appears to be a key player not only in HR-related cell death but also in regulating metabolomic responses and resistance. Based on the results obtained during the three types of interaction, a model is proposed of how NADPH oxidases and intracellular ROS interact to determine the outcome of pathogen defence responses.

Differential effects of nutritional folic acid deficiency and moderate hyperhomocysteinemia on aortic plaque formation and genome-wide DNA methylation in vascular tissue from ApoE-/- mice

Low folate intake is associated with vascular disease. Causality has been attributed to hyperhomocysteinemia. However, human intervention trials have failed to show the benefit of homocysteine-lowering therapies. Alternatively, low folate may promote vascular disease by deregulating DNA methylation. We investigated whether folate could alter DNA methylation and atherosclerosis in ApoE null mice. Mice were fed one of six diets (n = 20 per group) for 16 weeks. Basal diets were either control (C; 4% lard) or high fat (HF; 21% lard and cholesterol, 0.15%) with different B-vitamin compositions: (1) folic acid and B-vitamin replete, (2) folic acid deficient (−F), (3) folic acid, B6 and B12 deficient (−F−B). −F diets decreased plasma (up to 85%; P < 0.05), whole blood (up to 70%; P < 0.05), and liver folate (up to 65%; P < 0.05) and hepatic SAM/SAH (up to 80%; P < 0.05). −F−B diets reduced plasma (up to 76%; P < 0.05), whole blood (up to 72%; P < 0.05), and liver B12 (up to 39%; P < 0.05) and hepatic SAM/SAH (up to 90%; P < 0.05). −F increased homocysteine 2-fold, while −F−B increased homocysteine 3.6- and 6.8-fold in the C and HF groups (P < 0.05). Plaque formation was increased 2-fold (P < 0.0001) in mice fed a HF diet. Feeding a HF–F diet increased lesion formation by 17% (P < 0.05). There was no change in 5-methyldeoxycytidine in liver or vascular tissue (aorta, periadventitial tissue and heart). These data suggest that atherogenesis is not associated with genome-wide epigenetic changes in this animal model.

Atherosclerosis

Atherosclerosis is a very complex pathology. Over the past two decades we have come to appreciate that a major component of this pathological process is chronic inflammation. This inflammatory state is not only characterized by infiltration of lipid laden macrophages, the hallmark cell of this disease, into the vascular wall, but also is characterized by the infiltration of a host of other inflammatory cells. The complex interaction of inflammatory cells with the normal residents of the vascular wall (ie, endothelial and smooth muscle cells) directs the progression of the disease. Inflammatory cells secrete a variety of chemokines and cytokines which have a profound effect on the development and progression of the plaque. Moreover, many of these cytokines can mediate both pro- and antiatherogenic processes depending on the cellular milieu. The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies encompass numerous cytokines and receptors suggested to play a pivotal role in atherogenesis. Although many members of these families such as TNF-α and CD40 ligand and their putative receptors are considered to be proatherogenic,1–4 studies have also demonstrated an antiatherogenic role for others, including the p55 TNFR.5 TNF-like weak inducer of apoptosis (TWEAK) and its putative receptor, fibroblast growth factor-inducible 14 (Fn14), are also members of the TNF and TNFR superfamilies, respectively. Both TWEAK and Fn14 have been detected in human atherosclerotic plaques, suggesting that these molecules may also play a role in the atherogenic process. Clinical …

Abstract 5466: Nuclear Receptor Nurr1 in Vascular Smooth Muscle Cells is Protective in In-stent Restenosis

Introduction Coronary atherosclerosis is treated by percutaneous coronary intervention (PCI) in combination with stent placement. The major drawback of this intervention is (in-stent) restenosis, which is characterized by activation and excessive proliferation of smooth muscle cells (SMCs). In previous studies we have shown that nuclear receptor Nurr1 inhibits the inflammatory response of macrophages and reduces lipid loading of these cells. In this study we investigated nuclear receptor Nurr1 in SMCs in human and experimental vascular restenosis. Methods and results In a prospective cohort of 601 patients undergoing PCI including stent-placement, we report a strong association of Nurr1 haplotypes with angio-graphic and clinical in-stent restenosis risk (Hazard ratio (HR) 3.1; 95%CI:1.9–5.1 and HR 3.4; 95%CI 1.6–7.1, respectively). Furthermore, Nurr1 is expressed in human in-stent restenosis and induced in cultured human SMCs in response to serum or tumor necrosis factor (TNF)- α -mediated activation. Lentivirus-mediated gain and loss of function experiments in SMCs demonstrated that overexpression of Nurr1 inhibited proliferation, consistent with increased expression of the key cell-cycle inhibitor p27 Kip1 , whereas Nurr1 silencing enhanced SMC growth. The TNF α -induced pro-inflammatory response of SMCs is inhibited by Nurr1 as reflected by reduced interleukin-1 β , TNF α and monocyte chemo-attractant protein-1 expression. In subsequent mouse experiments, Nurr1 was locally expressed in the carotid artery of ApoE−/− mice after wire-induced injury, and we revealed that Nurr1 inhibits SMC-rich lesion formation. In line with this observation, silencing of murine Nurr1 in the vessel wall increased lesion formation. Conclusion Nurr1 haplotypes associate with human in-stent restenosis risk and Nurr1 is expressed in in-stent restenosis. In addition to the anti-inflammatory function of Nurr1 in macrophages, we show that Nurr1 inhibits proliferation and inflammatory responses in SMCs, which explains inhibition of SMC-rich lesion formation in mice. Since small-molecule drugs that enhance the activity of Nurr1 have been identified, we propose this nuclear receptor Nurr1 as an attractive novel target for (local) intervention in restenosis.

Arsenic exacerbates atherosclerotic lesion formation and inflammation in ApoE-/- mice

Exposure to arsenic-contaminated water has been shown to be associated with cardiovascular disease, especially atherosclerosis. We examined the effect of arsenic exposure on atherosclerotic lesion formation, lesion composition and nature in ApoE-/- mice. Early post-natal exposure (3-week-old mice exposed to 49 ppm arsenic as NaAsO 2 in drinking water for 7 weeks) increased the atherosclerotic lesion formation by 3- to 5-fold in the aortic valve and the aortic arch, without affecting plasma cholesterol. Exposure to arsenic for 13 weeks (3-week-old mice exposed to 1, 4.9 and 49 ppm arsenic as NaAsO 2 in drinking water) increased the lesion formation and macrophage accumulation in a dose-dependent manner. Temporal studies showed that continuous arsenic exposure significantly exacerbated the lesion formation throughout the aortic tree at 16 and 36 weeks of age. Withdrawal of arsenic for 12 weeks after an initial exposure for 21 weeks (to 3-week-old mice) significantly decreased lesion formation as compared with mice continuously exposed to arsenic. Similarly, adult exposure to 49 ppm arsenic for 24 weeks, starting at 12 weeks of age increased lesion formation by 2- to 3.6-fold in the aortic valve, the aortic arch and the abdominal aorta. Lesions of arsenic-exposed mice displayed a 1.8-fold increase in macrophage accumulation whereas smooth muscle cell and T-lymphocyte contents were not changed. Expression of pro-inflammatory chemokine MCP-1 and cytokine IL-6 and markers of oxidative stress, protein-HNE and protein-MDA adducts were markedly increased in lesions of arsenic-exposed mice. Plasma concentrations of MCP-1, IL-6 and MDA were also significantly elevated in arsenic-exposed mice. These data suggest that arsenic exposure increases oxidative stress, inflammation and atherosclerotic lesion formation.

Abstract 421: Aldose Reductase Protects Against the Early Atherosclerotic Lesion Formation InApoe-null Mice

Atherosclerotic lesion formation is associated with extensive oxidation of unsaturated lipids and the accumulation of lipid oxidation products. Products of lipid oxidation, particularly aldehydes, stimulate cytokine production and enhance monocyte adhesion. Aldehydes generated by oxidized lipids are metabolized by several biochemical pathways, of which aldose reductase (AR)-catalyzed reduction represents a metabolic fate common to both free and phospholipid esterified aldehydes. Herein, we tested the hypothesis that inhibition of AR could aggravate atherosclerotic lesion formation by preventing the removal and the detoxification of aldehydes generated by oxidized lipids. In atherosclerotic lesions of apoE-null mice, AR protein was associated with macrophage-rich regions and its abundance increased with lesion progression. Treatment of 8 week old apoE-null mice with AR inhibitors sorbinil or tolrestat for 4 weeks increased lesion formation in the aortic arch (P&lt;0.01) and the aortic sinus (P&lt;0.01). No change in lesion formation was observed when 24 week old mice were fed AR inhibitors for 12 weeks. To probe the role of AR in atherogenesis further, we generated AR −/− /apoE −/− mice. Lesions of 8 week old AR −/− /apoE −/− mice maintained on high fat diet for 4 or 12 weeks were significantly larger throughout the aortic tree (P&lt;0.01 for both the groups) when compared with age-matched AR +/+ /apoE −/− mice. Lesions in AR −/− /apoE −/− mice exhibited increased collagen (P&lt;0.01) and macrophage content (P&lt;0.01) and a decrease in smooth muscle cells (P&lt;0.01). GC-MS analysis showed that the concentration of AR substrates HNE and hexanal was increased by 2.5–3 fold (P&lt;0.01) in the plasma of AR −/− /apoE −/− mice as compared with AR +/+ /apoE −/− mice. Immunohistochemical analysis showed greater accumulation of protein-HNE adducts in arterial lesions of AR −/− /apoE −/− mice. These observations suggest that AR is up regulated during atherosclerosis and that this protein protects against early stages of atherosclerotic lesion formation by removing aldehydes generated by lipid oxidation.

The role of CD36 and Caveolin‐1 in atherosclerotic lesion formation

While CD36 has been found to have both positive and negative effects on the development of atherosclerosis, caveolin is thought to be cardioprotective. LDL receptor (LDLr) null mice form atherosclerotic lesions when fed a high fat (lard) diet. In this study, we examined the roles of CD36 and Caveolin‐1 (cav‐1) on the formation of atherosclerotic lesions in a LDLr null background. LDLr null, CD36/LDLr null, and Cav‐1/LDLr null mice were fed a high fat (lard) diet, myristic acid diet, linoleic acid diet, or a stearic acid diet for 9 – 13 weeks. At the end of the study, the mice were processed to quantify atherosclerotic lesions. On high fat (lard) diet, CD36/LDLr null mice had a dramatic increase in atherosclerotic lesions whereas the Cav‐1/LDLr null mice had a decrease compared to LDLr null mice. In contrast, on the myristic acid, linoleic acid, and stearic acid diets, the LDLr null mice produced significantly more lesions than both CD36/LDLr and Cav‐1/LDLr null mice. The stearic acid diet did not alter the extent of lesion formation between Cav‐1/LDLr and CD36/LDLr null mice however, the myristic acid and the linoleic acid diets increased lesion formation in the Cav‐1/LDLr null compared to the CD36/LDLr null mice. In conclusion, these data indicate that the role of CD36 in atherosclerosis is at least partially determined by the type of fat in the diets, whereas the absence of Cav‐1 seems to be protective.

Spontaneous Atherothrombosis and Medial Degradation in Apoe −/− , Npc1 −/− Mice

The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood. Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [Npc1] gene) were crossed with apolipoprotein E (Apoe) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe-/- littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1-/- macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls. A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.