Early Atherosclerotic Lesions Research Articles

Isoborneol Attenuates Low-Density Lipoprotein Accumulation and Foam Cell Formation in Macrophages.

PurposeIsoborneol has been used in the treatment of cardiovascular disease for several years in China. However, the mechanism is still unclear. The aim of this study was to identify the novel mechanism of isoborneol for its application in atherosclerotic disease.Materials and MethodsThe whole-genome gene expression profiles of MCF-7 cells treated with/or without isoborneol were detected by mRNA microarray analysis. The degree of similarity between the gene expression profiles was compared with the Connectivity Map (CMAP) database. An MTT assay was used to assess the toxicity of isoborneol on RAW 264.7 cells. Oil red O staining and a Dil-ox-LDL uptake assay in RAW 264.7 cells were also used to detect the accumulation of lipids in the macrophages and the uptake of oxidized low-density lipoprotein (ox-LDL).ResultsIsoborneol was proved to have mRNA expression profiles similar to that of ikarugamycin which can inhibit the uptake of ox-LDL. This process has proved to be an important cause of foam cell formation and early atherosclerotic lesions. It is speculated, therefore, that isoborneol may show similar activity to that shown by ikarugamycin. Subsequently, it was shown that RAW 264.7 cells reduced the absorption of ox-LDL and the accumulation of intracellular lipids after treatment with different concentrations of isoborneol.ConclusionThe results indicate that isoborneol inhibits macrophage consumption of ox-LDL, thereby preventing the accumulation of lipids in the macrophages. These results provide evidence for the application of isoborneol in atherosclerotic disease.

Histological variation of early stage atherosclerotic lesions in baboons after prolonged challenge with high-cholesterol, high-fat diet.

The baboon is a well-characterized model of human early stage atherosclerosis. However, histological and morphological changes involved in atherogenesis in baboons are not known. Previously, we challenged baboons with a high-cholesterol, high-fat diet for two years and observed fatty streak and plaque lesions in iliac arteries (RCIA). We evaluated histological and morphological changes of baboon arterial lesions and control arteries. In addition, we evaluated the vascular expression of CD68 and SMαA markers with progression of atherosclerosis. We observed changes that correlated with extent of atherosclerosis, including increased maximum intimal thickness. We demonstrated at molecular level the infiltration of smooth muscle cells and macrophages into the intimal layer. Further, we observed histological and morphological discordancy between the affected and adjacent areas of the same RCIA. Atherogenesis in baboons is accompanied by histological, morphological, and molecular changes, as in humans, providing insights to evaluate the mechanisms underlying early stage atherosclerosis in target tissues.

P7142-AG impacts on endothelial cell activation and endothelial cell viability in vitro and impairs endothelial repair in vivo

Abstract Background The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation and few studies have addressed its influence on myeloid cells in the context of atherogenesis. However, the impact of 2-AG on endothelial cell function has not been studied before. Methods Endothelial repair was studied in two treatment groups of wildtype mice following electrical denudation of the common carotid artery at a length of 3000 μm. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184 [5 mg/kg i.p.], which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received vehicle. The residual endothelial gap at five days in either group was visualized by Evan's blue staining. In vitro, the effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability was assessed by an XTT-based assay. Endothelial activation was studied by an adhesion assay of THP-1 monocytes to 2-AG-preconditioned HCAEC. HCAEC migration, ROS-production, expression of NADPH oxidases, and secretion of inflammatory cytokines were assessed by Boyden chamber, qPCR, and colorimetric assays. Results Treatment with JZL184 produced a significant increase in 2-AG levels and impaired reendothelialisation in wildtype mice following electrical injury of the common carotid artery. The residual denudation at 5 days yielded 2291±286 μm in JZL184-treated animals vs. 1505±223 μm in vehicle treated controls (n=18–19; p<0.05). In vitro, JZL184 significantly reduced viability of HCAEC at 24 hours (0.31±0.10 vs. 1.00±0.08; n=3; p<0.01). Finally, 2-AG promoted HCAEC activation resulting in a significant increase in THP-1 monocyte adhesion to HCAEC following pre-treatment of HCAEC with 2-AG (0.17±0.03 THP-1 cells per HCAEC vs. 0.07±0.01 THP-1 cells per HCAEC; n=3; p<0.05). Besides, HCAEC migration, ROS-production, expression of NADPH oxidases and secretion of inflammatory cytokines were unaffected by 2-AG. Conclusion Elevated 2-AG levels appear to hamper endothelial repair and to promote HCAEC activation and cell death. Our data suggest that besides its influence on myeloid cells, 2-AG is also adverse to endothelial integrity which might promote early atherosclerotic lesion formation. Thus, decreasing vascular 2-AG levels might represent a promising therapeutic strategy for the prevention of atherosclerosis and coronary heart disease.

Translocator protein localises to CD11b+ macrophages in atherosclerosis

Background and aimsAtherosclerosis is characterized by lipid deposition, monocyte infiltration and foam cell formation in the artery wall. Translocator protein (TSPO) is abundantly expressed in lipid rich tissues. Recently, TSPO has been identified as a potential diagnostic tool in cardiovascular disease. The purpose of this study was to determine if the TSPO ligand, 18F-PBR111, can identify early atherosclerotic lesions and if TSPO expression can be used to identify distinct macrophage populations during lesion progression. MethodsApoE−/− mice were maintained on a high-fat diet for 3 or 12 weeks. C57BL/6J mice maintained on chow diet served as controls. Mice were administered 18F-PBR111 intravenously and PET/CT imaged. After euthanasia, aortas were isolated, fixed and optically cleared. Cleared aortas were immunostained with DAPI, and fluorescently labelled with antibodies to TSPO, the tissue resident macrophage marker F4/80 and the monocyte-derived macrophage marker CD11b. TSPO expression and the macrophage markers were visualised in fatty streaks and established plaques by light sheet microscopy. ResultsWhile tissue resident F4/80 + macrophages were evident in the arteries of animals without atherosclerosis, no CD11b + macrophages were observed in these animals. In contrast, established plaques had high CD11b and low F4/80 expression. A ∼3-fold increase in the uptake of 18F-PBR111 was observed in the aortas of atherosclerotic mice relative to controls. ConclusionsImaging of TSPO expression is a new approach for studying atherosclerotic lesion progression and inflammatory cell infiltration. The TSPO ligand, 18F-PBR111, is a potential clinical diagnostic tool for the detection and quantification of atherosclerotic lesion progression in humans.

Abstract WMP39: Laser Angioscopy in Carotid Artery Disease: Imaging Findings and Histopathological Correlation

Our understanding and management of carotid artery disease has largely relied on angiography, US and MR. However, existing modalities cannot accurately identify the spectrum of thrombogenic lesions on the vascular surface or reliably detect structural markers of instability. Thus, the natural history of the different histopathological hallmarks of advanced or complicated plaques is unknown, and many patients with culprit substenotic plaques are misdiagnosed with cryptogenic stroke. An intraluminal approach to directly inspect the endovascular surfaces at unprecedented resolutions would enable detailed structural analysis of carotid plaques and the identification of the root cause of carotid-related strokes. A scanning fiber angioscope (SFA), a high-definition 1.2-mm forward-viewing camera that generates videos by spirally scanning low-power laser beams, was developed for clinical use and employed to analyze the endovascular surfaces of ex vivo human carotid arteries (n=95) and endarterectomized plaques (n=20). Specimens were then imaged with US and micro-CT, and all vascular diagnostic tests obtained in the patients undergoing carotid revascularization were accessed. Imaging findings in the vascular surfaces were then validated by histomorphological analysis of >300 lesions, and an angioscopic classifier for atherosclerosis was generated. Full-color videos at 30Hz and at a resolution >200,000 pixels were generated from the entire endovascular circumference in all specimens with depth-of-focus larger than 5cm in saline solution. The ability to analyze the surface anatomy with unparalleled detail allowed the identification of early, intermediate, advanced and complex atherosclerotic lesions, including ulceration of fibrotic cap, excavation of the necrotic core leading to pseudolumens, intimal flaps, exposed necrotic core to the lumen, erosions, recent and remote intimal hemorrhages, luminal thrombus, eruptive calcified nodules and calcified intimal sheaths and plates. SFA offers a direct view of the endoluminal surfaces with unparalleled resolution and has the potential to revolutionize research, diagnosis, prognosis and image-guided therapy in carotid atherosclerosis.

Functional pathways associated with human carotid atheroma: a proteomics analysis.

Advances in large-scale analysis are becoming very useful in understanding health and disease. Here, we used high-throughput mass spectrometry to identify differentially expressed proteins between early and advanced lesions. Carotid endarterectomy samples were collected and dissected into early and advanced atherosclerotic lesion portions. Proteins were extracted and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Differentially expressed proteins were identified and verified using multiple reaction monitoring (MRM), on which advanced systems biology and enrichment analyses were performed. The identified proteins were further compared to the transcriptomic data of 32 paired samples obtained from early and advanced atherosclerotic lesions. A total of 95 proteins were upregulated, and 117 proteins were downregulated in advanced lesions compared to early atherosclerotic lesions (p < 0.05). The upregulated proteins were associated with proatherogenic processes, whereas downregulated proteins were involved in extracellular matrix organization and vascular smooth muscle cytoskeleton. Many of the identified proteins were linked to various "upstream regulators", among which TGFβ had the highest connections. Specifically, a total of 19 genes were commonly upregulated, and 30 genes were downregulated at the mRNA and protein levels. These genes were involved in vascular smooth muscle cell activity, for which enriched transcription factors were identified. This study deciphers altered pathways in atherosclerosis and identifies upstream regulators that could be candidate targets for treatment.

Cardiovascular Effects of Cyclical Dietary Vitamin C Withdrawal in Mice Deficient in Internal Synthesis Vitamin C and producing human lipoprotein (a): Gulo(-/-); Lp(a)+

Background: Scientific knowledge of the impact of periodic dietary vitamin C intake and withdrawal on the development of cardiovascular disease has been limited. Our earlier study using a transgenic Gulo(-/-); Lp(a)+ mouse model mimicking human metabolism in respect to a lack of internal synthesis of vitamin C and expression of human lipoprotein (a) [Lp(a)], a recognized risk factor for cardiovascular disease, showed that vitamin C deficiency triggers vascular deposition of Lp(a) and atherosclerosis. In this study we used this mouse model to investigate the effect of cyclical vitamin C withdrawal and its continuous supplementation on metabolic factors related to cardiovascular health, such as lipid profile and vascular plaque development. Methods: Gulo (-/-); Lp (a)+ mice were subjected to 4 weeks of dietary vitamin C withdrawals followed by 4 weeks of a resupplementation pattern for 20 weeks in total. Mice supplemented with vitamin C continuously for 20 weeks served as reference control. Mice were harvested after 4, 8, 12, 16, and 20 weeks and analyzed. Serum ascorbic acid, lipid profile, lipoproteins, vascular lesion, and Lp(a) deposition were evaluated. Results: We observed that periodic vitamin C withdrawals resulted in a less healthy blood lipid profile and showed indications of metabolic adaptation to vitamin C withdrawals. In addition, mice experiencing recurring dietary vitamin C withdrawals were prone to the development of early atherosclerotic lesions with Lp(a) deposition in the structurally compromised areas of the vascular wall, compared to mice continuously supplemented with vitamin C. Conclusion: This study further supports the importance of consistent and sufficient intake of vitamin C in assuring optimum profile of blood risk factors, and maintaining vascular wall integrity and optimal cardiovascular health.

Emulating endothelial dysfunction by implementing an early atherosclerotic microenvironment within a microfluidic chip.

Recent studies on endothelial dysfunction in relation to vascular diseases including atherosclerosis have highlighted the key contribution of the microenvironment of endothelial cells (ECs). By mimicking the microenvironment of early atherosclerotic lesions, here, we replicate the pathophysiological phenotype and function of ECs within microchannels. Considering the elevated deposition of fibronectin (FN) in early atherosclerotic plaques and the close correlation between the vascular stiffness and the progression of atherosclerosis, we utilized FN coated hydrogels with increased stiffness for endothelial substrates within the microchannels. As a result, we demonstrated that endothelial integrity on FN coated microchannels is likely to be undermined exhibiting a random orientation in response to the applied fluid flow, notable disruption of vascular endothelial cadherins (VE-cadherins), and higher endothelial permeability as opposed to that on microchannels coated with collagen (CL), the atheroresistant vascular model.

Chondroitin sulfate N-acetylgalactosaminyltransferase-2 deletion alleviates lipoprotein retention in early atherosclerosis and attenuates aortic smooth muscle cell migration

Glycosaminoglycans (GAGs) play an integral role in low-density lipoprotein (LDL) retention in the vascular intimal layer and have emerged as attractive therapeutic targets for atherosclerosis. GAG biosynthesis involves the cooperation of numerous enzymes. Chondroitin sulfate N-acetylgalactosaminyltransferase-2 (ChGn-2) is a vital Golgi transferase that participates in enzymatic elongation of GAGs. Here, we investigated the effects of ChGn-2 gene deletion on the development of atherosclerosis. Partial carotid artery ligation was performed on ChGn-2−/−/LDLr−/− and ChGn-2+/+/LDLr−/− mice to induce diffuse intimal thickening (DIT). Aortic smooth muscle cells (ASMCs) were isolated to investigate cellular LDL binding and migration. Histological analysis of human coronary artery sections revealed that ChGn-2 was expressed in early and advanced atherosclerotic lesions. Deletion of the ChGn-2 gene significantly reduced LDL retention in the DIT mouse model. Furthermore, LDL binding, visualized using rhodamine-labeled LDLs, was dramatically reduced. Interestingly, a functional assay of ASMCs prepared from ChGn-2−/− mice displayed abrogation of platelet-derived growth factor (PDGF)-mediated migration via reduced PDGF receptor phosphorylation. Taken together, these findings indicate that ChGn-2 is functionally involved in the progression of atherosclerosis both in its early and advanced stages. Therefore, ChGn-2 may serve as a plausible target to treat atherosclerotic-related diseases in the future.

Overexpression of CTRP9 attenuates the development of atherosclerosis in apolipoprotein E-deficient mice.

This study was aimed to explore the role of C1q/TNF-related protein 9 (CTRP9) on atherosclerotic lesion formation. A recombinant lentiviral vector carrying mouse CTRP9 (Lv-CTRP9) was injected intravenously into apolipoprotein E knockout (ApoE-/-) mice given a high-fat diet (HFD). CTRP9 overexpression substantially attenuated atherosclerotic lesion size of mice. The accumulation of macrophages and smooth muscle cells (SMCs) was significantly decreased in atherosclerotic regions with CTRP9 overexpression by immunohistochemical analysis. In addition, CTRP9 downregulated the expressions of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α), two main proinflammatory cytokines in atherosclerosis. Furthermore, the autophagy level remarkably increased which was presented by microtubule-associated protein light chain 3B (LC3B) conversion and sequestosome 1 (SQSTM1/p62) degradation. Further study showed that CTRP9 increased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and decreased mammalian target of rapamycin (mTOR) phosphorylation in vivo. These observations reveal that CTRP9 exerts a protecting role in early atherosclerotic lesions and its anti-atherosclerotic effect is associated with autophagy induction through AMPK/mTOR signaling pathway.

Macrophage Inflammation, Erythrophagocytosis, and Accelerated Atherosclerosis in Jak2 V617F Mice.

The mechanisms driving atherothrombotic risk in individuals with JAK2 V617F ( Jak2 VF) positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. The goal of this study was to assess atherosclerosis and underlying mechanisms in hypercholesterolemic mice with hematopoietic Jak2 VF expression. Irradiated low-density lipoprotein receptor knockout ( Ldlr-/-) mice were transplanted with bone marrow from wild-type or Jak2 VF mice and fed a high-fat high-cholesterol Western diet. Hematopoietic functions and atherosclerosis were characterized. After 7 weeks of Western diet, Jak2 VF mice showed increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophil adhesion and content, correlating with lesion size. After 12 weeks of Western diet, Jak2 VF lesions showed increased complexity, with larger necrotic cores, defective efferocytosis, prominent iron deposition, and costaining of erythrocytes and macrophages, suggesting erythrophagocytosis. Jak2 VF erythrocytes were more susceptible to phagocytosis by wild-type macrophages and showed decreased surface expression of CD47, a "don't-eat-me" signal. Human JAK2VF erythrocytes were also more susceptible to erythrophagocytosis. Jak2 VF macrophages displayed increased expression and production of proinflammatory cytokines and chemokines, prominent inflammasome activation, increased p38 MAPK (mitogen-activated protein kinase) signaling, and reduced levels of MerTK (c-Mer tyrosine kinase), a key molecule mediating efferocytosis. Increased erythrophagocytosis also suppressed efferocytosis. Hematopoietic Jak2 VF expression promotes early lesion formation and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, Jak2 VF caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes promote accumulation of iron in plaques and increased necrotic core formation which, together with exacerbated proinflammatory responses, likely contribute to plaque instability.

Tumor Necrosis Factor-α and C-C Motif Chemokine Ligand 18 Associate with Atherosclerotic Lipid Accumulation In situ and In vitro.

Atherosclerosis is regarded as a chronic inflammatory disease associated with changes in the innate immune system functioning and cytokine disturbances. Local inflammation in the arterial wall is an important component in the development and growth of atherosclerotic plaques. Inside the lesions, both pro- and antiinflammatory cytokines were detected, highlighting the complexity of the atherosclerotic process. However, little is known about the expression of these signaling molecules in early human atherosclerotic lesions. In this study, we explored localization of a pro-inflammatory cytokine, tumor necrosis factor-α (TNFα), and anti-inflammatory chemokine, C-C motif chemokine ligand 18 (CCL18), in the arterial wall of human aorta. We noticed differences in the intensity of staining for TNFα and CCL18 in atherosclerotic lesions and grossly normal areas, as well as differences in their localization. While CCL18 prevailed in the areas close to the aortic lumen, TNFα was localized in deeper layers of the intima. We next studied the expression of TNFα and CCL18 mRNA in lesions corresponding to different stages of atherosclerosis progression and found that it was maximal in lipofibrous plaques that are most enriched in lipids. To test the hypothesis that cytokine expression can be associated with lipid accumulation, we studied the TNFα and CCL18 expression profiles in primary human monocyte-derived macrophages after inducing lipid accumulation by incubating cultured cells with atherogenic LDL. We found that intracellular cholesterol accumulation was associated with upregulation of both TNFα and CCL18, confirming our hypothesis. These results encourage further investigation of cytokine expression in human atherosclerotic lesions and its role in the atherosclerosis progression.

Progressive changes of elastic moduli of arterial wall and atherosclerotic plaque components during plaque development in human coronary arteries.

Stiffness of the arterial wall and atherosclerotic plaque components is a determinant of the stress field within plaques, which has been suggested to be an indicator of plaque vulnerability. The diversity and inhomogeneous structure of atherosclerotic lesions complicate the characterization of plaque components. In the present study, stiffness of the arterial wall and atherosclerotic plaque components in human coronary arteries was examined in early and developed atherosclerotic lesions. The force-spectroscopy mode of the atomic force microscope and histological examination were used for determination of elastic moduli at specified locations within samples. Fibrous cap (E = 14.1 ± 3.8kPa) showed lower stiffness than the fibrous tissue beneath the lipid pool (E = 17.6 ± 3.2kPa). Calcification zones (E = 96.1 ± 18.8kPa) and lipid pools (E = 2.7 ± 1.8kPa) were the stiffest and softest components of atherosclerotic lesions, respectively. The increase of media stiffness (%44.8) and reduction of the elastic modulus of the internal elastic lamina (%28.9) was observed in coronary arteries. Moreover, significant differences were observed between the stiffness of medial layer in diseased parts and free-plaque segments in incomplete plaques of coronary arteries. Our results can be used for better understanding of remodeling mechanisms of the arterial wall with plaque development. Graphical abstract Stiffness alteration of the arterial wall and atherosclerotic plaque components with plaque development in coronary arteries.

Hyperlipidemic mice as a model for a real‐time in vivo detection of atherosclerosis by gold nanorods‐based diffusion reflection technique

Atherosclerosis (AS), the leading cause of morbidity and mortality in cardiovascular disease, needs an early detection for treatment and prevention of fatal events. Here, for the first time, we applied gold nanorods (GNRs)-assisted diffusion reflection (DR), a noninvasive technique for in vivo detection of AS in a high-fat-diet-induced c57bl mouse model, which resembles the manifestation of AS in humans. DR simply detects the change in light reflection profile of tissue due to the accumulation of GNRs in the AS plaques and enables clear detection of AS lesions in carotid and femoral arteries of these hyperlipidemic mice. After 24 hours post-GNRs injection, DR showed the highest efficiency of AS detection. Moreover, the sensitivity of the DR method is much higher than computed tomography (CT) and is comparable to ex vivo high-resolution CT. Our results strongly suggest that the DR method can detect early atherosclerotic lesions in a sensitive and specific manner.

The role of UNC5b in ox-LDL inhibiting migration of RAW264.7 macrophages and the involvement of CCR7

The formation of macrophage foam cells by ingesting ox-LDL and focal retention in the subendothelial space are the hallmarks of the early atherosclerotic lesion. The C-C chemokine receptor type 7 (CCR7) is positively correlated with the macrophage migration. But the mechanism of CCR7 regulation is not fully clearness. In the present study, we demonstrates that expression in UNC5b and netrin-1 was enhanced in respond to ox-LDL in Raw264.7 macrophage and associated with decreasing cell migration. Interestingly, it was found that ox-LDL significantly downregulate CCR7 gene expression. The expression of CCR7 in mRNA and protein levels were decreased in ox-LDL treated Raw264.7 macrophage when we over expression of UNC5b with pcDNA3.1-UNC5b plasmid. We got the inverse results after silence UNC5b gene with siUNC5b. Meanwhile, the data show that in ox-LDL inducement, UNC5b down-regulated CCR7, and then inhibited macrophage migration. This novel phenomenon is of a crucial highlights to understand deeply the pathogenesis of atherosclerosis. The molecular mechanism of CCR7 regulation deserves intensive study.

Masscytometry identifies CD8 T-cell diversity in human atherosclerotic lesions

Aim: CD8+ T-cells are numerous in early and advanced atherosclerotic lesion, but their role in atherogenesis and plaque stability is still under debate. Recent work in murine models suggests the existence multiple CD8+ T-cell subsets that may differently impact the progression of disease. Here we aim to characterize the CD8+ T-cell population in human atherosclerotic lesions using a combination of histology, flow cytometry and mass cytometry (Cytof).

Proatherogenic effects of 4-hydroxynonenal

Among the various mechanisms involved in atherogenesis, the oxidative theory of atherosclerosis relies on the oxidation of low density lipoprotein (LDL) in the vascular wall and their implication in the formation of early atherosclerotic lesions. Reactive oxygen species (ROS) and oxidants generated by activated endothelium, initiate LDL oxidation in the intima, which generates a huge variety of lipid peroxidation products (LPPs), exhibiting atherogenic, pro-inflammatory and pro-apoptotic properties. Reactive carbonyl compounds (RCCs), are a family of highly reactive agents generated during polyunsaturated fatty acid (PUFA) peroxidation. RCCs covalently bind to nucleophilic group of proteins, peptides, phospholipids and nucleic acids, thereby generating a “carbonyl stress”. Among RCCs, 4-hydroxy-2-nonenal (HNE) exerts its atherogenic effects through several mechanisms, by targeting lipoproteins or cellular components. HNE generated during LDL oxidation is able to form HNE-apoB adducts, which are recognized by scavenger-receptors of macrophagic cells, thereby leading to foam cell formation. HNE can be released during the degradation of oxLDL, or generated through oxidative stress and PUFA peroxydation in cell membranes. The biological effects of HNE on vascular cells depend on its local concentration and on the expression of detoxifying systems, such as glutathione S-transferase, aldose reductase, and aldehyde dehydrogenase (ALDH), which rapidly neutralize and remove HNE from cells. Physiological concentrations (0.1-1 µmol/L) of HNE induce hormetic and adaptive responses, and transcription factors (Nrf2) that increase cell resistance to oxidative attack and other stresses, while moderate HNE concentrations, (1 to 10 µmol/L), trigger the accumulation of HNE-adducts and a variety of biological responses, such as inflammation and cell proliferation. Higher HNE concentrations, (above 10-20 µmol/L), induce cell dysfunction and apoptosis. However, important variations are observed in atherosclerotic lesions, from the lipid core to the periphery of the plaque, with very different local outcomes. HNE can modify signaling proteins involved in atherosclerotic plaque remodeling, particularly growth factor receptors (PDGFR, EGFR), cell cycle proteins, mitochondrial and endoplasmic reticulum components or extracellular matrix proteins, which progressively alters smooth muscle cell proliferation, angiogenesis and induces apoptosis. HNE-adducts accumulate in the lipidic necrotic core of advanced atherosclerotic lesions and may locally contribute to macrophage and smooth muscle cell apoptosis, which contributes to plaque destabilization and rupture, thereby increasing the risk of athero-thrombotic events.

TNF-alfa and CCL18 associate with atherosclerotic lipid accumulation in situ and in vitro

Aim: Although both pro- and anti-inflammatory cytokines have been detected in human atherosclerotic plaque, quantitative data on their expression in early atherosclerotic lesions remains to be reported.

CARDIOVASCULAR RISK FACTORS AND ARTERIAL STIFFNESS IN MEN AGED 41–43 YEARS

Objective: To study the relationship of individual cardiovascular risk factors with arterial stiffness and subclinical atherosclerosis. Design and method: The study is the part of a 32-year prospective cohort monitoring, beginning with childhood (11–12 years). The present cohort included 303 male subjects aged 41–43 years. Arterial stiffness (AS) was measured by aortic pulse wave velocity (PWV) and arterial wave reflections (augmentation index, Alx) with the use of SphygmoCor (AtCor Medical). Intima-media thickness (IMT) and subclinical atherosclerosis were estimateded by ultrasonography methods both in left and right carotid arteries. Results: Hypertension (H) was detected in 37,9%. PWV was significantly higher in the group with H. The risk of H development depended on hypertension presence in mother and did not depend on the presence of H in father. H was associated with the development of obesity, especially of the abdominal type and increased level of triglycerides. PWV was positively correlated with systolic (r = 0.248, p < 0.001) and diastolic blood pressure (BP) (r = 0.220, p < 0.001) measured on the brachial artery and with heart rate (r = 0.164, p < 0.01). A statistically significant positive correlation of the central aortic pressure (r = 0.326, p < 0.001) and central pulse pressure (r = 0.225, p < 0.001) with PWV was noted. When comparing arterial stiffness (AS) and duplex scan a correlation of mean IMT with the Alx (r = 0.134, p < 0.05) and augmentation pressure (r = 0.127, p < 0.05) was obtained, but no correlation between IMT and PWV was found. Correlation of AS with total cholesterol and glucose was also shown. Conclusions: The AS is closely related with H and develops already in the early stages, in a fairly young age. The AS in men group was not associated with the presence of dyslipidemia and diabetes melitus. The interrelation of arterial stiffness and the degree of early atherosclerotic vascular lesions is ambiguous.

Abstract 040: Accelerated Atherosclerosis and Thrombosis in Jak2v617f Mice

The mechanisms driving increased athero-thrombotic risk in individuals with JAK2V617F positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. This study was to assess atherosclerosis and thrombosis and associated mechanisms in hypercholesterolemic mice with hematopoietic JAK2V617F expression. Irradiated Ldlr -/- mice were transplanted with bone marrow from WT or JAK2V617F mice and fed a high fat high cholesterol diet (WD). After 7 weeks of WD JAK2V617F mice showed increased hematopoiesis, platelet activation, accelerated thrombosis and increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophils, correlating with lesion size and in association with increased rolling and adhesion of neutrophils on endothelial cells in carotid artery. After 12 weeks of WD JAK2V617F lesions were slightly larger than controls but showed additional complexity, with increased necrotic core area, prominent iron deposition and co-staining of erythrocytes and macrophages suggesting erythrophagocytosis. JAK2V617F erythrocytes were more susceptible to phagocytosis by WT macrophages and showed decreased surface expression of CD47, a “don’t eat me” signal. Human JAK2V617F erythrocytes were also more susceptible to erythrophagocytosis. JAK2V617F macrophages displayed increased expression of pro-inflammatory cytokines and chemokines, increased p38 map kinase signaling and increased cleavage and reduced levels of MerTK, a key molecule mediating phagocytosis of apoptotic cells (efferocytosis) in atherosclerotic lesions. Erythrophagocytosis also suppressed efferocytosis. As a result, JAK2V617F lesional efferocytosis was reduced in advanced lesions. In conclusion, JAK2V617F promotes early lesion formation, arterial thrombosis and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, JAK2V617F caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes appear to promote accumulation of iron and increased necrotic core formation which, together with exacerbated pro-inflammatory responses, may contribute to plaque instability.