NO-Donating Aspirin and Aspirin Partially Inhibit Age-Related Atherosclerosis but Not Radiation-Induced Atherosclerosis in ApoE Null Mice

BACKGROUND: We previously showed that irradiation to the carotid arteries of ApoE(-/-) mice accelerated the development of macrophage-rich, inflammatory atherosclerotic lesions, prone to intra-plaque hemorrhage. In this study we investigated the potential of anti-inflammatory and anti-coagulant intervention strategies to inhibit age-related and radiation-induced atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: ApoE(-/-) mice were given 0 or 14 Gy to the neck and the carotid arteries and aortic arches were harvested at 4 or 30 weeks after irradiation. Nitric oxide releasing aspirin (NCX 4016, 60 mg/kg/day) or aspirin (ASA, 30 or 300 mg/kg/day) were given continuously in the chow. High dose ASA effectively blocked platelet aggregation, while the low dose ASA or NCX 4016 had no significant effect on platelet aggregation. High dose ASA, but not NCX 4016, inhibited endothelial cell expression of VCAM-1 and thrombomodulin in the carotid arteries at 4 weeks after irradiation; eNOS and ICAM-1 levels were unchanged. After 30 weeks of follow-up, NCX 4016 significantly reduced the total number of lesions and the number of initial macrophage-rich lesions in the carotid arteries of unirradiated mice, but these effects were not seen in the brachiocephalic artery of the aortic arch (BCA). In contrast, high dose ASA lead to a decrease in the number of initial lesions in the BCA, but not in the carotid artery. Both high dose ASA and NCX 4016 reduced the collagen content of advanced lesions and increased the total plaque burden in the BCA of unirradiated mice. At 30 weeks after irradiation, neither NCX 4016 nor ASA significantly influenced the number or distribution of lesions, but high dose ASA lead to formation of collagen-rich "stable" advanced lesions in carotid arteries. The total plaque area of the irradiated BCA was increased after ASA, but the plaque burden was very low compared with the carotid artery. CONCLUSIONS/SIGNIFICANCE: The development and characteristics of radiation-induced atherosclerosis varied between different arteries but could not be circumvented by anti-inflammatory and anti-coagulant therapies. This implicates other underlying mechanistic pathways compared to age-related atherosclerosis

Impact of tissue adhesives on the prevention of anastomotic leakage of colonic anastomoses: an in vivo study

Background: Tissue adhesives (TA) may be useful to strengthen colorectal anastomoses, thereby preventing anastomotic leakage (AL). Previous studies have identified cyanoacrylate (CA) TAs as the most promising colonic anastomotic sealants. This stud

Ablation of CD8 alpha(+) dendritic cell mediated cross-presentation does not impact atherosclerosis in hyperlipidemic mice

Clinical complications of atherosclerosis are almost exclusively linked to destabilization of the atherosclerotic plaque. Batf3-dependent dendritic cells specialize in cross-presentation of necrotic tissue-derived epitopes to directly activate cytolytic CD8 Tcells. The mature plaque (necrotic, containing dendritic cells and CD8 Tcells) could offer the ideal environment for cross-presentation, resulting in cytotoxic immunity and plaque destabilization. Ldlr(-/-) mice were transplanted with batf3(-/-) or wt bone marrow and put on a western type diet. Hematopoietic batf3 deficiency sharply decreased CD8 alpha(+) DC numbers in spleen and lymph nodes (>80%;P < 0, 001). Concordantly, batf3(-/-) chimeras had a 75% reduction in OT-I cross-priming capacity in vivo. Batf3(-/-) chimeric mice did not show lower Tcell or other leukocyte subset numbers. Despite dampened cross-presentation capacity, batf3(-/-) chimeras had equal atherosclerosis burden in aortic arch and root. Likewise, batf3(-/-) chimeras and wt mice revealed no differences in parameters of plaque stability: plaque Tcell infiltration, cell death, collagen composition, and macrophage and vascular smooth muscle cell content were unchanged. These results show that CD8 alpha(+) DC loss in hyperlipidemic mice profoundly reduces cross-priming ability, nevertheless it does not influence lesion development. Taken together, we clearly demonstrate that CD8 alpha(+) DC-mediated cross-presentation does not significantly contribute to atherosclerotic plaque formation and stability

Adventitial lymphatic capillary expansion impacts on plaque T cell accumulation in atherosclerosis

During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE(-/-)mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3(+) T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3(+) T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.Peer reviewe

Whole body and hematopoietic ADAM8 deficiency does not influence advanced atherosclerotic lesion development, despite its association with human plaque progression

Although A Disintegrin And Metalloproteinase 8 (ADAM8) is not crucial for tissue development and homeostasis, it has been implicated in various inflammatory diseases by regulating processes like immune cell recruitment and activation. ADAM8 expression has been associated with human atherosclerosis development and myocardial infarction, however a causal role of ADAM8 in atherosclerosis has not been investigated thus far. In this study, we examined the expression of ADAM8 in early and progressed human atherosclerotic lesions, in which ADAM8 was significantly upregulated in vulnerable lesions. In addition, ADAM8 expression was most prominent in the shoulder region of human atherosclerotic lesions, characterized by the abundance of foam cells. In mice, Adam8 was highly expressed in circulating neutrophils and in macrophages. Moreover, ADAM8 deficient mouse macrophages displayed reduced secretion of inflammatory mediators. Remarkably, however, neither hematopoietic nor whole-body ADAM8 deficiency in mice affected atherosclerotic lesion size. Additionally, except for an increase in granulocyte content in plaques of ADAM8 deficient mice, lesion morphology was unaffected. Taken together, whole body and hematopoietic ADAM8 does not contribute to advanced atherosclerotic plaque development, at least in female mice, although its expression might still be valuable as a diagnostic/ prognostic biomarker to distinguish between stable and unstable lesions

The cholesterol derivative 27-hydroxycholesterol reduces steatohepatitis in mice.

<p>BACKGROUND & AIMS: Non-alcoholic steatohepatitis is characterized by hepatic steatosis with inflammation. Although steatosis is benign and reversible, inflammation can increase liver damage. Hepatic inflammation has been associated with accumulation of cholesterol in lysosomes of Kupffer cells. 27-Hydroxycholesterol (27HC), a derivative of cholesterol formed by CYP27A1, can mobilize cholesterol from the lysosomes to the cytoplasm. We investigated whether 27HC can change the intracellular distribution cholesterol and reduce hepatic inflammation in mice. METHODS: We transplanted bone marrow from irradiated wild-type or Cyp27a1(-/-) mice to mice that do not express the low density lipoprotein receptor (Ldlr(-/-)), which are hyperlipidemic; 9 weeks later, mice were fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 3 months. In a separate experiment, Ldlr(-/-) mice were given subcutaneous injections of 27HC and placed on regular chow or HFC diets for 3 weeks. Blood and liver tissues samples were collected and analyzed for intracellular cholesterol distribution and inflammation. RESULTS: In Ldlr(-/-) mice that received bone marrow transplants from Cyp27a1(-/-) mice, lysosomes of Kupfer cells had a greater accumulation of cholesterol than those of mice that received bone marrow from wild-type mice, after the HFC diet. Liver histology and gene expression analyses showed increased inflammation and liver damage in mice given bone marrow transplants from Cyp27a1(-/-) mice and placed on the HFC diet. Administration of 27HC to Ldlr(-/-) mice, following the HFC diet, reduced the accumulation of lysosomal cholesterol and hepatic inflammation, compared with mice that were not given 27HC. CONCLUSIONS: Accumulation of cholesterol in lysosomes of Kupfer cells promotes hepatic inflammation in mice. The cholesterol derivative 27HC reduces accumulation of cholesterol in lysosomes and might be used to treat non-alcoholic steatohepatitis.</p>

Quantification of carotid plaque composition with a multi-contrast atherosclerosis characterization (MATCH) MRI sequence

Background and purposeCarotid atherosclerotic plaques with a large lipid-rich necrotic core (LRNC), intraplaque hemorrhage (IPH), and a thin or ruptured fibrous cap are associated with increased stroke risk. Multi-sequence MRI can be used to quantify carotid atherosclerotic plaque composition. Yet, its clinical implementation is hampered by long scan times and image misregistration. Multi-contrast atherosclerosis characterization (MATCH) overcomes these limitations. This study aims to compare the quantification of plaque composition with MATCH and multi-sequence MRI.MethodsMATCH and multi-sequence MRI were used to image 54 carotid arteries of 27 symptomatic patients with ≥2 mm carotid plaque on a 3.0 T MRI scanner. The following sequence parameters for MATCH were used: repetition time/echo time (TR/TE), 10.1/4.35 ms; field of view, 160 mm × 160 mm × 2 mm; matrix size, 256 × 256; acquired in-plane resolution, 0.63 mm2× 0.63 mm2; number of slices, 18; and flip angles, 8°, 5°, and 10°. Multi-sequence MRI (black-blood pre- and post-contrast T1-weighted, time of flight, and magnetization prepared rapid acquisition gradient echo; acquired in-plane resolution: 0.63 mm2 × 0.63 mm2) was acquired according to consensus recommendations, and image quality was scored (5-point scale). The interobserver agreement in plaque composition quantification was assessed by the intraclass correlation coefficient (ICC). The sensitivity and specificity of MATCH in identifying plaque composition were calculated using multi-sequence MRI as a reference standard.ResultsA significantly lower image quality of MATCH compared to that of multi-sequence MRI was observed (p &lt; 0.05). The scan time for MATCH was shorter (7 vs. 40 min). Interobserver agreement in quantifying plaque composition on MATCH images was good to excellent (ICC ≥ 0.77) except for the total volume of calcifications and fibrous tissue that showed moderate agreement (ICC ≥ 0.61). The sensitivity and specificity of detecting plaque components on MATCH were ≥89% and ≥91% for IPH, ≥81% and 85% for LRNC, and ≥71% and ≥32% for calcifications, respectively. Overall, good-to-excellent agreement (ICC ≥ 0.76) of quantifying plaque components on MATCH with multi-sequence MRI as the reference standard was observed except for calcifications (ICC = 0.37–0.38) and fibrous tissue (ICC = 0.59–0.70).Discussion and conclusionMATCH images can be used to quantify plaque components such as LRNC and IPH but not for calcifications. Although MATCH images showed a lower mean image quality score, short scan time and inherent co-registration are significant advantages

Endothelial ADAM10 controls cellular response to oxLDL and its deficiency exacerbates atherosclerosis with intraplaque hemorrhage and neovascularization in mice

IntroductionThe transmembrane protease A Disintegrin And Metalloproteinase 10 (ADAM10) displays a “pattern regulatory function,” by cleaving a range of membrane-bound proteins. In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development in vivo.Methods and resultsEndothelial Adam10 deficiency (Adam10ecko) in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of Adam10ecko mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of Adam10ecko mice was significantly reduced compared to wildtypes.DiscussionCollectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases

Targeting macrophage Histone deacetylase 3 stabilizes atherosclerotic lesions

Macrophages are key immune cells found in atherosclerotic plaques and critically shape atherosclerotic disease development. Targeting the functional repertoire of macrophages may hold novel approaches for future atherosclerosis management. Here, we describe a previously unrecognized role of the epigenomic enzyme Histone deacetylase 3 (Hdac3) in regulating the atherosclerotic phenotype of macrophages. Using conditional knockout mice, we found that myeloid Hdac3 deficiency promotes collagen deposition in atherosclerotic lesions and thus induces a stable plaque phenotype. Also, macrophages presented a switch to anti-inflammatory wound healing characteristics and showed improved lipid handling. The pro-fibrotic phenotype was directly linked to epigenetic regulation of the Tgfb1 locus upon Hdac3 deletion, driving smooth muscle cells to increased collagen production. Moreover, in humans, HDAC3 was the sole Hdac upregulated in ruptured atherosclerotic lesions, Hdac3 associated with inflammatory macrophages, and HDAC3 expression inversely correlated with pro-fibrotic TGFB1 expression. Collectively, we show that targeting the macrophage epigenome can improve atherosclerosis outcome and we identify Hdac3 as a potential novel therapeutic target in cardiovascular disease

Internalization of Modified Lipids by CD36 and SR-A Leads to Hepatic Inflammation and Lysosomal Cholesterol Storage in Kupffer Cells

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis and inflammation, which can further progress into fibrosis and cirrhosis. Recently, we demonstrated that combined deletion of the two main scavenger receptors, CD36 and macrophage scavenger receptor 1 (MSR1), which are important for modified cholesterol-rich lipoprotein uptake, reduced NASH. The individual contributions of these receptors to NASH and the intracellular mechanisms by which they contribute to inflammation have not been established. We hypothesize that CD36 and MSR1 contribute independently to the onset of inflammation in NASH, by affecting intracellular cholesterol distribution inside Kupffer cells (KCs).Ldlr(-/-) mice were transplanted with wild-type (Wt), Cd36(-/-) or Msr1(-/-) bone marrow and fed a Western diet for 3 months. Cd36(-/-)- and Msr1(-/-)- transplanted (tp) mice showed a similar reduction in hepatic inflammation compared to Wt-tp mice. While the total amount of cholesterol inside KCs was similar in all groups, KCs of Cd36(-/-)- and Msr1(-/-)-tp mice showed increased cytoplasmic cholesterol accumulation, while Wt-tp mice showed increased lysosomal cholesterol accumulation.CD36 and MSR1 contribute similarly and independently to the progression of inflammation in NASH. One possible explanation for the inflammatory response related to expression of these receptors could be abnormal cholesterol trafficking in KCs. These data provide a new basis for prevention and treatment of NASH