Absence of p55 TNF Receptor Reduces Atherosclerosis, but Has No Major Effect on Angiotensin II Induced Aneurysms in LDL Receptor Deficient Mice

The aim of the current study was to investigate the role of p55 TNF Receptor (p55 TNFR), the main signaling receptor for the pro-inflammatory cytokine tumor necrosis factor (TNF), in the development of two vascular disorders: atherosclerosis and angiotensin (Ang) II-induced abdominal aortic aneurysms (AAA). p55 TNFR deficient mice were crossed to an LDL receptor deficient background and were induced for the development of either atherosclerosis or AngII-induced AAA, and compared to littermate controls, wild-type for p55 TNFR expression. p55 TNFR deficient mice developed 43% smaller atherosclerotic lesions in the aortic sinuses compared to controls. Moreover, expression of CD68, a macrophage specific marker, exhibited a 50% reduction in the aortic arches. Decreased atherosclerosis correlated with a strong down-regulation in the expression of adhesion molecules, such as VCAM-1 and ICAM-1, by p55 TNFR deficient endothelium. In addition, expression levels of the pro-inflammatory cytokines and chemokines TNF, IL-6, MCP-1 and RANTES were significantly reduced in aortas of p55 TNFR deficient mice. In contrast, in the AngII-induced model of AAA, p55 TNFR deficiency correlated with a slight trend towards increased aneurismal lethality, but the incidence of aortic rupture due to a dissecting aneurysm, and the expansion of the suprarenal aorta were not significantly different compared to controls. We found that p55 TNFR expression promotes atherosclerosis, among other mechanisms, by enhancing expression of endothelial adhesion molecules, while it seems to have no major role in the development of AngII-induced AA

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.</p

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

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

Objective: Angiopoietin-2 (Ang-2) blocking agents are currently undergoing clinical trials for use in cancer treatment. Ang-2 has also been associated with rupture-prone atherosclerotic plaques in humans, suggesting a role for Ang-2 in plaque stability. Despite the availability of Ang-2 blocking agents, their clinical use is still lacking. Our aim was to establish if Ang-2 has a role in atheroma development and in the transition of subclinical to clinically relevant atherosclerosis. We investigated the effect of antibody-mediated Ang-2 blockage on atherogenesis after in a mouse model of atherosclerosis. Methods: Hypercholesterolemic (low-density lipoprotein receptor(-/-) apolipoprotein B-100/100) mice were subjected to high-cholesterol diet for eight weeks, one group with and one group without Ang-2 blocking antibody treatment during weeks 4-8. To enhance plaque development, a peri-adventitial collar was placed around the carotid arteries at the start of antibody treatment. Aortic root, carotid arteries and brachiocephalic arteries were analyzed to evaluate the effect of Ang-2 blockage on atherosclerotic plaque size and stable plaque characteristics. Results: Anti-Ang-2 treatment reduced the size of fatty streaks in the brachiocephalic artery (-72%, p <0.05). In addition, antibody-mediated Ang-2 blockage reduced plasma triglycerides (-27%, p <0.05). In contrast, Ang-2 blockage did not have any effect on the size or composition (collagen content, macrophage percentage, adventitial microvessel density) of pre-existing plaques in the aortic root or collar-induced plaques in the carotid artery. Conclusions: Ang-2 blockage was beneficial as it decreased fatty streak formation and plasma triglyceride levels, but had no adverse effect on pre-existing atherosclerosis in hypercholesterolemic mice. (C) 2015 The Authors. Published by Elsevier Ireland Ltd.Peer reviewe

Development of a Supramolecular Hydrogel for Intraperitoneal Injections

Local intraperitoneal drug administration is considered a challenging drug delivery route. The therapeutic efficiency is low, mainly due to rapid clearance of drugs. To increase the intraperitoneal retention time of specific drugs, a pH-sensitive supramolecular hydrogel that can act as a drug delivery vehicle is developed. To establish the optimal formulation of the hydrogel and to study its feasibility, safety, and tissue compatibility, in vitro, postmortem, and in vivo experiments are performed. In vitro tests reveal that a hydrogelator formulation with pH ≥ 9 results in a constant viscosity of 0.1 Pa·s. After administration postmortem, the hydrogel covers the parietal and visceral peritoneum with a thin, soft layer. In the subsequent in vivo experiments, 14 healthy rats are subjected to intraperitoneal injection with the hydrogel. Fourteen and 28 days after implantation, the animals are euthanized. Intraperitoneal exposure to the hydrogel is not resulted in significant weight loss or discomfort. Moreover, no macroscopic adverse effects or signs of organ damage are detected. In several intra-abdominal tissues, vacuolated macrophages are found indicating a physiological degradation of the synthetic hydrogel. This study demonstrates that the supramolecular hydrogel is safe for intraperitoneal application and that the hydrogel shows good tissue compatibility in rats.</p

Treating colorectal peritoneal metastases with an injectable cytostatic loaded supramolecular hydrogel in a rodent animal model

Patients with peritoneal metastases (PM) of colorectal cancer have a very poor outcome. Intraperitoneal delivery of chemotherapy is the preferred route for PM treatment. The main limitation of the treatment options is the short residence time of the cytostatic, with subsequent short exposure of the cancer cells. To address this, a supramolecular hydrogel has been developed that allows both local and slow release of its encapsulated drug, mitomycin C (MMC) or cholesterol-conjugated MMC (cMMC), respectively. This experimental study investigates if drug delivery using this hydrogel improves the therapeutic efficacy against PM. PM was induced in WAG/Rij rats (n = 72) by intraperitoneally injecting syngeneic colon carcinoma cells (CC531) expressing luciferase. After seven days, animals received a single intraperitoneal injection with saline (n = 8), unloaded hydrogel (n = 12), free MMC (n = 13), free cMMC (n = 13), MMC-loaded hydrogel (n = 13), or cMMC-loaded hydrogel (n = 13). Primary outcome was overall survival with a maximum follow-up of 120 days. Intraperitoneal tumor development was non-invasive monitored via bioluminescence imaging. Sixty-one rats successfully underwent all study procedures and were included to assess therapeutic efficacy. After 120 days, the overall survival in the MMC-loaded hydrogel and free MMC group was 78% and 38%, respectively. A trend toward significance was found when comparing the survival curves of the MMC-loaded hydrogel and free MMC (p = 0.087). No survival benefit was found for the cMMC-loaded hydrogel compared to free cMMC. Treating PM with our MMC-loaded hydrogel, exhibiting prolonged MMC exposure, seems effective in improving survival compared to treatment with free MMC.</p

Targeting Histone Deacetylases in Myeloid Cells Inhibits Their Maturation and Inflammatory Function With Limited Effects on Atherosclerosis

Monocytes and macrophages are key drivers in the pathogenesis of inflammatory diseases. Epigenetic targets have been shown to control the transcriptional profile and phenotype of these cells. Since histone deacetylase protein inhibitors demonstrate profound anti-inflammatory activity, we wanted to test whether HDAC inhibition within monocytes and macrophages could be applied to suppress inflammation in vivo. ESM technology conjugates an esterase-sensitive motif (ESM) onto small molecules to allow targeting of cells that express carboxylesterase 1 (CES1), such as mononuclear myeloid cells. This study utilized an ESM-HDAC inhibitor to target monocytes and macrophages in mice in both an acute response model and an atherosclerosis model. We demonstrate that the molecule blocks the maturation of peritoneal macrophages and inhibits pro-inflammatory cytokine production in both models but to a lesser extent in the atherosclerosis model. Despite regulating the inflammatory response, ESM-HDAC528 did not significantly affect plaque size or phenotype, although histological classification of the plaques demonstrated a significant shift to a less severe phenotype. We hereby show that HDAC inhibition in myeloid cells impairs the maturation and activation of peritoneal macrophages but shows limited efficacy in a model of atherosclerosis

Evaluation of the Effect of an Intraperitoneal Cytostatic-Loaded Supramolecular Hydrogel on Intestinal Anastomotic Healing in an Animal Model

The prognosis of colorectal cancer patients with peritoneal metastases is very poor. Intraperitoneal drug delivery systems, like supramolecular hydrogels, are being developed to improve local delivery and intraperitoneal residence time of a cytostatic such as mitomycin C (MMC). In this study, we evaluate the effect of intraperitoneal hydrogel administration on anastomotic healing. Forty-two healthy Wistar rats received a colonic end-to-end anastomosis, after which 6 animals received an intraperitoneal injection with saline, 18 with unloaded hydrogel and 18 with MMC-loaded hydrogel. After 7 days, animals were euthanized, and the anastomotic adhesion and leakage score were measured as primary outcome. Secondary outcomes were bursting pressure, histological anastomosis evaluation and body weight changes. Twenty-two rats completed the follow-up period (saline: n = 6, unloaded hydrogel: n = 10, MMC-loaded hydrogel: n = 6) and were included in the analysis. A trend towards significance was found for anastomotic leakage score between the rats receiving saline and unloaded hydrogel after multiple-comparison correction (p = 0.020, α = 0.0167). No significant differences were found for all other outcomes. The main reason for drop-out in this study was intestinal blood loss. Although the preliminary results suggest that MMC-loaded or unloaded hydrogel does not influence anastomotic healing, the intestinal blood loss observed in a considerable number of animals receiving unloaded and MMC-loaded hydrogel implies that the injection of the hydrogel under the studied conditions is not safe in the current rodent model and warrants further optimalisation of the hydrogel.</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

Pneumococcal Immunization Reduces Neurological and Hepatic Symptoms in a Mouse Model for Niemann-Pick Type C1 Disease

Niemann-Pick type C1 (NPC1) disease is caused by a deleterious mutation in the Npc1 gene, causing lysosomal accumulation of unesterified cholesterol and sphingolipids. Consequently, NPC1 disease patients suffer from severe neurovisceral symptoms which, in the absence of effective treatments, result in premature death. NPC1 disease patients display increased plasma levels of cholesterol oxidation products such as those enriched in oxidized low-density lipoprotein (oxLDL), a pro-inflammatory mediator. While it has been shown that inflammation precedes and exacerbates symptom severity in NPC1 disease, it is unclear whether oxLDL contributes to NPC1 disease progression. In this study, we investigated the effects of increasing anti-oxLDL IgM autoantibodies on systemic and neurological symptoms in an NPC1 disease mouse model. For this purpose, Npc1 nih mice were immunized with heat-inactivated S. pneumoniae, an immunogen which elicits an IgM autoantibody-mediated immune response against oxLDL. Npc1 nih mice injected with heat-inactivated pneumococci displayed an improved hepatic phenotype, including liver lipid accumulation and inflammation. In addition, regression of motor skills was delayed in immunized Npc1 nih . In line with these results, brain analyses showed an improved cerebellar phenotype and neuroinflammation in comparison with control-treated subjects. This study highlights the potential of the pneumococcal immunization as a novel therapeutical approach in NPC1 disease. Future research should investigate whether implementation of this therapy can improve life span and quality of life of NPC1 disease patients