Heterozygosity for ADP-ribosylation factor 6 suppresses the burden and severity of atherosclerosis.

Atherosclerosis is the root cause of major cardiovascular diseases (CVD) such as myocardial infarction and stroke. ADP-ribosylation factor 6 (Arf6) is a ubiquitously expressed GTPase known to be involved in inflammation, vascular permeability and is sensitive to changes in shear stress. Here, using atheroprone, ApoE-/- mice, with a single allele deletion of Arf6 (HET) or wildtype Arf6 (WT), we demonstrate that reduction in Arf6 attenuates atherosclerotic plaque burden and severity. We found that plaque burden in the descending aorta was lower in HET compared to WT mice (p˂0.001) after the consumption of an atherogenic Paigen diet for 5 weeks. Likewise, luminal occlusion, necrotic core size, plaque grade, elastic lamina breaks, and matrix deposition were lower in the aortic root atheromas of HET compared to WT mice (all p≤0.05). We also induced advanced human-like complex atherosclerotic plaque in the left carotid artery using partial carotid ligation surgery and found that atheroma area, plaque grade, intimal necrosis, intraplaque hemorrhage, thrombosis, and calcification were lower in HET compared to WT mice (all p≤0.04). Our findings suggest that the atheroprotection afforded by Arf6 heterozygosity may result from reduced immune cell migration (all p≤0.005) as well as endothelial and vascular smooth muscle cell proliferation (both p≤0.001) but independent of changes in circulating lipids (all p≥0.40). These findings demonstrate a critical role for Arf6 in the development and severity of atherosclerosis and suggest that Arf6 inhibition can be explored as a novel therapeutic strategy for the treatment of atherosclerotic CVD.

Advancing age increases the size and severity of spontaneous atheromas in mouse models of atherosclerosis.

Using multiple mouse models, we explored the impact of aging on the size and severity of atherosclerotic lesions. In young, middle-aged and old apolipoprotein E knockout mice (ApoE-/-) fed an atherogenic diet (AD) for 3-8 weeks, plaque/atheroma formation in the descending aorta and aortic root, and atheroma development in the carotid in response to partial carotid ligation (PCL) were assessed. Total and LDL cholesterol, and triglycerides were higher in old compared to both other age groups, regardless of AD duration. Aortic plaque burden increased with AD duration in all ages. The size and plaque morphology grade of aortic root atheromas was higher with age; however, there was no effect of age on the size or severity of carotid atheromas after PCL. We additionally induced hyperlipidemia in young and old C57BL/6 mice by adeno-associated virus mediated upregulation of LDL receptor regulator, Pcsk9, and 5 weeks of AD. Despite lower cholesterol in old compared to young Pcsk9 mice, there was a greater size and severity of aortic root atheromas in old mice. However, like the ApoE-/- mice, there was no effect of age on size or severity of PCL-induced carotid artery atheromas in Pcsk9 mice. Together, these results suggest that aging increases the size and severity of spontaneous aortic atheromas.

Extreme lymphocytosis with myelomonocytic morphology in a horse with diffuse large B-cell lymphoma.

An 11-year-old, 443-kg Haflinger mare was presented to the North Carolina State University Veterinary Teaching Hospital with a 2-week history of lethargy and a 3-day duration of anorexia, pyrexia, tachycardia, and ventral edema. Severe pitting edema, peripheral lymphadenopathy, and a caudal abdominal mass were noted on physical examination. An extreme leukocytosis (154.3 × 103 /µL) and microscopic hematologic findings suggestive of myelomonocytic leukemia were observed. Serum protein electrophoresis revealed a monoclonal gammopathy and urine protein electrophoresis revealed a monoclonal light chain proteinuria. Necropsy and histopathology confirmed widespread neoplastic infiltration in many organs with a heterogenous population of cells; there was no apparent evidence of bone marrow involvement. Immunohistochemistry confirmed presence of a majority of B cells with a limited antigen expression, admixed with a lower number of T cells. Molecular clonality analysis of IgH2, IgH3, and kappa-deleting element (KDE, B cell) on whole blood and KDE on infiltrated tissues revealed clonal rearrangements, and the KDE intron clones that amplified in blood and in infiltrated tissue were identical. In contrast, the clonality analysis of T-cell receptor γ revealed no clonality on blood cells and infiltrated tissues. In conjunction with the histopathologic changes, the lesion was interpreted to be composed of neoplastic B cells with a reactive T-cell population. Polymerase chain reaction testing for equine herpes virus 5 was negative. The final diagnosis was diffuse large B-cell lymphoma with a marked hematogenous component.

Excess centrosomes disrupt endothelial cell migration via centrosome scattering.

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome-MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.

Responses of brown adipose tissue to diet-induced obesity, exercise, dietary restriction and ephedrine treatment.

Drug-induced weight loss in humans has been associated with undesirable side effects not present in weight loss from lifestyle interventions (caloric restriction or exercise). To investigate the mechanistic differences of weight loss by drug-induced and lifestyle interventions, we examined the gene expression (mRNA) in brown adipose tissue (BAT) and conducted histopathologic assessments in diet-induced obese (DIO) mice given ephedrine (18 mg/kg/day orally), treadmill exercise (10 m/min, 1-h/day), and dietary restriction (DR: 26% dietary restriction) for 7 days. Exercise and DR mice lost more body weight than controls and both ephedrine and exercise reduced percent body fat. All treatments reduced BAT and liver lipid accumulation (i.e., cytoplasmic lipids in brown adipocytes and hepatocytes) and increased oxygen consumption (VO2 ml/kg/h) compared with controls. Mitochondrial biogenesis/function-related genes (TFAM, NRF1 and GABPA) were up-regulated in the BAT of all groups. UCP-1 was up-regulated in exercise and ephedrine groups, whereas MFSD2A was up-regulated in ephedrine and DR groups. PGC-1α up-regulation was observed in exercise and DR groups but not in ephedrine group. In all experimental groups, except for ephedrine, fatty acid transport and metabolism genes were up-regulated, but the magnitude of change was higher in the DR group. PRKAA1 was up-regulated in all groups but not significantly in the ephedrine group. ADRß3 was slightly up-regulated in the DR group only, whereas ESRRA remained unchanged in all groups. Although our data suggest a common pathway of BAT activation elicited by ephedrine treatment, exercise or DR, mRNA changes were indicative of additional nutrient-sensing pathways in exercise and DR.

Sustained activation of AMPK ameliorates age-associated vascular endothelial dysfunction via a nitric oxide-independent mechanism.

Exercise restores endothelium-dependent dilation (EDD) in old mice by reducing oxidative stress and increasing nitric oxide (NO) bioavailability. Adenosine monophosphate protein kinase (AMPK) activation mimics some effects of exercise. Old (28-30 months) B6D2F1 mice had reduced arterial AMPK expression and superoxide-mediated suppression of EDD vs. young (3-6 months) controls. Pharmacological activation of AMPK by aminoimidazole carboxamide ribonucleotide (AICAR) for 2 weeks increased arterial AMPK and reversed this superoxide-induced impairment of EDD. The improvement in EDD was independent of NO or prostaglandin signaling, suggesting enhanced endothelium-dependent hyperpolarizing factor-related dilation. AMPK activation may represent a novel therapy for treating age-associated vascular dysfunction.

Aerobic exercise reverses arterial inflammation with aging in mice.

We tested the hypothesis that regular aerobic exercise reverses arterial inflammation with aging. When compared with young controls (6.2 ± 0.4 mo; n = 7), old (31.3 ± 0.5 mo; n = 11) male B6D2F1 cage-restricted mice demonstrated increased arterial activation of the proinflammatory transcription factor NF-κB, as indicated by greater aortic phosphorylation of both the inhibitor of NF-κB kinase (IKK) and the p65 subunit of NF-κB (both P < 0.05). Similarly, aortic expression of the proinflammatory cytokines IL-1 and IL-6, IFN-γ, and TNF-α were greater in the old mice (all P < 0.05). Macrophage and T lymphocyte abundance was unchanged with age in the aortic intima and media but was markedly increased in the adventitia and perivascular fat tissue of old mice (all P < 0.05). This proinflammatory arterial phenotype with aging was associated with vascular dysfunction, as reflected by impaired nitric oxide-mediated endothelium-dependent dilation. Voluntary wheel running (10-14 wk) normalized aortic IKK-NF-κB activation, cytokine expression, adventitial and perivascular macrophage infiltration, and vascular function in old mice (32.4 ± 0.3 mo; n = 8) while having no consistent effects in young mice. Short-term voluntary wheel running started late in life reverses arterial inflammation with aging in mice possibly via outside-in actions. These anti-inflammatory effects may play an important role in the amelioration of age-associated vascular dysfunction by regular aerobic exercise.

Salicylate treatment improves age-associated vascular endothelial dysfunction: potential role of nuclear factor kappaB and forkhead Box O phosphorylation.

We hypothesized that I kappa B kinase (IKK)-mediated nuclear factor kappa B and forkhead BoxO3a phosphorylation will be associated with age-related endothelial dysfunction. Endothelium-dependent dilation and aortic protein expression/phosphorylation were determined in young and old male B6D2F1 mice and old mice treated with the IKK inhibitor, salicylate. IKK activation was greater in old mice and was associated with greater nitrotyrosine and cytokines. Endothelium-dependent dilation, nitric oxide (NO), and endothelial NO synthase phosphorylation were lower in old mice. Endothelium-dependent dilation and NO bioavailability were restored by a superoxide dismutase mimetic. Nuclear factor kappa B and forkhead BoxO3a phosphorylation were greater in old and were associated with increased expression/activity of nicotinamide adenine dinucleotide phosphate oxidase and lower manganese superoxide dismutase expression. Salicylate lowered IKK phosphorylation and reversed age-associated changes in nitrotyrosine, endothelium-dependent dilation, NO bioavailability, endothelial NO synthase, nuclear factor kappa B and forkhead BoxO3a phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase, and manganese superoxide dismutase. Increased activation of IKK with advancing age stimulates nuclear factor kappa B and inactivates forkhead BoxO3a. This altered transcription factor activation contributes to a pro-inflammatory/pro-oxidative arterial phenotype that is characterized by increased cytokines and nicotinamide adenine dinucleotide phosphate oxidase and decreased manganese superoxide dismutase leading to oxidative stress-mediated endothelial dysfunction.