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.

SMYD1a protects the heart from ischemic injury by regulating OPA1-mediated cristae remodeling and supercomplex formation.

SMYD1, a striated muscle-specific lysine methyltransferase, was originally shown to play a key role in embryonic cardiac development but more recently we demonstrated that loss of Smyd1 in the murine adult heart leads to cardiac hypertrophy and failure. However, the effects of SMYD1 overexpression in the heart and its molecular function in the cardiomyocyte in response to ischemic stress are unknown. In this study, we show that inducible, cardiomyocyte-specific overexpression of SMYD1a in mice protects the heart from ischemic injury as seen by a > 50% reduction in infarct size and decreased myocyte cell death. We also demonstrate that attenuated pathological remodeling is a result of enhanced mitochondrial respiration efficiency, which is driven by increased mitochondrial cristae formation and stabilization of respiratory chain supercomplexes within the cristae. These morphological changes occur concomitant with increased OPA1 expression, a known driver of cristae morphology and supercomplex formation. Together, these analyses identify OPA1 as a novel downstream target of SMYD1a whereby cardiomyocytes upregulate energy efficiency to dynamically adapt to the energy demands of the cell. In addition, these findings highlight a new epigenetic mechanism by which SMYD1a regulates mitochondrial energetics and functions to protect the heart from ischemic injury.

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.

Sirt1 overexpression attenuates Western-style diet-induced aortic stiffening in mice.

Increased arterial stiffness is a cardiovascular disease risk factor in the setting of advancing age and Western diet (WD) induced obesity. Increases in large artery stiffness, as measured by pulse wave velocity (PWV), occur within 8 weeks of WD feeding in mice. Sirtuin-1 (Sirt1), a NAD-dependent deacetylase, regulates cellular metabolic activity and activation of this protein has been associated with vasoprotection in aged mice. The aim of the study was to elucidate the effect of global Sirt1 overexpression (Sirttg ) on WD-induced arterial stiffening. Sirt1 overexpression did not influence PWV in normal chow (NC) fed mice. However, PWV was higher in wild-type (WT) mice (p < 0.04), but not in Sirttg mice, after 12 weeks of WD and this effect was independent of changes in blood pressure or the passive pressure diameter relation in the carotid artery. Overexpression of Sirt1 was associated with lower collagen and higher elastin mRNA expression in the aorta of WD fed mice (both p < 0.05). Although MMP2 and MMP3 mRNA were both upregulated in WT mice after WD (both p < 0.05), this effect was reversed in Sirttg mice compared to WT mice fed WD (both p < 0.05). Surprisingly, histologically assessed collagen and elastin quality were unchanged in the aortas of WT or Sirttg mice after WD. However, Sirttg mice were protected from WD-induced glucose intolerance, although there was no difference in insulin tolerance between groups. These findings demonstrate a vasoprotective effect of Sirt1 overexpression that limits the increase in arterial stiffness in response to consumption of a WD.

The Thermal Safety Profile of a New Bipolar Vessel Sealing Device for Thyroid Surgery.

INTRODUCTION: Bipolar electrocautery devices used to achieve intraoperative hemostasis carry risk of imparting thermal energy to adjacent tissue, leading to postoperative morbidity. The aim of this study was to compare a new vessel sealing device, the CoolSeal™ Reveal (Bolder Surgical, Louisville, Colorado), with an established industry standard device, the LigaSure™ Exact Dissector (Valleylab, Boulder, Colorado), to assess their safety and the extent to which they impart thermal damage to tissue during thyroid surgery. MATERIALS AND METHODS: Vascular bundles associated with the thyroid gland in anesthetized sheep were exposed and sealed with a single activation of each device and excised en bloc. Additionally, vascular structures of the sheep were also sealed 0, 1, or 2mm adjacent to the recurrent laryngeal nerve (RLN). Vascular and RLN samples were processed for histopathologic evaluation and assessed for extent of thermal injury, seal width, and coagulative changes. RESULTS: The mean thermal injury extent across all sample sizes and vessel types was significantly lower for the CoolSeal™ Reveal device (547.2 ± 27.9µm) compared to the LigaSure™ device (802.7± 48.6µm) (p<0.001). Seal widths were significantly smaller in samples sealed with the CoolSeal™ Reveal device (899.0 ± 14.9µm) than samples sealed with the LigaSure™ device (1645.3 ± 160.3µm) (p<0.001). CONCLUSION: The CoolSeal™ Reveal device demonstrates significantly lower thermal spread in vivo compared to the LigaSure™ Exact Dissector. These results indicate that the CoolSeal™ Reveal is an effective tool for sealing blood vessels and minimizing thermal damage to adjacent structures during delicate surgeries or in narrow surgical fields associated with the thyroid gland.

Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking.

Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.

Genome-wide DNA copy number analysis and targeted transcriptional analysis of canine histiocytic malignancies identifies diagnostic signatures and highlights disruption of spindle assembly complex.

Canine histiocytic malignancies (HM) are rare across the general dog population, but overrepresented in certain breeds, such as Bernese mountain dog and flat-coated retriever. Accurate diagnosis relies on immunohistochemical staining to rule out histologically similar cancers with different prognoses and treatment strategies (e.g., lymphoma and hemangiosarcoma). HM are generally treatment refractory with overall survival of less than 6 months. A lack of understanding regarding the mechanisms of disease development and progression hinders development of novel therapeutics. While the study of human tumors can benefit veterinary medicine, the rarity of the suggested orthologous disease (dendritic cell sarcoma) precludes this. This study aims to improve the understanding of underlying disease mechanisms using genome-wide DNA copy number and gene expression analysis of spontaneous HM across several dog breeds. Extensive DNA copy number disruption was evident, with losses of segments of chromosomes 16 and 31 detected in 93% and 72% of tumors, respectively. Droplet digital PCR (ddPCR) evaluation of these regions in numerous cancer specimens effectively discriminated HM from other common round cell tumors, including lymphoma and hemangiosarcoma, resulting in a novel, rapid diagnostic aid for veterinary medicine. Transcriptional analysis demonstrated disruption of the spindle assembly complex, which is linked to genomic instability and reduced therapeutic impact in humans. A key signature detected was up-regulation of Matrix Metalloproteinase 9 (MMP9), supported by an immunohistochemistry-based assessment of MMP9 protein levels. Since MMP9 has been linked with rapid metastasis and tumor aggression in humans, the data in this study offer a possible mechanism of aggression in HM.

The pro-atherogenic response to disturbed blood flow is increased by a western diet, but not by old age.

Atherogenic remodeling often occurs at arterial locations with disturbed blood flow (i.e., low or oscillatory) and both aging and western diet (WD) increase the likelihood for pro-atherogenic remodeling. However, it is unknown if old age and/or a WD modify the pro-atherogenic response to disturbed blood flow. We induced disturbed blood flow by partial carotid ligation (PCL) of the left carotid artery in young and old, normal chow (NC) or WD fed male B6D2F1 mice. Three weeks post-PCL, ligated carotid arteries had greater intima media thickness, neointima formation, and macrophage content compared with un-ligated arteries. WD led to greater remodeling and macrophage content in the ligated artery compared with NC mice, but these outcomes were similar between young and old mice. In contrast, nitrotyrosine content, a marker of oxidative stress, did not differ between WD and NC fed mice, but was greater in old compared with young mice in both ligated and un-ligated carotid arteries. In primary vascular smooth muscle cells, aging reduced proliferation, whereas conditioned media from fatty acid treated endothelial cells increased proliferation. Taken together, these findings suggest that the remodeling and pro-inflammatory response to disturbed blood flow is increased by WD, but is not increased by aging.

Dopamine Receptor D5 is a Modulator of Tumor Response to Dopamine Receptor D2 Antagonism.

PURPOSE: Dopamine receptor D2 (DRD2) is a G protein-coupled receptor antagonized by ONC201, an anticancer small molecule in clinical trials for high-grade gliomas and other malignancies. DRD5 is a dopamine receptor family member that opposes DRD2 signaling. We investigated the expression of these dopamine receptors in cancer and their influence on tumor cell sensitivity to ONC201. EXPERIMENTAL DESIGN: The Cancer Genome Atlas was used to determine DRD2/DRD5 expression broadly across human cancers. Cell viability assays were performed with ONC201 in >1,000 Genomic of Drug Sensitivity in Cancer and NCI60 cell lines. IHC staining of DRD2/DRD5 was performed on tissue microarrays and archival tumor tissues of glioblastoma patients treated with ONC201. Whole exome sequencing was performed in RKO cells with and without acquired ONC201 resistance. Wild-type and mutant DRD5 constructs were generated for overexpression studies. RESULTS: DRD2 overexpression broadly occurs across tumor types and is associated with a poor prognosis. Whole exome sequencing of cancer cells with acquired resistance to ONC201 revealed a de novo Q366R mutation in the DRD5 gene. Expression of Q366R DRD5 was sufficient to induce tumor cell apoptosis, consistent with a gain-of-function. DRD5 overexpression in glioblastoma cells enhanced DRD2/DRD5 heterodimers and DRD5 expression was inversely correlated with innate tumor cell sensitivity to ONC201. Investigation of archival tumor samples from patients with recurrent glioblastoma treated with ONC201 revealed that low DRD5 expression was associated with relatively superior clinical outcomes. CONCLUSIONS: These results implicate DRD5 as a negative regulator of DRD2 signaling and tumor sensitivity to ONC201 DRD2 antagonism.

Oral Transmucosal Detomidine Gel in New Zealand White Rabbits (Oryctolagus cuniculus).

Handling and restraining rabbits for routine procedures may be impossible without prior sedation, result in unnecessary stress or injury to the rabbit or handler, and increase experimental variability. Parenteral administration of sedatives can cause stress also, as well as localized pain and tissue damage, especially in fractious animals. Detomidine hydrochloride, an α2-adrenergic receptor agonist, is commercially available in an oral transmucosal (OTM) gel formulation that is FDA-approved for sedation and restraint in horses. This study investigated the efficacy and safety of detomidine gel as an alternative to injectable sedation in rabbits. Eight adult male New Zealand White rabbits each received 0.6, 1.2, or 1.8 mg/kg OTM detomidine gel. Physiologic parameters and sedation scores (SS) were assessed at 10-min intervals from before administration until 100 min afterward. Histopathology of cardiac tissue was scored through 12 d after dosing. Gel administration increased the SS in all rabbits, but none of the animals developed clinically effective sedation (SS of 10 or greater, based on 5 reflex responses on a 3- or 4-point scale). The SS did not differ among dosage groups, and the time-dose interaction was not statistically significant. Heart rate decreased rapidly in all rabbits, with no difference among dosage groups, and there was no effect of time or dosage on peripheral capillary oxygen saturation. Minimal to mild degenerative changes were seen in the myocardium of all treated rabbits, but myocyte necrosis, inflammation, fibrosis, and mural thrombi-reported previously in rabbits that had received parenteral detomidine-did not occur. OTM detomidine gel was safely and easily administered to rabbits, but the duration and level of sedation were unpredictable. The use of OTM detomidine as a sole agent to facilitate handling and restraint of rabbits does not offer advantages over existing parenteral regimens.

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.

Retrobulbar pigmented peripheral nerve sheath tumor in a dog.

A 1-year-old male castrated Pug was referred for unilateral exophthalmos unresponsive to oral antibiotic and anti-inflammatory therapy. Clinical findings included exophthalmos of the left eye with lateral strabismus, resistance to retropulsion, and an elevated nictitans. Hematologic and biochemical analyses were within normal limits. Findings following computed tomography (CT) of the head included an expansile retrobulbar soft tissue mass with bony lysis extending into the left nasal cavity and nasopharynx. Ultrasound-guided fine-needle aspirates and biopsy samples obtained via rhinoscopy were nondiagnostic. Palliative exenteration was elected; the patient was euthanized 13 weeks following exenteration due to development of neurologic signs and perceived poor quality of life. The histopathologic diagnosis was a malignant pigmented peripheral nerve sheath tumor.

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.

Aging compounds western diet-associated large artery endothelial dysfunction in mice: prevention by voluntary aerobic exercise.

We tested the hypothesis that aging will exacerbate the negative vascular consequences of exposure to a common physiological stressor, i.e., consumption of a "western" (high fat/high sucrose) diet (WD), by inducing superoxide-associated reductions in nitric oxide (NO) bioavailability, and that this would be prevented by voluntary aerobic exercise. Incremental stiffness and endothelium-dependent dilation (EDD) were measured in the carotid arteries of young (5.4±0.3 mo, N=20) and old (30.4±0.2 mo, N=19) male B6D2F1 mice fed normal chow (NC: 17% fat, 0% sucrose) or a western diet (40% fat, 19% sucrose) and housed in either standard cages or cages equipped with running wheels for 10-14 weeks. Incremental stiffness was higher in old NC (P<0.05) and both young (P<0.01) and old (P<0.01) WD fed mice compared with young NC mice, but WD did not further increase stiffness in the old mice. In cage control mice, maximal EDD was 17% lower in both NC fed old mice and young WD fed mice (P<0.05). Consumption of WD by old mice led to a further 20% reduction in maximal EDD (P<0.05). Incremental stiffness was 28% lower and maximal EDD was 38% greater in old WD fed mice with access to running wheels vs. old WD fed control mice (P<0.05) and not different from young NC fed controls. Wheel running also tended to improve maximal EDD (+9%, P=0.11), but not incremental stiffness in young WD fed mice. Ex vivo treatment with the superoxide scavenger TEMPOL and NO inhibitor l-NAME abolished these respective effects of age, WD and voluntary running on EDD. Ingestion of a WD induces similar degrees of endothelial dysfunction in old and young adult B6D2F1 mice, and these effects are mediated by a superoxide-dependent impairment of NO bioavailability. However, the combination of old age and WD, a common occurrence in our aging society, results in a marked, additive reduction in endothelial function. Importantly, regular voluntary aerobic exercise reduces arterial stiffness and protects against the adverse influence of WD on endothelial function in old animals by preventing superoxide suppression of NO. These findings may have important implications for arterial aging and the prevention of age-associated cardiovascular diseases.

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.

SIRT-1 and vascular endothelial dysfunction with ageing in mice and humans.

We tested the hypothesis that reductions in the cellular deacetylase, sirtuin-1 (SIRT-1), contribute to vascular endothelial dysfunction with ageing via modulation of endothelial nitric oxide synthase (eNOS) acetylation/activation-associated nitric oxide (NO) production. In older (30 months, n = 14) vs. young (5-7 months, n = 16) B6D2F1 mice, aortic protein expression of SIRT-1 and eNOS phosphorylated at serine 1177 were lower (both P < 0.05), and acetylated eNOS was 6-fold higher (P < 0.05), whereas total eNOS did not differ (P = 0.65). Acetylcholine (ACh)-induced peak endothelium-dependent dilatation (EDD) was lower in isolated femoral arteries with ageing (P < 0.001). Incubation with sirtinol, a SIRT-1 inhibitor, reduced EDD in both young and older mice, abolishing age-related differences, whereas co-administration with l-NAME, an eNOS inhibitor, further reduced EDD similarly in both groups. Endothelium-independent dilatation to sodium nitroprusside (EID), was not altered by age or sirtinol treatment. In older (64 ± 1 years, n = 22) vs. young (25 ± 1 years, n = 16) healthy humans, ACh-induced forearm EDD was impaired (P = 0.01) and SIRT-1 protein expression was 37% lower in endothelial cells obtained from the brachial artery (P < 0.05), whereas EID did not differ. In the overall group, EDD was positively related to endothelial cell SIRT-1 protein expression (r = 0.44, P < 0.01). Reductions in SIRT-1 may play an important role in vascular endothelial dysfunction with ageing. SIRT-1 may be a key therapeutic target to treat arterial ageing.

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.