Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.

Nitrones have the general chemical formula X-CH=NO-Y. They were first used to trap free radicals in chemical systems and then subsequently in biochemical systems. More recently several nitrones including PBN (α-phenyl-tert-butylnitrone) have been shown to have potent biological activity in many experimental animal models. Many diseases of aging including stroke, cancer development, Parkinson’s disease and Alzheimer’s disease are known to have enhanced levels of free radicals and oxidative stress. Some derivatives of PBN are significantly more potent than PBN and have undergone extensive commercial development in stroke. Recent research has shown that PBN-related nitrones also have anti-cancer activity in several experimental cancer models and have potential as therapeutics in some cancers. Also in recent observations nitrones have been shown to act synergistically in combination with antioxidants in the prevention of acute acoustic noise induced hearing loss. The mechanistic basis of the potent biological activity of PBN-related nitrones is not known. Even though PBN-related nitrones do decrease oxidative stress and oxidative damage, their potent biological anti-inflammatory activity and their ability to alter cellular signaling processes can not readily be explained by conventional notions of free radical trapping biochemistry. This review is focused on our observations and others where the use of selected nitrones as novel therapeutics have been evaluated in experimental models in the context of free radical biochemical and cellular processes considered important in pathologic conditions and age-related diseases.

There is increasing evidence that vascular risk factors, including aging, hypertension, diabetes mellitus, and obesity, promote cognitive impairment; however, the underlying mechanisms remain obscure. Cerebral blood flow (CBF) is adjusted to neuronal activity via neurovascular coupling (NVC) and this mechanism is known to be impaired in the aforementioned pathophysiologic conditions. To establish a direct relationship between impaired NVC and cognitive decline, we induced neurovascular uncoupling pharmacologically in mice by inhibiting the synthesis of vasodilator mediators involved in NVC. Treatment of mice with the epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MSPPOH), the NO synthase inhibitor L-NGNitroarginine methyl ester (L-NAME), and the COX inhibitor indomethacin decreased NVC by over 60% mimicking the aging phenotype, which was associated with significantly impaired spatial working memory (Y-maze), recognition memory (Novel object recognition), and impairment in motor coordination (Rotarod). Blood pressure (tail cuff) and basal cerebral perfusion (arterial spin labeling perfusion MRI) were unaffected. Thus, selective experimental disruption of NVC is associated with significant impairment of cognitive and sensorimotor function, recapitulating neurologic symptoms and signs observed in brain aging and pathophysiologic conditions associated with accelerated cerebromicrovascular aging.

We report a general method for preparing nanoparticle clusters (NPCs) in an oil-in-water emulsion system mediated by cetyl trimethylammonium bromide (CTAB) where previously, only individual nanoparticles were obtained. NPCs of magnetic, metallic and semiconductor nanoparticles have been prepared to demonstrate the generality of the method. The NPCs were spherical and composed of densely packed individual nanoparticles. The number density of nanoparticles in the oil phase was found to be critical for the formation, morphology and yield of NPCs. The method developed here is scalable and can produce NPCs in nearly 100% yield at a concentration of 5 mg/ml in water which is approximately 5 times higher than the highest value reported in literature. The surface chemistry of NPCs can also be controlled by replacing CTAB with polymers containing different functional groups via a similar procedure. The reproducible production of NPCs with well defined shapes has allowed us to compare the properties of individual and clustered iron oxide nanoparticles including magnetization, magnetic moments and contrast enhancement in magnetic resonance imaging (MRI). We found that due to their collective properties, NPCs are more responsive to an external magnetic field and can potentially serve as better contrast enhancement agents than individually dispersed magnetic NPs in MRI.

Lipids are essential components of the nervous system. However, the functions of very long-chain fatty acids (VLC-FA; ≥ 28 carbons) in the brain are unknown. The enzyme ELOngation of Very Long-chain fatty acids-4 (ELOVL4) catalyzes the rate-limiting step in the biosynthesis of VLC-FA (Agbaga et al., Proc Natl Acad Sci USA 105(35): 12843–12848, 2008; Logan et al., J Lipid Res 55(4): 698–708, 2014), which we identified in the brain as saturated fatty acids (VLC-SFA). Homozygous mutations in ELOVL4 cause severe neuropathology in humans (Ozaki et al., JAMA Neurol 72(7): 797–805, 2015; Mir et al., BMC Med Genet 15: 25, 2014; Cadieux-Dion et al., JAMA Neurol 71(4): 470–475, 2014; Bourassa et al., JAMA Neurol 72(8): 942–943, 2015; Aldahmesh et al., Am J Hum Genet 89(6): 745–750, 2011) and are post-natal lethal in mice (Cameron et al., Int J Biol Sci 3(2): 111–119, 2007; Li et al., Int J Biol Sci 3(2): 120–128, 2007; McMahon et al., Molecular Vision 13: 258–272, 2007; Vasireddy et al., Hum Mol Genet 16(5): 471–482, 2007) from dehydration due to loss of VLC-SFA that comprise the skin permeability barrier. Double transgenic mice with homozygous knock-in of the Stargardt-like macular dystrophy (STDG3; 797-801_AACTT) mutation of Elovl4 with skin-specific rescue of wild-type Elovl4 expression (S + Elovl4 mut/mut mice) develop seizures by P19 and die by P21. Electrophysiological analyses of hippocampal slices showed aberrant epileptogenic activity in S + Elovl4 mut/mut mice. FM1-43 dye release studies showed that synapses made by cultured hippocampal neurons from S + Elovl4 mut/mut mice exhibited accelerated synaptic release kinetics. Supplementation of VLC-SFA to cultured hippocampal neurons from mutant mice rescued defective synaptic release to wild-type rates. Together, these studies establish a critical, novel role for ELOVL4 and its VLC-SFA products in regulating synaptic release kinetics and epileptogenesis. Future studies aimed at understanding the molecular mechanisms by which VLC-SFA regulate synaptic function may provide new targets for improved seizure therapies.Electronic supplementary materialThe online version of this article (10.1007/s12035-017-0824-8) contains supplementary material, which is available to authorized users.

Objective To test the hypothesis that high-fat (HF) diet-induced obesity increases pro-inflammatory cytokine expression, macrophage infiltration and M1 polarization in the infrapatellar fat pad (IFP) prior to knee cartilage degeneration. Methods We characterized the effect of HF feeding on knee OA pathology, body adiposity, and glucose intolerance in male C57BL/6J mice and identified a diet duration that induces metabolic dysfunction prior to cartilage degeneration. Magnetic resonance imaging and histomorphology were used to quantify changes in epididymal, subcutaneous, and infrapatellar fat pads and adipocyte sizes. Finally, we utilized targeted gene expression and protein arrays, immunohistochemsitry, and flow cytometry to quantify differences in fat pad inflammatory markers and immune cell populations. Results 20 weeks of HF diet treatment induced marked obesity, glucose intolerance, and early osteoarthritis (OA), including osteophytes and cartilage tidemark duplication. This duration of HF feeding increased IFP volume. However, it did not increase IFP inflammation, macrophage infiltration, or M1 macrophage polarization as observed in epididymal fat. Furthermore, leptin protein was reduced. This protection from obesity-induced inflammation corresponded with increased IFP fibrosis and the absence of adipocyte hypertrophy. Conclusion The IFP does not recapitulate classical abdominal adipose tissue inflammation during the early stages of knee OA in a high-fat diet-induced model of obesity. Consequently, these findings do not support the hypothesis that IFP inflammation is an initiating factor of obesity-induced knee OA. Furthermore, the pro-fibrotic and anti-hypertrophic responses of IFP adipocytes to high-fat feeding suggest that intra-articular adipocytes are subject to distinct spatial-temporal structural and metabolic regulation among fat pads.

Purpose: To evaluate the added value of non-contrastenhanced MR angiography (MRA) to conventional MR imaging for a detailed characterization of different rodent glioma models. Materials and Methods:Intracerebral tumor cell implantation and chemical induction methods were implemented to obtain rat C6, 9L/LacZ, F98, RG2, and ethyl-nitrosourea (ENU) -induced glioma models, a human U87 MG tumor model as well as a mouse GL261 glioma model. MR assessments were regularly conducted on a 7 Tesla Bruker BioSpin system. The tumor border sharpness and growth characteristics of each glioma model were assessed from T 2 -weighted images. Neovascularization and vascular alterations inherent to each model were characterized by assessing absolute blood volumes, vessel density, length, and diameter using Mathematica and Amira software.Results: The 9L/LacZ and ENU gliomas both presented flaws that hinder their use as reliable brain tumor models. C6 gliomas were slightly invasive and induced moderate vascular alterations, whereas GL261 tumors dramatically altered the brain vessels in the glioma region. F98, RG2, and U87 are infiltrative models that produced dramatic vascular alterations.Conclusion: MRI and MRA provided crucial in vivo information to identify a distinctive ''fingerprint'' for each of our seven rodent glioma models.

Purpose: To demonstrate that OKN007, a disulfonyl derivative of phenyl-tert-butyl nitrone (PBN), has anti-glioma activity in the clinically relevant C6 rat glioma model using multi-parametric magnetic resonance imaging.Materials and Methods: Twenty-one rats were intracerebrally implanted with C6 cells and administered OKN007 or kept as controls. Animals were monitored with MRI at 7 Tesla (T), using morphologic, diffusion-weighted and perfusion imaging, followed by histology and Western blots of angiogenesis and inflammatory markers.Results: OKN007 was found to decrease tumor volumes and increase survival. The glioma tissues of OKN007-treated rats were found to have longitudinal apparent diffusion coefficients (ADC z ) of 0.76 6 0.06 Â 10 À3 mm 2 /s, similar to the contralateral tissue and significantly smaller than untreated gliomas (0.97 6 0.13 Â 10 À3 mm 2 /s). They had higher perfusion rates (66 6 4 mL/100 gÁmin) than untreated gliomas (26 6 7 mL/100 gÁmin). All examined molecular markers were decreased in OKN007-treated rat gliomas, compared with elevated levels in untreated rats.Conclusion: MRI assessment was successfully used to monitor a decrease in tumor growth, and corresponding alterations in ADC and perfusion rates in rat C6 gliomas treated with the anti-glioma agent, OKN007.