Kenneth W. Fishbein

Resveratrol improves health and survival of mice on a high-calorie diet.

Resveratrol (3,5,4′-trihydroxystilbene) extends the lifespan of diverse species including Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In these organisms, lifespan extension is dependent on Sir2, a conserved deacetylase proposed to underlie the beneficial effects of caloric restriction. Here we show that resveratrol shifts the physiology of middle-aged mice on a high-calorie diet towards that of mice on a standard diet and significantly increases their survival. Resveratrol produces changes associated with longer lifespan, including increased insulin sensitivity, reduced insulin-like growth factor-1 (IGF-I) levels, increased AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) activity, increased mitochondrial number, and improved motor function. Parametric analysis of gene set enrichment revealed that resveratrol opposed the effects of the high-calorie diet in 144 out of 153 significantly altered pathways. These data show that improving general health in mammals using small molecules is an attainable goal, and point to new approaches for treating obesity-related disorders and diseases of ageing.

Glycogen synthase kinase-3β mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3β (GSK-3β). Alternatively, receptor tyrosine kinase or certain G protein–coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3β, via protein kinase B/Akt and mTOR/p70s6k pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3β on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.

Adenovirus-Mediated VEGF121 Gene Transfer Stimulates Angiogenesis in Normoperfused Skeletal Muscle and Preserves Tissue Perfusion After Induction of Ischemia

BACKGROUND Administration of angiogenic factors stimulates neovascularization in ischemic tissues. However, there is no evidence that angiogenesis can be induced in normoperfused skeletal muscles. We tested the hypothesis that adenovirus-mediated intramuscular (IM) gene transfer of the 121-amino-acid form of vascular endothelial growth factor (AdCMV.VEGF(121)) could stimulate neovascularization in nonischemic skeletal muscle and consequently attenuate the hemodynamic deficit secondary to surgically induced ischemia. METHODS AND RESULTS Rabbits and rats received IM injections of AdCMV.VEGF(121), AdCMV.Null, or saline in the thigh, 4 weeks (rabbits) or 2 weeks (rats) before femoral artery removal in the injected limb. In unoperated rats, at the site of injection of AdCMV.VEGF(121), we found 96% and 29% increases in length density of arterioles and capillaries, respectively. Increased tissue perfusion (TP) to the ischemic limb in the AdCMV.VEGF(121) group was documented, as early as day 1 after surgery, by improved blood flow to the ischemic gastrocnemius muscle measured by radioactive microspheres (AdCMV.VEGF(121)=5.69+/-0.40, AdCMV.Null=2.97+/-0.50, and saline=2.78+/-0.43 mL x min(-1) x 100 g(-1), P<0.001), more angiographically recognizable collateral vessels (angioscore) (AdCMV. VEGF(121)=50.58+/-1.48, AdCMV.Null=29.08+/-4.22, saline=11.83+/-1.90, P<0.0001), and improvement of the bioenergetic reserve of the gastrocnemius muscle as assessed by (31)P NMR spectroscopy. Follow-up studies showed that superior TP to the ischemic limb in the AdCMV.VEGF(121) group persisted until it was equalized by spontaneous collateral vessel development in untreated animals. CONCLUSIONS IM administration of AdCMV.VEGF(121) stimulates angiogenesis in normoperfused skeletal muscles, and the newly formed vessels preserve TP after induction of ischemia.

Multicomponent T2 relaxation analysis in cartilage.

MR techniques are sensitive to the early stages of osteoarthritis, characterized by disruption of collagen and loss of proteoglycan (PG), but are of limited specificity. Here, water compartments in normal and trypsin‐degraded bovine nasal cartilage were identified using a nonnegative least squares multiexponential analysis of T2 relaxation. Three components were detected: T2,1 = 2.3 ms, T2,2 = 25.2 ms, and T2,3 = 96.3 ms, with fractions w1 = 6.2%, w2 = 14.5%, and w3 = 79.3%, respectively. Trypsinization resulted in increased (P < 0.01) values of T2,2 = 64.2 ms and T2,3 = 149.4 ms, supporting their assignment to water compartments that are bound and loosely associated with PG, respectively. The T2 of the rapidly relaxing component was not altered by digestion, supporting assignment to relatively immobile collagen‐bound water. Relaxation data were simulated for a range of TE, number of echoes, and SNR to guide selection of acquisition parameters and assess the accuracy and precision of experimental results. Based on this, the expected experimental accuracy of measured T2s and associated weights was within 2% and 4% respectively, with precision within 1% and 3%. These results demonstrate the potential of multiexponential T2 analysis to increase the specificity of MR characterization of cartilage. Magn Reson Med, 2009.

Magnetic Resonance Imaging of Chondrocytes Labeled with Superparamagnetic Iron Oxide Nanoparticles in Tissue-Engineered Cartilage

The distribution of cells within tissue-engineered constructs is difficult to study through nondestructive means, such as would be required after implantation. However, cell labeling with iron-containing particles may prove to be a useful approach to this problem, because regions containing such labeled cells have been shown to be readily detectable using magnetic resonance imaging (MRI). In this study, we used the Food and Drug Administration-approved superparamagnetic iron oxide (SPIO) contrast agent Feridex in combination with transfection agents to label chondrocytes and visualize them with MRI in two different tissue-engineered cartilage constructs. Correspondence between labeled cell spatial location as determined using MRI and histology was established. The SPIO-labeling process was found not to affect the phenotype or viability of the chondrocytes or the production of major cartilage matrix constituents. We believe that this method of visualizing and tracking chondrocytes may be useful in the further development of tissue engineered cartilage therapeutics.

Effects of formalin fixation and collagen cross-linking on T2 and magnetization transfer in bovine nasal cartilage.

Endogenous collagen cross‐links influence cartilage biomechanical properties and resistance to degradation. Formalin fixation modifies collagen residues and forms new cross‐links in a dose‐dependent manner. We tested the hypothesis that magnetization transfer (MT) effects and T2 depend on collagen cross‐linking in cartilage. These parameters were measured in bovine nasal cartilage (BNC) prior to fixation, after 9 weeks of immersion in formalin solutions ranging in concentration from 0% to 10%, and after NaBH3CN reduction and washing. T2 decreased by 59.4% ± 1.1% upon fixation in 10% formalin, and was 32.2% ± 5.2% shorter than initial values after washing. The apparent MT rate increased 25.9% ± 3.7% and 52.8% ± 7.1% over baseline under these conditions. Biochemical assays showed no significant differences in water, proteoglycan, natural cross‐link, or collagen content between the 0% and 10% formalin‐treated samples, while amino acid analysis demonstrated losses in (hydroxy)lysine and tyrosine, and new peaks consistent with methylene cross‐links in fixed samples only. We conclude that formalin fixation of cartilage results in significant decreases in T2 and increases in MT parameters that persist after removal of unreacted formaldehyde. The collagen cross‐links thus created are associated with large changes in MT and T2, indicating that interpretation of T2 and MT values in terms of cartilage macromolecular content must be made with caution. Magn Reson Med 57:1000–1011, 2007. Published 2007 Wiley‐Liss, Inc.

Adiposity induces lethal cytokine storm after systemic administration of stimulatory immunotherapy regimens in aged mice

William Murphy’s group at UC Davis previously found that systemic administration of stimulatory immunotherapy (IT) in aged mice resulted in the rapid induction of cytokine storm culminating in multi-organ pathology and rapid lethality. They now show that in addition to age, increased body fat is critical to this adverse reaction, as aged calorie-restricted mice demonstrate protection from IT-induced toxicity. In contrast, young obese mice succumb to cytokine storm, multi-organ pathology, and lethality after systemic IT administration.

Automated quantification of muscle and fat in the thigh from water‐, fat‐, and nonsuppressed MR images

To introduce and validate an unsupervised muscle and fat quantification algorithm based on joint analysis of water‐suppressed (WS), fat‐suppressed (FS), and water and fat (nonsuppressed) volumetric magnetic resonance imaging (MRI) of the mid‐thigh region.

Analysis of Mitochondrial 3D-Deformation in Cardiomyocytes during Active Contraction Reveals Passive Structural Anisotropy of Orthogonal Short Axes

The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the isolated cell in an elongated cylindrical shape with an elliptical cross-section, even during contraction-relaxation cycles. Cardiomyocyte mitochondria are micron-sized, fluid-filled passive spheres distributed throughout the cell in a crystal-like lattice, arranged in pairs sandwiched between the sarcomere contractile machinery, both longitudinally and radially. Their shape represents the extant 3-dimensional (3D) force-balance. We developed a novel method to examine mitochondrial 3D-deformation in response to contraction and relaxation to understand how dynamic forces are balanced inside cardiomyocytes. The variation in transmitted light intensity induced by the periodic lattice of myofilaments alternating with mitochondrial rows can be analyzed by Fourier transformation along a given cardiomyocyte axis to measure mitochondrial deformation along that axis. This technique enables precise detection of changes in dimension of ∼1% in ∼1 µm (long-axis) structures with 8 ms time-resolution. During active contraction (1 Hz stimulation), mitochondria deform along the length- and width-axes of the cell with similar deformation kinetics in both sarcomere and mitochondrial structures. However, significant deformation anisotropy (without hysteresis) was observed between the orthogonal short-axes (i.e., width and depth) of mitochondria during electrical stimulation. The same degree of deformation anisotropy was also found between the myocyte orthogonal short-axes during electrical stimulation. Therefore, the deformation of the mitochondria reflects the overall deformation of the cell, and the apparent stiffness and stress/strain characteristics of the cytoskeleton differ appreciably between the two cardiomyocyte orthogonal short-axes. This method may be applied to obtaining a better understanding of the dynamic force-balance inside cardiomyocytes and of changes in the spatial stiffness characteristics of the cytoskeleton that may accompany aging or pathological conditions.

Noninvasive Assessment of Glycosaminoglycan Production in Injectable Tissue-Engineered Cartilage Constructs Using Magnetic Resonance Imaging

The glycosaminoglycan (GAG) content of engineered cartilage is a determinant of biochemical and mechanical quality. The ability to measure the degree to which GAG content is maintained or increases in an implant is therefore of importance in cartilage repair procedures. The gadolinium exclusion magnetic resonance imaging (MRI) method for estimating matrix fixed charge density (FCD) is ideally suited to this. One promising approach to cartilage repair is use of seeded injectable hydrogels. Accordingly, we assess the reliability of measuring GAG content in such a system ex vivo using MRI. Samples of the photopolymerizable hydrogel, poly(ethylene oxide) diacrylate, were seeded with bovine chondrocytes (approximately 2.4 million cells/sample). The FCD of the constructs was determined using MRI after 9, 16, 29, 36, 43, and 50 days of incubation. Values were correlated with the results of biochemical determination of GAG from the same samples. FCD and GAG were found to be statistically significantly correlated (R2 = 0.91, p < 0.01). We conclude that MRI-derived FCD measurements of FCD in injectable hydrogels reflect tissue GAG content and that this methodology therefore has potential for in vivo monitoring of such constructs.