Faculty Bibliography

BACKGROUND: Oxidative stress is an important pathophysiologic feature in chronic heart failure (CHF) and may in part result from the inability to counteract acute surges of circulating oxidant products. Oxidized low-density lipoprotein (oxLDL) is an emerging prognostic marker in CHF. Accordingly, we investigated the effect of exercise-induced oxidative stress on circulating levels of oxLDL and its association with clinical outcomes in CHF. METHODS AND RESULTS: Plasma levels of oxLDL and low-density lipoprotein cholesterol (LDL-c) were measured at rest and after maximal exercise in 48 subjects with CHF and 12 healthy controls. Subjects with CHF had a higher baseline oxLDL (77.7 +/- 3.2 U/L vs 57.9 +/- 5.0 U/L, P = .01) and a higher baseline oxLDL/LDL-c ratio (0.87 +/- 0.04 vs 0.49 +/- 0.04, P < or = .001). Exercise induced an increase in oxLDL in subjects with CHF (77.7 +/- 3.2 U/L to 85.3 +/- 3.0 U/L, P < or = .001) but not in controls (57.9 +/- 5.0 to 61.4 +/- 5.5, P = .17). In 39 subjects for whom follow-up data were available, an increase in oxLDL of more than 11.0 U/L was associated with an increased risk to meet a combined end point of death and need for ventricular assist device or heart transplant during a 19-month follow-up period (hazard ratio 8.6; 95% confidence interval 1.0-73.8, P = .05); this remained significant when adjusted for peak oxygen consumption, left ventricular ejection fraction, New York Heart Association class, sex, and age (hazard ratio 46.6, 95% confidence interval 1.5-1438.1, P = .02). CONCLUSION: Plasma oxLDL and the oxLDL/LDL-c ratio are elevated in subjects with CHF. Whether assessment of oxLDL during maximal exercise allows early identification of subjects at highest risk for adverse outcomes should be systematically investigated.

The vulnerability or destabilization of atherosclerotic plaques has been directly linked to plaque composition. Imaging modalities, such as magnetic resonance (MR) imaging, that allow for evaluation of plaque composition at a cellular and molecular level, could further improve the detection of vulnerable plaque and may allow for monitoring the efficacy of antiatherosclerotic therapies. In this review we focus on MR imaging strategies for the detection and evaluation of atherosclerotic plaques and their composition. We highlight recent advancements in the development of MR pulse sequences, computer image analysis, and the use of commercially available MR contrast agents, such as gadopentic acid (Gd-DTPA), for plaque characterization. We also discuss molecular imaging strategies that are currently being used to design specific imaging probes targeted to biochemical and cellular markers of atherosclerotic plaque vulnerability.

In higher plants, cellulose is synthesized at the plasma membrane by the cellulose synthase (CESA) complex. The catalytic core of the complex is believed to be composed of three types of CESA subunits. Indirect evidence suggests that the complex associated with primary wall cellulose deposition consists of CESA1, -3, and -6 in Arabidopsis thaliana. However, phenotypes associated with mutations in two of these genes, CESA1 and -6, suggest unequal contribution by the different CESAs to overall enzymatic activity of the complex. We present evidence that the primary complex requires three unique types of components, CESA1-, CESA3-, and CESA6-related, for activity. Removal of any of these components results in gametophytic lethality due to pollen defects, demonstrating that primary-wall cellulose synthesis is necessary for pollen development. We also show that the CESA6-related CESAs are partially functionally redundant.

Cytochrome bc(1) is a major component of biological energy conversion that exploits an energetically favourable redox reaction to generate a transmembrane proton gradient. Since the mechanistic details of the coupling of redox and protonation reactions in the active sites are largely unresolved, we have identified residues that undergo redox-linked protonation state changes. Structure-based Poisson-Boltzmann/Monte Carlo titration calculations have been performed for completely reduced and completely oxidised cytochrome bc(1). Different crystallographically observed conformations of Glu272 and surrounding residues of the cytochrome b subunit in cytochrome bc(1) from Saccharomyces cerevisiae have been considered in the calculations. Coenzyme Q (CoQ) has been modelled into the CoQ oxidation site (Q(o)-site). Our results indicate that both conformational and protonation state changes of Glu272 of cytochrome b may contribute to the postulated gating of CoQ oxidation. The Rieske iron-sulphur cluster could be shown to undergo redox-linked protonation state changes of its histidine ligands in the structural context of the CoQ-bound Q(o)-site. The proton acceptor role of the CoQ ligands in the CoQ reduction site (Q(i)-site) is supported by our results. A modified path for proton uptake towards the Q(i)-site features a cluster of conserved lysine residues in the cytochrome b (Lys228) and cytochrome c(1) subunits (Lys288, Lys289, Lys296). The cardiolipin molecule bound close to the Q(i)-site stabilises protons in this cluster of lysine residues.

Protein kinase C (PKC), a family of 12 distinct serine-threonine kinases, is an important intracellular signaling pathway involved in various cellular functions, such as proliferation, hypertrophy, apoptosis, and adhesion. PKC-epsilon, a novel PKC isoform that is activated in the diabetic kidney, has been demonstrated to have a central role in the underlying signaling infrastructure of myocardial ischemia and hypertrophy. The renal phenotype of PKC-epsilon(-/-) mice was studied with regard to renal hypertrophy and fibrosis. PKC-epsilon(-/-) deficient knockout mice were generated and then killed after 6, 16, and 26 wk of life. Kidney/body weight ratio did not show any significant group difference compared with appropriate wild-type controls. Urinary albumin/creatinine ratio remained normal in wild-type mice, whereas PKC-epsilon(-/-) mice after 6 and 16 wk showed elevated albuminuria. Masson-Goldner staining revealed that tubulointerstitial fibrosis and mesangial expansion were significantly increased in PKC-epsilon(-/-) mice. However, this profibrotic phenotype was not observed in other organs, such as liver and lung. Immunohistochemistry of the kidneys from PKC-epsilon(-/-) mice showed increased renal fibronectin and collagen IV expression that was further aggravated in the streptozotocin-induced diabetic stress model. Furthermore, TGF-beta(1), phospho-Smad2, and phospho-p38 mitogen-activate protein kinase expression was increased in PKC-epsilon(-/-) mice, suggesting a regulatory role of PKC-epsilon in TGF-beta(1) and its signaling pathway in the kidney. These results indicate that deletion of PKC-epsilon mediates renal fibrosis and that TGF-beta1 and its signaling pathway might be involved. Furthermore, these data suggest that activation of PKC-epsilon in the diabetic state may rather represent a protective response to injury than be a mediator of renal injury

Since the development of three-dimensional helical reconstruction methods in the 1960's, advances in Fourier-Bessel methods have facilitated structure determination to near-atomic resolution. A recently developed iterative helical real-space reconstruction (IHRSR) method provides an alternative that uses single-particle analysis in conjunction with the imposition of helical symmetry. In this work, we have adapted the IHRSR algorithm to work with frozen-hydrated tubular crystals of P-type ATPases. In particular, we have implemented layer-line filtering to improve the signal-to-noise ratio, Wiener-filtering to compensate for the contrast transfer function, solvent flattening to improve reference reconstructions, out-of-plane tilt compensation to deal with flexibility in three dimensions, systematic calculation of Fourier shell correlations to track the progress of the refinement, and tools to control parameters as the refinement progresses. We have tested this procedure on datasets from Na(+)/K(+)-ATPase, rabbit skeletal Ca(2+)-ATPase and scallop Ca(2+)-ATPase in order to evaluate the potential for sub-nanometer resolution as well as the robustness in the presence of disorder. We found that Fourier-Bessel methods perform better for well-ordered samples of skeletal Ca(2+)-ATPase and Na(+)/K(+)-ATPase, although improvements to IHRSR are discussed that should reduce this disparity. On the other hand, IHRSR was very effective for scallop Ca(2+)-ATPase, which was too disordered to analyze by Fourier-Bessel methods

In response to ER stress, the pancreatic endoplasmic reticulum kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha). IFN-gamma, which activates the ER stress response in oligodendrocytes, is believed to play a critical role in the immune-mediated CNS disorder multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE). Here we report that CNS delivery of IFN-gamma before EAE onset ameliorated the disease course and prevented demyelination, axonal damage, and oligodendrocyte loss. The beneficial effects of IFN-gamma were accompanied by PERK activation in oligodendrocytes and were abrogated in PERK-deficient animals. Our results indicate that IFN-gamma activation of PERK in mature oligodendrocytes attenuates EAE severity and suggest that therapeutic approaches to activate the ISR could prove beneficial in MS

Bone formation requires the coordinated activity of numerous proteins including the transcription factor core-binding factor alpha1 (Cbfa1). Deregulation of Cbfa1 results in metabolic bone diseases including osteoporosis and osteopetrosis. The retinoblastoma protein (pRb) that is required for osteogenesis binds Cbfa1. We reported earlier that the p200 family protein p204, which is known to be involved in the differentiation of skeletal muscle myotubes, cardiac myocytes, and macrophages, also serves as a cofactor of Cbfa1 and promotes osteogenesis. In this study we established that suppression of p204 expression by an adenovirus construct encoding p204 antisense RNA inhibited osteoblast-specific gene activation by Cbfa1 in an osteogenesis assay involving the pluripotent C2C12 mesenchymal cell line. Using protein-protein interaction assays we established that Cbfa1, pRb, and p204 form a ternary complex in which pRb serves as a linker connecting p204 and Cbfa1. Chromatin immunoprecipitation assays revealed the binding of such a p204-pRb-Cbfa1 transcription factor complex to the promoter of the osteocalcin gene. The pRb requirement of the stimulation of Cbfa1 activity by p204 was established in experiments involving p204 mutants lacking one or two pRb binding (LXCXE) motifs. Such mutants failed to enhance the Cbfa1-dependent transactivation of gene expression as well as osteogenesis. Furthermore, as revealed in reporter gene and in vitro osteogenesis assays p204 synergized with pRb in the stimulation of Cbfa1-dependent gene activation and osteoblast differentiation.

Using genome-wide approaches, we have elucidated the regulatory circuitry governed by the XBP1 transcription factor, a key effector of the mammalian unfolded protein response (UPR), in skeletal muscle and secretory cells. We identified a core group of genes involved in constitutive maintenance of ER function in all cell types and tissue- and condition-specific targets. In addition, we identified a cadre of unexpected targets that link XBP1 to neurodegenerative and myodegenerative diseases, as well as to DNA damage and repair pathways. Remarkably, we found that XBP1 regulates functionally distinct targets through different sequence motifs. Further, we identified Mist1, a critical regulator of differentiation, as an important target of XBP1, providing an explanation for developmental defects associated with XBP1 loss of function. Our results provide a detailed picture of the regulatory roadmap governed by XBP1 in distinct cell types as well as insight into unexplored functions of XBP1

PURPOSE OF REVIEW: Decades of literature have unambiguously demonstrated regression and remodeling of atherosclerotic lesions, including advanced plaques. Recent insights into underlying mechanisms are reviewed. RECENT FINDINGS: Factors promoting regression include decreased apolipoprotein B-lipoprotein retention within the arterial wall, efflux of cholesterol and other harmful lipids from plaques, and emigration of lesional foam cells followed by entry of healthy phagocytes that remove necrotic debris and other plaque components. Cellular lipid efflux and foam cell emigration can occur surprisingly rapidly once the plaque milieu is improved. Lipid efflux and foam cell emigration each involve specific molecular mediators, many of which have been identified. Necrotic debris removal can be surprisingly comprehensive, with essentially full disappearance documented in animal models. SUMMARY: The essential prerequisite for regression is robust improvement in plaque milieu, meaning large plasma reductions in atherogenic apolipoprotein B-lipoproteins or brisk enhancements in 'reverse' lipid transport from plaque into liver. Importantly, the processes of regression are consistent with rapid correction of features characteristic of the rupture-prone, vulnerable plaques responsible for acute coronary syndromes. New interventions to lower apolipoprotein B-lipoprotein levels and enhance reverse lipid transport may allow regression to become a widespread clinical goal. Strategies based on recent mechanistic insights may facilitate further therapeutic progress