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BBA - Molecular Basis of Disease (v.1772, #7)

Editorial Board (pp. ii).

Platelets: The universal killer? by Andrew D. Blann (pp. 715-717).

For many, the final terminal event in life is cessation of the heart beat. In turn, this is generally because this organ has been deprived of oxygen and glucose as the blood can no longer deliver these requirements to the myocardium. The principal reason for this is blockage of one or more coronary arteries or arterioles by platelet rich thrombus. A similar process exists for the pathophysiology of stroke—a disabilitating and often fatal event caused by occlusion or rupture of arteries in, or feeding, the brain. These scenarios are best developed in cardiovascular disease, but apply to almost all human disease. Therefore, the ultimate culprit for these major life events is the overactive platelet—too ready to form an inappropriate thrombus. Thus, one way forward in postponing an occlusive thrombotic event is to minimise platelet activation, new tools and treatments for which are eagerly sought.

Keywords: Platelets; Thrombosis; P selectin

Oxidized phospholipids: From molecular properties to disease by Gilbert O. Fruhwirth; Alexandra Loidl; Albin Hermetter (pp. 718-736).

Oxidized lipids are generated from (poly)unsaturated diacyl- and alk(en)ylacyl glycerophospholipids under conditions of oxidative stress. The great variety of reaction products is defined by the degree of modification, hydrophobicity, chemical reactivity, physical properties and biological activity. The biological activities of these compounds may depend on both, the recognition of the particular molecular structures by specific receptors and on the unspecific physical and chemical effects on their target systems (membranes, proteins). In this review, we aim at highlighting the molecular features that are essential for the understanding of the biological actions of pure oxidized phospholipids. Firstly, their chemical structures are described as a basis for an understanding of their physical and (bio)chemical properties in membrane- and protein-bound form. Secondly, the biological activities of oxidized phospholipids are discussed in terms of their unspecific effects on the membrane level as well as their potential interactions with specific targets (receptors) affecting a large set of (signaling) molecules. Finally, the role of oxidized phospholipids as important mediators in pathophysiology is discussed with emphasis on atherosclerosis.

Keywords: Phospholipid oxidation; Membrane; Receptors; Inflammation; Apoptosis

The ErbB4 receptor in fetal rat lung fibroblasts and epithelial type II cells by Washa Liu; Katja Zscheppang; Sandy Murray; Heber C. Nielsen; Christiane E.L. Dammann (pp. 737-747).

ErbB receptors are important regulators of fetal organ development, including the fetal lung. They exhibit diversity in signaling potential, acting through homo- and heterodimers to cause different biological responses. We hypothesized that ErbB receptors show cell-specific and stimuli-specific activation, heterodimerization, and cellular localization patterns in fetal lung. We investigated this using immunoblotting, co-immunoprecipitation, and confocal microscopy in primary isolated E19 fetal rat lung fibroblasts and epithelial type II cells, stimulated with epidermal growth factor, transforming growth factor α, neuregulin 1β, or treated with conditioned medium (CM) from the respective other cell type. Fetal type II cells expressed significantly more ErbB1, ErbB2, and ErbB3 protein than fibroblasts. ErbB4 was consistently identified by co-immunoprecipitation of all other ErbB receptors in both cell types independent of the treatments. Downregulation of ErbB4 in fibroblasts initiated cell–cell communication that stimulated surfactant phospholipid synthesis in type II cells. Confocal microscopy in type II cells revealed nuclear localization of all receptors, most prominently for ErbB4. Neuregulin treatment resulted in relocation to the extra-nuclear cytoplasmic region, which was distinct from fibroblast CM treatment which led to nuclear localization of ErbB4 and ErbB2, inducing co-localization of both receptors. We speculate that ErbB4 plays a prominent role in fetal lung mesenchyme–epithelial communication.

Keywords: Neuregulin; Epidermal growth factor; Transforming growth factor α; Conditioned medium; Lung development

Elevated small GTPase activation influences the cell proliferation signaling control in Niemann–Pick type C fibroblasts by Deborah A. Corey; Thomas J. Kelley (pp. 748-754).

Niemann–Pick type C (NPC) disease is characterized at the cellular level by the intracellular accumulation of free cholesterol. We have previously identified a similar phenotype in cystic fibrosis (CF) cell models that results in the activation of the small GTPase RhoA. The hypothesis of this study was that NPC cells would also exhibit an increase in small GTPase activation. An examination of the active, GTP-bound form of GTPases revealed a basal increase in the content of the active-form Ras and RhoA small GTPases in NPC fibroblasts compared to wt controls. To assess whether this increase in GTP-bound Ras and RhoA manifests a functional outcome, the expression of the proliferation control proteins p21/waf1 and cyclin D were examined. Consistent with increased GTPase signaling, p21/waf1 expression is reduced and cyclin D expression is elevated in NPC fibroblasts. Interestingly, cell growth rate is not altered in NPC fibroblasts compared to wt cells. However, NPC sensitivity to statin treatment is reversed by addition of the isoprenoid geranylgeranyl pyrophosphate (GGPP), a modifier of RhoA. It is concluded that Ras and RhoA basal activation is elevated in NPC fibroblasts and has an impact on cell survival pathways.

Keywords: Niemann–Pick type C; RhoA; Ras; Cyclin D; p21/waf1

Insulin resistance and the mitochondrial link. Lessons from cultured human myotubes by Michael Gaster (pp. 755-765).

In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects and exposed to mitochondrial inhibitors, either affecting the electron transport chain (Antimycin A), the ATP synthase (oligomycin) or respiratory uncoupling (2,4-dinitrophenol). Direct inhibition of the electron transport chain or the ATP synthase was followed by increased glucose uptake and lactate production, reduced glycogen synthesis, reduced lipid and glucose oxidation and unchanged lipid uptake. The metabolic phenotype during respiratory uncoupling resembled the above picture, except for an increase in glucose and palmitate oxidation. Antimycin A and oligomycin treatment induced insulin resistance at the level of glucose and palmitate uptake in all three study groups while, at the level of glycogen synthesis, insulin resistance was only seen in lean myotubes. Primary insulin resistance in diabetic myotubes was significantly worsened at the level of glucose and lipid uptake. The present study is the first convincing data linking functional mitochondrial impairment per se and insulin resistance. Taken together functional mitochondrial impairment could be part of the pathophysiology of insulin resistance in vivo.

Keywords: Abbreviations; AA; antimycin A; BSA; bovine serum albumin; DNP; 2,4-Dinitrophenol; FCS; foetal calf serum; FFA; Free fatty acids; GIR; glucose infusion rates; OA; oligomycin A; PA; palmitate; TAG; Triacylglycerol; T2D; Type 2 diabetic/type 2 diabetesGlucose metabolism; Insulin resistance; Lipid metabolism; Metabolic inhibition; Mitochondrial function; Myotube; Skeletal muscle; Type 2 diabetes

Pro-inflammatory cytokines from Kupffer cells downregulate hepatocyte expression of adrenomedullin binding protein-1 by Asha Jacob; Mian Zhou; Rongqian Wu; Vivienne J. Halpern; Thanjavur S. Ravikumar; Ping Wang (pp. 766-772).

Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late hypodynamic phase. Adrenomedullin (AM), a vasodilatory peptide, inhibits this transition from the early phase to the late phase. Adrenomedullin binding protein-1 (AMBP-1) enhances AM-mediated activities. The decrease of AMBP-1 levels in late sepsis reduces the vascular response to AM and produces the hypodynamic phase. Studies have indicated that the administration of LPS downregulates AMBP-1 production in the liver. Since hepatocytes are the primary source of AMBP-1 biosynthesis in the liver, we employed a co-culture strategy using hepatocyte and Kupffer cells to determine whether LPS directly or by increasing pro-inflammatory cytokines from Kupffer cells downregulates AMBP-1 production. Hepatocytes and Kupffer cells isolated from rats were co-cultured and treated with LPS for 24 h. LPS significantly attenuated AMBP-1 protein expression in a dose-dependent manner. Since AMBP-1 is basically a secretory protein, cell supernatants from co-culture cells treated with LPS were examined for AMBP-1 protein levels. LPS treatment caused a dose related decrease in AMBP-1 protein secretion. Similarly, LPS treatment produced a significant decrease in AMBP-1 protein expression in hepatocytes and Kupffer cells cultured using transwell inserts. LPS had no direct effect on AMBP-1 levels in cultured hepatocytes or Kupffer cells alone. To confirm that the observed effects in co-culture were due to the cytokines released from Kupffer cells, hepatocytes were treated with IL-1β or TNF-α for 24 h and AMBP-1 expression was examined. The results indicated that both cytokines significantly inhibited AMBP-1 protein levels. Thus, pro-inflammatory cytokines released from Kupffer cells are responsible for downregulation of AMBP-1.

Keywords: Adrenomedullin binding protein-1; LPS; Sepsis; TNF-α; IL-1β

Hemin rescues adrenodoxin, heme a and cytochrome oxidase activity in frataxin-deficient oligodendroglioma cells by Eleonora Napoli; Dexter Morin; Rita Bernhardt; Alan Buckpitt; Gino Cortopassi (pp. 773-780).

Mutations in the frataxin gene cause neurodegeneration and demyelination in Friedreich's ataxia. We showed earlier that frataxin deficiency causes primary iron–sulfur cluster defects, and later causes defects in heme and cytochrome c hemoprotein levels. Iron–sulfur (Fe/S) clusters are required in two enzymes of heme biosynthesis in humans i.e. in ferrochelatase and adrenodoxin. However, decreases in ferrochelatase activity have not been observed in frataxin-deficient HeLa cells or patient lymphoblasts. We knocked down frataxin in oligodendroglioma cells using siRNA, which produced significant defects in the activity of the Fe/S cluster enzymes adrenodoxin and aconitase, the adrenodoxin product heme a, and cytochrome oxidase, for which heme a serves as a prosthetic group. Exogenous hemin produced a significant rescue of adrenodoxin, aconitase, heme a levels and cytochrome oxidase activity. Thus hemin rescues iron–sulfur cluster defects that are the result of frataxin-deficiency, perhaps as a consequence of increasing the pool of bioavailable iron, and thus should be more fully tested for beneficial effects in Friedreich's ataxia models.

Keywords: RNAi; Frataxin; Iron–sulfur cluster; Heme; Adrenodoxin; Hemin; Theraphy

Molecular characterization of the first missense mutation in the fibrinogen Aalpha-chain gene identified in a compound heterozygous afibrinogenemic patient by Manuela Platé; Rosanna Asselta; Flora Peyvandi; Maria Luisa Tenchini; Stefano Duga (pp. 781-787).

Congenital afibrinogenemia is a rare coagulopathy characterized by extremely low levels of functional and immunoreactive fibrinogen in plasma, associated with a hemorrhagic phenotype of variable severity. It is transmitted as an autosomal recessive trait and is invariantly associated with mutations affecting 1 of the 3 fibrinogen genes ( FGA, FGB, and FGG, coding for Aα, Bβ, and γ chain, respectively). Most genetic defects causing afibrinogenemia are truncating mutations, whereas only few missense mutations (6) have been identified so far, all located in FGB.In this study, the mutational screening of an afibrinogenemic Italian male identified the first missense mutation (Met51Arg) in FGA leading to afibrinogenemia. The patient was a compound heterozygote for a previously described frameshift mutation (1215delT) in the same gene. Met51Arg involves a residue located at the very beginning of the coiled-coil domain, in a region demonstrated to play a pivotal role in hexamer formation. In-vitro expression experiments showed that Met51Arg strongly reduces secretion of hexameric fibrinogen, whereas traces of not completely assembled trimeric intermediate were found in conditioned media. Western blot analysis on the proband's plasma confirmed the presence in vivo of the trimeric fibrinogen, supporting the hypothesis that Met51Arg prevents the final step of fibrinogen assembly.

Keywords: Fibrinogen; Afibrinogenemia; Aα chain; Missense mutation; Protein ex-vivo expression

Increased plasma macrophage inflammatory protein (MIP)-1α and MIP-1β levels in type 1 Gaucher disease by Mariëlle J. van Breemen; Maaike de Fost; Jane S.A. Voerman; Jon D. Laman; Rolf G. Boot; Mario Maas; Carla E.M. Hollak; Johannes M. Aerts; Farhad Rezaee (pp. 788-796).

Pancytopenia, hepatosplenomegaly and skeletal complications are hallmarks of Gaucher disease. Monitoring of the outcome of therapy on skeletal status of Gaucher patients is problematic since currently available imaging techniques are expensive and not widely accessible. The availability of a blood test that relates to skeletal manifestations would be very valuable. We here report that macrophage inflammatory protein (MIP)-1α and MIP-1β, both implicated in skeletal complications in multiple myeloma (MM), are significantly elevated in plasma of Gaucher patients. Plasma MIP-1α of patients (median 78 pg/ml, range 21–550 pg/ml, n=48) is elevated (normal median 9 pg/ml, range 0–208 pg/ml, n=39). Plasma MIP-1β of patients (median 201 pg/ml, range 59–647 pg/ml, n=49) is even more pronouncedly increased (normal median 17 pg/ml, range 1–41 pg/ml, n=39; one outlier: 122 pg/ml). The increase in plasma MIP-1β levels of Gaucher patients is associated with skeletal disease. The plasma levels of both chemokines decrease upon effective therapy. Lack of reduction of plasma MIP-1β below 85 pg/ml during 5 years of therapy was observed in patients with ongoing skeletal disease. In conclusion, MIP-1α and MIP-1β are elevated in plasma of Gaucher patients and remaining high levels of MIP-1β during therapy seem associated with ongoing skeletal disease.

Keywords: Gaucher disease; MIP-1α; MIP-1β; Bone; Skeletal disease

Apolipoprotein e deficiency leads to altered brain uptake of alpha tocopherol injected into lateral cerebral ventricles by Govind T. Vatassery; Hung T. Quach; W. Ed Smith; Karen S. SantaCruz; Sabita Roy (pp. 797-803).

The incorporation of radioactive alpha tocopherol by various brain regions of wild type and apolipoprotein E (apoE)-deficient mice was investigated. Labeled tocopherol was injected into the lateral cerebral ventricles of 11 weeks old, male mice. Radioactive cholesterol injected simultaneously was used as an internal standard to account for experimental variability. Most areas of the brain of apoE-deficient mice took up less of alpha tocopherol per mg of protein than wild type animals. However, specific activity of alpha tocopherol was higher in cerebellum, pons, hypothalamus, midbrain and cerebral cortex in apoE-deficient brains than the wild type. This could be due to (a) the lower levels of alpha tocopherol in apoE-deficient brain and (b) reductions in the clearance and transport of tocopherol (possibly mediated by apoE). Tocopherol uptake by hippocampus was unusual since it was lower in apoE deficiency whether the data were expressed as specific activity or per mg of protein. Nearly all of the injected alpha tocopherol remained unchanged in the brains of both apoE-deficient and wild type animals suggesting low turnover. Overall, the current data reinforce the hypothesis that apoE is a key protein involved with the transport and/or retention of alpha tocopherol in brain.

Keywords: Vitamin E; Apolipoprotein E; Cerebral ventricles; Cholesterol; Deficiency; Uptake

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BBA - Molecular Basis of Disease (v.1772, #7)