Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia

Targeted gene disruption or overexpression of 12/15-lipoxygenase in mice on the genetic background of apolipoprotein E or low density lipoprotein-receptor (LDL-R) deficiency has implicated 12/15-lipoxygenase in atherogenesis. The data support indirectly a role for 12/15-lipoxygenase in the oxidative modification of low density lipoprotein. In this study we set out to explore other potential mechanisms for 12/15-lipoxygenase in atherosclerosis using apolipoprotein B mRNA editing catalytic polypeptide-1/LDL-R double-deficient mice, a model highly related to the human condition of familial hypercholesterolemia. 12/15-Lipoxygenase deficiency in this strain led to approximately 50% decrease in aortic lesions in male and female mice at 8 months on a chow diet in the absence of cholesterol differences. While studying 12/15-lipoxygenase-deficient macrophages in culture, we discovered a remarkable selective defect (75-90% decrease) in interleukin-12 production but not in tumor necrosis factor-alpha or nitric oxide release, in response to lipopolysaccharide in the presence or absence of interferon-gamma priming. The lipopolysaccharide/interferon-gamma response was associated with a 33-50% decrease in nuclear interferon consensus sequence-binding protein, which is consistent with interferon consensus sequence-binding protein containing protein complex-dependent regulation of the interleukin-12 p40 gene. The decrease in interleukin-12 production was recapitulated in vivo in mouse aortas of the triple knockout group and was reflected in a marked decrease in interferon-gamma expression. The data provide support for a novel mechanism linking the 12/15-lipoxygenase pathway to a known immunomodulatory Th1 cytokine in atherogenesis.     

Comparing the prognostic accuracy for all-cause mortality of frailty instruments: a multicentre 1-year follow-up in hospitalized older patients

Background

Frailty is a dynamic age-related condition of increased vulnerability characterized by declines across multiple physiologic systems and associated with an increased risk of death. We compared the predictive accuracy for one-month and one-year all-cause mortality of four frailty instruments in a large population of hospitalized older patients in a prospective multicentre cohort study.

Methods and Findings

On 2033 hospitalized patients aged ≥65 years from twenty Italian geriatric units, we calculated the frailty indexes derived from the Study of Osteoporotic Fractures (FI-SOF), based on the cumulative deficits model (FI-CD), based on a comprehensive geriatric assessment (FI-CGA), and the Multidimensional Prognostic Index (MPI). The overall mortality rates were 8.6% after one-month and 24.9% after one-year follow-up. All frailty instruments were significantly associated with one-month and one-year all-cause mortality. The areas under the receiver operating characteristic (ROC) curves estimated from age- and sex-adjusted logistic regression models, accounting for clustering due to centre effect, showed that the MPI had a significant higher discriminatory accuracy than FI-SOF, FI-CD, and FI-CGA after one month (areas under the ROC curves: FI-SOF = 0.685 vs. FI-CD = 0.738 vs. FI-CGA = 0.724 vs. MPI = 0.765, p<0.0001) and one year of follow-up (areas under the ROC curves: FI-SOF = 0.694 vs. FI-CD = 0.729 vs. FI-CGA = 0.727 vs. MPI = 0.750, p<0.0001). The MPI showed a significant higher discriminatory power for predicting one-year mortality also in hospitalized older patients without functional limitations, without cognitive impairment, malnourished, with increased comorbidity, and with a high number of drugs.

Conclusions

All frailty instruments were significantly associated with short- and long-term all-cause mortality, but MPI demonstrated a significant higher predictive power than other frailty instruments in hospitalized older patients.     

Hypertrophy and atrophy inversely regulate Caveolin-3 expression in myoblasts

Caveolin-3 (Cav-3) is a muscle-specific membrane protein crucial for myoblast differentiation, as loss of the protein due to mutations within the gene causes an autosomal dominant form of limb girdle muscular dystrophy 1-c. Here we show that along with p38 activity the PI3-kinase/AKT/mTOR pathway is required for proper Cav-3 up-regulation during muscle differentiation and hypertrophy, as confirmed by the marked increase of Cav-3 expression in hypertrophied C2C12 cells transfected with an activated form of AKT. Accordingly, Cav-3 expression was further increased during hypertrophy of L6C5 myoblasts treated with Arg(8)-vasopressin and in hypertrophic muscles of MLC/mIGF-1 transgenic mice. In contrast, Cav-3 expression was down-regulated in C2C12 myotubes exposed to atrophic stimuli such as starvation or treatment with dexamethasone. This study clearly suggests that Cav-3 expression is causally linked to the maturation of muscle phenotype and it is tightly regulated by hypertrophic and atrophic stimuli.     

The human tumor suppressor arf interacts with spinophilin/neurabin II, a type 1 protein-phosphatase-binding protein

The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16(INK4a) and p14(ARF) (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.     

Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), and biliverdin. We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression by Western blot in primary astroglial cells during differentiation and after exposure to glutamate (100microM). CLSM analysis of immunostained HO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and a decrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels. The distribution of HO-1 protein undergoes modification in the various cellular compartments. Furthermore, localization of the protein in untreated astrocytes at 7 days appeared prevalently localized in the cytosol and in the perinuclear region. In contrast, at 14 and 21 days, fluorescence detection suggests that HO-1 was present also in the nucleus, and in the nucleoli. Fluorescence intensity significantly increased in glutamate-treated astrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli. The involvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cells with GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors. Western blot analysis of HO-1 confirmed the CLSM results. Our results demonstrate that changes in HO-1 protein expression and localization in primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases.