CSRP3 mediates polyphenols-induced cardioprotection in hypertension

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure

Computational Insights on the Competing Effects of Nitric Oxide in Regulating Apoptosis

Despite the establishment of the important role of nitric oxide (NO) on apoptosis, a molecular- level understanding of the origin of its dichotomous pro- and anti-apoptotic effects has been elusive. We propose a new mathematical model for simulating the effects of nitric oxide (NO) on apoptosis. The new model integrates mitochondria-dependent apoptotic pathways with NO-related reactions, to gain insights into the regulatory effect of the reactive NO species N2O3, non-heme iron nitrosyl species (FeLnNO), and peroxynitrite (ONOO−). The biochemical pathways of apoptosis coupled with NO-related reactions are described by ordinary differential equations using mass-action kinetics. In the absence of NO, the model predicts either cell survival or apoptosis (a bistable behavior) with shifts in the onset time of apoptotic response depending on the strength of extracellular stimuli. Computations demonstrate that the relative concentrations of anti- and pro-apoptotic reactive NO species, and their interplay with glutathione, determine the net anti- or pro-apoptotic effects at long time points. Interestingly, transient effects on apoptosis are also observed in these simulations, the duration of which may reach up to hours, despite the eventual convergence to an anti-apoptotic state. Our computations point to the importance of precise timing of NO production and external stimulation in determining the eventual pro- or anti-apoptotic role of NO

Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of Lonomia obliqua

Apoptosis is a major problem in animal cell culture during production of biopharmaceuticals, such as recombinant proteins or viral particles. In the present work baculovirus-insect cell expression system (BEVS/IC) is used as model to produce rotavirus like-particles, composed by three layers of three different viral proteins (VP2, VP6 and VP7). In this model baculovirus infection also induces host cell death. Herein a new strategy to enhance cell life span and to increase recombinant rotavirus protein production of BEVS/IC system was developed. This strategy relies on hemolymph from Lonomia oblique (total extracts or a semi-purified fraction) medium supplementation. The total extract and a purified fraction from hemolymph of Lonomia obliqua were able to protect Sf-9 cell culture against apoptosis triggered by oxidative stress (using the pro-oxidant agents tert butylhydroperoxide and hydrogen peroxide) and by baculovirus infection. Furthermore, hemolymph enhance final recombinant protein production, as it was observed by the increased amounts of VP6 and VP7, which were measured by the semi-quantitative western blot method. In conclusion, hemolymph medium supplementation can be a promising strategy to improve cell viability and productivity of recombinant protein in BEVS/IC system

The localization and activity of sphingosine kinase 1 are coordinately regulated with actin cytoskeletal dynamics in macrophages

Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.The physiologic and pathologic functions of sphingosine kinase (SK) require translocation to specific membrane compartments. We tested the hypothesis that interactions with actin filaments regulate the localization of SK1 to membrane surfaces, including the plasma membrane and phagosome. Macrophage activation is accompanied by a marked increase in association of SK1 with actin filaments. Catalytically-inactive (CI)- and phosphorylation-defective (PD)-SK1 mutants exhibited reductions in plasma membrane translocation, colocalization with cortical actin filaments, membrane ruffling, and lamellipodia formation, compared with wild-type (WT)-SK1. However, translocation of CI- and PD-SK1 to phagosomes were equivalent to WT-SK1. SK1 exhibited constitutive- and stimulus-enhanced association with actin filaments and F-actin-enriched membrane fractions in both intact macrophages and a novel in vitro assay. In contrast, SK1 bound G-actin only under stimulated conditions. Actin inhibitors disrupted SK1 localization and modulated its activity. Conversely, reduction of SK1 levels or activity via RNA interference or specific chemical inhibition resulted in dysregulation of actin filaments. Thus, the localization and activity of SK1 are coordinately regulated with actin dynamics during macrophage activation.David J. Kusner, Christopher R. Thompson, Natalie A. Melrose, Stuart M. Pitson, Lina M. Obeid, and Shankar S. Iye

Modeling the behavior of human induced pluripotent stem cells seeded on melt electrospun scaffolds

Abstract Background Human induced pluripotent stem cells (hiPSCs) can form any tissue found in the body, making them attractive for regenerative medicine applications. Seeding hiPSC aggregates into biomaterial scaffolds can control their differentiation into specific tissue types. Here we develop and analyze a mathematical model of hiPSC aggregate behavior when seeded on melt electrospun scaffolds with defined topography. Results We used ordinary differential equations to model the different cellular populations (stem, progenitor, differentiated) present in our scaffolds based on experimental results and published literature. Our model successfully captures qualitative features of the cellular dynamics observed experimentally. We determined the optimal parameter sets to maximize specific cellular populations experimentally, showing that a physiologic oxygen level (∼ 5%) increases the number of neural progenitors and differentiated neurons compared to atmospheric oxygen levels (∼ 21%) and a scaffold porosity of ∼ 63% maximizes aggregate size. Conclusions Our mathematical model determined the key factors controlling hiPSC behavior on melt electrospun scaffolds, enabling optimization of experimental parameters