Disturbed flow induces a sustained, stochastic NF-κB activation which may support intracranial aneurysm growth in vivo

Intracranial aneurysms are associated with disturbed velocity patterns, and chronic inflammation, but the relevance for these findings are currently unknown. Here, we show that (disturbed) shear stress induced by vortices is a sufficient condition to activate the endothelial NF-kB pathway, possibly through a mechanism of mechanosensor de-activation. We provide evidence for this statement through in-vitro live cell imaging of NF-kB in HUVECs exposed to different flow conditions, stochastic modelling of flow induced NF-kB activation and induction of disturbed flow in mouse carotid arteries. Finally, CFD and immunofluorescence on human intracranial aneurysms showed a correlation similar to the mouse vessels, suggesting that disturbed shear stress may lead to sustained NF-kB activation thereby offering an explanation for the close association between disturbed flow and intracranial aneurysms

Rule-Based Cell Systems Model of Aging using Feedback Loop Motifs Mediated by Stress Responses

Investigating the complex systems dynamics of the aging process requires integration of a broad range of cellular processes describing damage and functional decline co-existing with adaptive and protective regulatory mechanisms. We evolve an integrated generic cell network to represent the connectivity of key cellular mechanisms structured into positive and negative feedback loop motifs centrally important for aging. The conceptual network is casted into a fuzzy-logic, hybrid-intelligent framework based on interaction rules assembled from a priori knowledge. Based upon a classical homeostatic representation of cellular energy metabolism, we first demonstrate how positive-feedback loops accelerate damage and decline consistent with a vicious cycle. This model is iteratively extended towards an adaptive response model by incorporating protective negative-feedback loop circuits. Time-lapse simulations of the adaptive response model uncover how transcriptional and translational changes, mediated by stress sensors NF-κB and mTOR, counteract accumulating damage and dysfunction by modulating mitochondrial respiration, metabolic fluxes, biosynthesis, and autophagy, crucial for cellular survival. The model allows consideration of lifespan optimization scenarios with respect to fitness criteria using a sensitivity analysis. Our work establishes a novel extendable and scalable computational approach capable to connect tractable molecular mechanisms with cellular network dynamics underlying the emerging aging phenotype

D017 Sonic hedgehog-mediated angiogenesis in the mouse ischemic limb requires osteopontin

PurposeSonic Hedgehog (Shh), a morphogen involved in embryonic development, stimulates repair of ischemic myocardium and skeletal muscle (Pola et al 2003 and Kusano et al 2005). Shh was shown to induce both the formation of new vessels and their muscularization. However the mechanisms involved in these processes are still poorly understood. We investigated the hypothesis that the angiogenic and chemotactic protein osteopontin (OPN) was involved in the angiogenic effect of Shh.Methods and ResultsWe used a plasmid transfection strategy to increase Shh expression in the ischemic region of the limb and we compared the effect of Shh overexpression in wild type (OPN+/+) and OPN deficient (OPN-/-) 12-weeks-old mice transfected either with Shh plasmid (pShh) or control plasmid (β-gal). Shh overexpression induced a significant increase in the number of new vessels (+ 22 %± 4.2) in OPN+/+ mice in comparaison with β-gal transfected wild type mice, moreover the Shh effect was lost in OPN-/- mice. Analysis of vessel muscularization showed that Shh mainly increased the number of small non-muscularized vessels. In parallel, we observed an increased number of OPN expressing cells in the ischemic lesion mainly localized close to or inside debasing/regenerating muscle fibres. These cells expressed Mac3 and F4/80 markers suggesting a macrophage phenotype. We suggested that shh could increase the migration of macrophages in an OPN-dependent manner in order to accelerate the repair of the ischemic tissue. The experiments related to the function of OPN in ischemic limb stimulated or not by Shh is currently under investigation.ConclusionOur results demonstrated that OPN is required for the Shh angiogenic effect in the ischemic tissue and suggest that osteopontin expression may be associated with a better migration of macrophages in the ischemic lesion under Shh stimulation.Kusano KF et al. Nat Med. 2005 ; 11:1197-1204.Pola R et al. Circulation. 2003 ; 108:479-485

H020 Chronic expression of osteopontin in cardiomyocytes led to dilated cardiomyopathy

Osteopontin (OPN) is not expressed in healthy heart while, its expression is dramatically increased in cardiomyocytes and inflammatory cells during cardiomyopathies and heart failure. However its role in the development of heart diseases is not known.To understand whether OPN is involved in cardiomyopathy, we created a transgenic mouse (MHC-OPN) expressing recombinant OPN (rOPN) specifically in cardiomyocytes using aMHC promoter-directed OPN expression and tTA technology. In these mice, rOPN expression could be regulated by doxycyclin oral administration.After birth, MHC-OPN young mice were phenotypically indistinguishable from their littermate controls, but most of them died early between the 8 th and 15 th weeks after birth with a half life of 12 weeks. However, less than 10 % MHC-OPN mice survived and were still alive 30 weeks after birth. Inhibition of recombinant OPN expression by doxycyclin at the beginning of T cell infiltration (5 weeks after birth) or when DCM was initiated (11 weeks after birth) reduced myocarditis and thus avoided the early death.Electrocardiography demonstrated atrio-ventricular and intra ventricular defects. Moreover, echocardiography showed left ventricular dilation without hypertrophy and a systolic dysfunction, as indicated by reduced left ventricular fractional shortening (control mice: 29,8±2,0 %, n=13 ; and MHC-OPN mice: 13,7±4,0 %, n=5 ; T test p<0.05). In vitro histology confirmed that mice died because of a dilated cardiomyopathy associated with a strong fibrosis.By immunohistology, we demonstrated that OPN expression in cardiomyocytes induced an important cell infiltration including some macrophages and a large number of fibroblasts and activated CD4+ and CD8+ T cells.All together these experiments suggested that chronic OPN expression is required for DCM development inducing Tcell activation and thus a chronic myocarditis resulting in the dilated cardiomyopathy

HDLs inhibit endoplasmic reticulum stress and autophagic response induced by oxidized LDLs

The apoptotic effect of oxidized LDLs (oxLDLs) is mediated through a complex sequence of signaling events involving a deregulation of the cytosolic Ca2+ homeostasis. OxLDLs also trigger ER stress that may lead to cellular dysfunction and apoptosis, through the activation of the IRE1α/c-Jun N-terminal kinase pathway. Moreover, ER stress and oxidized lipids have been shown to trigger autophagy. The antiatherogenic high-density lipoproteins (HDLs) display protective effects against oxLDLs toxicity. To more deeply investigate the mechanisms mediating the protective effects of HDLs, we examined whether ER stress and autophagy were implicated in oxLDLs-induced apoptosis and whether HDLs prevented these stress processes. We report that, in human endothelial cells, HDLs prevent the oxLDL-induced activation of the ER stress sensors IRE1α, eIF2α and ATF6 and subsequent activation of the proapoptotic mediators JNK and CHOP. OxLDLs also trigger the activation of autophagy, as assessed by LC3 processing and Beclin-1 expression. The autophagic process is independent of the proapoptotic arms of ER stress, but Beclin-1 contributes to PS exposure and subsequent phagocytosis of oxLDLs exposed cells. Induction of autophagy and PS exposure by oxLDLs is prevented by HDLs. Finally, the cytosolic Ca2+ deregulation triggered by oxLDLs is a common signaling pathway that mediates ER stress-induced cell death and autophagy, all these events being blocked by HDLs