Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy

The present study was undertaken to test whether inhibition of the proangiogenic inflammatory cytokine tumor necrosis factor (TNF)-alpha can modulate retinal hypoxia and preretinal neovascularization in a murine model of oxygen-induced retinopathy (OIR). OIR was produced in TNF-alpha-/- and wild-type (WT) control C57B6 neonatal mice by exposure to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Half of each WT litter was treated with the cytokine inhibitor semapimod (formerly known as CNI-1493) (5 mg/kg) by daily intraperitoneal injection from the time of reintroduction to room air at P12 until P17. The extent of preretinal neovascularization and intraretinal revascularization was quantified by image analysis of retinal flat-mounts and retinal hypoxia correlated with vascularization by immunofluorescent localization of the hypoxia-sensitive drug pimonidazole (hypoxyprobe, HP). HP adducts were also characterized by Western analysis and quantified by competitive enzyme-linked immunosorbent assay. TNF-alpha-/- and WT mice showed a similar sensitivity to hyperoxia-induced retinal ischemia at P12. At P13 some delay in early reperfusion was evident in TNFalpha-/- and WT mice treated with semapimod. However, at P17 both these groups had significantly better vascular recovery with less ischemic/hypoxic retina and preretinal neovascularization compared to untreated retinopathy in WT mice. Immunohistochemistry showed deposition of HP in the avascular inner retina but not in areas underlying preretinal neovascularization, indicating that such aberrant vasculature can reduce retinal hypoxia. Inhibition of TNF-alpha significantly, improves vascular recovery within ischemic tissue and reduces pathological neovascularization in OIR. HP provides a useful tool for mapping and quantifying tissue hypoxia in experimental ischemic retinopathy.

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER)(1). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes(2). Controversy exists, however, concerning whether ER has a role outside the nucleus(3), particularly in mediating the cardiovascular protective effects of oestrogen(4). Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ERa are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85 alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ERa with PI(3)K.

Expression of the counter-regulatory peptide intermedin is augmented in the presence of oxidative stress in hypertrophied cardiomyocytes.

Background: Intermedin (IMD), a novel cardiac peptide related to adrenomedullin (AM), protects against myocardial ischemia-reperfusion injury and attenuates ventricular remodelling. IMD’s actions are mediated by a calcitonin receptor-like receptor in association with receptor activity modifying proteins (RAMPs 1-3). Aim/method: using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rat at 20 weeks of age, to examine (i) the presence of myocardial oxidative stress and concentric hypertrophy; (ii) expression of IMD, AM and receptor components. Results: In left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only (i) oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%); (ii) IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered; (iii) abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. Conclusion: robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter-regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension. The local concentration and action of IMD may be further enhanced by down-regulation of NEP within the left ventricle.

Intermedin (Adrenomedullin-2) a novel counter-regulatory peptide in the cardiovascular and renal systems

Intermedin (IMD) is a novel peptide related to calcitonin gene-related peptide (CGRP) and adrenomedullin (AM). Proteolytic processing of a larger precursor yields a series of biologically active C-terminal fragments, IMD1–53, IMD1–47 and IMD8–47. IMD shares a family of receptors with AM and CGRP composed of a calcitonin-receptor like receptor (CALCRL) associated with one of three receptor activity modifying proteins (RAMP). Compared to CGRP, IMD is less potent at CGRP1 receptors but more potent at AM1 receptors and AM2 receptors; compared to AM, IMD is more potent at CGRP1 receptors but less potent at AM1 and AM2 receptors. The cellular and tissue distribution of IMD overlaps in some aspects with that of CGRP and AM but is distinct from both. IMD is present in neonatal but absent or expressed sparsely, in adult heart and vasculature and present at low levels in plasma. The prominent localization of IMD in hypothalamus and pituitary and in kidney is consistent with a physiological role in the central and peripheral regulation of the circulation and water-electrolyte homeostasis. IMD is a potent systemic and pulmonary vasodilator, influences regional blood flow and augments cardiac contractility. IMD protects myocardium from the deleterious effects of oxidative stress associated with ischaemia-reperfusion injury and exerts an anti-growth effect directly on cardiomyocytes to oppose the influence of hypertrophic stimuli. The robust increase in expression of the peptide in hypertrophied and ischaemic myocardium indicates an important protective role for IMD as an endogenous counter-regulatory peptide in the heart.

Vascular stem cells and ischaemic retinopathies

Retinal ischaemic disorders such as diabetic retinopathy and retinal vein occlusion are common. The hypoxia-related stimuli from oxygen-deprived neural and glial networks can drive expression of growth factors and cytokines which induce leakage from the surviving vasculature and/or pre-retinal and papillary neovascularisation. If left untreated, retinal vascular stasis, hypoxia or ischaemia can lead to macular oedema or fibro-vascular scar formation which are associated with severe visual impairment, and even blindness. Current therapies for ischaemic retinopathies include laser photocoagulation, injection of corticosteroids or VEGF-antibodies and vitreoretinal surgery, however they carry significant side effects. As an alternative approach, we propose that if reparative intra-retinal angiogenesis can be harnessed at the appropriate stage, ischaemia could be contained or reversed. This review provides evidence that reperfusion of ischaemic retina and suppression of sight-threatening sequelae is possible in both experimental and clinical settings. In particular, there is emphasis on the clinical potential for endothelial progenitor cells (EPCs) to promote vascular repair and reversal of ischaemic injury in various tissues including retina. Gathering evidence from an extensive published literature, we outline the molecular and phenotypic nature of EPCs, how they are altered in disease and provide a rationale for harnessing the vascular reparative properties of various cell sub-types. When some of the remaining questions surrounding the clinical use of EPCs are addressed, they may provide an exciting new therapeutic option for treating ischaemic retinopathies. (C) 2011 Elsevier Ltd. All rights reserved.

Preconditioning protects the guinea-pig urinary bladder against ischaemic conditions in vitro

Aims: To investigate the ability of ischaemic preconditioning (IPC) to protect guinea-pig detrusor from damage caused by a subsequent more prolonged exposure to ischaemic conditions.

Materials and Methods: Smooth muscle strips were mounted for tension recording in small organ baths continuously superfused with Krebs' solution at 37 degrees C. Ischaemia was mimicked by removing oxygen and glucose from the superfusing solution. Contractile responses to electrical field stimulation (EFS) and carbachol were monitored. Three regimes of preconditioning were examined: 15, 10, and 5 min of ischaemic conditions followed by 15, 10, and 5 min of normal conditions, respectively.

Results: Without preconditioning, nerve-mediated responses were significantly and proportionally reduced by periods of ischaemic conditions lasting for 45, 60, and 90 min, but recovered fully after exposure to ischaemic conditions for 30 min. The recovery of the responses to EFS was significantly improved in preconditioned strips when the period of ischaemic conditions was 45 or 60 min. However, no significant differences were seen with preconditioning when the period of ischaemic conditions was 90 min. The recovery of responses to carbachol was much greater than for the responses to EFS, and no significant differences were found between control and preconditioned strips.

Conclusions: It is suggested that in vivo short periods of transient ischaemia may be able to protect the guinea-pig bladder from the impairment associated with longer periods of ischaemia and reperfusion, which might happen in obstructed micturition. Our results also indicate that the phenomenon affects mainly the intrinsic nerves, which are more susceptible to ischaemic damage than the smooth muscle.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning:efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P <0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P <0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P <0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P <0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.

Paraquat-Induced Retinal Degeneration Is Exaggerated in CX3CR1-Deficient Mice and Is Associated with Increased Retinal Inflammation

PURPOSE:
To investigate the role of the Fractalkine receptor CX3CR1 pathway in oxidative insults-mediated retinal degeneration and immune activation.
METHODS:
A prooxidant, paraquat (0.75 µM) was injected into the vitreous of C57BL/6J, CX3CR1(gpf/+), and CX3CR1(gfp/gfp) mice. Retinal lesions were investigated clinically by topic endoscopic fundus imaging and fluorescence angiography, and pathologically by light- and electron microscopy. Retinal immune gene expression was determined by real-time RT-PCR. Microglial activation and immune cell infiltration were examined by confocal microscopy of retinal flatmounts.
RESULTS:
Intravitreal injection of paraquat (0.75 µM) resulted in acute retinal capillary nonperfusion within 2 days, which improved from 4 days to 4 weeks postinjection (p.i.). Panretinal degeneration was observed at 4 days p.i. and progressed further at 4 weeks p.i. In the absence of CX3CR1, retinal degeneration was exaggerated and was accompanied by increased TNF-a, iNOS, IL-1ß, Ccl2, and Casp-1 gene expression. Confocal microscopy of retinal flatmounts revealed microglial activation and CD44(+)MHC-II(+) monocyte and GR1(+) neutrophil infiltration in paraquat-injected eyes. The number of activated microglia and infiltrating leukocytes was significantly higher in CX3CR1(gfp/gfp) mice than in CX3CR1(gfp/+) mice.
CONCLUSIONS:
Our results suggest that the CX3CR1 signaling pathway may play an important role in controlling retinal inflammation under oxidative and ischemia/reperfusion conditions. In the absence of CX3CR1, uncontrolled retinal inflammation results in exaggerated retinal degeneration.

Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS.

Results: We found that in the heart, t½ of complex I deactivation during ischemia was 10?min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis.

Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting.

Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

Vascular Endothelial Cell Growth–Activated XBP1 Splicing in Endothelial Cells Is Crucial for Angiogenesis

BACKGROUND - : Vascular endothelial cell growth factor plays a pivotal role in angiogenesis via regulating endothelial cell proliferation. The X-box binding protein 1 (XBP1) is believed to be a signal transducer in the endoplasmic reticulum stress response. It is unknown whether there is crosstalk between vascular endothelial cell growth factor signaling and XBP1 pathway. 

METHODS AND RESULTS - : We found that vascular endothelial cell growth factor induced the kinase insert domain receptor internalization and interaction through C-terminal domain with the unspliced XBP1 and the inositol requiring enzyme 1 α in the endoplasmic reticulum, leading to inositol requiring enzyme 1 α phosphorylation and XBP1 mRNA splicing, which was abolished by siRNA-mediated knockdown of kinase insert domain receptor. Spliced XBP1 regulated endothelial cell proliferation in a PI3K/Akt/GSK3β/β- catenin/E2F2-dependent manner and modulated the cell size increase in a PI3K/Akt/GSK3β/β-catenin/E2F2-independent manner. Knockdown of XBP1 or inositol requiring enzyme 1 α decreased endothelial cell proliferation via suppression of Akt/GSK3β phosphorylation, β-catenin nuclear translocation, and E2F2 expression. Endothelial cell-specific knockout of XBP1 (XBP1ecko) in mice retarded the retinal vasculogenesis in the first 2 postnatal weeks and impaired the angiogenesis triggered by ischemia. Reconstitution of XBP1 by Ad-XBP1s gene transfer significantly improved angiogenesis in ischemic tissue in XBP1ecko mice. Transplantation of bone marrow from wild-type o XBP1ecko mice could also slightly improve the foot blood reperfusion in ischemic XBP1ecko mice. 

CONCLUSIONS - : These results suggest that XBP1 can function via growth factor signaling pathways to regulate endothelial proliferation and angiogenesis. 

Lipoxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions

Oxidative stress is implicated in the pathogenesis of numerous disease processes including diabetes mellitus, atherosclerosis, ischaemia reperfusion injury and rheumatoid arthritis. Chemical modification of amino acids in protein during lipid peroxidation results in the formation of lipoxidation products which may serve as indicators of oxidative stress in vivo. The focus of the studies described here was initially to identify chemical modifications of protein derived exclusively from lipids in order to assess the role of lipid peroxidative damage in the pathogenesis of disease. Malondialdehye (MDA) and 4-hydroxynonenal (HNE) are well characterized oxidation products of polyunsaturated fatty acids on low-density lipoprotein (LDL) and adducts of these compounds have been detected by immunological means in atherosclerotic plaque. Thus, we first developed gas chromatography-mass spectrometry assays for the Schiff base adduct of MDA to lysine, the lysine-MDA-lysine diimine cross-link and the Michael addition product of HNE to lysine. Using these assays, we showed that the concentrations of all three compounds increased significantly in LDL during metal-catalysed oxidation in vitro. The concentration of the advanced glycation end-product N epsilon-(carboxymethyl)lysine (CML) also increased during LDL oxidation, while that of its putative carbohydrate precursor the Amadori compound N epsilon-(1-deoxyfructose-1-yl)lysine did not change, demonstrating that CML is a marker of both glycoxidation and lipoxidation reactions. These results suggest that MDA and HNE adducts to lysine residues should serve as biomarkers of lipid modification resulting from lipid peroxidation reactions, while CML may serve as a biomarker of general oxidative stress resulting from both carbohydrate and lipid oxidation reactions.