Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis.

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). In this study we report the creation of a novel mouse model in which GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in Purkinje cells (PCs). GRP78-depleted PCs activate UPR including the induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2alpha phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding in which an abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78-null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells.

In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3'-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

Pathology of damaging electrical stimulation in the retina.

The goal of this study was to examine the characteristics of electrically induced retinal damage. A retinal prosthesis must be both effective and safe, but most research related to electrical stimulation of the retina has involved measures of efficacy (for example, stimulus threshold), while relatively little research has investigated the safety of electrical stimulation. In this study, a single platinum microelectrode was inserted into the vitreous cavity of normally-sighted adult Long Evans pigmented rats. In one group of animals, no contact was made between the electrode and the retina and current pulses of 0.05 (n=3) and 0.2 (n=6) microC/phase were applied. In a second group, visible contact (slight dimpling of the retina) was made between the electrode and the retina and current pulses of 0.09 (n=4) microC/phase were applied. In both cases, stimulus pulses (biphasic, cathodic first, 1 ms/phase) were applied for 1 h at 100 Hz. Also, control experiments were run with no electrical stimulation with retina contact (n=4) and with no retinal contact (n=3). After stimulation, the animal was survived for 2 weeks with ocular photography and electroretinography (ERG) to document changes. During the follow-up period, retinal changes were observed only when the electrode contacted the retina, with or without electrical stimulation. No difference was noted in ERG amplitude or latency comparing the test eye to the stimulated eye. Histological analysis was performed after sacrifice at 2 weeks. A semi-quantitative method for grading 18 features of retina/RPE/choroidal appearance was established and integer grades applied to both test and control eyes. Using this method and comparing the most severely affected area (highest grade), significant differences (p<0.05) were noted between experiments with retinal contact and without retinal contact in all features except inner nuclear layer thickness. No difference was noted within a group based on the intensity of electrical stimulus applied. The size of the affected area was significantly larger with both retinal contact and electrical stimulation compared to with retinal contact alone. We conclude that mechanical pressure alone and mechanical pressure with excessive electrical stimulation causes damage to the retina but that electrical stimulation coupled with mechanical pressure increases the area of the damage.

Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy.

AIMS: To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors. Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes. RESULTS: In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-beta. The TGF-beta is activated, upregulating expression of CTGF. Under the influence of TGF-beta and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury. CONCLUSIONS: HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.

Interferon beta affects retinal pigment epithelial cell proliferation via protein kinase C pathways.

BACKGROUND: The aim of this study is to see the effect of interferon beta (IFN-beta) on cell proliferation and the protein kinase C (PKC) signaling pathway. METHODS: Proliferation of cultured human retinal pigment epithelium (RPE) cells, with various concentrations of IFN-beta, and with or without 3% fetal calf serum (FCS), was assessed by cell counting. Effects of short (3 h) or prolonged (48 h) exposure of RPE cells to natural human IFN-beta were assessed by (3)H-thymidine uptake. Cytosolic and membranous PKC activity over time in cells treated with IFN-beta and calphostin C was also measured. RESULTS: IFN-beta inhibited the increased proliferation by FCS in the prolonged-exposure assay. The PKC inhibitor calphostin C also showed an inhibitory effect on RPE cell growth and (3)H-thymidine uptake in the chronic exposure with FCS. Short treatment with IFN-beta had no inhibitory or stimulatory effect on (3)H-thymidine uptake. Cytosolic and membranous PKC activity was strongly upregulated after short IFN-beta exposure but returned to original levels after 1 h. PKC activity was downregulated both in the cytosol and membrane after 24 or 48 h. CONCLUSION: IFN-beta inhibited RPE proliferation in vitro and the effect is mediated by upregulation of the PKC pathway.

Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors.

Angiogenesis is required for the progression of tumors from a benign to a malignant phenotype and for metastasis. Malignant tumor cells secrete factors such as vascular endothelial growth factor (VEGF), which bind to their cognate receptors on endothelial cells to induce angiogenesis. Here it is shown that several tumor types express VEGF receptors (VEGFRs) and that inhibition of VEGF (VEGF antisense oligonucleotide AS-3) or VEGFRs (neutralizing antibodies) inhibited the proliferation of these cell lines in vitro. Furthermore, this effect was abrogated by exogenous VEGF. Thus, VEGF is an autocrine growth factor for tumor cell lines that express VEGFRs. A modified form of VEGF AS-3 (AS-3m), in which flanking 4 nucleotides were substituted with 2-O-methylnucleosides (mixed backbone oligonucleotides), retained specificity and was active when given orally or systemically in vitro and in murine tumor models. In VEGFR-2-expressing tumors, VEGF inhibition may have dual functions: direct inhibition of tumor cell growth and inhibition of angiogenesis.

Systemically expressed soluble Tie2 inhibits intraocular neovascularization.

Retinal and choroidal neovascularization are the most frequent causes of severe and progressive vision loss. Studies have demonstrated that Tie2, an endothelial-specific receptor tyrosine kinase, plays a key role in angiogenesis. In this study, we determined whether adenovirus-mediated gene delivery of extracellular domain of the Tie2 receptor (ExTek) could inhibit experimental retinal and choroidal neovascularization. Immunofluorescence histochemistry with a monoclonal antibody to human Tie2 showed that Tie2 expression is prominent around and within the base of newly formed blood vessels of retinal and choroidal neovascular lesions. A single intramuscular injection of adenovirus expressing ExTek genes achieved plasma levels of ExTek exceeding 500 microg/ml in mice for 10 days (in neonates) and 7 days (in adults). This treatment inhibited retinal neovascularization by 47% (p < 0.05) in a murine model of ischemia-induced retinopathy. The same treatment reduced the incidence and extent of sodium fluorescein leakage from choroidal neovascular lesions by 52% (p < 0.05) and 36% (p < 0.01), respectively, in a laser-induced murine choroidal neovascularization model. The same mice showed a 45% (p < 0.001) reduction of integrated area of the choroidal neovascularization. These findings indicate that Tie2 signaling is a common component of the angiogenic pathway in both retinal and choroidal neovascularization, providing a potentially useful target in the treatment of intraocular neovascular diseases.

Response of experimental retinal neovascularization to thiazolidinediones.

OBJECTIVE: To determine the effect of thiazolidinediones (TZDs) on experimental retinal neovascularization. METHODS: The ability of the TZDs troglitazone and rosiglitazone maleate (1-20 micromol/L) to inhibit retinal endothelial cell (REC) proliferation, migration, tube formation, and signaling was determined in response to vascular endothelial growth factor (VEGF). In vivo studies were performed using the oxygen-induced ischemia model of retinal neovascularization. Neonatal mice were treated with intravitreous injection of 0.5 microL of troglitazone (100 micromol/L) or rosiglitazone maleate (100 micromol/L), or vehicle, and retinal neovascularization was assayed qualitatively and quantitatively by means of angiography and histological examination. RESULTS: Expression of the TZD receptor, peroxisome proliferator-activated receptor gamma, was confirmed in RECs by means of Western immunoblotting. Rosiglitazone and troglitazone inhibited VEGF-induced migration (P< .05), proliferation (P< .05), and tube formation (P< .01) by RECs in vitro beginning at 10 micromol/L. Rosiglitazone and troglitazone inhibited phosphorylation of extracellular signal-regulated mitogen-activated protein kinase 1 in RECs. Intravitreous injection of rosiglitazone or troglitazone inhibited development of retinal neovascularization (P< .01) but did not significantly inhibit VEGF overexpression in the ganglion cell layer of the ischemic retina. CONCLUSION: The TZDs inhibit experimental retinal neovascularization with an effect that is primarily downstream of VEGF expression. CLINICAL RELEVANCE: The TZDs are widely prescribed and should be evaluated for their potential to inhibit the progression of diabetic retinopathy.

Angiopoietin-1 upregulation by vascular endothelial growth factor in human retinal pigment epithelial cells.

PURPOSE: To determine whether vascular endothelial growth factor (VEGF) regulates angiopoietin (Ang)-1 and -2 expression in retinal pigment epithelial (RPE) cells. METHODS: Expression of VEGF, Ang1, and Ang2 in surgically removed human choroidal neovascular membranes (CNVMs) was analyzed by double-label confocal immunofluorescence microscopy. Total RNA was extracted from cultured human RPE cells treated with VEGF for mRNA analysis. Northern blot analysis was performed to examine the time course and dose response of Ang1 and Ang2 mRNA expression. mRNA stability and nuclear run-on analyses were performed. Secreted Ang1 and Ang2 protein levels in conditioned media from RPE cells were examined by Western blot analysis. RESULTS: Ang1 and Ang2 immunostaining colocalized with VEGF-positive stromal cells in human CNVMS: Ang1 and Ang2 mRNAs were expressed by cultured serum-starved RPE cells. VEGF upregulated Ang1 mRNA in a time- and dose-dependent manner without a significant change in Ang2 mRNA. Ang1 and Ang2 mRNAs in RPE cells were as stable as that of S18. VEGF stimulation further increased the half-life of Ang1 mRNA, but did not alter its transcription rate. VEGF increased the amount of Ang1, but not Ang2, protein secreted into the medium. CONCLUSIONS: The colocalization of Ang1 and Ang2 with VEGF in CNVM stromal cells and the upregulation of Ang1 expression by VEGF in cultured RPE cells suggest that VEGF may selectively modulate Ang expression during CNV.

Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice.

Targeting cytocidal vectors to tumors and associated vasculature in vivo is a long-standing goal of human gene therapy. In the present study, we demonstrated that intravenous infusion of a matrix (i.e., collagen)-targeted retroviral vector provided efficacious gene delivery of a cytocidal mutant cyclin G1 construct (designated Mx-dnG1) in human cancer xenografts in nude mice. A nontargeted CAE-dnG1 vector (p = 0.014), a control matrix-targeted vector bearing a marker gene (Mx-nBg; p = 0.004), and PBS served as controls (p = 0.001). Enhanced vector penetration and transduction of tumor nodules (35.7 +/- 1.4%, mean +/- SD) correlated with therapeutic efficacy without associated toxicity. Kaplan-Meier survival studies were conducted in mice treated with PBS placebo, the nontargeted CAE-dnG1 vector, and the matrix-targeted Mx-dnG1 vector. Using the Tarone log-rank test, the overall p value for comparing all three groups simultaneously was 0.003, with a trend that was significant to a level of 0.004, indicating that the probability of long-term control of tumor growth was significantly greater with the matrix-targeted Mx-dnG1 vector than with the nontargeted CAE-dnG1 vector or PBS placebo. The present study demonstrates that a matrix-targeted retroviral vector deployed by peripheral vein injection (1) accumulated in angiogenic tumor vasculature within 1 hr, (2) transduced tumor cells with high-level efficiency, and (3) enhanced therapeutic gene delivery and long-term efficacy without eliciting appreciable toxicity.

Viral induced demyelination.

Viral induced demyelination, in both humans and rodent models, has provided unique insights into the cell biology of oligodendroglia, their complex cell-cell interactions and mechanisms of myelin destruction. They illustrate mechanisms of viral persistence, including latent infections in which no infectious virus is readily evident, virus reactivation and viral-induced tissue damage. These studies have also provided excellent paradigms to study the interactions between the immune system and the central nervous system (CNS). Although of interest in their own right, an understanding of the diverse mechanisms used by viruses to induce demyelination may shed light into the etiology and pathogenesis of the common demyelinating disorder multiple sclerosis (MS). This notion is supported by the persistent view that a viral infection acquired during adolescence might initiate MS after a long period of quiescence. Demyelination in both humans and rodents can be initiated by infection with a diverse group of enveloped and non-enveloped RNA and DNA viruses (Table 1). The mechanisms that ultimately result in the loss of CNS myelin appear to be equally diverse as the etiological agents capable of causing diseases which result in demyelination. Although demyelination can be a secondary result of axonal loss, in many examples of viral induced demyelination, myelin loss is primary and associated with axonal sparing. This suggests that demyelination induced by viral infections can result from: 1) a direct viral infection of oligodendroglia resulting in cell death with degeneration of myelin and its subsequent removal; 2) a persistent viral infection, in the presence or absence of infectious virus, resulting in the loss of normal cellular homeostasis and subsequent oligodendroglial death; 3) a vigorous virus-specific inflammatory response wherein the virus replicates in a cell type other than oligodendroglia, but cytokines and other immune mediators directly damage the oligodendroglia or the myelin sheath; or 4) infection initiates activation of an immune response specific for either oligodendroglia or myelin components. Virus-induced inflammation may be associated with the processing of myelin or oligodendroglial components and their presentation to the host's own T cell compartment. Alternatively, antigenic epitopes derived from the viral proteins may exhibit sufficient homology to host components that the immune response to the virus activates autoreactive T cells, i.e. molecular mimicry. Although it is not clear that each of these potential mechanisms participates in the pathogenesis of human demyelinating disease, analysis of the diverse demyelinating viral infections of both humans and rodents provides examples of many of these potential mechanisms.

Promotion of adhesion and migration of RPE cells to provisional extracellular matrices by TNF-alpha.

PURPOSE: Adhesion and migration of retinal pigment epithelial (RPE) cells to provisional extracellular matrices (ECM) is important in the development of epiretinal membranes found in proliferative vitreoretinopathy (PVR). Tumor necrosis factor alpha (TNF-alpha) is found in PVR membranes and regulates many functions of RPE cells. In this study, the effects of TNF-alpha on adhesion and migration of RPE cells to various components of ECM were examined and elucidation of the mechanism of the response was attempted. METHODS: Mitogen activated protein kinase (ERK1/2; MAPK) activation was measured by immunoblot. RPE cells pretreated with TNF-alpha (10 ng/ml) or TNF-alpha + PD98059 (a specific inhibitor of MAPK, 30 microM) for 24 hours were compared with control RPE. Attachment was measured by modified MTT assay on fibronectin and collagen types I and IV. Spreading was measured by staining with fluo3-AM and confocal laser scanning microscopy. Migration of RPE cells on substrates was determined by Boyden chamber assay using PDGF-BB (20 ng/ml) as a chemotactic factor. Integrin expression was determined by flow cytometry and RT-PCR. RESULTS: TNF-alpha rapidly activated MAPK and increased the extent of attachment, spreading and migration on fibronectin and collagen type I (P < 0.01) but not on collagen type IV. TNF-stimulated RPE cells showed increased mRNA and surface protein expression for alpha1 and alpha5 integrin (P < 0.01) but not alpha3 integrin subunit. Neutralizing the anti-alpha1 antibody inhibited migration on collagen type I, whereas alpha5 antibody inhibited fibronectin-induced migration. Treatment with both TNF and PD98095 reduced attachment and migration on provisional ECM and reduced the upregulated integrin expression to control levels. CONCLUSIONS: After treatment with TNF-alpha, there is increased expression of specific integrins associated with increased adhesion and migration on provisional ECM (fibronectin and collagen type I). This effect is mediated, at least in part, by activation of MAPK signaling pathway.

Inhibition of metastatic tumor growth in nude mice by portal vein infusions of matrix-targeted retroviral vectors bearing a cytocidal cyclin G1 construct.

Tumor invasion and associated angiogenesis evoke a remodeling of extracellular matrix components. Retroviral vectors bearing auxiliary matrix-targeting motifs (ie., collagen-binding polypeptides) accumulate at sites of newly exposed collagen, thus promoting tumor site-specific gene delivery. In this study, we assessed the antitumor effects of serial portal vein infusions of matrix-targeted vectors bearing a mutant cyclin G1 (dnG1) construct in a nude mouse model of liver metastasis. The size of tumor foci was dramatically reduced in dnG1 vector-treated mice compared with that in control vector- or PBS-treated animals (P = 0.0002). These findings represent a definitive advance in the development of targeted injectable vectors for metastatic cancer.

Peroxisome proliferator-activated receptor-gamma ligands inhibit choroidal neovascularization.

PURPOSE: To determine the antiangiogenic effects of peroxisome proliferator-activated receptor (PPAR)-gamma agonists on ocular cells involved in the pathogenesis of choroidal neovascularization (CNV) in vitro and on experimental laser photocoagulation-induced CNV in vivo. METHODS: PPAR-gamma expression in human retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (CECs) was determined using an RNase protection assay and Western blot analysis. Two PPAR-gamma ligands, troglitazone (TRO) and rosiglitazone (RSG; 0.1-20 microM), were used to assess effects on RPE and CEC proliferation and migration and CEC tube formation in response to vascular endothelial growth factor (VEGF). The effects of intravitreal injection of TRO on laser photocoagulation-induced CNV lesions in rat eyes (15 experimental, 15 control, nine burns per eye) and cynomolgus monkey eyes (two experimental, two control, seven paramacular burns per eye) was assessed by fluorescein angiography and histologic evaluation. RESULTS. PPAR-gamma1 was expressed in both RPE and CEC. PPAR-gamma ligands significantly inhibited VEGF-induced migration and proliferation in both cell types and tube formation of CEC in a dose-response manner. CNV in rats was markedly inhibited by intravitreous injection of TRO (P < 0.001). Lesions showed significantly less fluorescein leakage and were histologically thinner in the TRO-treated animals. Similar findings were present in the TRO-treated lesions in two monkey eyes. The drug showed no apparent adverse effects in the adjacent retina or in control eyes. CONCLUSIONS: The inhibition of VEGF-induced choroidal angiogenesis in vitro, and CNV in vivo by PPAR-gamma ligands suggests the potential application of these agents in the large group of patients with age-related macular degeneration complicated by CNV.

Selective killing of RPE with a vascular endothelial growth factor chimeric toxin.

PURPOSE: To determine the sensitivity of retinal pigment epithelial (RPE) cells to a vascular endothelial growth factor (VEGF) chimeric toxin. METHODS: A targeted toxin was developed using recombinant methods to fuse VEGF165 to the diphtheria toxin (DT) translocation and enzymatic domain (DT390-VEGF165). Human RPE cells, choroidal endothelial cells (CECs), and scleral fibroblasts were isolated, and a dose-response for DT390-VEGF165 was determined by measurement of cell proliferation and cell number. In parallel experiments, cultures were pretreated with transforming growth factor (TGF)-beta2. VEGF-receptor (VEGFR-1 and -2) expression was determined using reverse transcription-polymerase chain reaction and fluorescence-activated cell sorting, and affinity was measured using Scatchard analysis. RESULTS: RPE cells and CECs were similarly prone to killing by the VEGF-toxin, but scleral fibroblasts were unaffected. Pretreatment with TGF-beta2 selectively increased the sensitivity of RPE cells to the VEGF-toxin. RPE cells expressed both VEGFR-1 and -2 in vitro; however, the expression of VEGFR-1 was very low. Pretreatment with TGF-beta2 (10 ng/ml) was associated with increased expression of the VEGFR-1 in RPE cells and increased receptor affinity for VEGF detected by Scatchard analysis. CONCLUSIONS: Dose-dependent killing of RPE cells by the DT390-VEGF165 conjugate is selectively enhanced by pretreatment with TGF-beta2. This study provides further strong support for the presence of functional VEGFRs on human RPE cells, and demonstrates for the first time the ability to target a normal nonendothelial cell type through VEGFR expression.