Distal retinal ganglion cell axon transport loss and activation of p38 MAPK stress pathway following VEGF-A antagonism.

There is increasing evidence that VEGF-A antagonists may be detrimental to neuronal health following ocular administration. Here we investigated firstly the effects of VEGF-A neutralization on retinal neuronal survival in the Ins2(Akita) diabetic and JR5558 spontaneous choroidal neovascularization (CNV) mice, and then looked at potential mechanisms contributing to cell death. We detected elevated apoptosis in the ganglion cell layer in both these models following VEGF-A antagonism, indicating that even when vascular pathologies respond to treatment, neurons are still vulnerable to reduced VEGF-A levels. We observed that retinal ganglion cells (RGCs) seemed to be the cells most susceptible to VEGF-A antagonism, so we looked at anterograde transport in these cells, due to their long axons requiring optimal protein and organelle trafficking. Using cholera toxin B-subunit tracer studies, we found a distal reduction in transport in the superior colliculus following VEGF-A neutralization, which occurred prior to net RGC loss. This phenomenon of distal transport loss has been described as a feature of early pathological changes in glaucoma, Alzheimer's and Parkinson's disease models. Furthermore, we observed increased phosphorylation of p38 MAPK and downstream Hsp27 stress pathway signaling in the retinas from these experiments, potentially providing a mechanistic explanation for our findings. These experiments further highlight the possible risks of using VEGF-A antagonists to treat ocular neovascular disease, and suggest that VEGF-A may contribute to the maintenance and function of axonal transport in neurons of the retina.

Targeted deletion of p97 (VCP/CDC48) in mouse results in early embryonic lethality.

The highly conserved AAA ATPase p97 (VCP/CDC48) has well-established roles in cell cycle progression, proteasome degradation and membrane dynamics. Gene disruption in Saccromyces cerevisiae, Drosophila melanogaster and Trypanosoma brucei demonstrated that p97 is essential in unicellular and multicellular organisms. To explore the requirement for p97 in mammalian cell function and embryogenesis, we disrupted the p97 locus by gene targeting. Heterozygous p97+/- mice were indistinguishable from their wild-type littermates, whereas homozygous mutants did not survive to birth and died at a peri-implantation stage. These results show that p97 is an essential gene for early mouse development.

Centrosome disorganization in fibroblast cultures derived from R6/2 Huntington's disease (HD) transgenic mice and HD patients.

Huntington's disease (HD) is a progressive neurological disorder caused by a CAG/polyglutamine repeat expansion. We have previously generated the R6/2 mouse model that expresses exon 1 of the human HD gene containing CAG repeats in excess of 150. These mice develop a progressive neurological phenotype with a rapid onset and progression. We show here that it is impossible to establish fibroblast lines from these mice at 12 weeks of age, whilst this can be achieved without difficulty at 6 and 9 weeks. Cultures derived from mice at 12 weeks contained a high frequency of dysmorphic cells, including cells with an aberrant nuclear morphology and a high frequency of micronuclei and large vacuoles. All of these features were also present in a line derived from a juvenile HD patient. Fibroblast lines derived from R6/2 mice and from HD patients were found to have a high frequency of multiple centrosomes which could account for all of the observed phenotypes including a reduced mitotic index, high frequency of aneuploidy and persistence of the midbody. We were unable to detect large insoluble polyglutamine aggregates in either the mouse or human lines. We have identified a novel progressive HD pathology that occurs in cells of non-central nervous system origin. An investigation of the pathological consequences of the HD mutation in these cells will provide insight into cellular basis of the disease.

Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum.

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589-601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP-Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.

Vascular developmental biology: getting nervous.

First described in the developing nervous system, Semaphorin III/Neuropilin, Ephrin/Eph, and Delta/Notch signaling relays have now been implicated in the elaboration of the blood vessel network during embryogenesis.

An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion.

The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, we have introduced a mutation into mammalian NSF that, in Drosophila dNSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs.

Segregation of COPI-rich and anterograde-cargo-rich domains in endoplasmic-reticulum-to-Golgi transport complexes.

Membrane traffic between the endoplasmic reticulum (ER) and the Golgi complex is regulated by two vesicular coat complexes, COPII and COPI. COPII has been implicated in the selective packaging of anterograde cargo into coated transport vesicles budding from the ER [1]. In mammalian cells, these vesicles coalesce to form tubulo-vesicular transport complexes (TCs), which shuttle anterograde cargo from the ER to the Golgi complex [2] [3] [4]. In contrast, COPI-coated vesicles are proposed to mediate recycling of proteins from the Golgi complex to the ER [1] [5] [6] [7]. The binding of COPI to COPII-coated TCs [3] [8] [9], however, has led to the proposal that COPI binds to TCs and specifically packages recycling proteins into retrograde vesicles for return to the ER [3] [9]. To test this hypothesis, we tracked fluorescently tagged COPI and anterograde-transport markers simultaneously in living cells. COPI predominated on TCs shuttling anterograde cargo to the Golgi complex and was rarely observed on structures moving in directions consistent with retrograde transport. Furthermore, a progressive segregation of COPI-rich domains and anterograde-cargo-rich domains was observed in the TCs. This segregation and the directed motility of COPI-containing TCs were inhibited by antibodies that blocked COPI function. These observations, which are consistent with previous biochemical data [2] [9], suggest a role for COPI within TCs en route to the Golgi complex. By sequestering retrograde cargo in the anterograde-directed TCs, COPI couples the sorting of ER recycling proteins [10] to the transport of anterograde cargo.

The mouse p97 (CDC48) gene. Genomic structure, definition of transcriptional regulatory sequences, gene expression, and characterization of a pseudogene.

Here we present the first description of the genomic organization, transcriptional regulatory sequences, and adult and embryonic gene expression for the mouse p97(CDC48) AAA ATPase. Clones representing two distinct p97 genes were isolated in a genomic library screen, one of them likely representing a non-functional processed pseudogene. The coding region of the gene encoding the functional mRNA is interrupted by 16 introns and encompasses 20.4 kilobase pairs. Definition of the transcriptional initiation site and sequence analysis showed that the gene contains a TATA-less, GC-rich promoter region with an initiator element spanning the transcription start site. Cis-acting elements necessary for basal transcription activity reside within 410 base pairs of the flanking region as determined by transient transfection assays. In immunohistological analyses, p97 was widely expressed in embryos and adults, but protein levels were tightly controlled in a cell type- and cell differentiation-dependent manner. A remarkable heterogeneity in p97 immunostaining was found on a cellular level within a given tissue, and protein amounts in the cytoplasm and nucleus varied widely, suggesting a highly regulated and intermittent function for p97. This study provides the basis for a detailed analysis of the complex regulation of p97 and the reagents required for assessing its functional significance using targeted gene manipulation in the mouse.

Synergistic roles for Pim-1 and c-Myc in STAT3-mediated cell cycle progression and antiapoptosis.

The activation of STAT3 by the cytokine receptor gp130 is required for both the G1 to S cell cycle transition and antiapoptosis. We found that Pim-1 and Pim-2 are targets for the gp130-mediated STAT3 signal. Expression of a kinase-defective Pim-1 mutant attenuated gp130-mediated cell proliferation. Constitutive expression of Pim-1 together with c-myc, another STAT3 target, fully compensated for loss of the STAT3-mediated cell cycle progression, antiapoptosis, and bcl-2 expression. We also identified valosine-containing protein (VCP) as a target gene for the Pim-1-mediated signal. Expression of a mutant VCP led cells to undergo apoptosis. These results indicate that Pim-family proteins play crucial roles in gp130-mediated cell proliferation and explain the synergy between Pim and c-Myc proteins in cell proliferation and lymphomagenesis.

Inheritance of the mammalian Golgi apparatus during the cell cycle.

The creation and propagation of the intricate Golgi architecture during the cell cycle poses a fascinating problem for biologists. Similar to the inheritance process for nuclear DNA, the inheritance of the Golgi apparatus consists of biogenesis (replication) and partitioning (mitosis/meiosis) phases, in which Golgi components must double in unit mass, then be appropriately divided between nascent daughter cells during cytokinesis. In this article we focus discussion on the recent advances in the area of Golgi inheritance, first outlining our current understanding of the behaviour of the Golgi apparatus during cell division, then concluding with a more conceptual discussion of the Golgi biogenesis problem. Throughout, we attempt to integrate ultrastructural and biochemical findings with more recent information obtained using live cell microscopy and morphological techniques.

An ordered inheritance strategy for the Golgi apparatus: visualization of mitotic disassembly reveals a role for the mitotic spindle.

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1-3-micron fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.

An isoform of the Golgi t-SNARE, syntaxin 5, with an endoplasmic reticulum retrieval signal.

The early Golgi t-SNARE (target-membrane-associated soluble-N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 5 is thought to specify the docking site for both COPI and COPII coated vesicles originating from the endoplasmic reticulum (ER) and COPI vesicles on the retrograde pathway. We now show that there are two forms of syntaxin 5 that appear to be generated from the same mRNA by alternative initiation of translation. The short form (35 kDa) corresponds to the published sequence. The long form (42 kDa) has an N-terminal cytoplasmic extension containing a predicted type II ER retrieval signal. When grafted onto a reporter molecule, this signal localized the construct to the ER. Biochemical fractionation and immunofluorescence microscopy showed that there was less of the long form in the Golgi apparatus and more in peripheral punctate structures, some of which colocalized with markers of the intermediate compartment. The predicted absence of the long form in budding yeast points to a function unique to higher organisms.

Partitioning of the Golgi apparatus during mitosis in living HeLa cells.

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I-GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.

Up-regulation of vascular endothelial growth factor production by iron chelators.

Agents that modulate cellular iron availability have been studied for their antitumor activity. Based on encouraging in vitro studies, the iron chelator deferoxamine (DFO) has been used in clinical studies to treat cancer patients. The observation that DFO induced macular edema in several cancer patients led to the present investigation of vascular endothelial growth factor (VEGF) as a possible mediator of the encountered side effects. Both normal and malignant cell lines were incubated with DFO and a variety of other iron chelators. DFO, at concentrations achievable in humans, induced a 3-5-fold increase in VEGF mRNA expression in all cell lines studied. This increased VEGF mRNA expression was dose and time dependent. A panel of structurally different iron chelators induced an even more potent increase in VEGF mRNA expression. The DFO-induced increase in VEGF mRNA expression translated into 6- and 4-fold increases in VEGF protein secretion in conditioned media of retinal pigment epithelial and C6 glioblastoma cells, respectively. These findings suggest that VEGF may act as a mediator of the side effects induced by iron chelation therapy. In addition, because VEGF is an important regulator of angiogenesis, iron chelators should be given with caution to cancer patients.

Cloning and mRNA expression of vascular endothelial growth factor in ischemic retinas of Macaca fascicularis.

PURPOSE: To identify and isolate cDNAs for the alternatively spliced vascular endothelial growth factor (VEGF) mRNAs present in retina and to compare the relative levels of the splice variants and localization of VEGF mRNA in nonischemic and ischemic adult simian retinas. METHODS: Retinas of cynomolgous monkeys were made ischemic by laser occlusion of the main branch retinal veins. Reverse transcription-polymerase chain reaction was used to amplify the VEGF coding region of RNA from ischemic and control retinas, and amplification products were analyzed by agarose gel electrophoresis, Southern blot, and nucleotide sequencing. Analysis of VEGF mRNA expression was accomplished by in situ hybridization. RESULTS: Control and ischemic retinas produce mRNAs that correspond to the 121 and 165 amino acid diffusible isoforms of VEGF, and relatively low levels of VEGF189, the heparin-binding isoform. There was no significant difference in the levels of the alternatively spliced VEGF transcripts between control and ischemic retinas. Cloning and sequencing revealed that simVEGF cDNAs are 99% identical to human VEGFs and are predicted to encode proteins identical to their respective human homologues. In situ hybridization of nonischemic retinas revealed a low level of VEGF mRNA in retinal ganglion cells and in the inner nuclear layer. VEGF mRNA levels were elevated in ischemic retinas as early as 1 day after laser vein occlusion, when there was a small but reproducible increase in signal. The expression peaked at approximately 13 days, coincident with maximal iris neovascularization, and was significantly reduced by 28 days, when the iris vessels largely regressed. CONCLUSIONS: The elevation of simVEGF121 and VEGF165 in ischemic retinas is consistent with a role for diffusible, retina-derived angiogenic factors in the development of ocular neovascularization.

Tumor angiogenesis: a physiological process or genetically determined?

Continued tumor growth is dependent upon the growth of new blood vessels. This commentary reviews the mechanisms whereby tumors become vascularized and examines whether tumor angiogenesis is solely an example of a normal physiologic process or is part of the genetic program of the tumor. The likelihood that neovascularization of tumors combines both of these components, that is, utilizing tumor-specific elements as well as capacities common to all cells, is discussed.

Matrix metalloproteinases and their inhibitors in aqueous humor.

Matrix metalloproteinase activity is the rate-limiting step in extracellular matrix degradation. One mechanism by which metalloproteinases are regulated is through the activity of their endogenous inhibitors, the tissue inhibitors of metalloproteinases. Since metalloproteinase activity is a key component of the angiogenic process and many anterior segment structures are largely avascular, we became interested in examining aqueous humor for the presence of metalloproteinases and their endogenous inhibitors. Using zymography, we have identified the presence of several metalloproteinases in normal aqueous humor. Treatment with 4-aminophenylmercuric acetate, an organomercurial which activates latent metalloproteinases, revealed that all metalloproteinases were in their active state. By Western blot analysis, normal aqueous humor was also found to contain at least two tissue inhibitors of metalloproteinases. Subsequent partial purification by two successive chromatographic steps revealed the presence of inhibitory activity against collagenase, endothelial cell DNA synthesis, and angiogenesis on the chick chorioallantoic membrane. The presence of metalloproteinases and their inhibitors in normal aqueous humor, a fluid which bathes avascular ocular structures, suggests that future studies should examine whether an imbalance in this protease/inhibitor family may contribute to the anterior chamber extracellular matrix alterations associated with diseases such as ocular neovascularization and glaucoma.

The mouse gene for vascular endothelial growth factor. Genomic structure, definition of the transcriptional unit, and characterization of transcriptional and post-transcriptional regulatory sequences.

We describe the genomic organization and functional characterization of the mouse gene encoding vascular endothelial growth factor (VEGF), a polypeptide implicated in embryonic vascular development and postnatal angiogenesis. The coding region for mouse VEGF is interrupted by seven introns and encompasses approximately 14 kilobases. Organization of exons suggests that, similar to the human VEGF gene, alternative splicing generates the 120-, 164-, and 188-amino acid isoforms, but does not predict a fourth VEGF isoform corresponding to human VEGF206. Approximately 1. 2 kilobases of 5'-flanking region have been sequenced, and primer extension analysis identified a single major transcription initiation site, notably lacking TATA or CCAT consensus sequences. The 5'-flanking region is sufficient to promote a 7-fold induction of basal transcription. The genomic region encoding the 3'-untranslated region was determined by Northern and nuclease mapping analysis. Investigation of mRNA sequences responsible for the rapid turnover of VEGF mRNA (mRNA half-life, <1 h) (Shima, D. T. , Deutsch, U., and D'Amore, P. A. (1995) FEBS Lett. 370, 203-208) revealed that the 3'-untranslated region was sufficient to trigger the rapid turnover of a normally long-lived reporter mRNA in vitro. These data and reagents will allow the molecular and genetic analysis of mechanisms that control the developmental and pathological expression of VEGF.

Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate.

OBJECTIVE: To determine if the angiogenic peptide vascular endothelial growth factor (VEGF) is required for retinal ischemia-associated iris neovascularization in a nonhuman primate. METHODS: Laser retinal vein occlusion was used to produce retinal ischemia in 16 eyes of eight animals (Macaca fascicularis). Eyes were randomized to treatment every other day with intravitreal injections of either a neutralizing anti-VEGF monoclonal antibody or a control monoclonal antibody of the same isotype. Serial iris fluorescein angiograms were assessed using a standardized grading system and masked readers. Retinal VEGF and placental growth factor expression were assessed by Northern blotting. The specificity of the antibodies was determined in capillary endothelial cell proliferation assays prior to intravitreal injection. RESULTS: Zero of eight eyes receiving the neutralizing anti-VEGF antibodies developed iris neovascularization. Five of eight control antibody-treated eyes developed iris neovascularization. The difference was statistically significant (P = .03). Intravitreal antibody injection did not impair the ability of the ischemic retina to increase VEGF messenger RNA expression. The anti-VEGF antibodies specifically inhibited VEGF-driven capillary endothelial cell proliferation in vitro. CONCLUSION: These data demonstrate that VEGF is required for iris neovascularization in an adult nonhuman primate eye. The inhibition of VEGF is a new potential therapeutic strategy for the treatment of ocular neovascularization.