Nonvascular role for VEGF: VEGFR-1, 2 activity is critical for neural retinal development.
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Angiogenesis Inhibitors↗
Biomedical subjects
G S Robinson
Publications and source records attributed to G S Robinson.
Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor.
Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.
Animals↗
Oligonucleotide-based inhibition of embryonic gene expression.
We describe a technique to define gene function using antisense oligonucleotide (AS-ODN) inhibition of gene expression in mice. A single intravenous injection of an AS-ODN targeting vascular endothelial growth factor (VEGF) into pregnant mice between E7.5-8.5 resulted in a lack of primary angiogenesis. This enabled us to define the critical window required to inhibit VEGF expression and recapitulate the primary loss of function phenotype observed in VEGF (-/-) embryos. This phenotype was sequence-specific and time- and dose-dependent. Injection of an AS-ODN targeting a second gene, E-cadherin, into pregnant mice at E10 confirmed a hypothesized secondary phenotype. This is the first report of AS-ODN inhibition of gene expression in utero and provides a new strategy for target validation in functional genomics.
Animals↗
A new class of auditory warning signals for complex systems: auditory icons.
This simulator-based study examined conventional auditory warnings (tonal, nonverbal sounds) and auditory icons (representational, nonverbal sounds), alone and in combination with a dash-mounted visual display, to present information about impending collision situations to commercial motor vehicle operators. Brake response times were measured for impending front-to-rear collision scenarios under 6 display configurations, 2 vehicle speeds, and 2 levels of headway. Accident occurrence was measured for impending side collision scenarios under 2 vehicle speeds, 2 levels of visual workload, 2 auditory displays, absence/presence of mirrors, and absence/presence of a dash-mounted iconic visual display. For both front-to-rear and side collision scenarios, auditory icons elicited significantly improved driver performance over conventional auditory warnings. Driver performance improved when collision warning information was presented through multiple modalities. Brake response times were significantly faster for impending front-to-rear collision scenarios using the longer headway condition. The presence of mirrors significantly reduced the number of accidents for impending side collision scenarios. Subjective preference data indicated that participants preferred multimodal displays over single-modality displays. Actual or potential applications for this research include auditory displays and warnings, information presentation, and the development of alternative user interfaces.
Accidents, Traffic↗
Transcription factors Sp1 and Sp3 alter vascular endothelial growth factor receptor expression through a novel recognition sequence.
Kinase domain receptor (KDR) is a high affinity, endothelial cell-specific, autophosphorylating tyrosine kinase receptor for vascular endothelial growth factor. This transcriptionally regulated receptor is a critical mediator of endothelial cell (EC) growth and vascular development. In this study, we identify a DNA element modulating KDR promoter activity and evaluate the nuclear binding proteins accounting for a portion of the cell-type specificity of the region. KDR promoter luciferase activity was retained within -85/+296 and was 10-30-fold higher in EC than non-EC. Electrophoretic mobility shift assays demonstrated specific nuclear protein binding to -85/-64, and single point mutations suggested important binding nucleotides between -79/-68 with five critical bases between -74/-70 (5'-CTCCT-3'). DNA-protein complexes were displaced by Sp1 consensus sequence oligodeoxynucleotides and supershifted by Sp1- and Sp3-specific antibodies. Sp1 and Sp3 protein in EC nuclear extracts bound the -79/-68 region even when all surrounding classic Sp1 recognition sites were removed. Sp1 protein in nuclear extracts was 4-24-fold higher in EC than non-EC, whereas Sp3 was 3-7-fold higher. Sp1/Sp3 ratios in EC were 2-10-fold higher. Overexpression of Sp1 protein increased KDR promoter activity 3-fold in both EC and non-EC, whereas simultaneous co-expression of Sp3 attenuated this response. An Sp1 consensus sequence cis element "decoy" reduced EC KDR promoter activity and mRNA expression by 85 and 69%, respectively. An antisense phosphorothioate oligodeoxynucleotide to Sp1 inhibited Sp1 and KDR protein expression by 66 and 68%, respectively, without changing Sp3 protein expression. These data illustrate that Sp1 and Sp3 modulate KDR promoter activity through a novel recognition binding sequence. However, since Sp1-mediated promoter activation is attenuated by Sp3, endothelial selective KDR promoter activity may be partially regulated by variations in the Sp1/Sp3 ratio.
Base Sequence↗
Angiogenic factors in diabetic ocular disease: mechanisms of today, therapies for tomorrow.
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Adult↗
Antisense oligonucleotides inhibit vascular endothelial growth factor/vascular permeability factor expression in normal human epidermal keratinocytes.
In psoriatic lesions, epidermal keratinocytes overexpress vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) and transforming growth factor alpha (TGF-alpha). TGF-alpha has been shown to induce VEGF/VPF in normal human epidermal keratinocytes in vitro. By using a 19-mer antisense phosphorothioate oligodeoxynucleotide (PS-ODN) complementary to bases 6-24 relative to the translational start site of the VEGF/VPF mRNA, the control sense and mismatched PS-ODNs, we examined modulation of VEGF/VPF induction by TGF-alpha in vitro. Normal human epidermal keratinocytes were treated with PS-ODNs and Lipofectin for 8 h prior to the addition of TGF-alpha. Inhibition was assayed at the level of secreted protein by capture ELISA and mRNA expression was assayed by Northern blot analysis. The anti-sense PS-ODN was capable of inhibiting VEGF/VPF RNA and protein to near-basal levels. This inhibition was concentration dependent. No effect was observed with the sense or mismatch control PS-ODNs. These studies suggest that antisense oligonucleotide technology may be a potential therapy for the inhibition of angiogenesis associated with certain skin disorders such as psoriasis.
Down-Regulation↗
Characterization of vascular endothelial growth factor's effect on the activation of protein kinase C, its isoforms, and endothelial cell growth.
Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen which mediates its effects by binding to tyrosine kinase receptors. We have characterized the VEGF-activated intracellular signal transduction pathway in bovine aortic endothelial cells and correlated this to its mitogenic effects. VEGF induced concentration- and time-dependent increases in protein kinase C (PKC) activation with a maximum of 2.2-fold above the basal level at 5 x 10(-10) M within 10 min as measured both by in situ and translocation assays. Immunoblotting analysis of PKC isoforms in cytosolic and membrane fractions indicated that after VEGF stimulation the content of Ca(2+)-sensitive PKC isoforms (alpha and betaII) was increased in the membrane fractions, whereas no changes were observed for PKC isoforms delta and epsilon. The stimulation of PKC activity by VEGF was preceded by the activation of phospholipase Cgamma (PLCgamma). This was demonstrated by parallel increases in PLCgamma tyrosine phosphorylation, [3H]inositol phosphate production, and [3H]arachidonic acid-labeled diacylglycerol formation in bovine aortic endothelial cells. In addition, VEGF increased phosphatidylinositol 3-kinase activity 2.1-fold which was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, without decreasing the VEGF-induced increase in PKC activity or endothelial cell growth. Interestingly, genistein, a tyrosine kinase inhibitor, and GFX or H-7, PKC inhibitors, abolished both VEGF-induced PKC activation and endothelial cell proliferation. VEGF's mitogenic effect was inhibited by a PKC isoform beta-selective inhibitor, LY333531, in a concentration-dependent manner. In contrast, antisense PKC-alpha oligonucleotides enhanced VEGF-stimulated cell growth with a simultaneous decrease of 70% in PKC-alpha protein content. Thus, VEGF appears to mediate its mitogenic effects partly through the activation of the PLCgamma and PKC pathway, involving predominately PKC-beta isoform activation in endothelial cells.
Animals↗
Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.
Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.
Animals↗
Regulation of VEGF/VPF expression in tumor cells: consequences for tumor growth and metastasis.
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF) is a multifunctional cytokine which potently stimulates angiogenesis in vivo. VEGF/VPF expression is elevated in pathological conditions including cancer, proliferative retinopathy, psoriasis and rheumatoid arthritis. The angiogenesis associated with human tumors is likely a central component in promoting tumor growth and metastatic potential. The regulation of VEGF/VPF expression during tumor progression may involve diverse mechanisms including activated oncogenes, mutant or deleted tumor suppressor genes, cytokine activation, hormonal modulators, and a particularly effective activator, hypoxia. Understanding the diverse mechanisms by which tumor cells overexpress VEGF/VPF, and which mechanisms are operating in specific tumor types is important for the design of effective anti-cancer therapies.
Animals↗
Adenosine mediates hypoxic induction of vascular endothelial growth factor in retinal pericytes and endothelial cells.
PURPOSE: To determine the mechanistic role for adenosine and adenosine receptors in the hypoxic induction of vascular endothelial growth factor (VEGF) in retinal microvascular cells. METHODS: Bovine retinal capillary endothelial cells and microvascular pericytes were studied under normoxic (95% air, 5% CO2) or hypoxic conditions (0% to 2% O2, 5% CO2, 93% to 95% N2) using a variety of well-characterized adenosine and adenosine receptor agonists and antagonists. Vascular endothelial growth factor mRNA expression was evaluated by Northern blot analysis, VEGF protein levels were determined by Western blot analysis, and cyclic adenosine monophosphate (cAMP) accumulation was measured by radioimmunoassay. RESULTS: Inhibitors of oxidative respiration increased VEGF mRNA 5 +/- 3 times (P < 0.001) after 3 hours. Adenosine A1 receptor (A1R) agonist N6-cyclopentyl-adenosine did not increase VEGF mRNA at A1R stimulatory concentrations; however, adenosine A2 receptor (A2R) agonists DPMA, NECA, and CGS21680 increased VEGF mRNA in a dose-dependent manner with elevations of 2 +/- 0.3 (P < 0.001), 2.3 +/- 0.5 (P = 0.016), and 2 +/- 0.2 (P = 0.002) times, respectively. A2R antagonist CSC and adenosine degradation by adenosine deaminase reduced hypoxic stimulation of VEGF mRNA 68% +/- 18% (P = 0.038) and 37% +/- 6% (P = 0.025), respectively, in a dose-dependent manner. A1R antagonists DPCPX and 8-PT had no significant effect. Hypoxia and NECA increased VEGF protein secretion 4.7 times, whereas CSC inhibited hypoxia-induced VEGF protein secretion by 96%. NECA and CGS21680 increased cAMP production within 10 minutes, and cAMP stimulation increased VEGF mRNA 4.8 +/- 2.6 times (P = 0.034). CSC suppressed the hypoxic elevation of cAMP (P < 0.05). Inhibition of protein kinase A using H-89 reduced hypoxia-induced VEGF expression 61% +/- 6.3% (P = 0.043) in a dose-dependent manner. CONCLUSIONS: These data suggest that the hypoxia-induced accumulation of adenosine stimulates VEGF gene expression through stimulation of adenosine A2a receptor and subsequent activation of the cAMP-dependent protein kinase A pathway in retinal vascular cells.
Adenosine↗
Audibility of reverse alarms under hearing protectors for normal and hearing-impaired listeners.
The question of whether or not an individual suffering from a hearing loss is capable of hearing an auditory alarm or warning is an extremely important industrial safety issue. The ISO Standard that addresses auditory warnings for workplaces requires that any auditory alarm or warning be audible to all individuals in the workplace including those suffering from a hearing loss and/or wearing hearing protection devices (HPDs). Research was undertaken to determine how the ability to detect an alarm or warning signal changed for individuals with normal hearing and two levels of hearing loss as the levels of masking noise and alarm were manipulated. Pink noise was used as the masker and a heavy-equipment reverse alarm was used as the signal. The rating method paradigm of signal detection theory was used as the experimental procedure to separate the subjects' absolute sensitivities to the alarm from their individual criteria for deciding to respond in an affirmative manner. Results indicated that even at a fairly low signal-to-noise ratio (0 dB), subjects with a substantial hearing loss [a pure-tone average (PTA) hearing level of 45-50 dBHL in both ears] were capable of hearing the reverse alarm while wearing a high-attenuation earmuff in the pink noise used in the study.
Adult↗
Identification of multiple genes in bovine retinal pericytes altered by exposure to elevated levels of glucose by using mRNA differential display.
Loss of capillary pericytes, a characteristic finding in diabetic retinopathy, is strongly associated with hyperglycemia. The pathologic aberrations associated with diabetic retinopathy are localized primarily in the retinal capillaries and are only poorly reversed by subsequent euglycemic control. Since hyperglycemia significantly inhibits pericyte growth in culture, we investigated the regulation of gene expression in retinal pericytes exposed to physiologic (5.5mM) and pathologic (20 mM) glucose concentrations. By utilizing modifications of the mRNA differential display technique, over 14,000 mRNA species were screened, and 35 candidate clones were obtained. Partial DNA sequence demonstrated that 25 of these were distinct genes, including 7 known, 16 previously unreported, and 2 sequences with known homologues. Northern blot analysis demonstrated altered gene expression in 10 (40%), undetectable signals in 12 (48%), and nonregulation in 3 (12%). Genes with glucose-regulated expression included those encoding fibronectin (51% +/- 15%, P = 0.003; mean percentage of control +/- SD), caldesmon (68% +/- 18%; P = 0.026), two ribosomal proteins (201% +/- 72%, P = 0.011; 136% +/- 16%, P = 0.036), Rieske FeS reductase (66% +/- 17%; P = 0.029), three previously unreported sequences (57%, 167%, 271%), and molecules homologous to autoantigens (213%) and tyrosine kinases (down 16- to 33-fold). Caldesmon protein concentrations in pericytes and smooth muscle cells demonstrated decreases by Western blot analysis concordant with mRNA levels. These studies identify genes whose expression is significantly altered after 7 days of exposure to elevated glucose levels and provide new targets for understanding the adverse effects of hyperglycemia on vascular cells. In addition, this study provides strong support for the use of differential mRNA display as a method to rapidly isolate differentially expressed genes in metabolic systems.
Amino Acid Sequence↗
Volumetric and visual assessment of the mesial temporal structures in Alzheimer's disease.
BACKGROUND: Alzheimer's disease is the commonest cause of dementia. Clinical diagnosis of Alzheimer's disease may be difficult. Magnetic resonance imaging has a role to play in diagnosis. AIM: To assess whether volumetric and/or visual assessment of the mesial temporal structures is useful in separating patients with Alzheimer's disease from age matched controls. METHODS: Twenty-four patients with Alzheimer's disease diagnosed by NINCDS/ADRDA criteria and 15 age matched controls were studied with magnetic resonance imaging (MRI) and volumetric techniques. Segmented volumes of the mesial temporal structures were assessed visually and volumetrically. RESULTS: Volumetric analysis demonstrated significant (p < .001) differences between the two groups, but showed overlap in individual cases. Discriminant function analysis predicted correct group membership (patient or control) in 85% of cases. Visual assessment alone demonstrated a sensitivity of 92% and a specificity of 93% in distinguishing the Alzheimer patients from controls. CONCLUSION: Volumetric and visual assessment of the mesial temporal structures is useful in separating Alzheimer patients from controls. Overlap is present in individual cases. Visual assessment was as useful in separating the two groups as the volumetric analysis.
Aged↗
Transiently and stably introduced CCAAT/enhancer-binding-protein genes are constitutively expressed in cultured cells.
CCAAT/enhancer-binding protein (C/EBP) is expressed in certain cell types including hepatocytes and adipocytes. In order to understand the mechanisms that control the expression of the mouse C/EBP gene in the liver as well as in adipocytes, we have studied both the endogenous gene and transfected C/EBP gene constructs. The initiation site of transcription was identified and a strong liver-specific DNase-I hypersensitive site located at -3 kb, which does not appear to contribute functionally to the regulation of the gene in a variety of either transiently or stably transfected cells with constructs which include sequences up to 6-kb upstream of the transcription start. C/EBP gene expression during the transition from preadipocytes to adipocytes was shown to be controlled at the level of transcription. However, adipocytes stably transfected with constructs that include -3.3 kb upstream of the C/EBP gene do not express the reporter genes in a differentiation-specific manner. We detected several DNA-binding proteins that interact with the upstream sites of the C/EBP gene. Those include two labile and two heat-stable site-specific DNA-binding proteins that are present in nuclear extracts from several tissues and cultured cell lines.
Adipose Tissue↗
Fatty acid regulation of gene expression. Transcriptional and post-transcriptional mechanisms.
Fatty acids are important metabolic substrates and may also be involved in pathological syndromes such as the insulin resistance of diabetes and obesity. We demonstrate here that fatty acids can regulate specific gene expression; mRNAs encoding the fatty acid binding protein adipocyte P2 (aP2) and the Fos-related transcription factor Fra1 are specifically induced at least 20-fold upon treatment of preadipocytes with oleate. For aP2, the effect requires long chain fatty acids and occurs without a generalized activation of the genes linked to adipocyte differentiation. Other fibroblastic cells without preadipocyte characteristics do not induce aP2 mRNA in response to fatty acids. Unlike aP2, Fra1 induction by fatty acids also can be detected in NIH 3T3 and 3T3-C2 fibroblasts. Nuclear transcription assays in 3T3-F442A preadipocytes demonstrate that fatty acids elicit no transcriptional increase in the aP2 gene. Fra1, on the other hand, shows a 3-4-fold increase in transcription. These results demonstrate at least two distinct mechanisms by which fatty acids may influence gene expression.
3T3 Cells↗
Yeast GCN4 as a probe for oncogenesis by AP-1 transcription factors: transcriptional activation through AP-1 sites is not sufficient for cellular transformation.
The Jun and Fos oncoproteins belong to the AP-1 family of transcriptional activators and are believed to induce cellular transformation by inappropriately activating genes involved in cell replication. To determine whether transcriptional activation through AP-1 sites is sufficient for transforming activity, we examined the properties of an autonomous and heterologous AP-1 protein, yeast GCN4, in rat embryo fibroblasts. GCN4 induces transcriptional activation through AP-1 sites but, unlike Jun and Fos, fails to induce cellular transformation, in cooperation with Ha-ras. Jun-GCN4 and Fos-GCN4 homodimers independently induce cellular transformation indicating that the amino-terminal regions of Jun and Fos each contain regulatory functions that are required for oncogenesis but are distinct from generic transcriptional activation domains. In addition, these observations have implications for the nature of the oncogenically relevant target genes that respond to Jun and Fos.
Base Sequence↗
Effects of extracellular matrix on hepatocyte growth and gene expression: implications for hepatic regeneration and the repair of liver injury.
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Animals↗