Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.
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PMID:Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies. 1102 22

Accumulating evidence suggests the involvement of TGF-beta in the process of corneal opacity, which is one of the serious causes of visual loss. However, whether TGF-beta is indeed critical for the pathogenesis remains unknown. We constructed an adenovirus expressing an entire ectodomain of the human type II TGF-beta receptor fused to Fc portion of human IgG (AdTbeta-ExR): this soluble receptor is secreted from AdTbeta-ExR-infected cells, binds to TGF-beta and inhibits TGF-beta signaling. When AdTbeta-ExR was injected into the femoral muscle of Balb/c mice, a high level of the soluble receptor protein (2.0-3.5 x 10(3) pM) was detectable in the serum and in the ocular fluid for at least 10 days. In the mice subjected to corneal injury with silver nitrate and to intramuscular injection with either saline or a control adenovirus expressing beta-galactosidase (AdLacZ), corneal opacification composed of extracellular matrix (ECM) accumulation, of infiltration of neutrophils and monocytes/macrophages, and of angiogenesis were all induced. In contrast, they were markedly reduced in the mice injected with AdTbeta-ExR. Immunohistochemical analysis revealed that TGF-beta, fibronectin, macrophage chemoattractant protein-1, and vascular endothelial growth factor were densely stained in the edge of wounded cornea, but they were scarcely present in the injured-cornea of AdTbeta-ExR-treated mice. Our results demonstrate that TGF-beta indeed plays a critical role in the process of cornea opacification, and that adenovirus-mediated expression of a soluble TGF-beta receptor can be therapeutically useful.
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PMID:Blockade of TGF-beta by in vivo gene transfer of a soluble TGF-beta type II receptor in the muscle inhibits corneal opacification, edema and angiogenesis. 1112 79

VPF/VEGF acts selectively on the vascular endothelium to enhance permeability, induce cell migration and division, and delay replicative senescence. To understand the changes in gene expression during endothelial senescence, we investigated genes that were differentially expressed in early vs. late passage (senescent) human dermal endothelial cells (HDMEC) using cDNA array hybridization. Early passage HDMEC cultured with or without VPF/VEGF overexpressed 9 and underexpressed 6 genes in comparison with their senescent counterparts. Thymosin beta-10 expression was modulated by VPF/VEGF and was strikingly down-regulated in senescent EC. The beta-thymosins are actin G-sequestering peptides that regulate actin dynamics and are overexpressed in neoplastic transformation. We have also identified senescent EC in the human aorta at sites overlying atherosclerotic plaques. These EC expressed senescence-associated neutral beta-galactosidase and, in contrast to adventitial microvessel endothelium, exhibited weak staining for thymosin beta-10. ISH performed on human malignant tumors revealed strong thymosin beta-10 expression in tumor blood vessels. This is the first report that Tbeta-10 expression is significantly reduced in senescent EC, that VPF/VEGF modulates thymosin beta-10 expression, and that EC can become senescent in vivo. The reduced expression of thymosin beta-10 may contribute to the senescent phenotype by reducing EC plasticity and thus impairing their response to migratory stimuli.
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PMID:Differential expression of thymosin beta-10 by early passage and senescent vascular endothelium is modulated by VPF/VEGF: evidence for senescent endothelial cells in vivo at sites of atherosclerosis. 1115 61

Experimental studies indicate that administration of angiogenic proteins or genes by the epicardial or intracoronary route can stimulate development of new collateral vessels and improve myocardial perfusion. An endocardial catheter-based approach to this therapy would obviate the need for surgery, while preserving the effectiveness of direct intramyocardial administration. Fluoroscopic guidance and prototype, preformed, coaxial catheters were used to examine the feasibility of percutaneous catheter-based adenovirus (Ad)-mediated gene transfer and expression in normal swine myocardium. The feasibility of intramyocardial administration (100 microl/injection) of a radiocontrast agent and black tissue dye to all regions of the left ventricle (septum, anterior, lateral, and inferior wall) was confirmed fluoroscopically and on postmortem examination. Injections of replication-deficient adenovirus (10 injections of 10(11) particle units/100 microl each) coding for beta-galactosidase (Adbetagal) or vascular endothelial growth factor (Ad(GV)VEGF121.10) were administered to the left ventricular free wall to examine endocardial based gene transfer and expression. beta-Galactosidase activity was detected by histochemical staining and quantitative assay in targeted regions of the myocardium. Regional VEGF expression was found to be significantly greater in targeted regions (1.3 +/- 0.4 ng/mg protein) as compared with non-targeted regions (0.3 +/- 0.1 ng/mg protein) or regions injected with control (Adbetagal) virus (0.2 +/- 0.03 ng/mg protein, P < 0.001). Catheter-based Ad mediated endocardial gene transfer and expression is feasible using percutaneous, fluoroscopically guided, preformed, coaxial catheters. This approach should be clinically useful to administer angiogenic genes to the ischemic myocardium.
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PMID:Percutaneous endocardial transfer and expression of genes to the myocardium utilizing fluoroscopic guidance. 1197 42

Inflammatory breast cancer (IBC) is a distinct and aggressive form of locally advanced breast cancer. IBC is highly angiogenic, invasive, and metastatic at its inception. Previously, we identified specific genetic alterations of IBC that contribute to this highly invasive phenotype. RhoC GTPase was overexpressed in 90% of archival IBC tumor samples, but not in stage-matched, non-IBC tumors. To study the role of RhoC GTPase in contributing to an IBC-like phenotype, we generated stable transfectants of human mammary epithelial cells overexpressing the RhoC gene, and studied the effect of RhoC GTPase overexpression on the modulation of angiogenesis in IBC. Levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6), and interleukin-8 (IL-8) were significantly higher in the conditioned media of the HME-RhoC transfectants than in the untransfected HME and HME-beta-galactosidase control media, similar to the SUM149 IBC cell line. Inhibition of RhoC function by introduction of C3 exotransferase decreased production of angiogenic factors by the HME-RhoC transfectants and the SUM149 IBC cell line, but did not affect the control cells. These data support the conclusion that overexpression of RhoC GTPase is specifically and directly implicated in the control of the production of angiogenic factors by IBC cells.
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PMID:RhoC GTPase overexpression modulates induction of angiogenic factors in breast cells. 1119 Nov 8

Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.
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PMID:Inhibition of angiogenesis by adenovirus-mediated sFlt-1 expression in a rat model of corneal neovascularization. 1144 Jun 23

Angiogenesis represents a compensatory response targeted to preserve the integrity of tissues subjected to ischemia. The aim of the present study was to examine whether reparative angiogenesis is impaired in spontaneously hypertensive rats (SHR), as a function of progression of hypertension. In addition, the potential of gene therapy with human tissue kallikrein (HK) in revascularization was challenged in SHR and normotensive Wistar-Kyoto rats (WKY) that underwent excision of the left femoral artery. Expression of vascular endothelial growth factor and HK was upregulated in ischemic hindlimb of WKY but not of SHR. Capillary density was increased in ischemic adductor muscle of WKY (from 266+/-20 to 633+/-73 capillaries/mm(2) at 28 days, P<0.001), whereas it remained unchanged in SHR (from 276+/-20 to 354+/-48 capillaries/mm(2), P=NS), thus compromising perfusion recovery as indicated by reduced plantar blood flow ratio (0.61+/-0.08 versus 0.92+/-0.07 in WKY at 28 days, P<0.05). In separate experiments, saline or 5x10(9) pfu adenovirus containing the HK gene (Ad.CMV-cHK) or the beta-galactosidase gene (Ad.CMV-LacZ) was injected intramuscularly at 7 days after the induction of ischemia. Ad.CMV-cHK augmented capillary density and accelerated hemodynamic recovery in both strains, but these effects were more pronounced in SHR (P<0.01). Our results indicate that native angiogenic response to ischemia is impaired in SHR, possibly as a result of defective modulation of endothelial cell mitogens. Supplementation with kallikrein, one of the growth factors found to be deficient in SHR, restores physiological angiogenic response utilitarian for tissue healing. Our discoveries may have important implications in vascular medicine for therapeutic benefit.
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PMID:Rescue of impaired angiogenesis in spontaneously hypertensive rats by intramuscular human tissue kallikrein gene transfer. 1146 74

An efficient gene delivery system is a prerequisite for myocardial gene therapy. Among the various procedures studied so far, catheter-based percutaneous gene delivery to the myocardium through the coronary vessels seems the most relevant to routine clinical practice; however, the optimal conditions remain to be determined. We selectively infused adenoviral vectors encoding luciferase (1 x 10(9) PFU) or beta-galactosidase (1 x 10(10) PFU) into coronary arteries of adult rabbits in various experimental conditions. Coronary artery occlusion for 30 sec, during and after adenovirus delivery, was required to observe luciferase activity in the target area of the circumflex artery (4.0 +/- 1.0 x 10(5) vs. 1.1 +/- 0.2 x 10(4) RLU/mg with and without coronary occlusion, respectively, p < 0.01, and 1.0 +/- 0.1 x 10(3) RLU/mg using nonselective infusion). When adenoviruses were delivered using high-pressure infusion (82 +/- 12 vs. 415 +/- 25 mmHg before and during infusion, respectively, p < 0.01), luciferase activity increased to 8.5 +/- 2.5 x 10(5) RLU/mg (p < 0.05 vs coronary occlusion alone). Coronary venous sinus occlusion with saline buffer retroinfusion starting before and during anterograde adenovirus delivery resulted in a further 4.7-fold increase in luciferase activity (4.4 +/- 0.8 x 10(6) RLU/mg, p < 0.01) with 5-25% blue-stained myocytes in the target area, compared with 0-5% with the other procedures. Histamine or VEGF-A(165) pretreatment, used to increase vascular permeability, slightly increased gene transfer efficiency (8.5 +/- 2.0 x 10(5) and 9.0 +/- 2.5 x 10(5) RLU/mg respectively, p < 0.05 vs. coronary occlusion alone). We conclude that catheter-mediated adenoviral gene transfer to cardiac myocytes through coronary vessels can be a very efficient procedure for myocardial gene therapy, particularly when the vector residence time and perfusion pressure in the vessels are increased.
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PMID:How to optimize in vivo gene transfer to cardiac myocytes: mechanical or pharmacological procedures? 1153 64

Hypoxia initiates an adaptive physiological response in all organisms and plays a role in the pathogenesis of several human diseases. The hypoxia/HIF-inducible factor-1 (HIF-1) transcription factor mediates transcriptional responses to hypoxia by binding to a cis-acting hypoxia-responsive element (HRE) present within target genes. The use of the HIF-1/HRE system of gene regulation can be utilized as a mechanism to target expression of therapeutic genes to hypoxic cells or cells that have a constitutively active HIF-1/HRE pathway due to cell transformation. Given the rapid resistance of tumors to single therapeutic strategies, new vector systems need to be developed that can deliver multimodal therapy. Here we show that HREs function as classical enhancer elements and function bidirectionally to co-regulate the expression of two genes. We designed a large series of novel bidirectional hypoxia/HIF-responsive expression vectors using HREs derived from the human vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We measured the ability of these constructs to express the luciferase and LacZ/beta-galactosidase (beta-gal) reporter genes bidirectionally under normoxic (21% O(2)) versus hypoxic (1, 3, 5, and 10% O(2)) conditions by transient transfection in three human glioma cell lines (LN229, U251MG and U138MG). Nine constructs were identified that exhibited moderate to high inducibility at 1% O(2) while maintaining tight regulation under normoxic conditions. Moreover, the level of activation was a function of O(2) concentration and was exponential at O(2) levels below 5%. These vectors will be valuable tools in a variety of gene therapy applications targeting pathological activation of the HIF-1/HRE pathway.
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PMID:Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells. 1180

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 die of vascular overgrowth, and we are interested in how flt-1 normally prevents this outcome. Our results support a model whereby aberrant endothelial cell division is the cellular mechanism resulting in vascular overgrowth, and they suggest that VEGF-dependent endothelial cell division is normally finely modulated by flt-1 to produce blood vessels. Flt-1(-/-) embryonic stem cell cultures had a 2-fold increase in endothelial cells by day 8, and the endothelial cell mitotic index was significantly elevated before day 8. Flt-1 mutant embryos also had an increased endothelial cell mitotic index, indicating that aberrant endothelial cell division occurs in vivo in the absence of flt-1. The flt-1 mutant vasculature of the cultures was partially rescued by mitomycin C treatment, consistent with a cell division defect in the mutant background. Analysis of cultures at earlier time points showed no significant differences until day 5, when flt-1 mutant cultures had increased beta-galactosidase(+) cells, indicating that the expansion of flt-1 responsive cells occurs after day 4. Mitomycin C treatment blocked this early expansion, suggesting that aberrant division of angioblasts and/or endothelial cells is a hallmark of the flt-1 mutant phenotype throughout vascular development. Consistent with this model is the finding that expansion of platelet and endothelial cell adhesion molecule(+) and VE-cadherin(+) vascular cells in the flt-1 mutant background first occurs between day 5 and day 6. Taken together, these data show that flt-1 normally modulates vascular growth by controlling the rate of endothelial cell division both in vitro and in vivo.
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PMID:Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. 1189 72


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