The Human ICAM-2 Promoter is Endothelial Cell-specific in Vitro and in Vivo and Contains Critical Sp1 and GATA Binding Sites

Cowan, Peter J.; Tsang, Denise; Pedic, Christopher M.; Abbott, Lucy R.; Shinkel, Trixie A.; d’Apice, Anthony J.F.; Pearse, Martin J.

doi:10.1074/jbc.273.19.11737

Cited by 74 publications

(58 citation statements)

References 47 publications

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“…These results differ from those published previously. 8,20 In transgenic animals, the same ICAM-2 promoter demonstrated high-level consistent transgene expression in ECs of all blood vessels in the heart, kidney, lung, liver, and pancreas, with negligible expression in other cell types, except neutrophils and monocytes. 8 Another study using the same fragment in plasmid vectors showed high-level activity in bovine ECs but not in COS cells, although no human ECs were tested.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Cell-Specific Promoters for Viral Gene Therapy Targeted at the Vascular Endothelium

et al. 2001

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Abstract-The use of viral vectors for vascular gene therapy targeted at the endothelium is limited by the promiscuous tropism of vectors and nonspecificity of viral promoters, resulting in high-level transgene expression in multiple tissues.To evaluate suitable endothelial cell (EC)-specific promoters for vascular gene therapy, we directly compared the ability of the fms-like tyrosine kinase-1 (FLT-1), intercellular adhesion molecule-2 (ICAM-2), and von Willebrand factor (vWF) promoters to drive EC-restricted transcription after cloning into adenoviral vectors upstream of lacZ. Vastly different expression profiles were observed. Whereas both FLT-1 and ICAM-2 promoters generated transgene expression levels similar to cytomegalovirus in ECs in vitro, vWF expression levels were extremely low. Analysis of non-EC types revealed that ICAM-2 but not FLT-1 evoked leaky transgene expression, thus identifying FLT-1 as the most selective promoter. With an ex vivo human gene therapy model, the FLT-1 promoter demonstrated EC-specific transgene expression in intact human vein but no detectable expression from infected exposed smooth muscle cells in EC-denuded vein. Furthermore, when adenoviruses were systemically administered to mice, the FLT-1 promoter demonstrated extremely low-level gene expression in the liver, the major target organ for adenoviral transduction in vivo. This study highlights the potential of using the FLT-1 promoter for local and systemic human gene therapy in hypertension and its complications.

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Section: Discussionmentioning

confidence: 99%

Analysis of Cell-Specific Promoters for Viral Gene Therapy Targeted at the Vascular Endothelium

et al. 2001

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“…Electrophoretic Mobility Shift Assays (EMSAs)-Nuclear extracts were prepared as described previously (31). EMSAs were performed using the following double-stranded oligonucleotides as probes: Ϫ1306C, 5Ј-ACCCAGCACTCCACCTCTTTAGCT-3Ј; Ϫ1306T, 5Ј-AC-CCAGCACTCTACCTCTTTAGCT-3Ј; and Sp1cons, 5Ј-ATTCGATCGG-GGCGGGGCGAGC-3Ј.…”

Section: Isolation Of the Human Mmp-2 Promoter-the Human Genomementioning

confidence: 99%

Identification of Novel, Functional Genetic Variants in the Human Matrix Metalloproteinase-2 Gene

Price¹,

Greaves

Watkins³

2001

Journal of Biological Chemistry

375

313

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Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix and nonmatrix proteins, particularly basement membrane constituents. Thus, any naturally occurring genetic variants that directly affect gene expression and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling. We scanned a 2-kilobase pair promoter region and all 13 exons of the human MMP-2 gene, from a panel of 32 individuals, and we identified the position, nature, and relative allele frequencies of 15 variant loci as follows: 6 in the promoter, 1 in the 5-untranslated region, 6 in the coding region, 1 in intronic sequence, and 1 in the 3-untranslated region. The majority of coding region polymorphisms resulted in synonymous substitutions, whereas three promoter variants (at ؊1306, ؊790, and ؉220) mapped onto cis-acting elements. We functionally characterized all promoter variants by transient transfection experiments with 293, RAW264.7, and A10 cells. The common C 3 T transition at ؊1306 (allele frequency 0.26), which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with the T allele. Electrophoretic mobility shift assays confirmed that these differences in allelic expression were attributable to abolition of Sp1 binding. These data suggest that this common functional genetic variant influences MMP-2 gene transcription in an allele-specific manner and is therefore an important candidate to test for association in a wide spectrum of pathologies for which a role for MMP-2 is implicated, including atherogenesis and tumor invasion and metastasis.

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“…Extensive in vitro analysis of the ICAM-2 promoter fragment originally reported to function in transgenic mice 34 demonstrated the necessity (for transient activity in bovine endothelial cells) of two GATA-2 binding sites, an Sp1 site and an 8 bp palindrome (P 8 ). 38 The importance of four Ets sites conserved between mouse and human promoters has not been investigated. All of the aforementioned sites are present in the region incorporated into our vector.…”

Section: Discussionmentioning

confidence: 99%

Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity

2004

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Transcriptional targeting is an important aspect of developing gene therapy vectors in order to restrict transgene expression to selected target cells. One approach, when using retroviral vectors, is to replace viral transcriptional control elements within the long terminal repeat (LTR) with sequences imparting the desired specificity. We have developed such hybrid LTR retroviruses, incorporating sequences from each of the human promoters for flt-1, ICAM-2 and KDR, as part of our antivascular cancer gene therapy strategy targeting tumour endothelial cells. The chosen fragments were used to replace the enhancer or combined enhancer and proximal promoter regions of the viral LTR. All showed activity in primary human breast microvascular endothelial cells, with viruses incorporating ICAM-2 sequences exhibiting the greatest specificity versus nonendothelial cells in vitro and a marked alteration of specificity towards endothelial cells in a subcutaneous xenograft model in vivo. Moreover, our study documents the effect of enhancer and/or proximal promoter deletion on LTR activity and reports that differential dependence in different cell lines can give the false impression of specificity if experiments are not adequately controlled. This finding also has implications for other retroviral vector designs seeking to provide transcriptional specificity and for their safety with respect to prevention of gene activation at sites of proviral integration.

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The Human ICAM-2 Promoter is Endothelial Cell-specific in Vitro and in Vivo and Contains Critical Sp1 and GATA Binding Sites

Cited by 74 publications

References 47 publications

Analysis of Cell-Specific Promoters for Viral Gene Therapy Targeted at the Vascular Endothelium

Analysis of Cell-Specific Promoters for Viral Gene Therapy Targeted at the Vascular Endothelium

Identification of Novel, Functional Genetic Variants in the Human Matrix Metalloproteinase-2 Gene

Retroviral hybrid LTR vector strategy: functional analysis of LTR elements and generation of endothelial cell specificity

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