Efficient Expression of Exogenous Genes in Primary Vascular Cells Using IRES-Based Retroviral Vectors

Garton, Kyle J.; Ferri, Nicola; Raines, Elaine W.

doi:10.2144/02324rr01

Cited by 43 publications

(39 citation statements)

References 15 publications

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“…Plasmids-mAktER and A2 AktER (16) were cloned into the retroviral vector pBMN IRES puro (17). IGF1R wild type and YF mutant (18) were cloned into pBMN IRES puro (17).…”

Section: Methodsmentioning

confidence: 99%

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Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3

Allard

Figg

Bennett

et al. 2008

Journal of Biological Chemistry

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Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis.Insulin-like growth factor 1 (IGF1) 2 is a ubiquitous factor exhibiting pleiotropic effects on different cell types. Stimulation of the IGF1 receptor (IGF1R) initiates signaling pathways involved in cell proliferation, differentiation, transformation, and survival. IGF1R-dependent signaling is crucial for the survival of many cell types including vascular smooth muscle cells (VSMCs). VSMCs are the principle source of collagen and extracellular matrix that maintain the tensile strength of atherosclerotic plaques, and VSMC loss induces multiple features of plaque instability (1). In humans, rupture or erosion of the atherosclerotic plaque underlie the majority of myocardial infarctions, stroke, and sudden death (2). We have previously shown that VSMCs derived from atherosclerotic plaques (pVSMCs) are more sensitive to apoptosis than cells derived from non-diseased vessels (3) and exhibit a defect in IGF1-dependent survival signaling (4). Oxidative stress is increasingly implicated in the development of atherosclerosis (5) and increased oxidative damage, and elevated levels of DNA strand breaks occur in human atherosclerotic plaques (6). Oxidative stress reduces IGF1R expression and induces VSMC apoptosis in culture (7-10). Reduced IGF1R expression is also seen within plaques, suggesting that IGF1R-dependent survival regulates apoptosis in vivo (11,12). However, plaque VSMCs also show increased sensitivity to multiple proapoptotic stimuli, including the tumor suppressor gene P53 and death receptor ligation (13,14). It is therefore not known whether defective IGF1R expression alone is an important cause of reduced survival of pVSMCs.A major downstream effector of IGF1R ...

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“…Plasmids-mAktER and A2 AktER (16) were cloned into the retroviral vector pBMN IRES puro (17). IGF1R wild type and YF mutant (18) were cloned into pBMN IRES puro (17).…”

Section: Methodsmentioning

confidence: 99%

“…IGF1R wild type and YF mutant (18) were cloned into pBMN IRES puro (17). Dominant negative Akt (DN-Akt, T308A, S473A (19)) was cloned into pCDNA3, and the wild type and the A3 mutant of FoxO3a (20) were cloned into pCDNA3.1.…”

Section: Methodsmentioning

confidence: 99%

Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3

Allard

Figg

Bennett

et al. 2008

Journal of Biological Chemistry

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“…All PCR-generated constructs were verified by DNA sequencing. Retroviral expression plasmids were constructed using the pBM-IRES-EGFP vector (G. Nolan, Stanford University) or the pBM-IRES-PURO vector (20). High titer retrovirus was prepared as previously described (21).…”

Section: Generation Of Expression Constructs and Retroviralmentioning

confidence: 99%

Stimulated Shedding of Vascular Cell Adhesion Molecule 1 (VCAM-1) Is Mediated by Tumor Necrosis Factor-α-converting Enzyme (ADAM 17)

Garton

Gough

Philalay

et al. 2003

Journal of Biological Chemistry

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247

186

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A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-␣-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.The proteolytic cleavage and release of transmembrane cell surface proteins, termed ectodomain shedding, has emerged as an important post-translational mechanism for regulating the function of cell surface proteins (1). A wide variety of structurally diverse proteins including cytokines, growth factors, and adhesion molecules can be shed from the cell surface. In many cases, these shed ectodomains are biologically active. Ectodomain shedding can be mediated by both membrane-bound as well as soluble proteases. To date, members of the Zn 2ϩ -dependent protease superfamily, including the matrix metalloproteinases (MMPs), 1 membrane-tethered MMPs (MT-MMPs), and the disintegrin metalloproteinases (ADAMs), have been shown to be responsible for the cleavage of the majority of shed proteins identified. In addition, soluble neutrophil-derived proteases including neutrophil elastase, cathepsin G, and proteinase-3 have also been implicated in the shedding of cell surface proteins (2). Of the disintegrin and metalloproteinase (ADAM) family of proteases, tumor necrosis factor-␣-converting enzyme (TACE; ADAM 17) has emerged as a central mammalian ectodomain sheddase (3). TACE-deficient mice are not viable and show multiple developmental defects (4). Furthermore, cells isolated from TACE-deficient mice lack shedding of several unrelated cell surface proteins including tumor necrosis factor-␣, tumor necrosis factor-␣ receptor, several epidermal growth factor receptor ligands, Notch-1, amyloid precursor protein,...

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“…Gene transduction, expression and selection should be rapid for use in primary cells with a limited life span in vitro. Retroviral vectors appear to fulfill these requirements [19], and efficient expression of exogenous genes in primary human vascular cells, including EC, was demonstrated using internal ribosomal entry site-containing retroviral vectors [20]. HIV-1-based lentiviral vectors can stably transduce a variety of non-dividing cells in vitro and in vivo [21,22], which may be of particular importance for the transduction of confluent, slowly proliferating monolayers of EC.…”

Section: Introductionmentioning

confidence: 99%

Aberrant expression of α‐Gal on primary human endothelium does not confer susceptibility to NK cell cytotoxicity or increased NK cell adhesion

He¹,

Ehrnfelt²,

Kumagai‐Braesch³

et al. 2004

Eur J Immunol

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The contribution of Gal § 1,3Gal ( § -Gal) to cell-mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine § 1,3 galactosyltransferase ( § 1,3GalT) to obtain § -Gal-expressing primary human aortic endothelial cells (HAEC) at a frequency of 70-90%. These cells were compared to non-transduced and mock-transduced HAEC with regard to their susceptibility to human NK cell-mediated lysis, ability to stimulate IFN-+ production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 posttransduction, and at a multiplicity of infection of 2.5, it reached 98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF- § and IFN-+ stimulation. No differences were detected between non- § -Gal-and § -Galexpressing HAEC in terms of their susceptibility to NK cell-mediated lysis, ability to stimulate IFN-+ production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the § -Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.

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Efficient Expression of Exogenous Genes in Primary Vascular Cells Using IRES-Based Retroviral Vectors

Cited by 43 publications

References 15 publications

Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3

Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3

Stimulated Shedding of Vascular Cell Adhesion Molecule 1 (VCAM-1) Is Mediated by Tumor Necrosis Factor-α-converting Enzyme (ADAM 17)

Aberrant expression of α‐Gal on primary human endothelium does not confer susceptibility to NK cell cytotoxicity or increased NK cell adhesion

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