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1.
Gene Ther ; 18(5): 501-8, 2011 May.
Article in English | MEDLINE | ID: mdl-21179172

ABSTRACT

The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here, we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin (IL)-10) in large-vessel endothelial cells (ECs) at levels as high as obtained with the cytomegalovirus (CMV) immediate early promoter, while retaining EC specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely four copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining--and possibly enhancing--EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large-vessel EC, while minimizing systemic effects.


Subject(s)
Adenoviridae/genetics , Carotid Arteries , Gene Transfer Techniques , Interleukin-10/genetics , Animals , Cattle , Cell Line , Cytomegalovirus/genetics , Defective Viruses , Endothelial Cells , Genetic Vectors , Mice , Organ Specificity/genetics , Promoter Regions, Genetic , Rabbits , Regulatory Sequences, Nucleic Acid , Transgenes
2.
Arterioscler Thromb Vasc Biol ; 21(11): 1777-82, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11701465

ABSTRACT

Adenoviral vectors are promising agents for vascular gene transfer. Their use, however, is limited by inflammatory host responses, neointima formation, and brevity of transgene expression. Inclusion of the immunomodulatory adenoviral E3 genes in a vector might prevent inflammation and neointima formation and prolong transgene expression. We compared 2 adenoviral vectors in a model of in vivo gene transfer to rabbit arteries. Both vectors expressed a luciferase reporter gene. One vector (AdE3Luc) contained the adenovirus early 3 (E3) region and the other (AdRSVLuc) lacked E3. Expression of E3 genes by AdE3Luc was confirmed in vitro and in vivo. Arteries transduced with AdE3Luc had substantially and significantly less inflammation (fewer T cells and lower levels of vascular cell adhesion molecule-1 and intercellular adhesion molecule 1 expression) and decreased neointima formation 14 days after gene transfer. Luciferase expression from the 2 vectors was equivalent, however, at both 3 and 14 days after gene transfer. Expression of E3 had no systemic immunosuppressive effects, as measured by peripheral blood counts and by assays for serum antibodies to adenovirus. We conclude that expression of E3 significantly decreases adenovirus-induced arterial wall inflammation and neointima formation. Because inflammation and neointima formation are major barriers to the clinical application of adenoviral vectors, use of E3-containing vectors improves the promise of adenovirus-mediated arterial gene transfer.


Subject(s)
Adenoviridae/genetics , Adenovirus E3 Proteins/genetics , Arterial Occlusive Diseases/therapy , Arteritis/therapy , Genetic Therapy/methods , Adenoviridae/immunology , Adenoviridae/metabolism , Adenovirus E3 Proteins/biosynthesis , Animals , Antibodies, Viral/biosynthesis , Arterial Occlusive Diseases/metabolism , Arterial Occlusive Diseases/pathology , Arteries/metabolism , Arteries/pathology , Arteritis/immunology , Arteritis/pathology , CHO Cells , Cell Adhesion Molecules/metabolism , Cell Line , Cricetinae , DNA, Viral/genetics , Genetic Vectors , Luciferases/biosynthesis , Luciferases/genetics , RNA, Viral/biosynthesis , Rabbits , Transduction, Genetic
3.
Circulation ; 104(16): 1972-1, 2001 Oct 16.
Article in English | MEDLINE | ID: mdl-11602503

ABSTRACT

BACKGROUND: Elevated plasma levels of plasminogen activator inhibitor type 1 (PAI-1) are associated with myocardial infarction, atherosclerosis, and restenosis. PAI-1 is increased in atherosclerotic arteries and failed vein grafts. No experimental data, however, support a causal relationship between elevated PAI-1 expression and vascular lesions. Paradoxically, data generated in PAI-1 knockout mice suggest that PAI-1 might decrease lesion formation after arterial injury and that PAI-1 gene transfer might prevent restenosis. METHODS AND RESULTS: Using the rat carotid balloon injury model and a PAI-1-expressing adenoviral vector, we tested whether elevated arterial PAI-1 expression would alter neointima formation. Compared with control-transduced arteries, neointima formation in PAI-1-transduced arteries was initially retarded. By 14 days, however, the intimas of PAI-1-transduced arteries were significantly larger than intimas of control-transduced arteries (1.6+/-0.1x10(5) versus 1.2+/-0.1x10(5) micrometer(2), n=18 to 19, P<0.03). PAI-1 expression in individual arteries correlated with increased cell proliferation at 4 and 8 days after injury (R=0.6, P<0.02 and P<0.006). PAI-1 expression also correlated with fibrin(ogen) accumulation (R=0.77, P<0.001), and fibrin(ogen) accumulation correlated strongly with proliferation (R=0.86, P<0.00001). CONCLUSIONS: Increased expression of PAI-1 in the artery wall promotes neointima growth after balloon injury. Therefore, despite encouraging data generated in other animal models, PAI-1 is not a promising agent for gene therapy to prevent restenosis. Moreover, our data associate elevated PAI-1 expression with fibrin(ogen) accumulation and increased cell proliferation. These data suggest a mechanism to explain the association between elevated PAI-1 expression and the progression of arterial disease.


Subject(s)
Angioplasty, Balloon , Carotid Artery Diseases/pathology , Plasminogen Activator Inhibitor 1/administration & dosage , Tunica Intima/drug effects , Tunica Intima/pathology , Adenoviridae/genetics , Angioplasty, Balloon/adverse effects , Animals , Carotid Artery Diseases/etiology , Carotid Artery Diseases/prevention & control , Cell Division/drug effects , Disease Models, Animal , Disease Progression , Fibrin/metabolism , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Immunohistochemistry , Plasminogen Activator Inhibitor 1/genetics , Rats , Transduction, Genetic , Tunica Intima/metabolism , Tunica Media/drug effects , Tunica Media/pathology
4.
Mol Ther ; 2(5): 496-504, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11082323

ABSTRACT

The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a "null" (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P<0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens.


Subject(s)
Adenoviridae/genetics , Adenoviridae/immunology , Defective Viruses/genetics , Genetic Vectors/immunology , Lymphocyte Activation , Adenoviridae/metabolism , Animals , Antibodies, Viral/biosynthesis , Antigens, Viral/immunology , Cells, Cultured , DNA, Viral/analysis , Defective Viruses/immunology , Defective Viruses/metabolism , Immune Tolerance , Mice , Mice, Transgenic , Spleen/cytology , Spleen/immunology , Transcriptional Activation , Transgenes , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolism
5.
J Pept Sci ; 6(9): 432-9, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11016879

ABSTRACT

The generation of the broad specificity serine protease plasmin in the pericellular environment is regulated by binding of the urokinase-type plasminogen activator (uPA) to its specific glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor, uPAR. This interaction potentiates the reciprocal activation of the cell-associated zymogens pro-uPA and plasminogen. To further study the role of uPAR in this mechanism, we have expressed two directly membrane-anchored chimeric forms of uPA, one anchored by a C-terminal GPI-moiety (GPI-uPA), the other with a C-terminal transmembrane peptide (TM-uPA). These were expressed in the monocyte-like cell lines U937 and THP-1, which are excellent models for kinetic and mechanistic studies of cell-surface plasminogen activation. In both cell-lines, GPI-uPA activated cell-associated plasminogen with characteristics both qualitatively and quantitatively indistinguishable from those of uPAR-bound uPA. By contrast, TM-uPA activated cell-associated plasminogen less efficiently. This was due to effects on the K, for plasminogen activation (which was increased up to five-fold) and the efficiency of pro-uPA activation (which was decreased approximately four-fold). These observations suggest that uPAR serves two essential roles in mediating efficient cell-surface plasminogen activation. In addition to confining uPA to the cell-surface, the GPI-anchor plays an important role by increasing accessibility to substrate plasminogen and, thus, enhancing catalysis. However, the data also demonstrate that, in the presence of an alternative mechanism for uPA localization, uPAR is dispensable and, therefore, unlikely to participate in any additional interactions that may be necessary for the efficiency of this proteolytic system. In these experiments zymogen pro-uPA was unexpectedly found to be constitutively activated when expressed in THP-1 cells, suggesting the presence of an alternative plasmin-independent proteolytic activation mechanism in these cells.


Subject(s)
Cell Membrane/metabolism , Plasminogen Activators/metabolism , Plasminogen/metabolism , Receptors, Cell Surface/metabolism , Urokinase-Type Plasminogen Activator/metabolism , Cells, Cultured , Enzyme Precursors/metabolism , Fibrinolysin/metabolism , Genetic Vectors , Glycosylphosphatidylinositols/metabolism , Humans , Kinetics , Monocytes/metabolism , Peptide Fragments/metabolism , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Urokinase Plasminogen Activator , Recombinant Proteins/metabolism , Time Factors , Transfection
6.
Transplantation ; 69(9): 1813-7, 2000 May 15.
Article in English | MEDLINE | ID: mdl-10830216

ABSTRACT

BACKGROUND: Fas ligand (FasL) mediates apoptosis of susceptible Fas-expressing lymphocytes, and may contribute to the maintenance of peripheral tolerance. In transplantation models, however, artificial expression of FasL on cellular as well as islet transplants results in accelerated rejection by neutrophils. The mechanism of the neutrophilic response to FasL expression is unknown. FasL, like other members of the tumor necrosis factor family, is cleaved to a soluble form by metalloproteases. We tested the hypothesis that soluble FasL (sFasL) was responsible for neutrophil migration by creating a non-cleavable mutant of FasL. METHODS: Three mutants of FasL with serial deletions in the putative proteolytic cleavage site of human FasL were made using inverse polymerase chain reaction. The relative fractions of sFasL and membrane-bound FasL were assessed by Western blot and immunoprecipitation, as well as by cytotoxicity assay using Fas-expressing target cells. The fully non-cleavable mutant was transduced into murine islets as well as myoblasts and tumor cell lines, and tested in a murine transplantation model. RESULTS: Serial deletions in the putative metalloprotease site of FasL resulted in a fully non-cleavable mutant of FasL (ncFasL). Expression of ncFasL in tumor lines induced higher levels of apoptosis in Fas bearing targets than wild-type FasL. Transplantation of ncFasL-expressing islets under the kidney capsule of allogenic mice resulted in accelerated rejection identical to that seen with wild-type Fas ligand-expressing islets. Myoblasts and tumor cell lines expressing ncFasL also induced neutrophil infiltration. CONCLUSIONS: Membrane-bound Fas ligand is fully capable of inducing a neutrophilic response to transplants, suggesting an activation by Fas ligand of neutrophil chemotactic factors.


Subject(s)
Islets of Langerhans Transplantation , Islets of Langerhans/pathology , Membrane Glycoproteins/physiology , Neutrophils/physiology , Animals , COS Cells , Fas Ligand Protein , Humans , Jurkat Cells , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/therapeutic use , Mice , Mutation , Structure-Activity Relationship
7.
Arterioscler Thromb Vasc Biol ; 20(6): 1452-8, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10845857

ABSTRACT

The utility of adenoviral vectors for arterial gene transfer is limited by the brevity of their expression and by inflammatory host responses. As a step toward circumventing these difficulties, we used a rabbit model of in vivo arterial gene transfer to test 3 second-generation vectors: a vector containing a temperature-sensitive mutation in the E2A region, a vector deleted of E2A, and a vector that expresses the immunomodulatory 19-kDa glycoprotein (gp19k) from adenovirus 2. Compared with similar first-generation vectors, the second-generation vectors did not significantly prolong beta-galactosidase transgene expression or decrease inflammation in the artery wall. Although cyclophosphamide ablated the immune and inflammatory responses to adenovirus infusion, it only marginally prolonged transgene expression (94% drop in expression between 3 and 14 days). In experiments performed with "null" adenoviral vectors (no transgene), loss of vector DNA from the arterial wall was also rapid (>99% decrease between 1 hour and 14 days), unrelated to dose, and only marginally blunted by cyclophosphamide. Thus, the early loss of transgene expression after adenoviral arterial gene transfer is due primarily to loss of vector DNA, is not correlated with the presence of local vascular inflammation, and cannot be prevented by use of E2A-defective viruses, expression of gp19k, or cyclophosphamide-mediated immunosuppression. Adenovirus-induced vascular inflammation can be prevented by cyclophosphamide treatment or by lowering the dose of infused virus. However, stabilization of adenovirus-mediated transgene expression in the arterial wall is a more elusive goal and will require novel approaches that prevent the early loss of vector DNA.


Subject(s)
Adenoviridae/genetics , Arteries/metabolism , DNA-Binding Proteins , DNA/metabolism , Gene Expression , Gene Transfer Techniques , Genetic Vectors , Animals , Basic Helix-Loop-Helix Transcription Factors , Cyclophosphamide/pharmacology , Genetic Vectors/adverse effects , Immunosuppressive Agents/pharmacology , Male , Rabbits , Transcription Factors/genetics , Vasculitis/etiology , beta-Galactosidase/genetics
8.
Circ Res ; 86(10): 1024-30, 2000 May 26.
Article in English | MEDLINE | ID: mdl-10827131

ABSTRACT

Transforming growth factor-beta(1) (TGF-beta(1)) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-beta(1) expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-beta(1) in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-beta(1) is expressed from the SM22alpha promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22alpha-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22alpha-beta-galactosidase transgene. SM22alpha- beta-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22alpha-TGF-beta(1) transgenic mice died before E11.5. SM22alpha-TGF-beta(1) transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-beta(1) from the SM22alpha promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-beta(1) expression in adults may require a conditional transgenic approach. Moreover, because the SM22alpha promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22alpha promoter to drive expression of "vasculoactive" transgenes may be particularly likely to cause embryonic death.


Subject(s)
Cardiovascular System/metabolism , Fetal Death/etiology , Transforming Growth Factor beta/metabolism , Yolk Sac/blood supply , Animals , Blood Vessels/embryology , Embryo, Mammalian/physiology , Fetal Resorption/etiology , Gene Expression/genetics , Heart/embryology , Hematopoietic Stem Cells/cytology , Mice , Mice, Transgenic/genetics , Microfilament Proteins/genetics , Muscle Proteins/genetics , Promoter Regions, Genetic/physiology , Transforming Growth Factor beta/genetics , Transgenes/genetics , Yolk Sac/cytology
9.
Arterioscler Thromb Vasc Biol ; 20(2): 298-308, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10669624

ABSTRACT

Fas ligand (FasL) is expressed by cells of the arterial wall and is present in human atherosclerotic lesions. However, the role of FasL in modifying the initiation and progression of atherosclerosis is unclear. To investigate the role of arterial FasL expression in the development of atherosclerosis, we first established a model of primary lesion formation in rabbit carotid arteries. In this model, infusion of adenoviral vectors into surgically isolated, nondenuded arteries of hypercholesterolemic rabbits leads to the formation of human-like early atherosclerotic lesions. Expression of FasL in arterial endothelium in this model decreased T-cell infiltration and expression of vascular cell adhesion molecule-1 but did not affect expression of intercellular adhesion molecule-1. Intimal lesions grew more rapidly in FasL-transduced arteries than in arteries transduced with a control adenovirus that did not express a transgene. Total intimal macrophage accumulation was increased in FasL-transduced arteries; however, the proportion of lesion area occupied by macrophages was not elevated. The accelerated lesion growth was primarily due to the accumulation of intimal smooth muscle cells with a synthetic proliferative phenotype. There was no significant apoptosis in FasL-transduced or control arteries and no granulocytic infiltrates. Thus, the net result of elevated FasL expression is to accelerate atherosclerotic lesion growth by increasing lesion cellularity. Vascular expression of FasL may contribute to the progression of atherosclerosis.


Subject(s)
Arteries/metabolism , Arteriosclerosis/etiology , Hypercholesterolemia/complications , Hypercholesterolemia/metabolism , Membrane Glycoproteins/metabolism , Adenoviridae/physiology , Animals , Apoptosis/physiology , Arteriosclerosis/metabolism , Arteriosclerosis/pathology , Cell Division/drug effects , Endothelium, Vascular/metabolism , Fas Ligand Protein , Gene Expression , Macrophages/pathology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/pharmacology , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Rabbits , T-Lymphocytes/physiology , Transgenes/genetics , Vascular Cell Adhesion Molecule-1/metabolism
10.
Circ Res ; 85(9): e25-32, 1999 Oct 29.
Article in English | MEDLINE | ID: mdl-10532959

ABSTRACT

We developed a murine model of arterial gene transfer and used it to test the role of antigen-specific immunity in the loss of adenovirus-mediated transgene expression. Adenoviral vectors encoding either beta-galactosidase (beta-gal) or green fluorescent protein were infused to the lumen of normal common carotids of CD-1 and C57BL/6 mice and atherosclerotic carotids of Apoe(-/-) mice. At 3 days after gene transfer, significant reporter gene expression was detected in all strains. Transgene expression was transient, with expression undetectable at 14 days. Next, a beta-gal-expressing vector was infused into carotids of ROSA26 mice (transgenic for, and therefore tolerant of, beta-gal) and RAG-2(-/-) mice (deficient in recombinase-activating gene [RAG]-2 and therefore lacking in antigen-specific immunity). beta-Gal expression was again high at 3 days but declined substantially (>90%) by 14 days. In vivo labeling with bromodeoxyuridine revealed that carotid endothelial proliferation was increased dramatically by the gene-transfer procedure alone, likely leading to the loss of episomal adenoviral DNA. Gene transfer to normal and atherosclerotic mouse carotids can be accomplished; however, elimination of antigen-specific immune responses does not prevent the early loss of adenovirus-mediated transgene expression. Efforts to prolong adenovirus-mediated transgene expression in the artery wall must be redirected. These efforts will likely include strategies to avoid the consequences of increased cell turnover. Nevertheless, despite the brevity of expression, this mouse model of gene transfer to normal and severely atherosclerotic arteries will likely be useful for investigating the genetic basis of vascular disease and for developing gene therapies.


Subject(s)
Adenoviridae/immunology , Arteriosclerosis/genetics , Carotid Artery, Common/metabolism , Gene Transfer Techniques , Genetic Vectors/immunology , Animals , Apolipoproteins E/genetics , Arteriosclerosis/pathology , Arteriosclerosis/virology , Azacitidine/pharmacology , Carotid Artery, Common/pathology , Carotid Artery, Common/virology , Cell Division , Endothelium, Vascular/cytology , Epitopes , Gene Expression , Genes, Reporter , Immune Tolerance , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Time Factors , Transfection , beta-Galactosidase/metabolism
11.
Gene Ther ; 6(3): 385-92, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10435088

ABSTRACT

We constructed a hirudin cDNA cassette, HV-1.1, that encodes mature hirudin variant-1 fused to the signal peptide of human tissue-type plasminogen activator (t-PA). The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively. Transduced cells secreted up to 35 +/- 2 ng/10(6) cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks. Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 +/- 0.2 antithrombin units per microgram (ATU/microgram), compared with specific activities of approximately 12 ATU/microgram for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity. We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone. Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 +/- 0.2 ATU/microgram. Protein sequencing and mass spectroscopic analysis demonstrated proper cleavage of the growth hormone signal peptide. Thus, we achieved high level retrovirus-mediated secretion of biologically active hirudin from endothelial cells in vitro. Use of these vectors may permit sustained local antagonism of thrombin activity in vivo.


Subject(s)
Antithrombins/genetics , Endothelium, Vascular/metabolism , Genetic Therapy/methods , Genetic Vectors , Hirudins/genetics , Retroviridae/genetics , Cells, Cultured , Gene Expression , Growth Hormone/metabolism , Humans , Mass Spectrometry , Protein Sorting Signals/metabolism , Tissue Plasminogen Activator/metabolism , Transfection/methods
12.
Hum Gene Ther ; 10(9): 1469-78, 1999 Jun 10.
Article in English | MEDLINE | ID: mdl-10395372

ABSTRACT

The vessel wall fibrinolytic system plays an important role in maintaining the arterial phenotype and in regulating the arterial response to injury. Plasminogen activator inhibitor type 1 (PAI-1) regulates tissue fibrinolysis and is expressed in arterial tissue; however, its biological role remains uncertain. To help elucidate the role of PAI-1 in the artery wall, and to begin to clarify whether manipulation of vascular PAI-1 expression might be a target for gene therapy, we used adenoviral vectors to increase expression of rat PAI-1 in rat carotid arteries. Infusion of an adenoviral vector in which PAI-1 expression was driven by a promoter derived from the Rous sarcoma virus (RSV) did not increase PAI-1 expression above endogenous levels. To improve PAI-1 expression, we modified the vector by (1) truncating the 3' untranslated region of PAI-1 to increase the mRNA half-life, (2) substituting the SRalpha or the cytomegalovirus (CMV) promoter for the RSV promoter, (3) including an intron in the expression cassette, and (4) altering the direction of transcription of the transgene cassette. The optimal expression vector, revealed by in vitro studies, contained the CMV promoter, an intron, and a truncated PAI-1 mRNA. This vector increased PAI-1 expression by 30-fold over control levels in vitro and by 1.6 to 2-fold over endogenous levels in vivo. This vector will be useful for elucidating the role of PAI-1 in arterial pathobiology. Because genes that are important in maintaining the vascular phenotype are likely to be expressed in the vasculature, the technical issues of how to increase in vivo expression of endogenous genes are highly relevant to the development of genetic therapies for vascular disease.


Subject(s)
Adenoviruses, Human , Carotid Arteries , Gene Transfer Techniques , Genetic Vectors , Plasminogen Activator Inhibitor 1/genetics , Animals , Avian Sarcoma Viruses/genetics , Cells, Cultured , Culture Techniques , Cytomegalovirus/genetics , Gene Transfer Techniques/standards , Humans , Male , Muscle, Smooth, Vascular/cytology , Plasminogen Activator Inhibitor 1/metabolism , Promoter Regions, Genetic , Rats , Rats, Sprague-Dawley
13.
J Vasc Surg ; 29(3): 543-50, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10069919

ABSTRACT

PURPOSE: Adenovirus-mediated arterial gene transfer is a promising tool in the study of vascular biology and the development of vascular gene therapy. However, intraluminal delivery of adenoviral vectors causes vascular inflammation and neointimal formation. Whether these complications could be avoided and gene transfer efficiency maintained by means of delivering adenoviral vectors via the adventitia was studied. METHODS: Replication-defective adenoviral vectors encoding a beta-galactosidase (beta-gal) gene (AdRSVnLacZ) or without a recombinant gene (AdNull) were infused into the lumen or the adventitia of rabbit carotid arteries. Two days after infusion of either AdRSVnLacZ (n = 8 adventitial, n = 8 luminal) or AdNull (n = 4 luminal), recombinant gene expression was quantitated by histochemistry (performed on tissue sections) and with a beta-gal activity assay (performed on vessel extracts). Inflammation caused by adenovirus infusion was assessed 14 days after infusion of either AdNull (n = 6) or vehicle (n = 6) into the carotid adventitia. Inflammation was assessed by means of examination of histologic sections for the presence of neointimal formation and infiltrating T cells and for the expression of markers of vascular cell activation (ICAM-1 and VCAM-1). To measure the systemic immune response to adventitial infusion of adenovirus, plasma samples (n = 3) were drawn 14 days after infusion of AdNull and assayed for neutralizing antibodies. RESULTS: Two days after luminal infusion of AdRSVnLacZ, approximately 30% of luminal endothelial cells expressed beta-gal. Similarly, 2 days after infusion of AdRSVnLacZ to the adventitia, approximately 30% of adventitial cells expressed beta-gal. beta-gal expression was present in the carotid adventitia, the internal jugular vein adventitia, and the vagus nerve perineurium. Elevated beta-gal activity (50- to 80-fold more than background; P <.05) was detected in extracts made from all AdRSVnLacZ-transduced arteries. The amount of recombinant protein expression per vessel did not differ significantly between vessels transduced via the adventitia (17.1 mU/mg total protein [range, 8.1 to 71.5]) and those transduced via a luminal approach (10.0 mU/mg total protein [range, 3.9 to 42.6]). Notably, adventitial delivery of AdNull did not cause neointimal formation. In addition, vascular inflammation in arteries transduced via the adventitia (ie, T-cell infiltrates and ICAM-1 expression) was confined to the adventitia, sparing both the intima and media. Antiadenoviral neutralizing antibodies were present in all rabbits after adventitial delivery of AdNull. CONCLUSION: Infusion of adenoviral vectors into the carotid artery adventitia achieves recombinant gene expression at a level equivalent to that achieved by means of intraluminal vector infusion. Because adventitial gene transfer can be performed by means of direct application during open surgical procedures, this technically simple procedure may be more clinically applicable than intraluminal delivery. Moreover, despite the generation of a systemic immune response, adventitial infusion had no detectable pathologic effects on the vascular intima or media. For these reasons, adventitial gene delivery may be a particularly useful experimental and clinical tool.


Subject(s)
Adenoviridae/genetics , Arteritis/prevention & control , Carotid Arteries , DNA, Viral/genetics , Elastic Tissue , Gene Transfer Techniques , Genetic Vectors/administration & dosage , Animals , Antibodies, Viral/blood , Arteritis/pathology , Carotid Arteries/enzymology , Carotid Arteries/pathology , DNA, Recombinant/genetics , Elastic Tissue/enzymology , Elastic Tissue/pathology , Endothelium, Vascular/enzymology , Endothelium, Vascular/pathology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Viral , Genetic Vectors/adverse effects , Histocytochemistry , Infusions, Intra-Arterial/adverse effects , Intercellular Adhesion Molecule-1/genetics , Jugular Veins/enzymology , Male , Pharmaceutical Vehicles , Rabbits , T-Lymphocytes/pathology , Tunica Intima/pathology , Tunica Media/pathology , Vagus Nerve/enzymology , Vascular Cell Adhesion Molecule-1/genetics , beta-Galactosidase/analysis , beta-Galactosidase/genetics
14.
Proc Natl Acad Sci U S A ; 95(12): 6983-8, 1998 Jun 09.
Article in English | MEDLINE | ID: mdl-9618525

ABSTRACT

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor beta1 (TGF-beta1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-beta1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-beta1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-beta1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-beta1 in vascular disease.


Subject(s)
Apoptosis/genetics , Carotid Arteries/pathology , Carotid Arteries/physiology , Cartilage/pathology , Endothelium, Vascular/pathology , Endothelium, Vascular/physiology , Transforming Growth Factor beta/genetics , Animals , Cell Differentiation/genetics , Cell Division/genetics , Chondrocytes/pathology , Gene Expression , Gene Transfer Techniques , Rats , Rats, Sprague-Dawley
15.
Circ Res ; 82(8): 871-8, 1998 May 04.
Article in English | MEDLINE | ID: mdl-9580553

ABSTRACT

To study the role of vascular cell adhesion molecule-1 (VCAM-1) in monocyte recruitment and atherogenesis, we constructed a recombinant adenovirus, AdRSVrVCAM-1, carrying the rabbit VCAM-1 cDNA. We have previously shown that AdRSVrVCAM-1-transduced human umbilical vein endothelial cells (HUVECs) support the adhesion of CD4+ CD45RO+ memory T lymphocytes under laminar flow conditions. We now demonstrate that AdRSVrVCAM-1-transduced HUVECs support the adhesion of peripheral blood monocytes at a shear stress of < or = 1.5 dyne/cm2. Although VCAM-1 supported only firm adhesion of lymphocytes, it was able to mediate monocyte rolling, firm adhesion, and transmigration when expressed in the context of otherwise unactivated vascular endothelium. VCAM-1-transduced HUVECs supported the adhesion of as many as 4-fold more monocytes than T cells under laminar flow. The greater monocyte adhesion was explained at least in part by leukocyte-leukocyte interactions (secondary adhesions), which were not seen with T cells. These secondary monocyte interactions were specifically blocked by monoclonal antibodies to L-selectin and P-selectin glycoprotein ligand-1. These data demonstrate that VCAM-1 expressed in the context of unactivated vascular endothelium supports the adhesion of the leukocyte populations present in atherosclerotic plaque and may contribute to the predominance of monocytes over lymphocytes.


Subject(s)
Cell Adhesion/physiology , Endothelium, Vascular/physiology , Monocytes/physiology , Vascular Cell Adhesion Molecule-1/physiology , Adenoviruses, Human , Animals , Antigens, CD/analysis , Cell Line , Cells, Cultured , Endothelium, Vascular/cytology , Gene Transfer Techniques , Genetic Vectors , Humans , Integrin alpha4 , Integrin beta1/analysis , Jurkat Cells , Kidney , L-Selectin/analysis , Membrane Glycoproteins/analysis , Monocytes/cytology , P-Selectin/analysis , Rabbits , T-Lymphocytes/immunology , T-Lymphocytes/physiology , Transfection , Umbilical Veins , Vascular Cell Adhesion Molecule-1/biosynthesis
16.
Hum Gene Ther ; 9(6): 815-21, 1998 Apr 10.
Article in English | MEDLINE | ID: mdl-9581904

ABSTRACT

Arterial gene transfer with adenoviral vectors is a promising approach for the treatment and prevention of vascular disorders. However, in small animals such as rats and rabbits adenoviral vectors can have deleterious effects on the artery wall. The effects of adenovirus in primate arteries have not been studied. AdRSVn-LacZ, a replication-defective adenoviral vector, was delivered to the left brachial arteries of six hypercholesterolemic cynomolgus monkeys; right brachial arteries received vehicle only. Serum was collected before gene transfer and at vessel harvest 9 or 10 days later. Recombinant gene expression was present in occasional endothelial cells of transduced arteries, and all animals generated neutralizing antibodies. In transduced arteries, immunostaining revealed a fourfold increase in intimal and medial macrophage accumulation (p < 0.05); intimal cellularity was also significantly increased (twofold; p < 0.05). T cell density and total cellular proliferation (determined by bromodeoxyuridine labeling) were unaffected. In hypercholesterolemic nonhuman primates, adenoviral vectors increase vessel wall inflammation and promote the progression of early atherosclerotic lesions. The long-term consequences of these observations remain unclear; however, a better understanding of host responses to specific vector systems appears necessary for the development of safe and effective approaches to human vascular gene therapy.


Subject(s)
Brachial Artery/pathology , Gene Transfer Techniques , Genetic Therapy , Hypercholesterolemia/therapy , Adenoviridae , Animals , Antibody Formation , Cell Division , Endothelium, Vascular/pathology , Female , Humans , Hypercholesterolemia/pathology , Macaca fascicularis , Macrophages/pathology , Rabbits , Rats , T-Lymphocytes/pathology , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
17.
Hum Gene Ther ; 9(4): 507-20, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9525312

ABSTRACT

The ability of adenoviral vectors to mediate efficient gene delivery both in vitro and in vivo is limited by the availability of specific cell surface receptors and alpha(v)-containing integrins. We tested whether this limitation could be overcome by enhancing viral entry with cationic liposomes. In cultured vascular smooth muscle cells, delivery of adenoviral vectors in the presence of cationic liposomes increased vector-encoded transgene expression up to 20-fold. The increase in transgene expression was associated with the formation of adenovirus-lipid aggregates and an increase in the amount of vector DNA in the cells, suggesting that enhanced viral entry was responsible for the increase in gene expression. Treatment of the cells with an RGD-containing peptide or adenovirus type 5 fiber protein did not diminish liposome enhancement of transgene expression, indicating that liposomes increase viral entry via a pathway independent of the fiber receptor and of alpha(v) integrin-assisted endocytosis. Liposomes also significantly enhanced transgene expression from adenoviral vectors delivered to cells deficient in alpha(v)-containing integrins. The magnitude of liposome enhancement of transgene expression in cultured smooth muscle cells was greatest during brief periods of virus-cell contact and at low concentrations of virus. Despite these promising in vitro results, addition of liposomes did not improve in vivo adenoviral gene delivery into injured rat carotid arteries. Liposomes can improve adenoviral gene delivery in vitro; however, application of this observation to accomplish improved in vivo gene delivery remains a challenge.


Subject(s)
Adenoviridae/genetics , Capsid Proteins , Gene Transfer Techniques , Genetic Vectors/genetics , Liposomes , Animals , Antigens, CD/physiology , COS Cells , Capsid/pharmacology , Carotid Arteries , Cations , Cells, Cultured , Cytochalasin B/pharmacology , DNA, Recombinant/analysis , DNA, Viral/analysis , Drug Carriers , Gene Expression/drug effects , Genetic Vectors/ultrastructure , Integrin alphaV , Male , Muscle, Smooth, Vascular/chemistry , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/virology , Oligopeptides/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Virus/physiology , Transgenes/genetics , Virion/ultrastructure
18.
Circ Res ; 82(3): 306-13, 1998 Feb 23.
Article in English | MEDLINE | ID: mdl-9486658

ABSTRACT

The application of gene therapy techniques to the clinical problem of coronary restenosis has generated tremendous attention and enthusiasm. Use of gene transfer technology to prevent a common intractable illness would represent a watershed event for human gene therapy. However, the time is not yet right to initiate gene therapy trials for restenosis. The biology of restenosis is incompletely understood, catheter-based gene delivery is poorly adapted to the coronary circulation, and current gene transfer vectors are ill-suited for safe and effective gene delivery to the coronary artery wall. Basic research designed to overcome these obstacles is currently more appropriate than the initiation of clinical trials.


Subject(s)
Coronary Disease/therapy , Genetic Therapy , Animals , Genetic Therapy/methods , Genetic Vectors , Humans , Recurrence
19.
Nat Med ; 3(7): 738-43, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9212099

ABSTRACT

Fas ligand is believed to mediate immune privilege in a variety of tissues, including the eye, testis, and a subset of tumors. We tested whether expression of Fas ligand on pancreatic islets either following adenoviral or germline gene transfer could confer immune privilege after transplantation. Islets were infected with an adenoviral vector containing the murine Fas ligand cDNA (AdFasL), and were transplanted into allogenic diabetic hosts. Paradoxically, AdFasL-infected islets underwent accelerated neutrophilic rejection. The rejection was T cell and B cell independent and required Fas protein expression by host cells, but not on islets. Similarly, transgenic mice expressing Fas ligand in pancreatic beta cells developed massive neutrophilic infiltrates and diabetes at a young age. Thus, Fas ligand expression on pancreatic islets results in neutrophilic infiltration and islet destruction. These results have important implications for the development of Fas ligand-based immunotherapies.


Subject(s)
Cell Transplantation , Graft Rejection/immunology , Islets of Langerhans/cytology , Islets of Langerhans/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Adenoviridae , Animals , B-Lymphocytes/immunology , Diabetes Mellitus, Experimental , Fas Ligand Protein , Gene Transfer Techniques , Genetic Vectors , Islets of Langerhans/metabolism , Mice , Mice, Transgenic , Neutrophils/immunology , T-Lymphocytes/immunology , Transformation, Genetic , Transplantation Immunology , fas Receptor/immunology
20.
J Clin Invest ; 99(2): 209-19, 1997 Jan 15.
Article in English | MEDLINE | ID: mdl-9005989

ABSTRACT

Preclinical arterial gene transfer studies with adenoviral vectors are typically performed in laboratory animals that lack immunity to adenovirus. However, human patients are likely to have prior exposures to adenovirus that might affect: (a) the success of arterial gene transfer; (b) the duration of recombinant gene expression; and (c) the likelihood of a destructive immune response to transduced cells. We confirmed a high prevalence (57%) in adult humans of neutralizing antibodies to adenovirus type 5. We then used a rat model to establish a central role for the immune system in determining the success as well as the duration of recombinant gene expression after adenovirus-mediated gene transfer into isolated arterial segments. Vector-mediated recombinant gene expression, which was successful in naive rats and prolonged by immunosuppression, was unsuccessful in the presence of established immunity to adenovirus. 4 d of immunosuppressive therapy permitted arterial gene transfer and expression in immune rats, but at decreased levels. Ultraviolet-irradiated adenoviral vectors, which mimic advanced-generation vectors (reduced viral gene expression and relatively preserved capsid function), were less immunogenic than were nonirradiated vectors. A primary exposure to ultraviolet-irradiated (but not nonirradiated) vectors permitted expression of a recombinant gene after redelivery of the same vector. In conclusion, arterial gene transfer with current type 5 adenoviral vectors is unlikely to result in significant levels of gene expression in the majority of humans. Both immunosuppression and further engineering of the vector genome to decrease expression of viral genes show promise in circumventing barriers to adenovirus-mediated arterial gene transfer.


Subject(s)
Adenoviridae Infections/immunology , Adenoviruses, Human/genetics , Carotid Artery, Common/virology , Gene Transfer Techniques , Adenoviruses, Human/radiation effects , Animals , Antibodies, Viral/blood , Arteritis , Gene Expression , Genetic Vectors/radiation effects , Humans , Immunization , Immunosuppression Therapy , Male , Neutralization Tests , Rats , Rats, Sprague-Dawley , Ultraviolet Rays
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