Biomaterial Database

Vascular inducible nitric oxide synthase gene therapy: requirement for guanosine triphosphate cyclohydrolase I. 1996

E Tzeng, and T Yoneyama, and K Hatakeyama, and L L Shears, and T R Billiar

Department of Surgery, University of Pittsburgh, Pa 15261, USA.

BACKGROUND Human inducible nitric oxide synthase (iNOS) gene transfer inhibits myointimal hyperplasia in vitro. However, unstimulated vascular smooth muscle cells (SMC) do not synthesize tetrahydrobiopterin (BH4), an essential cofactor for iNOS, which may be an obstacle to successful vascular iNOS gene therapy. We investigated the capacity of gene transfer of guanosine triphosphate (GTP) cyclohydrolase I (GTPCH), the rate-limiting enzyme for BH4 biosynthesis, to supply cofactor for iNOS activity. METHODS A human GTPCH expression plasmid (pCIS-GTPCH) was transfected into rat aortic SMC (RAOSMC) and BH4-deficient NIH3T3 cells engineered to stably express human iNOS (3T3-iNOS). GTPCH activity and intracellular biopterins were assessed as a measure of successful transfection, and the capacity of GTPCH to reconstitute iNOS activity was used to determine whether BH4 was made available to the iNOS protein. RESULTS The pCIS-GTPCH-transfected 3T3 cells had demonstrable GTPCH activity as compared with control cells (169.3 +/- 6.6 pmol/hr/mg versus 0, p < 0.001). Intracellular biopterin levels were also increased in transfected 3T3 and SMC (60.6 +/- 2.6 and 101.7 +/- 28.3 pmol/mg, respectively, versus less than 4 in control cells). GTPCH reconstituted near-maximal iNOS activity in 3T3-iNOS cells despite a gene transfer efficiency of less than 1%. GTPCH and iNOS enzymes did not have to coexist in the same cell for the synthesized BH4 to support iNOS activity. CONCLUSIONS GTPCH gene transfer reconstitutes iNOS activity in BH4-deficient cells despite poor transfer efficiency. GTPCH can deliver a cofactor to targeted cells even if it is synthesized in neighboring cells, and may be a means to concurrently deliver BH4 with iNOS in vivo.

UI	MeSH Term	Description	Entries
D009131	Muscle, Smooth, Vascular	The nonstriated involuntary muscle tissue of blood vessels.	Vascular Smooth Muscle,Muscle, Vascular Smooth,Muscles, Vascular Smooth,Smooth Muscle, Vascular,Smooth Muscles, Vascular,Vascular Smooth Muscles
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D000975	Antioxidants	Naturally occurring or synthetic substances that inhibit or retard oxidation reactions. They counteract the damaging effects of oxidation in animal tissues.	Anti-Oxidant,Antioxidant,Antioxidant Activity,Endogenous Antioxidant,Endogenous Antioxidants,Anti-Oxidant Effect,Anti-Oxidant Effects,Anti-Oxidants,Antioxidant Effect,Antioxidant Effects,Activity, Antioxidant,Anti Oxidant,Anti Oxidant Effect,Anti Oxidant Effects,Anti Oxidants,Antioxidant, Endogenous,Antioxidants, Endogenous
D001011	Aorta	The main trunk of the systemic arteries.	Aortas
D001708	Biopterins	Pterin derivatives based on 2-amino-6-(1,2-dihydroxypropyl)-4(1H)-pteridinone. Biopterins are natural products that have been considered as growth factors for some insects. Biopterins are cofactors for the AROMATIC AMINO ACID hydroxylases and NITRIC OXIDE SYNTHASE. Deficiencies in BIOPTERINS metabolism (e.g., lowered TETRAHYDROBIOPTERIN) are associated with neurological deterioration (e.g., HYPERPHENYLALANINAEMIA).	2-Amino-6-((1S,2R)-1,2-dihydroxypropyl)-4(1H)-pteridinone,2-Amino-6-((1S,2S)-1,2-dihydroxypropyl)-4(1H)-pteridinone,2-Amino-6-(1,2-dihydroxypropyl)-4(8H)-pteridinone,2-amino-6-((1R,2R)-1,2-dihydroxypropyl)-4(3H)-pteridinone,4(1H)-Pteridinone, 2-amino-6-(1,2-dihydroxypropyl)-, (S-(R,S))-,6-Biopterin,Biopterin,D-threo-Biopterin,L-Biopterin,L-erythro-Biopterin,L-threo-Biopterin,2-Amino-6-(1,2-dihydroxypropyl)-4(1H)-pteridinone,Dictyopterin,Orinapterin,6 Biopterin,D threo Biopterin,L Biopterin,L erythro Biopterin,L threo Biopterin
D014162	Transfection	The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES.	Transfections
D015152	Blotting, Northern	Detection of RNA that has been electrophoretically separated and immobilized by blotting on nitrocellulose or other type of paper or nylon membrane followed by hybridization with labeled NUCLEIC ACID PROBES.	Northern Blotting,Blot, Northern,Northern Blot,Blots, Northern,Blottings, Northern,Northern Blots,Northern Blottings
D015316	Genetic Therapy	Techniques and strategies which include the use of coding sequences and other conventional or radical means to transform or modify cells for the purpose of treating or reversing disease conditions.	Gene Therapy,Somatic Gene Therapy,DNA Therapy,Gene Therapy, Somatic,Genetic Therapy, Gametic,Genetic Therapy, Somatic,Therapy, DNA,Therapy, Gene,Therapy, Somatic Gene,Gametic Genetic Therapies,Gametic Genetic Therapy,Genetic Therapies,Genetic Therapies, Gametic,Genetic Therapies, Somatic,Somatic Genetic Therapies,Somatic Genetic Therapy,Therapies, Gametic Genetic,Therapies, Genetic,Therapies, Somatic Genetic,Therapy, Gametic Genetic,Therapy, Genetic,Therapy, Somatic Genetic
D015971	Gene Expression Regulation, Enzymologic	Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control of gene action in enzyme synthesis.	Enzymologic Gene Expression Regulation,Regulation of Gene Expression, Enzymologic,Regulation, Gene Expression, Enzymologic