GLP-1-(7-36 GLP-1-(7-36)-amide (GLP-1)1 is an incretin hormone that is secreted from the gastrointestinal tract in response to food intake and increases insulin secretion from pancreatic beta cells (1). The physiological action of GLP-1 gained considerable interest following the demonstration that GLP-1 acts on the pancreatic islet beta cells as a potent glucose-dependent insulin secretagogue (2-4). Despite previous structure-function studies of GLP-1 (5-8), no native analog of GLP-1 has been shown to possess potent antagonist activity.The GLP-1 receptor is a putative seven-transmembrane domain receptor (9) and belongs to the family of G-protein-coupled receptors that includes glucagon-secretin-vasoactive intestinal peptide receptors. GLP-1 binding to the pancreatic beta cell receptor induces an increase in intracellular cAMP levels (10).Exendin-4-(1-39) (exendin) was originally isolated from Gila monster venom (11) and is a member of the glucagon-secretinvasoactive intestinal peptide family of peptides. It has 48% amino acid sequence homology to glucagon and 50% homology to human GLP-1. An antagonist, exendin-4-(9 -39) (Ex9), was created by the deletion of 8 N-terminal amino acids from exendin (12). Subsequent work by Thorens et al. (13) showed that exendin acts directly on the GLP-1 receptor as an agonist, whereas Ex9 acts as an antagonist of the GLP-1 receptor and provided the first high potency antagonist of GLP-1. The purpose of this study is 2-fold: first, to analyze the transition from agonist to antagonist between exendin and Ex9, and second, to characterize the peptide domains of exendin that confer binding and thus antagonist activity by constructing exendin-(3-39)/GLP-1-(9 -36)-amide chimeras and by testing them for the retention of antagonist activity. MATERIALS AND METHODSPeptide Synthesis-Peptides were synthesized on a PAL resin solidphase support using activated Fmoc (N-(9-fluorenyl)methoxycarbonyl)-amino acids on a Milligen 9050 automated peptide synthesizer. Cleavage and deprotection of peptides were performed using 90% trifluoroacetic acid, 5% thioanisole, 3% anisole, and 2% ethanedithiol. Crude synthetic peptide mixtures were individually purified by preparative high pressure liquid chromatography. Purified peptides were quantitated by amino acid analysis.Plasmid Constructs-Full-length GLP-1 receptor cDNA isolated from rat pancreas (gift from Dr. Bernard Thorens, University of Lausanne, Lausanne, Switzerland) was subcloned in pSVbeta (CLONTEC, Palo Alto, CA) downstream of the SV40 promoter after replacing the ␤-galactosidase gene with a full-length GLP-1 receptor cDNA to obtain pSVGLPR.Cell Culture and Transfection-CHO cells that overexpress the human insulin receptor (CHO/HIRc cells) were trypsinized and resuspended in Ham's F-12 medium. Cells (10 6 cells in 800 l) were cotransfected with 10 g of HindIII-linearized pSVGLPR plasmid and 1 g of BamHI-linearized pSVHPH plasmid (conferring hygromycin resistance; American Type Culture Collection, Rockville, MD) by electroporation. Electroporation ...

Ischemic heart disease (IHD) and heart failure (HF) are major causes of morbidity and mortality in the Western society. Advances in understanding the molecular pathology of these diseases, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed these conditions within the reach of gene-based therapy. One of the cornerstones of limiting the effectiveness of gene therapy is the establishment of clinically relevant methods of genetic transfer. Recently there have been advances in direct and transvascular gene delivery methods with the use of new technologies. Current research efforts in IHD are focused primarily on the stimulation of angiogenesis, modify the coronary vascular environment and improve endothelial function with localized gene-eluting catheters and stents. In contrast to standard IHD treatments, gene therapy in HF primarily targets inhibition of apoptosis, reduction in adverse remodeling and increase in contractility through global cardiomyocyte transduction for maximal efficacy. This article will review a variety of gene-transfer strategies in models of coronary artery disease and HF and discuss the relative success of these strategies in improving the efficiency of vector-mediated cardiac gene delivery.

Heart failure (HF) is a complex multifaceted problem of abnormal ventricular function and structure. In recent years, new information has been accumulated allowing for a more detailed understanding of the cellular and molecular alterations that are the underpinnings of diverse causes of HF, including myocardial ischemia, pressure-overload, volume-overload or intrinsic cardiomyopathy. Modern pharmacological approaches to treat HF have had a significant impact on the course of the disease, although they do not reverse the underlying pathological state of the heart. Therefore gene-based therapy holds a great potential as a targeted treatment for cardiovascular diseases. Here, we survey the relative therapeutic efficacy of genetic modulation of β-adrenergic receptor signaling, Ca2+ handling proteins and angiogenesis in the most common extrinsic models of HF.

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