Michael J. Mann

Expression of Inducible Nitric Oxide Synthase in Human Heart Failure

BACKGROUND There is increasing evidence that alterations in nitric oxide synthesis are of pathophysiological importance in heart failure. A number of studies have shown altered nitric oxide production by the endothelial constitutive isoform of nitric oxide synthase (NOS), but there is very little information on the role of the inducible isoform. METHODS AND RESULTS We analyzed inducible NOS (iNOS) expression in ventricular myocardium taken from 11 control subjects (who had died suddenly from noncardiac causes), from 10 donor hearts before implantation, and from 51 patients with heart failure (24 with dilated cardiomyopathy [DCM], 17 with ischemic heart disease [IHD], and 10 with valvular heart disease [VHD]). Reverse transcription-polymerase chain reaction was used to confirm the presence of intact mRNA and to detect expression of iNOS and atrial natriuretic peptide (ANP). ANP was used as a molecular phenotypic marker of ventricular failure. iNOS was expressed in 36 of 51 biopsies (71%) from patients with heart failure and in none of the control patients (P<.0001). iNOS expression could also be detected in 50% of the donor hearts. All samples that expressed iNOS also expressed ANP. iNOS gene expression occurred in 67% of patients with DCM, 59% of patients with IHD, and 100% of patients with VHD. To determine whether iNOS protein was expressed in failing ventricles, immunohistochemistry was performed on three donor hearts and nine failing hearts with iNOS mRNA expression. Staining for iNOS was almost undetectable in the donor myocardium and in control sections, but all failing hearts showed diffuse cytoplasmic staining in cardiac myocytes. Expression of iNOS could be observed in all four chambers. Western blot analysis with the same primary antibody showed a specific positive band for iNOS protein in the heart failure specimens; minimal iNOS protein expression was seen in donor heart samples. CONCLUSIONS iNOS expression occurs in failing human cardiac myocytes and may be involved in the pathophysiology of DCM, IHD, and VHD.

Isolation and primary structure of human peptide YY

The isolation, primary structure and chemical synthesis of human peptide YY (PYY) are described. The peptide was purified from human colonic extracts using a chemical method which detected the C-terminal tyrosine amide structure of PYY. Human PYY consists of 36 amino acid residues and the complete amino acid sequence is: Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu- Asp-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu- Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2. The differences between the structures of porcine and human PYY are at positions 3 (Ala/Ile replacement) and 18 (Ser/Asn). Synthetic human PYY prepared using a solid-phase synthetic technique was found to be structurally identical to the natural peptide.

DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HJV)-liposomes

Manipulation of the genetic machinery of cells both in vitro and in vivo is becoming an ever more important means of elucidating pathways of molecular and cellular biochemistry. In addition, gene therapy has been proposed as a novel and potentially powerful treatment for both inherited and acquired diseases. Successful gene transfer and gene blockade generally depend on high efficiency delivery of exogenous DNA or RNA into living cells, and much effort has therefore been focused on the development of methods for achieving this delivery in a safe and effective manner. We describe here our application of fusigenic Sendai virus (HVJ)-liposome technology toward the effective delivery of DNA into vascular smooth muscle cells (VSMC) in cell culture. Cellular uptake and intracellular distribution of oligodeoxynucleotide (ODN) after transfection with HVJ-liposome complexes was characterized using fluorescent (FITC)-labeled ODN, and the biologic effect of HVJ-liposome mediated transfection was demonstrated via inhibition of DNA synthesis in cultured VSMC using antisense ODN against basic fibroblast growth factor.

901-90 In Vivo Genetic Engineering of Cardiac Cells: Intracoronary Administration of Antisense (AS) Oligonucleotides (ODN)

We have previously documented that transfection of antisense ODN by a highly efficient Sendai virus (HVJ)-liposome delivery system can be utilized to modify lesion formation within the peripheral vasculature in vivo. In this study, we defined the feasibility of modifying cardiac cell gene expression via a catheter-based coronary infusion of AS ODN in rabbits. The coronary artery was cannulated via an over-the-wire approach from the carotid artery. Fluorescein (F)-Iabeled ODN were utilized to evaluate the cellular distribution and kinetics of ODN uptake within the myocardium after a single intraluminal bolus of HVJ-liposomes containing ODN. Cellular uptake of F-ODN was primarily localized in the microvasculature and significant staining was also observed in conduit vessels and cardiac myocytes. Immunohistochemical analysis verified prominent localization of F-ODN within the microvascular endothelium. Expression of F-ODN was observed within 10 minutes, peaked at 1 day, and remained evident for up to one week after transfection by the HVJ-liposome method. In contrast, F-ODN infused within liposomes without the viral particle exhibited transient expression that was undetectable within 3 days. These findings indicate that a single intracoronary bolus infusion of ODN within HVJ-liposomes is a reproducible methodology for delivery of AS ODN to targeted cells within the myocardium. Future studies will characterize the feasibility of using this approach to modify cardiac structure and function via regulating myocardial cell gene expression.