pr-39 and Myocardial-Infarction

Cotransfer of angiogenic and antiapoptotic genes could be the basis of new gene therapy strategies for myocardial infarction. In this study, rAAV-PR39-ADM, coexpressing antimicrobial peptide (PR39) and adrenomedullin (ADM), was designed with the mediation of recombinant adeno-associated virus. In vitro, CRL-1730 cells were divided into four groups, namely, the sham group, the AAV-null group, the NS (normal saline) group, and the PR39-ADM group. Immunocytochemistry analysis, CCK-8 assays, Matrigel assays, and apoptotic analysis were performed; in vivo, myocardial infarction model was established through ligation of the left coronary artery on rats, and treatment groups corresponded to those used in vitro. Myocardial injury, cardiac performance, and the extent of myocardial apoptosis were assessed. Results suggested that rAAV-PR39-ADM administration after myocardial infarction improved cell viability and cardiac function, attenuated apoptosis and myocardial injury, and promoted angiogenesis. Subsequently, levels of 6×His, HIF-1

Topics: Adrenomedullin; Animals; Antimicrobial Cationic Peptides; Apoptosis; bcl-2-Associated X Protein; Cell Line; Dependovirus; Disease Models, Animal; Genetic Vectors; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Vascular Endothelial Growth Factor A

PR39 is an angiogenic masterswitch protein, belonging to the second generation of angiogenic growth factors. However, the role of recombinant adeno-associated virus (AAV) carrying the PR39 fusion gene (AAV-PR39) in acute myocardial infarction remains unclear. Therefore, in this study, we investigated the role of AAV-PR39 in an experimental animal model of acute myocardial infarction. The PR39 gene was fused with the transmembrane peptide, TAT, 6xHis‑tag and NT4 signal sequences. AAV-PR39 was then obtained by calcium phosphate co-precipitation. A total of 18 healthy Chinese mini pigs were randomly divided into an experimental groups (the AAV-PR39-treated group) and a control group [phosphated-buffered saline (PBS)-treated group]. Following the induction of myocardial infarction, enhanced 3.0T MR imaging was performed to observe the changes in myocardial signal intensity at 0 h, 1, 2 and 3 weeks. The expression of hypoxia-inducible factor‑1α (HIF-1α) in the myocardial tissues was determined by SABC immunohistochemistry. In addition, in vitro experiments using CRL-1730 endothelial cells transfected with AAV vector containing NT4-TAT-His-PR39 revealed that the AAV-PR39-treated group had a significantly higher expression of HIF-1α compared with the control group. Moreover, PR39 regulated the HIF-1α-induced expression of angiogenic growth factors. Under hypoxic conditions, the anti-apoptotic effects in the AAV-PR39 group were more pronounced than those observed in the control (PBS-treated) group. In vivo, the enforced expression of recombinant PR39 elevated the level of HIF-1α under hypoxic conditions and decreased the size of the infarcted areas by upregulating the expression of HIF-1α in the areas surrounding the infarct area. Taken together, our data demonstrate that the recombinant AAV-PR39-mediated HIF-1α expression attenuates myocardial infarction, indicating that AAV-PR39 may serve as a novel therapeutic agent for the treatment of myocardial infarction.

Topics: Animals; Antimicrobial Cationic Peptides; Apoptosis; Cell Line; Dependovirus; Gene Expression; Genetic Therapy; Genetic Vectors; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Myocardial Infarction; Myocardium; Recombinant Proteins; Swine; Swine, Miniature

Although tissue injury and inflammation are considered essential for the induction of angiogenesis, the molecular controls of this cascade are mostly unknown. Here we show that a macrophage-derived peptide, PR39, inhibited the ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1alpha protein, resulting in accelerated formation of vascular structures in vitro and increased myocardial vasculature in mice. For the latter, coronary flow studies demonstrated that PR39-induced angiogenesis resulted in the production of functional blood vessels. These findings show that PR39 and related compounds can be used as potent inductors of angiogenesis, and that selective inhibition of hypoxia-inducible factor-1alpha degradation may underlie the mechanism of inflammation-induced angiogenesis.

Topics: Animals; Antimicrobial Cationic Peptides; Aorta; Capillaries; Cattle; Cell Hypoxia; Cells, Cultured; Coronary Vessels; Cysteine Endopeptidases; DNA-Binding Proteins; Endothelium, Vascular; Heart; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multienzyme Complexes; Myocardial Infarction; Myocardial Ischemia; Neovascularization, Physiologic; Nuclear Proteins; Peptides; Proteasome Endopeptidase Complex; Recombinant Proteins; Swine; Transcription Factors; Ubiquitins; Umbilical Veins; von Willebrand Factor

Induction of NF-kappaB-dependent gene expression plays an important role in a number of biological processes including inflammation and ischemia-reperfusion injury. However, few attempts aimed at selective regulation of this transcription factor have been successful. We report here that a naturally occurring antibacterial peptide PR39 reversibly binds to the alpha 7 subunit of the 26S proteasome and blocks degradation of NF-kappa B inhibitor I kappa B alpha by the ubiquitin-proteasome pathway without affecting overall proteasome activity. I kappa B alpha phosphorylation and ubiquitination occur normally after PR39 treatment, and binding of valosin-containing proteins is not impaired. The inhibition of I kappa B alpha degradation abolishes induction of NF-kappa B-dependent gene expression in cell culture and in mouse models of acute pancreatitis and myocardial infarction, including upregulation of endothelial adhesion proteins VCAM-1 and ICAM-1. In the latter model, sustained infusion of PR39 peptide resulted in significant reduction of myocardial infarct size. PR39 and related peptides may provide novel means to regulate cellular function and to control of NF-kappa B-dependent gene expression for therapeutic purposes.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Cells, Cultured; Cysteine Endopeptidases; DNA-Binding Proteins; Gene Expression; Humans; I-kappa B Proteins; Male; Mice; Mice, Inbred ICR; Mice, Transgenic; Multienzyme Complexes; Myocardial Infarction; NF-kappa B; NF-KappaB Inhibitor alpha; Pancreatitis; Peptides; Proteasome Endopeptidase Complex; Swine; Ubiquitins

We investigated the effects of PR-39, a recently discovered neutrophil inhibitor, in a murine model of myocardial ischemia-reperfusion injury. Mice were given an intravenous injection of vehicle (n = 12) or PR-39 (n = 9) and subjected to 30 min of coronary artery occlusion followed by 24 h of reperfusion. In addition, the effects of PR-39 on leukocyte rolling and adhesion were studied utilizing intravital microscopy of the rat mesentery. The area-at-risk per left ventricle was similar in vehicle- and PR-39-treated mice. However, myocardial infarct per risk area was significantly (P < 0.01) reduced in PR-39 treated hearts (21.0 +/- 3.8%) compared with vehicle (47.1 +/- 4.8%). Histological analysis of ischemic reperfused myocardium demonstrated a significant (P < 0.01) reduction in polymorphonuclear neutrophil (PMN) accumulation in PR-39-treated hearts (n = 6, 34.3 +/- 1.7 PMN/mm(2)) compared with vehicle-treated myocardium (n = 6, 59.7 +/- 3.1 PMN/mm(2)). In addition, PR-39 significantly (P < 0.05) attenuated leukocyte rolling and adherence in rat inflamed mesentery. These results indicate that PR-39 inhibits leukocyte recruitment into inflamed tissue and attenuated myocardial reperfusion injury in a murine model of myocardial ischemia-reperfusion.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Adhesion; Coronary Vessels; Hemostatics; Ileum; Ligation; Male; Mice; Mice, Inbred Strains; Microscopy; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Neutrophils; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Thrombin