pr-39 and Hypoxia

To investigate the impact of AAV-encoding NT4-TAT-His-PR39 fusion gene expression on HIF-1alpha level in ECV304 cultured under hypoxic condition (1%O(2)) and on angiogenesis in hypoxic chick embryo.. PR39 cDNA was connected with NT4, TAT, 6 x His cDNA by molecular biology methods. The recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells. Then ECV304 were respectively infected with AAV-NT4-TAT-His-PR39, 6 x His expression and HIF-1alpha level in ECV304 were detected by immunocytochemistry. The chicken embryos were randomized into the AAV-PR39, EV and PBS groups (n = 10 each) subject to hypoxia (5%O(2), n = 15) or normoxia environments (n = 15), the vessel density of the chicken chorioallantoic membrane (CAM) were measured by Image Pro Plus (IPP) software.. The expression of 6 x His protein was detected in AAV-PR39 infected ECV304 cells. HIF-1alpha protein activity was significantly increased in AAV-PR39 infected ECV304 underwent hypoxia compared to PBS and non-infected ECV304 groups (P < 0.05). The vessel density of chicken CAM in hypoxia environment but not in normoxia environment was also significantly higher in AAV-PR39 group than in EV group and PBS group (all P < 0.05).. AAV-encoding NT4-TAT-His-PR39 fusion gene expression significantly increased HIF-1alpha level in ECV304 exposed to hypoxia and promoted angiogenesis in hypoxic chicken embryo.

Topics: Animals; Antimicrobial Cationic Peptides; Cell Line; Chick Embryo; Dependovirus; Gene Fusion; Gene Transfer Techniques; Genes, Viral; Hypoxia; Neovascularization, Physiologic

Endotoxin (a lipopolysaccharide (LPS) component of the Gram negative bacterial cell wall) induces sepsis in laboratory animals and is the cause of septic shock in patients. Tissues often develop necrotic regions, particularly in kidney and liver, thought to be directly the result of endotoxin-induced release of nitric oxide (NO). These studies investigated the potential of PR-39, an antibacterial peptide, as an alternative treatment for sepsis. Our rationale for these experiments was based on the knowledge that PR-39 inhibits the superoxide-producing NADH/NADPH-oxidase system, and also inhibits NOS. In a mouse model of sepsis, we carried out EPR measurements of liver pO2 and NO simultaneously in vivo. Physiological parameters were also measured in these animals (blood pressure, heart rate). NO levels in blood were measured by EPR analysis of red blood cell nitrosyl-hemoglobin. We found PR-39 alleviated endotoxin-induced liver hypoxia 6 hrs after treatment. Tissue NO was higher in the PR-39 + LPS group compared to LPS alone. Circulating levels of NO were the same in these groups. Taken together, these results suggest PR-39 is effective in improving survival following a septic episode. The exact mechanism is unclear, but increased NO as a result of decreased superoxide production seems to play an important role in alleviating tissue hypoxia.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Infections; Hypoxia; Male; Mice; Mice, Inbred BALB C; Sepsis; Spin Labels