Pancreatic ascites haemoglobin up-regulates the HIF/VEGF pathway in the lung in severe acute pancreatitis.

Extracellular haemoglobin (EHb) is considered a toxic molecule due to its cytotoxicity and peroxidase activity. EHb may release free hemin that increases vascular permeability, leukocyte recruitment, and adhesion molecule expression. Pancreatitis-associated ascitic fluid is reddish and may contain cell-free hemoglobin. Our aim was to determine the role of EHb in the local and systemic inflammatory response during severe acute pancreatitis in rats. To this end, taurocholate-induced necrotizing pancreatitis in rats was used. EHb levels were quantified in ascites and plasma and the hemolytic action of ascitic fluid was tested. Furthermore, we assessed if peritoneal lavage prevented the increase in EHb levels in plasma during pancreatitis. Finally, hemoglobin was purified from rat erythrocytes and administered i.p. to assess the local and systemic effects of ascitic-associated EHb during acute pancreatitis. EHb levels markedly increased in ascitic fluid and plasma during necrotizing pancreatitis. Peroxidase activity was very high in ascites. The peritoneal lavage abrogated the increase in cell-free hemoglobin in plasma. The administration of EHb enhanced ascites, dramatically increased abdominal fat necrosis, up-regulated tumor necrosis factor-a, interleukin 1ß and interleukin 6 gene expression and decreased expression of interleukin 10 in abdominal adipose tissue during pancreatitis. EHb enhanced the gene expression and protein levels of vascular endothelial growth factor (VEGF) and other hypoxia inducible factor-related genes [inducible nitric oxide synthase (inos), endothelial nitric oxide synthase (enos) and hexokinase 2] in the lung. EHb also increased myeloperoxidase activity in the lung. In conclusion, extracellular hemoglobin contributes to the inflammatory response in severe acute pancreatitis through abdominal fat necrosis and inflammation and increasing VEGF and leukocyte infiltration in the lung.