Acute Hemolysis Induces Pro-Angiogenic Molecule Production and Neovascularization In Vivo

Background: The intravascular hemolysis associated with hemolytic disorders, such as sickle cell anemia, results in the release of cell-free hemoglobin (Hb) and heme in the circulation, in turn, inducing inflammatory processes, vascular damage and endothelial activation. Angiogenesis, or the formation of new blood vessels, involves the proliferation, migration, and reorganization of endothelial cells in response to diverse physiological or pathological stimuli. Although angiogenesis is important for tissue growth and regeneration, uncontrolled angiogenesis can result in the accumulation of inflammatory cells, together with fibrosis and ischemia, and could play a role in some of the complications of hemolytic disorders. Aim: The aim of this study was to characterize angiogenic and inflammatory responses to the hemolytic process using an experimental in vivo model of acute hemolysis. Methods: C57BL/6J male mice were submitted, or not, to an osmotic hemolytic stimulus by intravascular injection of 150μL of sterile water (HEM group) or saline (CON group). After 1, 24 and 120 h, blood was collected by cardiac puncture for cell counts and plasma Hb and total heme quantification (colorimetric assays). Hemopexin, haptoglobin, inflammatory cytokines (Interleukin [IL]-6, IL-1β and IL-10) and angiogenic factors (Angiopoietin-2, Fibroblast growth factor [FGF]-basic, Platelet-derived growth factor [PDGF]-AA, PDGF-BB, Trombospondin-4, Vascular endothelial growth factor [VEGF], VEGFR2) were quantified in plasma by immunoassay. To evaluate in vivo neovascularization, a Matrigel®/ heparin mixture was injected subcutaneously into the dorsal region of the mice, two days before the administration of the hemolytic stimulus. After five more days, the Matrigel® plugs were removed, photographed and neovascularization quantified by colorimetric measurement of Hb in the plug. Results: At 1 h after the acute hemolytic stimulus, significant increases were observed in plasma Hb and heme (0.1±0.02 vs 0.2±0.03 g/L Hb, p