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<0.001; 32.9±1.9 vs 50.04±4.6 µM heme, p<0.01, for CON and HEM [1h], respect., n=5), indicative of the induction of hemolysis. Haptoglobin levels were almost completely depleted at 1 h after hemolysis, but recovered and were even higher at 24 h (14.2±2.9 vs 1.1±0.3 and 34.6±2.1 ng/mL for CON, HEM [1h] and HEM [24h], respect., p<0.01, n=5), before normalizing at 120 h. In contrast, circulating levels of hemopexin were not altered at any time post hemolysis (data not shown, p>0.05). Hemolysis also elevated the white blood cell count (2.2±0.2 vs 3.5±0.3 10³/µL for CON and HEM [1h], respect., p<0.05, n=5) and plasma IL-10 (4.8±0.6 vs 12.4±1.7 pg/mL for CON and HEM [1h] respect., p<0.001, n=10 and n=5) within 1h, suggesting that systemic inflammation accompanied this hemolysis. The red blood cell count did not change in the HEM group at any of the time points, nor did plasma IL-1β or IL-6 levels (p>0.05). A balance of angiogenic mediators, including growth factors and inflammatory cytokines, regulates angiogenesis; at 1 h after hemolysis, plasma levels of angiopoietin-2 were decreased (2.75±0.1 vs 2.1±0.15 ng/mL for CON and HEM [1h], respect., p<0.05, n=5), while pro-angiogenic VEFG and trombospondin-4 were significantly increased (163.1±8.9 vs 233.3±15.8 pg/mL and 148.6±4.1 vs 169.4±7.1 ng/mL for CON and HEM [1h], respect., p<0.05, n=5), by 24 h levels of angiogenic markers were normalized. In association with the alterations in the molecular angiogenic profile of mice after hemolysis, the formation of new blood vessels in dorsal Matrigel® plugs was significantly elevated during the 5 days following the hemolytic stimulus, as quantified by plug Hb content (2.0±0.3 vs 3.0±0.04 µg/mL for CON and HEM mice, respect., p<0.05, n=10, 11). Conclusions: Acute intravascular hemolysis was associated with rapid alterations in circulating angiogenic and inflammatory markers in mice. In association with this pro-angiogenic profile, in vivo neovascularization was accelerated in animals following hemolysis. These data suggest that hemolysis may be a significant stimulus for angiogenic processes, which in turn may contribute to some of the clinical complications of hemolytic diseases, including pulmonary hypertension, stroke and leg ulcers. Furthermore, the angiogenic process may represent a target for the development of therapeutic strategies in disorders characterized by hemolysis.