Several diseases and pathological events incur intravascular hemolysis (IVH), a pathophysiological mechanism characterized by the destruction of red blood cells and the release of hemoglobin (Hb) and heme into the circulation. Hemolytic diseases, such as sickle cell disease (SCD), result in chronic inflammation and vascular damage due to the accumulation of plasma Hb and heme, which overload the homeostatic defense system. Hemolytic disorders are often associated with dysregulated angiogenic processes, contributing to complications that include proliferative retinopathy, pulmonary hypertension, leg ulcers and neovascularization of ischemic tissues. However, the role of hemolytic events in generating angiogenic factors and mechanisms is unclear. Therefore, the aim of this study was to investigate the effects of hemolysis on neovascularization and angiogenic parameters in an in vivo experimental model of IVH. C57BL/6J mice were subjected to chronic IVH, induced by the administration of low doses of phenylhydrazine (LDPHZ; 10 mg/kg.day i.p twice a week) for 4 weeks, while control mice (CON) received saline at the same frequency and during the same experimental period. Blood samples were collected 48 hours and plasma separated after the last induction. Matrigel® plugs pre-inoculated with 50 μL plasma from mice subjected to hemolysis, or not, were injected subcutaneously in mice for evaluation of neovascularization. Chronic IVH significantly increased plasma vascular endothelial growth factor (VEGF) levels (p = 0.04; n = 5) and liver VEGF expression (p = 0.02; n = 4) compared to saline mice. No significant differences were found in other angiogenic factors evaluated. Furthermore, factors present in the plasma of mice subjected to chronic IVH stimulated neovascularization and accelerated the formation of new vessels, as demonstrated by a significant increase in Hb levels quantified in the plug (p = 0.04; n = 5), compared to control plugs. Concurrently with hemolysis induction, an experimental group received treatment with hydroxyurea (LDPHZ+HU; 50 mg/kg.day i.p. five times a week) during the 4 weeks. LDPHZ+HU mice presented a trend towards increased VEGF protein expression in the liver (p = 0.06; n = 4) and the plasma factors of these HU treated mice reduced (although not significantly) Matrigel-plug neovascularization, when compared to neovascularization induced by LDPHZ plasma (p = 0.06; n = 5). An in vivo wound healing assay was also performed on the dorsal skin of mice subjected to IVH and treated or not with HU (n = 2). Preliminary results showed that wound healing was slower in the LDPHZ group when compared to the CON group, while treatment with HU improved the wound-healing process. Therefore, this study provides important perspectives for understanding the angiogenic mechanisms associated with IVH and may contribute to the development of therapies aimed at modulating neovascularization and minimizing complications associated with dysregulated angiogenic processes in patients with clinical complications of hemolytic diseases.

FAPESP.