Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

Abstract

ABSTRACTas a therapy in SCD. 25 Hydroxyurea is a cytostatic drug that inhibits ribonucleotide reductase, arresting cell division in the S-phase; additionally, data suggest that hydroxyurea can generate nitric oxide following administration. 26 We recently reported that hydroxyurea exerts anti-angiogenic effects in VEGF-dependent EC angiogenic in vitro assays and inhibits VEGF-dependent neovascularization in Matrigel implants in C57BL/6 mice. 27 This anti-angiogenic activity appears to be mediated by the down-regulation of endothelial hypoxia-inducible factor-1α (HIF-1α) expression, and consequent modulation of miRNA 221 expression, leading to the inhibition of EC proliferation and invasion/migration. 27To lend further support to the hypothesis of an unbalanced angiogenic state in SCD, we investigated the in vitro effects of plasma from patients with SCD on key steps of EC angiogenic behavior, namely, proliferative capability, invasive capacity and the formation of capillary-like EC structures. The plasma contents of angiogenic factors were associated with these data, as was the incidence of proliferative retinopathy in HbSC patients, a clinical manifestation thought to reflect augmented angiogenesis. 28The angiogenic activity of plasma from patients on hydroxyurea therapy was also examined. Finally, to confirm that SCD pathophysiology is associated with exacerbated angiogenesis, in vivo neovascularization was examined in a SCD mouse model, compared with non-SCD mice, utilizing a Matrigel plug angiogenesis assay. Methods PatientsPatients with HbSS or HbSC disease (collectively termed SCD) were recruited, during steady-state, at the Hematology Center, UNICAMP (Table 1). Details on the diagnosis of HbSS/HbSC, our definition of steady-state and criteria for commencing hydroxyurea therapy (15-30 mg hydroxyurea/kg/day) are given in the Online Supplementary Information. All patients had undergone ophthalmoscopy and fluorescein angiography for detection of proliferative retinopathy. A total of 29 HbSS patients, who did not have proliferative retinopathy, were recruited into the study. A total of 33 HbSC patients were recruited, of whom 16 did not have proliferative retinopathy (SC group) and 17 did (SCR group). One HbSC patient had been prescribed hydroxyurea following an ischemic stroke. Homozygous hemoglobin A (HbAA) healthy controls were age-and gender-matched, where possible. Informed written consent was obtained from all participants and the study was approved by the UNICAMP Ethics Committee, in accordance with national guidelines. Bioplex assayThe Fluorokine ® MAP, Human Angiogenesis Custom Premix Kit A (R&D Systems, Minneapolis, USA) was used to quantify Ang1, basic fibroblast growth factor (bFGF), PlGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, VEGF, VEGF-D, endostatin and thrombospondin-2 (TSP-2) in plasma, according to the manufacturer's instructions. The preparation of the plasma for the assay is described in the Online Supplementary Information. In vitro culture of human umbilical vein endothelial c...

Bone marrow microvessel density and plasma angiogenic factors in myeloproliferative neoplasms: clinicopathological and molecular correlations

“…Limitation of our study is that the grading of fibrosis was performed for reticular but not collagen fibrosis, due to the small number of patients with grades of fibrosis 2 and 3 [36]. Antiangiogenic effect of HU has been reported in sickle cell disease [37]. After 6 months of treatment with HU, plasma angiogenic factors and MVD significantly decreased in MPNs, except bFGF in PV and ET, according to our study.…”

Role of innate immunity-triggered pathways in the pathogenesis of Sickle Cell Disease: a meta-analysis of gene expression studies

“…In this regard, several details on how the immune system ultimately responds to HbS polymerization - the initial trigger of the pathogenic cascade of SCD - have been described. This complex immune response involves endothelial and coagulation activation 18 19 , increased cellular adhesion 20 , expression of pro-inflammatory and pro-angiogenic cytokines 21 , neutrophil activation 22 and increased oxidative stress 23 . Although critical to our understanding of SCD pathogenesis, these studies do not explain how exactly inflammation is elicited in the first place, nor the hierarchical relationship between all these elements.…”

Ischemia-Reperfusion Injury in Sickle Cell Disease

“…18 SCD microvasculature is also highly proangiogenic, which has been attributed to the hypoxic environment and increased levels of various proangiogenic factors, including vascular endothelial growth factor, placental growth factor, angiopoietin-1, angiopoietin-2, and erythropoietin in the circulation. 18,21 For further reading about the role of the endothelium in SCD, please refer to the review by Hebbel et al 18…”