Increased Angiogenic Activity of Plasma From Sickle Cell Disease Patients and Anti-Angiogenic Effects of Hydroxyurea: Evaluation of Capillary-Like Structure Formation of Human Umbilical Vein Endothelial Cells on Matrigel
Authors: Mascia Lopes Flávia Cristine , Fertrin Kleber Yotsumoto , Olalla-Saad Sara T. , Costa Fernando Ferreira , Conran Nicola 
Published: October 2019

Abstract
Abstract 899
Sickle cell disease (SCD) results from mutations in the β chain of hemoglobin and is associated with a complex pathophysiological process that involves endothelial activation, inflammation and oxidative stress. Several clinical aspects of SCD such as pulmonary hypertension, moyamoya vasculopathy, proliferative retinopathy and leg ulcers suggest the involvement of an unbalanced angiogenesis. Plasma levels of vascular endothelial growth factor, placenta growth factor, angiopoietin-1 and angiopoietin-2 are reportedly elevated in SCD, although the effects of these alterations on angiogenic mechanisms in SCD are unclear. Hydroxyurea (HU) has been used successfully for SCD therapy and may act through several mechanisms. The purpose of this study was to evaluate the effects of plasma obtained from SCD patients upon capillary-like structure formation of human umbilical vein endothelial cell (HUVEC) cultures on Matrigel. Patients (in steady state) and controls were divided into six groups: HbSS phenotype not treated with HU (SSHU−), HbSS treated with HU (SSHU+), HbSC phenotype with retinopathy not treated with HU (SCR+HU−), HbSC without retinopathy and not treated with HU (SCR-HU−), HbSC with retinopathy and treated with HU (SCR+HU+) and control individuals (HbAA). Patients on HU had been taking 20–30mg/kg/day for at least 3 months. To assess tube formation, HUVECs (5× 104cells/mL) were cultured on growth factor reduced-Matrigel coated plates in the presence of 10% plasma and the number of cord-like structures quantified. Photomicrographs were taken using a phase-contrast inverted microscope. HUVEC were also incubated with HU (50–200 μM) and migration (double-chamber assay), proliferation (BrdU assay) and capillary-like structure formation (Matrigel) were addressed to compare effects of plasma from patients treated with HU to medium containing HU alone. After 17 H of incubation, a small decrease in HUVEC tube formation (4.1 ±4.9%, n=5, P>0.05 comp. to control, HUVEC in medium alone) was observed in the presence of HbAA group plasma, in agreement with reports suggesting an antiangiogenic activity of human plasma in absence of diseases. In contrast, angiogenesis of HUVEC increased significantly in the presence of plasma from all SCD patients, independently of retinopathy, as demonstrated by increases in tube formation indexes of 32.5 ±7.5%, 15.8 ±9.2%, 30.0 ±4.4% (N=5, P<0.05 comp. control, ANOVA-Dunnet) for the SSHU−, SCR-HU− and SCR+HU−, respectively. Notably, HUVEC cultures containing plasma from patients treated with HU presented considerably less angiogenesis compared to control cultures. The ability of HUVEC to form branching and thick anastomosing capillaries on Matrigel was inhibited by SSHU+ and SCR+HU+ plasma; furthermore, tube formation indexes decreased by 61.7 ± 8.8% and 92.3 ±2.9% in the presence of these plasmas (P<0.001, n= 5/2 respect.). In experiments conducted with just HU (200μM, n=5) in the HUVEC medium, significant decreases in cell migration (51.1 ± 3.45%; P<0.001), proliferation capacity (49.4 ± 4.66%; P<0.001) and capillary-like structure formation 75.8 ± 4.1% (P<0.001) were observed. Importantly, MTT (tetrazolium salt) assays showed that none of the plasmas nor HU was cytotoxic to HUVEC at the concentrations used. Angiogenesis is a complex process, where multiple contributing factors can have opposing effects. Plasma is rich in proteins that regulate angiogenesis and our data indicate that plasma from SCD patients is highly pro-angiogenic at a biologically-relevant level. Interestingly, we detected no significant difference between SC and SS individuals, although plasma from SC individuals with retinopathy was significantly more angiogenic than that of SC individuals without retinopathy. The highly anti-angiogenic capacity of plasma from patients treated with HU suggests that one of the beneficial effects of this drug may be inhibition of angiogenesis. Data suggest that the HU drug itself may be capable of regulating angiogenesis by attenuating migration, proliferation and tube formation; however, since the half-life of HU is about 3–4 H, these anti-angiogenic effects are probably not mediated directly by this drug, but by newly-generated molecule(s) with a longer half-life. In conclusion, our study highlights the possible role of angiogenesis in SCD pathophysiology and suggests that SCD patients could be candidates for anti-angiogenic therapy.

Disclosures:
No relevant conflicts of interest to declare.