Hydroxyurea Therapy Reduces the Gene Expression of Adhesion Molecules VLA-4 and CD36 in Sickle Erythrocytes, with a Concomitant Decrease in Adhesion to Fibronectin.

Abstract

Adhesive interactions between sickle red cells, leukocytes and the endothelium are thought to initiate vaso-oclusive events in the microcirculation. An increased expression of adhesion molecules such as CD 36 (thrombospondin receptor) and VLA-4 integrin (α4β1, CD49d/CD29) has been observed on the surface of sickle reticulocytes and the increased adhesive properties of sickle erythrocytes are well documented. Gene expression of adhesion molecules and their modulation by drugs such as hydroxyurea (HU) have yet to be fully explored. We compared the expression of the CD36 gene and the VLA-4 integrin subunit genes, CD29 and CD49d, in the reticulocytes of steady-state SCD patients and patients on HU therapy (20–30 mg/Kg/day) using Real Time PCR and also compared the basal adhesion of erythrocytes from these subjects using static adhesion assays. Basal adhesion of sickle erythrocytes (2x108 cells/ml) to fibronectin (20mg/ml) - coated plates was significantly higher than normal erythrocyte adhesion (15.4 ± 2.8% compared to 5.9 ± 1.1%, n=9, p=0.008). In contrast, the adhesion of erythrocytes from SS patients on HU therapy to fibronectin was significantly lower than that of SS erythrocytes of patients not on HU (5.8 ± 0.7%, n=9, p=0.006). Pre-treatment of cells, in vitro, with varying concentrations of HU (0.1 → 250μM, 30 minutes, 37oC), had no significant effect on either normal or sickle cell erythrocyte adhesion under our experimental conditions, suggesting that HU may not reduce sickle cell adhesion by altering adhesion molecule function due to affinity changes. We next used real time PCR to investigate the expression of the mRNA of the CD36 gene and the genes for the VLA-4 subunits, CD49d and CD29, in the reticulocytes of steady-state SCD patients and patients on HU therapy. CD 36 gene expression was reduced by 62.5% ± 10.4% in the reticulocytes of patients on HU compared to the reticulocytes of SCD patients not on HU (n=8, p < 0.001). The β1 (CD29) integrin subunit expression was reduced by 87.9 ± 8.7% (n=8, p < 0.001) in patients on HU compared to SS reticulocytes; likewise α4 integrin (CD49d), expression was reduced by 50.0% ± 2.6% (n=8, p <0.001). In addition, reticulocyte mRNA was collected from one steady-state SCD patient before and 3 months after initiation of HU therapy and decreases of 99.5%, 98.0% and 98.0% were observed in the expressions of CD36, CD29 and CD49d mRNA, respectively, in the reticulocytes of this patient following HU therapy. We speculate that the decrease in adhesive properties observed in the erythrocytes of sickle cell patients during HU therapy may be the consequence of a down-regulation of the gene expressions of adhesion molecules and, consequently, protein expression by sickle cells rather than as a result of alterations in adhesion molecule function.