Effects of density and of dehydration of sickle cells on their adhesion to cultured endothelial cells

Abstract

Abnormal adhesion of sickle cells to vascular endothelium may be a factor in the initiation of painful vaso-occlusive crisis. The sickle cell population contains an unusually large number of less dense reticulocytes that are known to be more adhesive than mature red cells, but there is contradictory evidence regarding the adhesiveness of dense sickle cells. We used a flow-based assay of adhesion to cultured human umbilical vein endothelial cells to test the properties of density fractions of sickle cells, prepared either by density gradient or by centrifugation of packed cells. We also examined the effects of incubating sickle cells with or without cyclical deoxygenation on their adhesion. After fractionation on a Percoll-lsopaque gradient, the less dense 10% (reticulocyte-rich) cells and the most dense 10% cells adhered in greater number than the remainder (by about twofold). However, after centrifugation of packed cells, the less dense 10% were again more adhesive than the "middle" cells, but the most dense were not. Exposing sickle cells to constituents of the gradient had no consistent effect on adhesion, while centrifugal packing induced a degree of hemolysis, and tended to reduce adhesiveness of the dense fraction previously obtained from a gradient. Incubation in air at 37°C for 15 hr reduced the number of reticulocytes and the adhesiveness of less dense sickle cells compared to those held at 4°C. On the other hand, incubation at 37°C for 15 hr with cyclical deoxygenation caused formation of dense cells and increased adhesiveness compared to incubation without cyclical deoxygenation. We conclude that young, less dense sickle cells are unusually adhesive, but that this adhesiveness is reduced during maturation. However, repeated sickling in vivo causes formation of an abnormally dense subpopulation of cells which either redevelop an increased tendency to adhere to endothelial cells or preserve their initial adhesiveness. Both adhesive cell populations may be implicated in promoting vascular obstruction.

Efficacy and safety of the Gardos channel blocker, senicapoc (ICA-17043), in patients with sickle cell anemia

“…In addition to the decreased markers of hemolysis and increased hemoglobin concentrations that likely resulted from an improvement in the hydration state of the sickle erythrocyte, it is quite possible that the observed decrease in dense RBCs could provide further beneficial effects due to a reduction in the adhesivity of sickle RBCs to both the vascular endothelium and to the proteins of the subendothelial matrix. [14][15][16] Furthermore, because reticulocytes are particularly adhesive and express the integrin complex ␣ 4 ␤ 1 that binds to both fibronectin 17 and vascular-cell adhesion molecule-1, 18,19 it is conceivable that a decrease in circulating reticulocytes could also reduce the adhesion of sickle erythrocytes to the vascular endothelium.…”

Role of erythrocyte phosphatidylserine in sickle red cell–endothelial adhesion

“…However, a previous study has demonstrated that the maximum plasma effect on sickle RBC-endothelial adhesion occurs in vitro at a 10% plasma dilution. 37 A static adhesion assay was performed for the erythrocyte-microendothelial adhesion experiments and relationships described. Although it has been suggested that RBC adhesion mechanisms identified under conditions of flow are of more pathophysiologic relevance, it has been also noted that this view does not give consideration to the fact that microvessel blood flow can be intermittent, 38 and that stasis may be enhanced by slowmoving and relatively large granulocytes or cellular aggregates, 39 thus simulating more closely adhesion assays where flow is absent.…”

Hydroxyurea therapy decreases the in vitro adhesion of sickle erythrocytes to thrombospondin and laminin

“…Although the decrease in sickle erythrocyte adhesion to endothelial cells (Bridges et al , 1996), TSP and laminin and the decrease in integrin α 4 β 1 and CD36 expression (Styles et al , 1997) all preceded major increases in Hb F or F cells, most patients in these studies did have early increases in MCV, suggesting that cell volume may have an impact upon the adhesive phenotype. The hydration status of the erythrocyte has been reported to affect the adhesive phenotype of sickle erythrocytes to both endothelial cells (Hebbel et al , 1989; Francis et al 1991; Stone et al , 1996) and immobilized TSP and laminin (Punzalan et al , 1997), possibly by exposing or altering adhesive components of the erythrocyte membrane. A significant proportion of sickle red cells are inherently dehydrated, primarily because of intracellular K + and water losses via the erythrocyte Ca 2+ ‐dependent K + (Gardos) channel (Brugnara et al , 1986) and the K/Cl co‐transport system (Canessa et al , 1986).…”