Introduction: anti-adhesion therapy in sickle cell disease.

In this issue of Blood, Kaul et al1 report that stimulation by platelet-activating factor (PAF) of artificially perfused rat mesocecum ex vivo promotes the adhesion of human sickle red blood cells (SS RBC) to postcapillary endothelium. Notably, this adhesive interaction was blocked by two different monoclonal antibodies (MoAbs) to endothelial aVb3 integrin receptor, with resulting improvement in microvascular hemodynamics. This study is an elegant and important contribution to our understanding of SS RBC interactions with the vessel wall and, hence, the mechanisms of vasoocclusion. Vasoocclusion of small and sometimes large vessels is the hallmark of sickle cell disease, accounting for much of its morbidity and mortality. The pathophysiology of the vasoocclusive episodes is complex, involving not only the polymerization of the mutant hemoglobin, but also interactions between SS RBC, endothelium, platelets, leukocytes, and plasma constituents. Intracapillary sickling and vasoocclusion occur when transit time through capillaries is longer than the lag time for deoxygenationinduced polymerization of sickle hemoglobin. Thus, processes that delay passage of SS RBC through the microvasculature may participate in the initiation and propagation of vasoocclusion. In particular, an increase in SS RBC adhesion to postcapillary endothelium could initiate vasoocclusion by impairing flow, thereby delaying transit time of less deformable SS RBC and propagating intracapillary sickling.2 Factors such as inflammatory mediators that activate endothelial cells3 and enhance endothelial adhesivity for SS RBC might, therefore, promote vasoocclusion. Conversely, anti-adhesive or anti-inflammatory therapies might attenuate vasoocclusion. Seminal studies by Hebbel et al4 and Hoover et al5 two decades ago first demonstrated that SS RBC showed increased adherence to endothelial cells in vitro. Moreover, Hebbel et al6 showed that vasoocclusive severity correlated with adhesivity of SS RBC in vitro. Subsequently, these and many other investigators have defined adhesion pathways involved in SS RBC adhesion to cultured endothelium under static and flow conditions (Table; also reviewed in references 2 and 7). Adhesion receptors on SS RBC include a4b1 integrin8,9 and CD368,10 whose expression is increased on sickle versus normal reticulocytes8-10 and is downregulated by treatment with hydroxyurea.11 Aggregated, membranous band 312 and sulfated glycolipids exposed on the surface of damaged RBC also have been implicated. On the endothelial side, cytokine-induced VCAM-1,9,13,14 a ligand for a4b1, and avb3 integrin,10 which binds von Willebrand factor (vWf) and thrombospondin (TSP), have been demonstrated to mediate SS RBC adhesion. GpIb and CD36 are also potential endothelial adhesion receptors. The adhesive protein TSP,15,16 released by platelets, and vWf,17 released by endothelium, promote SS RBC adhesion to cultured endothelium, serving as bridging molecules between endothelial and SS RBC receptors. SS RBC interactions with the vessel wall also may involve interactions with subendothelial matrix components such as laminin (LN),18,19 TSP,18 vWf, or fibronectin20 (Table). Matrix components may be exposed by vascular injury or by endothelial retraction induced by stimuli such as thrombin.21 A sulfated glycolipid isolated from SS RBC was shown to bind to LN and TSP,18 and sulfated glycolipids also have been reported to bind to vWf.22 B-CAM/LU (basal cell adhesion molecule/lutheran protein) was recently shown to be a major LN receptor on SS RBC.19 The majority of studies of SS RBC interactions with endothelium or subendothelial matrix have used in vitro approaches. Studies with cultured cells or purified matrix components, under static or flow conditions, have been invaluable in identifying potential adhesive interactions. Ultimately, however, it is necessary to validate concepts and pathways in animal models. Fabry et al23 used an intact rat model with gamma camera imaging and visualization of the microvasculature by silicone injection. They demonstrated that desmopressin, possibly by releasing vWf, increased the retention of arterially injected deformable SS discocytes without producing overt obstruction, and suggested that narrowing of postcapillary venules by the adhesion of deformable SS RBC facilitated trapping of less deformable SS RBC, triggering vasoocclusion. The century-old technique of intravital microscopy, coupled with modern video image analysis, allows precise quantification of blood cell interactions with the vessel wall. This approach has proven to be a powerful tool in defining leukocyte-endothelial interactions, leading to the formulation of the multistep model of leukocyte emigration with selectin-mediated tethering/rolling and integrin-dependent sticking/transmigration.24 Kaul and colleagues have been leaders in applying intravital microscopy to elucidate SS RBC interactions with the vessel wall, using the rat mesocecum ex vivo25 and the sickle transgenic mouse in vivo.26 Their studies demonstrated that deformable SS RBC are more likely to adhere than dense SS RBC25 and that adhesion was limited to postcapillary venules.25,26 They also showed that desmopressin-stimulated human SS RBC adhesion to postcapillary venules in the ex vivo rat mesocolon was inhibited by anti-vWf—but not anti-TSP— antibody, providing the first trial of adhesion blockade with specific reagents in an animal model of sickle disease.27 Previous studies in vitro10,28 suggested that aVb3 integrin receptor, which is expressed on the luminal surface of endothelial cells,29 may be involved in SS RBC adhesion to endothelium. Kumar et al28 found that a conformationally constrained RGDcontaining peptide that inhibits aVb3 significantly reduced plasmainduced SS RBC adhesion to cultured endothelial cells under flow. Sugihara et al10 reported that the anti-aVb3 MoAb LM609 and anti-aIIbb3 MoAb 7E3, which cross-reacts with aVb3 but not the non–cross-reactive anti-aIIbb3 MoAb 10E5, reduced plasmadependent SS RBC static adhesion to cultured human endothelial cells. In the current study, Kaul and colleagues used the same