Milena Batchvarova

Sickle red cells induce adhesion of lymphocytes and monocytes to endothelium.

Infusion of epinephrine-activated human sickle erythrocytes (SS RBCs) into nude mice promotes both SS RBC and murine leukocyte adhesion to vascular endothelium in vivo. We hypothesized that interaction of epinephrine-stimulated SS RBCs with leukocytes leads to activation of leukocytes, which then adhere to endothelial cells (ECs). In exploring the underlying molecular mechanisms, we have found that coincubation in vitro of epinephrine-treated SS RBCs with human peripheral blood mononuclear cells (PBMCs) results in robust adhesion of PBMCs to ECs. Sham-treated SS RBCs had a similar but less pronounced effect, whereas neither sham- nor epinephrine-treated normal RBCs activated PBMC adhesion. PBMC activation was induced via at least 2 RBC adhesion receptors, LW and CD44. In response to SS RBCs, leukocyte CD44 and beta2 integrins mediated PBMC adhesion to ECs, a process that involved endothelial E-selectin and fibronectin. SS RBCs activated adhesion of both PBMC populations, lymphocytes and monocytes. Thus, our findings reveal a novel mechanism that may contribute to the pathogenesis of vaso-occlusion in sickle cell disease, in which SS RBCs act via LW and CD44 to stimulate leukocyte adhesion to endothelium, and suggest that RBC LW and CD44 may serve as potential targets for antiadhesive therapy designed to prevent vaso-occlusion.

Effect of Propranolol as Antiadhesive Therapy in Sickle Cell Disease

Sickle red blood cells (SSRBCs) adhere to both endothelial cells (ECs) and the extracellular matrix. Epinephrine elevates cyclic adenosine monophosphate in SSRBCs and increases adhesion of SSRBCs to ECs in a β‐adrenergic receptor and protein kinase A‐dependent manner. Studies in vitro as well as in vivo have suggested that adrenergic stimuli like epinephrine may contribute to vaso‐occlusion associated with physiologic stress. We conducted both animal studies and a Phase I dose‐escalation study in sickle cell disease (SCD) patients to investigate whether systemically administered propranolol inhibits SSRBC adhesion and to document the safety of propranolol in SCD. Systemically administered propranolol prevented SSRBC adhesion and associated vaso‐occlusion in a mouse model. In patients receiving a single oral dose of 10, 20, or 40 mg propranolol, SSRBC adhesion to ECs was studied before and after propranolol, with and without stimulation with epinephrine. Propranolol administration significantly reduced epinephrine‐stimulated SSRBC adhesion in a dose dependent manner (p = 0.03), with maximum inhibition achieved at 40 mg. Adverse events were not severe, did not show dose dependence, and were likely unrelated to drug. No significant heart rate changes occurred. These results imply that β‐blockers may have a role as antiadhesive therapy for SCD. Clin Trans Sci 2012; Volume 5: 437–444

Sevuparin binds to multiple adhesive ligands and reduces sickle red blood cell-induced vaso-occlusion

Sevuparin is a novel drug candidate in phase II development as a treatment for vaso‐occlusive crises (VOC) in patients with sickle cell disease (SCD). As a heparin‐derived polysaccharide, sevuparin has been designed to retain anti‐adhesive properties, while the antithrombin‐binding domains have been eliminated, substantially diminishing its anticoagulant activity. Here, we demonstrate that sevuparin inhibits the adhesion of human sickle red blood cells (SS‐RBCs) to stimulated cultured endothelial cells in vitro. Importantly, sevuparin prevents vaso‐occlusion and normalizes blood flow in an in vivo mouse model of SCD vaso‐occlusion. Analyses by surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) demonstrate that sevuparin binds to P‐ and L‐selectins, thrombospondin, fibronectin and von Willebrand factor, all of which are thought to contribute to vaso‐occlusion in SCD. Despite low anticoagulation activity, sevuparin has anti‐adhesive efficacy similar to the low molecular weight heparin tinzaparin both in vitro and in vivo. These results suggest that the anti‐adhesive properties rather than the anticoagulant effects of heparinoids are critical for the treatment of vaso‐occlusion in SCD. Therefore, sevuparin is now being evaluated in SCD patients hospitalized for treatment of VOC.

RNA Aptamer Therapy for Vaso-Occlusion in Sickle Cell Disease

Patients with sickle cell disease (SCD) often suffer painful vaso-occlusive episodes caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium. To investigate inhibition of SS-RBC adhesion as a possible treatment for vaso-occlusion, 2 adhesion molecules, α(v)β(3) and P-selectin, were targeted by high-affinity RNA aptamers. An in vitro flow chamber assay was used to test the antiadhesion activity of α(v)β(3) aptamer clone 17.16. Human SS-RBC were passed across a confluent monolayer of thrombin-stimulated human umbilical vein endothelial cells (HUVEC) at a constant rate. α(v)β(3) aptamer reduced SS-RBC adhesion to activated endothelial cells to the level seen with untreated HUVEC. An aptamer reactive with complement component 8 was used as a negative control and exerted no inhibition, confirming the specificity of α(v)β(3) aptamer (P=0.04). At 2 dyn/cm(2) shear stress, 30 nM α(v)β(3) aptamer showed maximal effect in decreasing SS-RBC adhesion to HUVEC. The antiadhesive activity of the P-selectin aptamer clone PF377 was also tested using HUVEC pretreated with IL-13 to upregulate expression of P-selectin as seen in activated endothelial cells. At 1 dyn/cm(2) shear stress, 60 nM of P-selectin aptamer had antiadhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin. A negative control did not prevent adhesion (P=0.05). These data show the potential utility of aptamers to block endothelial adhesion molecules to prevent or treat vaso-occlusion in SCD.