Testing Rapamycin As an Anti-Adhesion Therapy for Sickle Cell Disease

Background: Patients with sickle cell disease (SCD) suffer from recurrent vaso-occlusion leading to numerous debilitating acute and chronic complications including painful vaso-occlusive crisis, stroke, and multi-organ degeneration. The vaso-occlusive process is promoted by an increased adhesion of red blood cells (RBCs) and leukocytes to each other and to the vascular endothelium via activated surface adhesion molecules. Preventing blood cell adhesion by decreasing the adhesive properties of individual adhesion molecules was proven to be an effective therapeutic strategy for SCD. Key cell surface adhesion molecules associated with vaso-occlusion in SCD, include P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressed by endothelial cells, and Lutheran basal cell adhesion molecule (Lu/BCAM) expressed by RBCs. For the past few years, the drug rapamycin has been described as a potential effective fetal hemoglobin inducer. In studies not related to SCD, rapamycin was also shown to decrease the expression and activation of VCAM-1 and P-selectin as well as leukocytes adhesion in vitro and in vivo. Therefore we hypothesize that rapamycin may decrease the activation of key endothelial adhesion molecules in SCD. In addition, in RBCs, Lu/BCAM adhesive properties are activated by phosphorylation by protein kinase A-dependent (PKA) and oxidative stress. Because mTOR activates PKA, and mTOR inhibitors decrease oxidative stress, we hypothesize that mTOR inhibition with rapamycin may prevent Lu/BCAM activation. In this study, we tested rapamycin's effect on key erythroid and endothelial adhesion molecules involved in SCD pathophysiology.

Methods: To determine rapamycin's effect on VCAM-1 induction mediated by TNF-α in human endothelial cells, we pre-treated Human umbilical vein endothelial cells (HUVECs) and Blood Outgrowth Endothelial Cells (BOECs) derived from patients with SCD with DMSO, rapamycin (100 nM) or hydroxyurea (HU) (30 μM) for 24h in their culture media. TNF-α (10 ng/ml) was then added for 18h to stimulate the VCAM-1 that was then quantified by flow cytometry. To assess rapamycin's impact on RBCs adhesion to laminin, patients' RBCs were treated with DMSO, rapamycin (100 nM) or Lu/BCAM blocking antibody prior to Lu/BCAM activation with epinephrine. Cells were then perfused in a microfluidic chamber coated with laminin, Lu/BCAM's endothelial receptor, at 0.5 dyn/cm² for 5 min. The chamber was washed with PBS at 2 dyn/cm² for 5 min to remove non-adherent cells and adherent cells were counted. Finally, we assessed rapamycin's effect on key adhesion molecules in Townes mice with SCD. To do so, we treated 8-week-old mice with 4 mg/kg of rapamycin or vehicle by intraperitoneal injection for 4 weeks and measure VCAM-1, P-selectin, E-selectin and ICAM-1 plasma levels by ELISA.

Results: Our results show that: (1) Rapamycin significantly decreases VCAM-1 expression induced by TNF-α in both endothelial cells in vitro, derived or not from patients with SCD; (2) Rapamycin significantly reduces Lu/BCAM activation on patients' RBCs after stimulation with epinephrine ; (3) Initial results indicate that rapamycin decreases VCAM-1, E-selectin and ICAM-1 plasma levels.

Conclusions: We showed that rapamycin decreases the expression and activation on both erythroid and endothelial adhesion molecules involved in SCD pathophysiologic process. These results build the foundation to further investigate rapamycin as an anti-adhesion therapy for SCD that simultaneously targets several adhesion molecules. Important next steps include the evaluation of rapamycin's long-term safety by analyzing its effect on erythropoiesis, blood count, organ damage and potential toxicity.

Weiss:Cellarity Inc., Novartis, and Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

We are testing rapamycin as an anti-adhesion therapy in mice with sickle cell disease