Simvastatin Reduces the in Vitro Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers

Abstract

Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation, leukocyte and red cell adhesion. Markers of neutrophil activation are also increased in SCD, in association with increased levels of circulating cytokines and increased leukocyte adhesion. In animal models, vaso-occlusion causes hypoxia/reperfusion, leading to vascular endothelium damage and an inflammatory response. We postulate that anti-inflammatory agents may reduce the participation of activated endothelium in the vaso-occlusive process. Statins are commonly used to treat arteriosclerosis and have anti-inflammatory effects that include a regulatory action on endothelial function, reduced oxidative stress and inflammation. The objective of this study was to investigate the in vitro effect of simvastatin on the adhesion of sickle neutrophils to activated endothelial cell layers (HUVEC). Neutrophils (Neu) were isolated from the peripheral blood of healthy controls (ConNeu) and SCD (SCDNeu) individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106 cell/ml in Ham's F12 K) to cultured human umbilical vein endothelial cells (HUVEC) grown to confluence was assessed using static adhesion assays. HUVEC cells were treated with or without 1 μg/ml simvastatin for 6 hours in the absence or presence of a 10nM TNF-α activating stimulus (3 hours) before allowing adhesion of Neu to the cell layers (30 min, 37°C, 5%CO2). Neu from SCD patients demonstrated a significantly greater adhesion to HUVEC than ConNeu (20.5 ± 1.9% compared to 13.8 ± 1.7 %; n=15; p<0.02; Mann Whitney test). Subsequently, Neu from patients and controls were allowed to adhere to endothelial layers previously treated with simvastatin; adhesion was not significantly different to the adhesion of Neu to nonsimvastatin treated HUVEC (16.7 ± 3.2% for ConNeu; n=8, p>0.05 and 19.8 ±2.7% for SCDNeu; n=11, p>0.05, paired t test). Pre-treatment of HUVEC with the cytokine TNF-α increased the adhesion of SCD and Con Neu to HUVEC (40.9 ± 5.4%; 28.9 ± 5.0%, respect, N>8, P<0.01 compared to adhesion to non-activated HUVEC). Interestingly, when the endothelium layer was protected with simvastatin and then stimulated with TNF-α, SCDNeu adhesion was significantly diminished (reduced to 31.3% ± 3.6%; n=11, p<0.005 comp. to adhesion to non-simvastatin-treated HUVEC); in contrast, no difference in the adhesion of ConNeu to HUVEC treated with TNF-α and simvastatin was observed (31.9 ± 5.8%, n=8, p>0.05 for ConNeu). In conclusion, data indicate that under in vitro inflammatory conditions, simvastatin appears to protect endothelium layers and reduces SCD leukocyte adhesion. We speculate that statins may have anti-inflammatory properties and, as such, may be useful for diminishing endothelial activation and, in turn, preventing the adhesion of leukocytes adhesion to the vascular wall in SCD, a mechanism that is essential to the vaso-occlusive process.