Adhesive and Inflammatory Properties Are Increased in Blood Outgrowth Endothelial Cells From Patients with Sickle Cell Anemia

Abstract 2133

The endothelium plays an important role in the pathophysiology of sickle cell anemia (SCA) due to interactions of endothelial cells with other cell types in the vaso-occlusive process and endothelial dysfunction caused by chronic inflammation and decreased nitric oxide bioavailability. Inflammatory molecules, such as cytokine TNF-α, may alter the endothelial cell surface and, thus, modulate the adhesion molecules that bind red blood cells (RBC). Damaged or activated endothelium produces cytokines and chemokines that result in the high levels of circulating inflammatory molecules found in the SCA. Blood Outgrowth Endothelial Cells (BOEC) are circulating endothelial cells isolated from the peripheral blood of adults, they have a high proliferative capacity in vitro, with the characteristic phenotype of mature cells. BOEC cultures were established to serve as a model for an in vitro study of endothelial properties and function in SCA, to evaluate RBC adhesion, expression of adhesion molecules and production of inflammatory cytokines in BOEC from healthy controls (Con) and SCA patients. Three cultures of BOEC from Con and three cultures from SCA patients in steady state were established and identification confirmed by flow cytometry (CD31+; CD146+; VEGF R2+; CD45-; CD133-). BOEC were used under basal conditions or following treatment with 10ng/ml TNF-α (3 H). RBC were isolated from Con, SCA and SCA patients on hydroxyurea treatment (SCAHU; 20–30 mg/Kg/day) and their adhesion to confluent Con and SCA BOEC was evaluated (4×10⁶ cell/ml in EGM-2), using static adhesion assays. BOEC expression of adhesion molecules VCAM-1, E-selectin, P-selectin, CD36, the Vitronectin receptor and BCAM/Lu was evaluated by flow cytometry. The production and release of GM-CSF and IL-8 to the supernatant of BOEC in the presence and absence of an antiinflammatory stimulus (1μM Simvastatin, 3.5H) was quantified by ELISA. Notably, Con RBC adhered significantly more to SCA BOEC (16.6 ± 4.6%; n=14; p<0.001; Wilcoxon Test) than to Con BOEC (9.4 ± 3.2%; n=14). Furthermore, TNF-α stimulation further increased the adhesion of Con RBC to both Con BOEC (16.6 ± 5.6%; n=14 p<0.001 comp. to non-stimulated Con BOEC) and SCA BOEC (20.6 ±7.0%; n=14; p<0.05 comp. to non-stimulated SCA BOEC). Similarly, SCA RBC adhered significantly more to SCA BOEC (18.5 ± 4.8%; n=10; p<0.001; Wilcoxon Test) than to Con BOEC (14.3 ± 4.2%; n=10) and TNF-α stimulation further increased SCA RBC adhesion to Con BOEC (18.3 ± 5.4%; n=10; p<0.01) and SCA BOEC (20.9 ± 6.4%; n=10; p<0.05; Wilcoxon). SCAHU RBC also adhered more to SCA BOEC (19.5 ± 7.4%; p<0.01; Wilcoxon) compared to Con BOEC (14.3 ± 4.2%; n=10) and to TNF-α stimulated Con BOEC (19.1 ± 6.0%; n=10; p<0.01) and SCA BOEC (23.8 ± 6.3%; n=10; p<0.01), compared to respective non-stimulated BOEC. Con and SCA BOEC presented low-level expression of the P-selectin and CD36 adhesion molecules, even following TNF-α inflammatory stimulus; VCAM-1 was increased on both Con and SCA BOEC under TNF-α conditions, but no difference in expression was observed between the Con and SCA BOEC (data not shown); however, after TNF-α stimulus the expression of E-selectin on SCA BOEC was significantly higher (6613 ± 729 MFI; n=3; p=0.03; Unpaired t Test) than on Con BOEC (3645 ± 547 MFI; n=3). Vitronectin receptor and BCAM/Lu expression was slightly higher on SCA BOEC (6248.3 ± 552.9MFI; 1152 ± 51.4MFI respect; n=3; p<0.05, Unpaired t Test) than on Con BOEC (5059.7 ± 426.2MFI; 904.7 ± 70.9MFI; respect; n=3). Following TNF-α stimulus, SCA BOEC released significantly more GM-CSF (271.5 ± 8.7pg/ml; n=3; p<0.01; Unpaired t Test) than Con BOEC (175.2 ± 18.4pg/ml; n=3); where co-incubation with Simvastatin reduced the release of GM-CSF from TNF-α stimulated SCA (189.0 ± 7.1pg/ml; n=3; p<0.05 comp. to TNF-α SCA BOEC; Paired t Test) and Con BOEC (118.9 ± 21.9pg/ml; p<0.01; Paired t Test). SCA BOEC released more IL-8 (10977.4 ± 379.7pg/ml) than BOEC Con (8913.7 ± 234.7pg/ml) under TNF-α inflammatory conditions; however Simvastatin reduced the production of IL-8 only from Con BOEC (data not shown). Results suggest that SCA BOEC present inherent endothelial abnormalities that promote increased adhesion of red cells and augmented inflammatory properties, which are maintained during in vitro culture and following inflammatory stimulus. These cells may serve as a model for in vitro study of aberrant endothelial function in SCA.

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