Endothelial activation by platelets from sickle cell anemia patients

Abstract

Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD.

Figure 1. Platelets as inflammatory cells in SCA.

Release of (A) IL-1β, (B) IL-8, (C) platelet factor 4 (PF4) from platelets of healthy control individuals (CON) and SCA patients in steady state (SCA). Cytokine release was determined by ELISA in platelet suspensions (1×10⁸ PLT/ml) after incubation for 4 h (37°C, 5% CO₂). Expressions of (D) P-selectin and (E) activated α_IIbβ₃ integrin on the surface of platelets from healthy control individuals (CON) and SCA patients in steady state (SCA). Medians and interquartile ranges are depicted. Adhesion molecule expression was determined by flow cytometry. Some of the data included in the data sets depicted in graphs 1D and 1E have been previously published in table form . Closed black square symbols represent SCA patients not on HU therapy; open grey square symbols represent patients on HU (15–30 mg/kg/day) therapy.

Figure 2. Adhesive properties of platelets.

A microfluidic assay was used to determine the adhesion of platelets from healthy control individuals (CON) and SCA patients in steady state (SCA) to (A) 50 µg/ml fibrinogen and (B) 100 µg/ml collagen. Platelets (1×10⁷ PLTs/mL) were perfused over microchannels (400 µm width) coated with immobilized ligands at a shear stress of 0.3 dyne/cm² for 3 min at 37°C; adhesion was detected using brightfield inverted microscopy. The adhesion of platelets to duplicate microchannels was recorded at three positions in each channel and analyzed using the DucoCell analysis program, recording the mean number of platelets adhered to an area of 0.08 mm². Medians and interquartile ranges are depicted. Closed black square symbols represent SCA patients not on HU therapy; open grey square symbols represent patients on HU (15–30 mg/kg/day) therapy.

Figure 3. Effects of platelets on endothelial adhesion molecule expression.

Surface expressions of (A) ICAM-1 (CD54), (B) E-selectin (CD62E) and (C) VCAM-1 (CD106) adhesion molecules on HUVEC following co-culture in direct contact with healthy control (CON; N≥15) PLTs, steady-state sickle cell anemia (SCA; N≥25) PLTs or TNF-α (N≥11). HUVEC (1×10⁶ cells/well) were incubated with CON or SCA PLTs (1×10⁸ PLTs/well) for 4 h (37°C, 5% CO₂), or with TNF-α, (10 ng/ml; 3 h, 37°C, 5% CO₂). Expression of ICAM-1, E-selectin or VCAM-1 was analyzed on HUVEC (10 000 cells) by flow cytometry using anti-CD54-PE, CD62E-APC and CD106-FITC. **, p<0.01; ***, p<0.001, compared to basal (N≥16).

Figure 4. Transwell inserts reverse the effects of SCA platelets on endothelial adhesion molecules expression.

Surface expressions of (A) ICAM-1 (CD54) and (B) E-selectin (CD62E) adhesion molecules on HUVEC (1×10⁶ cells/well) following culture in the presence of healthy control (CON; N≥6; 1×10⁸ PLTs/well) PLTs or steady-state sickle cell anemia (SCA; N ≥12; 1×10⁸ PLTs/well) PLTs and in the presence and absence of transwell inserts (0.4 µm pores). Expressions of ICAM-1 and E-selectin were evaluated by flow cytometry using anti-CD54-PE and CD62E-APC. *, p<0.01; ***, p<0.001, compared to basal (N = 9).

Figure 5. Release of IL-8 from co-cultures of HUVEC and PLTs.

Levels of IL-8 (ng/ml) were quantified in supernatants of co-cultures of HUVEC (1×10⁶ cells/well) (4 h in suspension, 37°C, 5% CO₂) in direct contact with CON (n = 27) or SCA PLTs (1×10⁸ PLTs/well; n = 44), (4 h, 37°C, 5% CO₂); or following pre-incubation of HUVEC with TNF-α (10 ng/ml; N = 24; 3 h, 37°C, 5% CO₂). ***, P<0.001, compared to basal (N = 24); +++, P<0.001, compared to all groups.

Figure 6. Effect of SCA platelets on endothelial gene expression.

Relative gene expression of (A) ICAM1, (B) NFKB1, (C) RELA, (D) CHUK and (E) IKKBETA in HUVEC after co-culture with PLTs. HUVEC (1×10⁶ cells/well) were co-cultured in direct contact with CON (N = 10) or SCA PLTs (N≥24; 1×10⁸ PLTs/well) (4 h, 37°C, 5% CO₂); or pre-incubated with HUVEC with TNF-α (10 ng/ml; N≥11; 3 h, 37°C, 5% CO₂). Relative gene expressions were determined by qPCR and expressed relative to ACTB and GAPDH expressions. *, P<0.05; **,P<0.01, compared to basal (N≥11). +++, P<0.001, compared to all groups.

Figure 7. Effect of inhibition of the NF-κB pathway on the induction of endothelial adhesion molecule expression by SCA PLTs.

The expressions of (A) ICAM-1 (CD54) and (B) E-selectin (CD62E) were determined by flow cytometry on HUVEC (1×10⁶ cells/well) following co-culture in direct contact with CON PLTs (N≥13) or SCA PLTs (1×10⁸ PLTs/well; N≥14) in the presence or absence of BAY 11-7082 (20 µM; 4 h, 37°C, 5% CO₂). *, P<0.05; **, P<0.01, compared to basal (N≥12).