Sickle red cell-endothelium interactions

Abstract

Periodic recurrence of painful vaso-occlusive crisis is the defining feature of sickle cell disease. Among multiple pathologies associated with this disease, sickle red cell-endothelium interaction has been implicated as a potential initiating mechanism in vaso-occlusive events. This review focuses on various interrelated mechanisms involved in human sickle red cell adhesion. We discuss in vitro and microcirculatory findings on sickle red cell adhesion, its potential role in vaso-occlusion, and the current understanding of receptor-ligand interactions involved in this pathological phenomenon. In addition, we discuss the contribution of other cellular interactions (leukocytes recruitment and leukocyte-red cell interaction) to vaso-occlusion, as observed in transgenic sickle mouse models. Emphasis is given to recently discovered adhesion molecules that play a predominant role in mediating human sickle red cell adhesion. Finally, we analyze various therapeutic approaches for inhibiting sickle red cell adhesion by targeting adhesion molecules and also consider therapeutic strategies that target stimuli involved in endothelial activation and initiation of adhesion.

Figure 1

Adhesion of sickle red cells to HUVEC under flow conditions is maximal for reticulocyte-rich fraction (top fraction), while it shows a marked decrease for ISC-rich dense sickle red cell fraction (bottom fraction). Modified from Barabino et al. (3).

Figure 2

Models of vaso-occlusion in sickle cell disease. (A) Initial adhesion of deformable sickle red cells in small-diameter postcapillary venules. (B) Adhesion of deformable sickle cells is followed by selective trapping of dense or sickled red cells among adherent red cells. (C) Alternatively, recruitment of leukocytes in inflamed venules may trigger selective trapping of sickled and dense red cells. Modified from Kaul et al. (43).

Figure 3

Potential mechanisms in sickle red cell adhesion to endothelium. IAP = integrin-associated protein; ICAM-4/LW = intercellular adhesion molecule-4/Landsteiner-Weiner protein; PS = phosphatidyl serine.

Figure 4

Videomicrographs showing inhibition of PAF-induced sickle erythrocytes adhesion in the ex vivo mesocecum microvasculature in the presence of ICAM-4 peptide ATSR. (A-D) Ex vivo preparation treated with PAF. (A) Clear vessel lumen (a = arteriole, v = venule) during perfusion with Ringer-albumin solution; (B and C) sickle erythrocytes bolus (arrows in B indicate flow direction) is followed by adhesion of these cells exclusively in the venule but not in the arteriole; (D) Maximal adhesion is observed in small-diameter postcapillaty venules (arrow-heads), frequently resulting in occlusion. (E-H) Ex vivo preparation treated with PAF and an ICAM-4 F strand control peptide AWSS; (E and F) Infusion of sickle erythrocytes bolus results in adhesion in the venules (v), but not in the arteriole (b); (G) adherent sickle erythrocytes in venules; (H) adhesion results in frequent blockage of small-diameter venules (arrow-head). (I-L) Ex vivo preparation treated with PAF and ICAM-4 peptide ATSR; (I) clear vessel lumens during perfusion with Ringer-albumin; (J and K) after a bolus infusion of sickle erythrocytes, rapid flow is observed in both arteriole and venules (arrows indicate flow direction), resulting in no adhesion; (L) scanning of the vasculature revealed little or no adhesion in venules. From Kaul et al. (50).

Figure 5

Regression plots for the number of adhered sickle erythrocytes (SS RBC)/100 μm² relative to venular diameters in ex vivo preparations treated as follows: (A) PAF alone, (B) PAF and peptide FWV, (C) PAF and peptide ATSR and (D) PAF and control peptide AWSS. The regression lines represent the multiplicative equation of the form Y = aX^-b for the best fit. In preparations treated with PAF alone, adhesion of sickle erythrocytes showed a strong correlation with the venular diameter. Preparations treated with peptide FWV or ATSR showed a marked inhibition of sickle erythrocyte adhesion in venules of all diameters, with ATSR having the maximal inhibitory effect, especially in small-diameter venules, the sites of frequent blockage. In contrast, in the presence of the control peptide AWSS, the resulting adhesion was essentially similar to that observed in PAF-treated preparations. Modified from Kaul et al. (50).

Figure 6

Top panel: Colocalization of fluoresceinated ATSR peptide with vascular endothelium of the ex vivo preparation pretreated with PAF (A-C). (A) The presence of the fluorescent peptide is shown in green. (B) Blood vessels were identified by a polyclonal primary antibody to vWF and a secondary TRITC-conjugated antibody (red). (C) Merged image signals showed colocalization (yellow) of ATSR with the endothelial lining. No fluorescence staining was noted when the control peptide was infused (images not shown). Middle panel: The effect of a control antibody OC125 (D-F) on the colocalization of fluoresceinated ATSR peptide with vascular endothelium of the ex vivo preparation pretreated with PAF. (D) The presence of the fluorescent peptide is shown in green. (E) Blood vessels were identified by a polyclonal antibody to vWF as in B (red). (F) Merged image signals showed colocalization (yellow) of ATSR with the vessel wall. Bottom panel: The effect of 7E3 antibody to αVβ3 (G-I) on the colocalization of fluoresceinated ATSR peptide with vascular endothelium in PAF treated ex vivo preparation. (G) In the presence of 7E3 antibody, there was a marked decrease in ATSR localization with the vessel wall. ATSR infusion resulted in weak green staining likely attributable to autofluorescence or a low level of binding of ATSR peptide. (H) Vessel was identified by immunofluorescent staining for vWF (red). (I) No colocalization of ATSR with vessel wall in the presence of 7E3. From Kaul et al. (50).

Figure 7

Regression plots for the number of adherent SS RBC/100 :m² relative to venular diameter in the ex vivo mesocecum treated as follows: (A) PAF alone, (B) PAF and peptide EMD 270179 (cRGDF-ACHA [α-amino cyclohexyl carboxylic acid]), (C) PAF and peptide EMD 66203 (cRGDFV), (D) PAF and control peptide EMD 135981 (cRβ-ADFV). The regression lines represent the multiplicative equation of the form Y = aX^-b for the best fit. In preparations treated PAF alone, adhesion of SS RBCs showed a strong correlation with the venular diameter. Preparations treated with αVβ3 antagonist EMD 270179 or EMD 66203 showed a marked inhibition of SS RBC adhesion in venules of all diameters, with EMD 66203 having a greater inhibitory effect, especially in small-diameter venules, the site of frequent blockage. In contrast, in the presence of the control peptide EMD 135981, the resulting adhesion was essentially similar to that observed with PAF alone. Modified from Finnegan et al. (22).

Figure 8

The effect of ∀V∃3 antagonists (EMD 66203 and EMD 270179) on SS RBC adhesion to HUVEC. The human endothelial monolyers were incubated with either peptide (100 :g/ml) for 30 min before the perfusion of the endothelialized flow chamber with SS RBC suspension (Hct 1%) at 1 dyne/cm². The HUVEC treated with control peptide (n=7) showed no significant difference in adhesion of SS RBC/mm² as compared to the baseline adhesion of these cells to untreated control HUVEC (n=6) (P>0.51). In contrast, both EMD 66203 (n=6) and EMD 270179 (n=4), caused marked inhibition of adhesion (i.e., 55% and 62%, respectively) of SS RBCs to HUVEC as compared with the control peptide group (P<0.018 and P<0.015, respectively). Modified from Finnegan et al. (22).