White, J., Callaghan, M.U., Gao, X., Liu, K., Zaidi, A., Tarasev, M. and Hines, P.C. (2021), Longitudinal assessment of adhesion to vascular cell adhesion molecule-1 at steady state and during vaso-occlusive crises in sickle cell disease. Br J Haematol.
The objective of this study was to assess the variability and clinical predictive value of FA-WB-VCAM in a six-month longitudinal, observational study (ELIPSIS) in SCD subjects during at-home, steady-state and self-reported VOCs, and following VOC resolution
Hines, P.C., Callaghan, M.U., Zaidi, A.U., Gao, X., Liu, K., White, J. and Tarasev, M. (2021), Flow adhesion of whole blood to P-selectin: a prognostic biomarker for vaso-occlusive crisis in sickle cell disease. Br J Haematol, 194: 1074-1082. https://doi.org/10.1111/bjh.17643
Blood cell adhesion to P-selectin and vascular cell adhesion molecule-1(VCAM-1) contributes to the pathophysiology of vaso-occlusion crisis(VOC) events in individuals with sickle cell disease (SCD). We evaluated theuse of standardized ﬂow adhesion biomarkers in a six-month, 35-subjectslongitudinal study (ELIPSIS). Flow adhesion of whole blood on P-selectin(FA-WB-Psel) and VCAM1 (FA-WB-V CAM), and of isolated white bloodcells on P-selectin (FA-WBC-Psel) and VCAM-1 (FA-WBC-VCAM) wereelevated on VOC days compared with non-VOC days, but only FA-WB-Pselreached statistical signiﬁcance (P = 0015). Optimal cut-off values wereestablished with Cox regression models for FA-WB-Psel [46 cells/mm; haz-ard ratio (HR): 23; 95% conﬁdence inte rval (CI):14–40; P = 001] and FA-WB-VCAM (408 cells/mm, HR:18; 95% CI: 09–345; P = 001). A com-bined (FA-WB-Psel and FA-WB-VCAM) mu ltimarker risk score was also sig-niﬁcantly (P = 00006) correlated with VOC risk that was two-fold higherfor intermediate and 564-fold higher for high score. The concordance (C)-index for the multimarker score was 063 in the six-month period (95% CI:056–070), indicating a better ability to distinguish patient risk of VOC,compared to individual biomarkers FA-WB-VCAM (C-index: 0 57; 95% CI:049–065) or FA-WB-Psel (C-index: 058; 95% CI: 053–062). The presentedmultimarker score can be used to risk-stratify individuals with SCD duringtheir steady state into low, intermediate, and high-risk strata for self-reportedVOCs. Such risk stratiﬁcation could help focus healthcare resources moreefﬁciently to maintiain health, personalize treatment selection to eachpatient’s individual needs, and potentially reduce healthcare costs.
Keywords: ﬂow adhesion, P-selectin, vascular cell adhesion molecule-1,biomarker, sickle cell disease
White J, Lancelot M, Gao X, McGraw BJ, Tabb C 2nd, Hines P. Cross-sectional analysis of adhesion in individuals with sickle cell disease using a standardized whole blood adhesion bioassay to VCAM-1. Blood Cells Mol Dis. 2020 Mar;81:102397. doi: 10.1016/j.bcmd.2019.102397. Epub 2019 Dec 6. PMID: 31864103.
Sickle cell disease (SCD) is characterized by frequent and unpredictable vaso-occlusive episodes (VOEs) that lead to severe pain, organ damage, and early death. Lack of reliable biomarkers that objectively define VOEs remains a critical barrier to improving the care for SCD patients. VOEs result from a complex interplay of cell-cell interactions that promote micro-vascular occlusion. Earlier studies demonstrated that sickle erythrocytes are more adherent than non-sickle erythrocytes and established a direct link between adhesion and frequency of VOEs. We developed a standardized, flow-based adhesion bioassay to assess the adhesive properties of SCD blood samples. The current study provides a cross-sectional analysis of steady state adhesion in SCD patients presenting at monthly out-patient hematology visits. Steady state adhesion varied from patient-to-patient. Adhesion positively correlated with reticulocyte percent and WBC count although there was no significant relationship between adhesion and platelets or hemoglobin in this study. Additionally, steady state adhesion indices were significantly lower in SCD subjects receiving hydroxyurea therapy when compared to the untreated group. The well-plate based microfluidic flow adhesion bioassay described in this report may provide a platform to identify SCD subjects with severe disease phenotypes, predict impending VOEs, and monitor response to current and developing therapies.
Pittman DD, Hines PC, Beidler D, Rybin D, Frelinger AL, Michelson AD, Liu K, Gao X, White J, Zaidi AU, Charnigo RJ, Callaghan MU. Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers. Blood. 2021 Apr 15;137(15):2010-2020. doi: 10.1182/blood.2020006020. PMID: 33067606;
Clinical trials in sickle cell disease (SCD) often focus on health care utilization for painful vaso-occlusive crises (VOCs). However, no objective, quantifiable pain biomarkers exist, pain is not specific to VOCs, health care utilization varies between patients, unreported at-home VOCs likely contribute to long-term outcomes, and patient-reported outcomes are seldom considered. This noninterventional, longitudinal, 6-month study aimed to develop tools to identify VOCs in SCD patients with or without health care utilization. Participants wore an actigraph device, tracking sleep and activity. Patients with SCD used an electronic patient-reported outcome (ePRO) tool to collect data on pain, medication, fatigue, and daily function. Patients self-reported when they experienced VOC pain (VOC day). Biomarkers were collected every 3 weeks (non-VOC). Self-reported VOCs triggered at-home or in-hospital blood collection. The study enrolled 37 participants with SCD; 35 completed the study. Participants reported 114 VOC events and 346 VOC days, of which 62.3% and 78.3%, respectively, were self-treated at home. The ePRO and actigraphy captured end points of pain, functionality, fatigue, activity, and sleep; each was significantly altered on VOC days compared with non-VOC days. Biomarkers collected at home or in the hospital on VOC days were significantly altered compared with non-VOC baseline values, including leukocyte-platelet aggregates, microfluidic-based blood cell adhesion, interleukin-6, C-reactive protein, interleukin-10, tumor necrosis factor-α, and thrombin-antithrombin. The Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) trial shows the feasibility of accurately monitoring out-of-hospital pain by using patient-reported VOC days as potential end points for clinical trials in SCD; it describes the changes in biomarkers and activity measured by actigraphy that may enable improved identification and assessment of VOCs.
White J, Lindgren M, Liu K, Gao X, Jendeberg L, Hines P. Sevuparin blocks sickle blood cell adhesion and sickle-leucocyte rolling on immobilized L-selectin in a dose dependent manner. Br J Haematol. 2019 Mar;184(5):873-876. doi: 10.1111/bjh.15188. Epub 2018 May 16. PMID: 29767405.
Adhesion of sickle red blood cells (SSRBC) to the vascular endothelium may initiate and propagate vascular obstruction in sickle cell disease (SCD) (Hoover et al, 1979; Hebbel et al, 1980). Hebbel et al (1980) were the first to report a correlation between erythrocyte adherence and disease severity. Subsequent studies demonstrated that the pathological adhesion of SSRBCs involves red cell receptors, adhesive bridging proteins and endothelial receptors (Joneckis et al, 1993; Swerlick et al, 1993; Udani et al, 1998; Hillery et al, 2000). These complex and multimodal mechanisms of SSRBC adhesion may require a multi‐targeted approach to achieve the best clinical outcome.
Lancelot M, White J, Sarnaik S, Hines P. Low molecular weight heparin inhibits sickle erythrocyte adhesion to VCAM-1 through VLA-4 blockade in a standardized microfluidic flow adhesion assay. Br J Haematol. 2017 Aug;178(3):479-481. doi: 10.1111/bjh.14137. Epub 2016 Jun 24. PMID: 27341635.
The vaso-occlusive events in sickle cell disease (SCD) begin in early childhood, warranting the need for more preventative and therapeutic interventions for those affected. Vaso-occlusion is partly caused by adhesion of sickle erythrocytes (SSRBCs) to components of the vascular wall and to circulating leucocytes (WBCs). SSRBCs have greater adhesion to the vascular endothelium and sub-endothelial matrix (SEM) compared to RBCs from unaffected individuals.
White J, Krishnamoorthy S, Gupta D, Lancelot M, Moore N, Sarnaik S, Hobbs WE 2nd, Light DR, Hines P. VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow. Br J Haematol. 2016 Sep;174(6):970-82. doi: 10.1111/bjh.14158. Epub 2016 Jun 12. PMID: 27291690.
Very Late Antigen-4 (VLA-4, α4β1-integrin, ITGA4) orchestrates cell-cell and cell-endothelium adhesion. Given the proposed role of VLA-4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA-4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA-4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA-4, while VLA-4 staining of non-SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA-4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM-1) was blocked by natalizumab in a dose-dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF-α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 μg/ml). This indicates that VLA-4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM-1 and that the VLA-4 adhesion to VCAM-1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA-4 blockade may be beneficial in sickle cell disease.
Keywords: adhesion; microfluidics; natalizumab; sickle cell; very late antigen-4.
White J, Lancelot M, Sarnaik S, Hines P. Increased erythrocyte adhesion to VCAM-1 during pulsatile flow: Application of a microfluidic flow adhesion bioassay. Clin Hemorheol Microcirc. 2015;60(2):201-13. doi: 10.3233/CH-141847. PMID: 24898561;
Sickle cell disease (SCD) is characterized by microvascular occlusion mediated by adhesive interactions of sickle erythrocytes (SSRBCs) to the endothelium. Most in vitro flow adhesion assays measure SSRBC adhesion during continuous flow, although in vivo SSRBC adhesive interactions occur during pulsatile flow. Using a well-plate microfluidic flow adhesion system, we demonstrate that isolated SSRBCs adhere to vascular cell adhesion molecule (VCAM-1) at greater levels during pulsatile versus continuous flow. A significant increase in adhesive interactions was observed between all pulse frequencies 1 Hz to 2 Hz (60-120 beats/min) when compared to non-pulsatile flow. Adhesion of isolated SSRBCs and whole blood during pulsatile flow was unaffected by protein kinase A (PKA) inhibition, and exposure of SSRBCs to pulsatile flow did not affect the intrinsic adhesive properties of SSRBCs. The cell type responsible for increased adhesion of whole blood varied from patient to patient. We conclude that low flow periods of the pulse cycle allow more adhesive interactions between sickle erythrocytes and VCAM-1, and sickle erythrocyte adhesion in the context of whole blood may better reflect physiologic cellular interactions. The microfluidic flow adhesion bioassay used in this study may have applications for clinical assessment of sickle erythrocyte adhesion during pulsatile flow.
Keywords: Pulsatile flow; VCAM-1; VLA-4; adhesion; erythrocyte; microfluidics; sickle cell disease; variable shear.
Hines PC, Zen Q, Burney SN, Shea DA, Ataga KI, Orringer EP, Telen MJ, Parise LV. Novel epinephrine and cyclic AMP-mediated activation of BCAM/Lu-dependent sickle (SS) RBC adhesion. Blood. 2003 Apr 15;101(8):3281-7. doi: 10.1182/blood-2001-12-0289. Epub 2002 Dec 27. PMID: 12506027.
The vasoocclusive crisis is the major clinical feature of sickle cell anemia, which is believed to be initiated or sustained by sickle (SS) red blood cell (RBC) adhesion to the vascular wall. SS RBCs, but not unaffected (AA) RBCs, adhere avidly to multiple components of the vascular wall, including laminin. Here we report a novel role for epinephrine and cyclic adenosine monophosphate (cAMP) in the regulation of human SS RBC adhesiveness via the laminin receptor, basal cell adhesion molecule/Lutheran (BCAM/Lu). Our data demonstrate that peripheral SS RBCs contain greater than 4-fold more cAMP than AA RBCs under basal conditions. Forskolin or the stress mediator epinephrine further elevates cAMP in SS RBCs and increases adhesion of SS RBCs to laminin in a protein kinase A (PKA)-dependent manner, with the low-density population being the most responsive. Epinephrine-stimulated adhesion to laminin, mediated primarily via the beta 2-adrenergic receptor, occurred in SS RBC samples from 46% of patients and was blocked by recombinant, soluble BCAM/Lu, implicating this receptor as a target of cAMP signaling. Thus, these studies demonstrate a novel, rapid regulation of SS RBC adhesion by a cAMP-dependent pathway and suggest that components of this pathway, particularly PKA, the beta 2-adrenergic receptor, and BCAM/Lu, should be further explored as potential therapeutic targets to inhibit SS RBC adhesion.
Lee SP, Cunningham ML, Hines PC, Joneckis CC, Orringer EP, Parise LV. Sickle cell adhesion to laminin: potential role for the alpha5 chain. Blood. 1998 Oct 15;92(8):2951-8. PMID: 9763582.
Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the alpha1, alpha2, beta1, and gamma1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin alpha5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin alpha5 chain.