Integrin VLA-4 as a PET imaging biomarker of hyper-adhesion in transgenic sickle mice

Abstract

In sickle cell disease (SCD), very late antigen-4 (VLA-4 or integrin α4β1) mediates the adhesion of reticulocytes to inflamed, proinflammatory endothelium, a key process in promoting vaso-occlusive episodes (VOEs). We hypothesized that a radionuclide tracer targeting VLA-4 could be harnessed as a positron emission tomography (PET) imaging biomarker of VOEs. We tested the VLA-4 peptidomimetic PET tracer 64Cu-CB-TE1A1P-PEG4-LLP2A (64Cu-LLP2A) for imaging hyper-adhesion–associated VOEs in the SCD Townes mouse model. With lipopolysaccharide (LPS)-induced VOEs, 64Cu-LLP2A uptake was increased in the bone marrow of the humeri and femurs, common sites of VOEs in SCD mice compared with non-SCD mice. Treatment with a proven inhibitor of VOEs (the anti-mouse anti-P-selectin monoclonal antibody [mAb] RB40.34) during LPS stimulation led to a reduction in the uptake of 64Cu-LLP2A in the humeri and femurs to baseline levels, implying blockade of VOE hyper-adhesion. Flow cytometry with Cy3-LLP2A demonstrated an increased percentage of VLA-4–positive reticulocytes in SCD vs non-SCD mice in the bone and peripheral blood after treatment with LPS, which was abrogated by anti-P-selectin mAb treatment. These data, for the first time, show in vivo imaging of VLA-4–mediated hyper-adhesion, primarily of SCD reticulocytes, during VOEs. PET imaging with 64Cu-LLP2A may serve as a valuable, noninvasive method for identifying sites of vaso-occlusion and may provide an objective biomarker of disease severity and anti-P-selectin treatment efficacy in patients with SCD.

P-selectin deficiency promotes liver senescence in sickle cell disease mice

“…16,17 Therefore, we hypothesized that the large iron deposits in SS-Selp 2/2 mice are the result of dysregulated iron clearance, secondary to impaired recruitment of myeloid cells in absence of P-selectin. Interestingly, we 11 have previously reported that the circulating myeloid cells (neutrophils and monocytes) are significantly elevated in SS-Selp 2/2 mice, 11,18 most likely because of impaired recruitment to tissues and bone marrow. Indeed, we found significant reduction in messenger RNA (mRNA) and expression of myeloid cell markers (Ly6G, CXCR4, CD62L, and CD11b) in the liver of SS-Selp 2/2 than SS mice (Figure 2E), which was further confirmed by the reduction in immunofluorescence staining for neutrophil marker (Ly6g) in SS-Selp 2/2 compared with SS liver (Figure 2F).…”

Flow adhesion of whole blood to P‐selectin: a prognostic biomarker for vaso‐occlusive crisis in sickle cell disease

“…A decrease in VLA‐4 expression on reticulocytes in SCD subjects treated with HC, a therapy known to reduce the clinical severity of SCD, was previously reported 32 . Recent positron emission tomography data indicate VLA‐4‐mediated hyper‐adhesion, primarily of SCD reticulocytes, during VOCs in the SCD Townes mouse model 33 . We have demonstrated that pulsatile flow increased VCAM‐1 – VLA‐4 adhesive interactions as compared to non‐pulsatile flow, 34 likely due to an increase in interaction time allowing for the development of additional adhesive points of attachment 5 .…”

Adhesion to VCAM1 and P-selectin Predict Time-to-Resolution (TTR) of Vaso-Occlusive Crisis

“…Blood cell adhesion to the vascular endothelium is a crucial event in the pathophysiology of VOCs and other clinical manifestations of SCD [19]. VCAM-1/VLA-4 and P-Selectin/PSGL-1 interactions were reported as both independently and cumulatively contributing to risks of vaso-occlusive events [18,20].…”

“…Blood cell adhesion to the vascular endothelium is a crucial event in the pathophysiology of VOCs and other clinical manifestations of SCD [19]. VCAM-1/VLA-4 and P-Selectin/PSGL-1 interactions were reported as both independently and cumulatively contributing to risks of vaso-occlusive events [18, 20]. High levels of VLA-4 and VCAM-1 have been associated with vaso-occlusive adhesive events and severe disease phenotypes, and with a response to SCD-modifying therapy [21-26].…”