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N. Luebbering et al.
present in low concentrations outside the cell.12 Actin is the most abundant protein in the human body and exists in two forms- monomeric or globular actin (G-actin) and a polymeric form, F-actin. F-actin is known to be angiopath- ic, and an effective actin scavenger system exists to rapidly clear F-actin that enters the circulation.13 Extracellular F- actin is depolymerized into G-actin by plasma gelsolin. Monomeric G-actin then binds to the highly abundant cir- culating protein, vitamin D binding protein (VDBP), and the actin-VDBP complex is removed from the circulation by the reticulo-endothelial system, primarily in the liver.14 VDBP has been shown to be consumed in other clinical circumstances of significant cell lysis such as acute liver failure and major trauma.15,16
VDBP is an abundant protein, related to albumin and α-fetoprotein. VDBP is conserved throughout evolution, and no humans deficient in VDBP have been identified, indicating an important biological function for VDBP. VDBP is a highly polymorphic molecule, with more than 70 different alleles described, and the frequency of three major alleles, Gc1s, Gc1f and Gc2 varies by race.17 VDBP transports the major circulating form of vitamin D, 25-hydroxyvitamin D, and other vitamin D metabolites in the circulation, but only about 4% of VDBP is conjugated to vitamin D metabolites, indicating other important func- tions for the molecule. A deglycosylated form of VDBP has been shown to have immunomodulatory activity, serving as a so-called “macrophage-activating factor”,18,19 but also demonstrating capacity for regulation of angio- genesis.20 These properties of VDBP, in addition to actin scavenging, have the potential to significantly modify out- comes of HSCT.
We hypothesized that the release of F-actin would be associated with adverse clinical outcomes of transplant,
Table 1. Demographics of patients included in assessment of clinical outcomes and vitamin D binding protein, divided according to whether cases developed or did not develop ransplant-associated thrombotic microangiopathy.
and that higher levels of VDBP would be associated with improved outcomes after transplant. Our data indicate a significant and beneficial role for VDBP in HSCT which appears to involve both actin scavenging and modulation of the macrophage phenotype.
Methods
Patients
Patient samples were obtained from the Cincinnati Children’s Hospital Medical Center HSCT repository, approved by the Institutional Review Board. Demographics of 190 patients includ- ed in the study are shown in Table 1. Careful attention was paid to prospective phenotyping for TA-TMA including monitoring for schistocytes, lactate dehydrogenase, haptoglobin, proteinuria, ele- vation of creatinine, new and excessive thrombocytopenia and anemia, as previously reported.21
Ultra-high performance liquid chromatography
Serum vitamin D metabolites by ultra-high performance liquid chromatography were determined as previously described. 22
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) testing serum VDBP levels were measured using a polyclonal antibody (Genway Bio, DM3741), and commercial ELISA kits were used to measure serum F-actin (MyBioSource, 702018), IL6 (R&D Systems Quantikine, D6050), TNF (R&D Systems Quantikine, DTA00C), IL10 (R&D Systems Quantikine, D1000B), and gelsolin (LSBio, F22526).
Calculation of bioavailable 25-hydroxyvitamin D
Calculation of bioavailable 25-hydroxyvitamin D (25-OH-D) is described in the Online Supplementary Appendix in Dahl et al.17
Vitamin D-binding protein genotyping genotyping
Participants were genotyped for two common single-nucleotide polymorphisms (SNP) in the coding region of the VDBP gene (Applied Biosystems, rs4588 and rs7041). TaqMan SNP Genotyping assay protocol was performed following the manu- facture’s guidelines (Applied Biosystems, 4371355).
Western blot analysis
Serum proteins were electrophoresed under native conditions through a precast 10% polyacrylamide gel (Bio-Rad, #456-1034) and transferred using conventional techniques. The polyvinyli- dene fluoride membrane was probed with a mouse monoclonal antibody against native full-length, human plasma derived, VDBP (1:4000, Abcam, ab23480), then incubated with an horse radish peroxidase conjugated, anti-mouse immunoglobulin G (IgG) anti- body (1:5,000, Cell Signalling, 7076P2). Results were visualized by chemiluminescence (Amersham, RPN2235) using a BioRad ChemiDoc Touch imager. A positive actin-VDBP complex band (~250 kDa) was scored visually.
Measurements of ATP levels
Measurement of ATP levels by quantitative mass spectrometry is detailed in the Online Supplementary Appendix.
RNA sequencing studies
RNA sequencing (RNAseq) studies were performed as described previously.23 Briefly, normal peripheral blood mononu- clear (PBMC) were incubated overnight with sera from two HSCT recipients, collected at days 0 and 100 (four wells total). RNA was
Variable
Sex Male Female
Age
Race
Caucasian Non-Caucasian
Preparative regimen intensity Myeloablative
Reduced intensity
Donor type Related Unrelated
Stem cell source
Bone marrow
Peripheral blood stem cells
Cordblood 2 9
TA-TMA
39 30
10.2 (5.6-15.4)
70 51
7.4 (3.5-13.3)
0.042
No TA-TMA P 0.88
58 105 0.67 11 16
43 81
26 40
20 39 49 82
0.53
0.75
0.32
0.25
50 77 17 35
HLA match Match Mismatch
45 89
24 32
Patients demographics included in the assessment of clinical outcomes and vitamin D binding protein (VDBP),divided according to whether cases developed transplant- associated thrombotic microangiopathy (TA-TMA) or did not develop TA-TMA (No TA-TMA). HLA: human leukocyte antigen.
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haematologica | 2021; 106(5)