The adhesive protein von Willebrand factor (VWF) plays an essential role in physiological hemostasis, mediating platelet adhesion and aggregation under high shear stress conditions.21 The VWF-cleaving protease ADAMTS13 precisely down-regulates VWF activity to avoid pathological intravascular thrombosis in the microvasculature, including arterial capillaries, where blood flow typically creates high shear stress.31 Indeed, the functional deficiency of ADAMTS13 is known to cause thrombotic thrombocytopenic purpura, a typical thrombotic occlusion of the microvasculature.542 Thus, the proper equilibrium between VWF and ADAMTS13 is necessary for robust microcirculation in vivo. In this context, we hypothesized that ADAMTS13 might contribute to better donor cell homing and engraftment in various cell therapy approaches, in which fluent blood flow in the microcirculation system could be critical. To test this hypothesis, we investigated the role of ADAMTS13 on donor cell engraftment using a bone marrow transplantation (BMT) model in ADAMTS13 gene-deleted (Adamts13 −/−) mice.
Adamts13 −/− (KO) mice were back-crossed for more than 15 generations to the C57BL/6 background, as described.6 Wild-type (WT) mice (C57BL/6-background) were purchased from Japan SLC (Shizuoka, Japan). All mice used in this study were 8–12 weeks old with body weights of 25–30 grams. Mouse experiments were performed in accordance with protocols approved by the Ethics Review Committee for Animal Experimentation of Nara Medical University. In the BMT experiment, recipient WT or KO mice were conditioned for cellular transplantation with lethal total body irradiation (TBI: 5.5 × 2 = total 11 Gray) using a cesium irradiator (MBR-1520, Hitachi, Tokyo, Japan). Bone marrow cells to be transplanted were collected from femurs and tibias of donor green fluorescence protein (GFP) mice7 (purchased from Japan SLC: C57BL/6-background), as described.8 After removing the red blood cells by lysing with Tris-buffered ammonium chloride, suspended donor bone marrow mononuclear cells were transplanted into irradiated sex-matched recipient mice via tail vein. In some indicated experiments, recombinant human ADAMTS13 (rhADAMTS13), which was prepared as previously described,9 was added to the donor bone marrow cell suspension prior to cellular transplantation. The VWF-cleaving activity of rhADAMTS13 was determined by in vitro FRETS-VWF73 assay.10
Kaplan-Meier analysis showed that the mean survival rate of KO mice receiving TBI and subsequent BMT was significantly lower than that of WT mice starting at Day 14 after BMT, and recombinant ADAMTS13 restored the survival rate of KO mice to that of WT mice (Figure 1A). Since all WT and KO mice that underwent TBI without BMT died within 21 days (results not shown), the mortality rates under our experimental conditions most likely depended upon the cell engraftment efficacy during BMT and indicate an important contribution of ADAMTS13 in this regard. Indeed, peripheral blood analysis following BMT revealed the longer nadir period in KO mice with regard to neutrophils and platelets (Figure 1B), which was shortened significantly by recombinant ADAMTS13, with the resulting nadir periods comparable to those of WT mice (Figure 1B).
In addition to the above long-term observation experiment, some recipient mice were sacrificed at Days 1, 7, and 14 after BMT to check the extent of donor cell engraftment to the bone marrow and to assess the pathohistological conditions of major organs. After removing the red blood cells, the recipients’ bone marrow was collected from the femurs and tibias and used to assess donor cell engraftment efficacy based on the percentage of GFP-positive cells relative to total mononuclear cells using flow cytometer (BD LSR-II: Nippon Becton Dickinson Company Ltd., Tokyo, Japan). Consistent with the findings in the peripheral blood, flow cytometric analysis of recipient bone marrow revealed the reduction of donor GFP-positive cells in KO mice that was already significant at Day 1 after BMT (Figure 2A). The population of GFP-positive cells in the bone marrow of KO mice expands gradually in a time-dependent manner similar to that of WT mice (Figure 2A), suggesting that ADAMTS13 is likely to play a role in the initial donor cell homing rather than cell propagation in the bone marrow cell engraftment. Thus, our results could verify the initial hypothesis that ADAMTS13 may contribute to better donor cell homing to the target recipient marrow, a process that requires fluent blood flow in the microvasculature including arterial capillaries.
Thrombotic microangiopathy (TMA) is a well-recognized serious complication of BMT, especially in the liver in the form of sinusoidal obstruction syndrome (SOS), and is known to be associated with functional ADAMTS13 deficiency.11 Our histological studies, however, have only confirmed mild congestion and sinusoidal dilatation in the liver as well as significant splenic enlargement and congestion in KO mice, without typical thrombotic or SOS lesions of microvessels (Figure 2B and C). These histological findings may be consistent with possible portal hypertension, perhaps reflecting transient occlusion of the microvasculature by enhanced leukocyte plugging or platelet microaggregate formation that may occur in systemic microcirculation. Thus, the reduced local microcirculation could result in the poor donor cell homing to bone marrow that was observed in KO mice. Indeed, some clinical symptoms of TMA with functional deficiency of ADAMTS13 are known to be labile and variable,5 suggesting the existence of transient microvasculature occlusion that cannot be reproducibly demonstrated in final tissue sample sections.
Recent mouse model studies by us and others demonstrated that proper functional regulation of VWF by ADAMTS13 significantly ameliorates the severity of fatal arterial thrombosis in conditions such as cerebrovascular accident or myocardial infarction.1512 ADAMTS13 reduces VWF-dependent platelet microaggregate formation as well as inflammatory responses such as leukocyte accumulation at ischemic sites, both of which may result in local microvasculature occlusion.5 Thus, this property of ADAMTS13 can protect against impaired microcirculation in vivo, and may also contribute to better donor cell homing and engraftment in various cell therapy approaches that require fluent blood flow in the microvasculature.
In conclusion, our results illustrate that the regulation of VWF-mediated thrombotic or inflammatory responses by ADAMTS13 may contribute to the improved systemic microcirculation critical for efficient donor cell homing and engraftment in BMT, suggesting a clinical therapeutic potential of ADAMTS13 in cell therapy approaches.
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