Zhiling Yan

Endothelial Injury, an Intriguing Effect of Methotrexate and Cyclophosphamide During Hematopoietic Stem Cell Transplantation in Mice

OBJECTIVE Elevated circulating endothelial cells (EC) in peripheral blood are an important indicator of endothelial damage and graft-versus-host disease (GVHD). However, the injured endothelial vasculature may in turn promote GVHD. In this study, we investigated whether methotrexate or cyclophosphamide, two conventional chemotherapeutic agents used in hematopoietic stem cell transplantation, caused endothelial injury and what were the functional consequences of this injury on GVHD. METHODS Six to 8-week-old female mice were randomly separated into three groups, including methotrexate (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11), cyclophosphamide (60 mg/kg; days 1, 2), and PBS saline alone (control). Circulating EC (CD31+CD133(-)CD45low) and the CD4+/CD8+ T lymphocytes in the peripheral blood were estimated by flow cytometry on days 1, 3, 5, 7, 9, 11, 16, 21, and 26. The morphologic changes of the endothelium were examined by phase contrast light microscopy to determine the integrity of the endothelial vasculature. RESULTS Elevated EC were detected at day 1 or 3 in mice receiving cyclophosphamide or methotrexate, respectively, with a peak increase at day 5 or day 3, respectively. The ratio of CD4+/CD8+ T lymphocytes showed a delayed increase in the peak to day 11 for both groups. In the cyclophosphamide group, there was significant apomorphosis with necrosis/thrombosis under light microscopy, whereas only apomorphosis was noticed in the methotrexate group. In both groups, EC showed hydropsia and cytomembrane damage. CONCLUSIONS Circulating EC increase during the early phases of cyclophosphamide or methotrexate conditioning, suggesting that both chemotherapeutic drugs induce endothelial damage, which occurs a little earlier than suppression of the immune system.

Irradiation is an early determinant of endothelial injury during hematopoietic stem cell transplantation.

OBJECTIVE We investigated the degree and the time course of endothelial injury in mice pretreated with lethal or reduced-intensity irradiation administered before transplantation. MATERIALS AND METHODS Six- to eight-week-old female mice were randomly allocated into three groups: lethal-intensity irradiation (8.5 Gy, group 1), reduced-intensity irradiation (5.0 Gy, group 2), or nonirradiated controls (group 3). After conditioning, circulating endothelial cells (CD31+, CD133(-), and CD45low) and peripheral blood CD4+ or CD8+ T-lymphocyte subpopulations were enumerated using flow cytometry at various times. The morphologic changes in endothelium were examined at phase-contrast light microscopy. RESULTS Circulating endothelial cells showed an earlier and higher peak in the lethal irradiation group compared with the reduced-intensity irradiation group, which exhibited a protean elevation in cell numbers. There were no visible histopathologic changes during the early stage of endothelial damage. CONCLUSIONS Lethal and reduced doses of irradiation induced endothelial injury in a dose-dependent manner. Endothelial damage may occur before graft-vs-host disease and its related complications.

CXCR4-transduced mesenchymal stem cells protect mice against graft-versus-host disease

Mesenchymal stem cells (MSCs) possessing immunoregulatory activities have been evaluated in the treatment of graft-versus-host disease (GVHD). However, the immunomodulatory effects of MSCs are not always successfully achieved in some animal models, and this deficiency may be caused in part by poor homing of these cells to hematopoietic tissues. In this study, we assessed the immunsuppressive capacity of lentiviral vector transduced MSCs expressing CXCR4 in a major histocompatibility complex (MHC)-mismatched mouse model of bone marrow (BM) transplantation from C57BL/6 donors to BALB/c recipients. The survival, body weight and clinical score of GVHD in transplanted mice were monitored. Liver, intestine and skin from mice in each group were obtained for histological examination. Plasma concentrations of interleukin (IL)-2, IL-4, IL-6, IL-10, IFN-γ, TNF-α and IL-17A were also determined using a Cytometric Bead Array. CXCR4 over-expressing MSCs maintained their immunsuppressive capacity and showed enhanced migration capacity in vitro. In the mouse GVHD model, treatment with CXCR4 over-expressing MSCs decreased the mortality rate and attenuated clinical and pathological GVHD scores. Moreover, compared with control groups, the plasma IL-2, IL-6, IFN-γ and TNF-α levels in recipients infused with CXCR4 over-expressing MSCs were significantly decreased, while those of IL-4 and IL-10 were increased. In conclusion, our report reveals that CXCR4-transduced MSCs effectively controlled the occurrence of mouse GVHD following allogeneic BM transplantation.

MiR-15a/16 regulates the growth of myeloma cells, angiogenesis and antitumor immunity by inhibiting Bcl-2, VEGF-A and IL-17 expression in multiple myeloma

miRNAs have been reported to be involved in the pathogenesis of many cancers. In this article, we investigated the role and the mechanisms of miR-15a/16 in the pathogenesis of multiple myeloma (MM). We found that miR-15a/16 was down-regulated in bone marrow-derived mononuclear cells (BM-MNCs) of newly diagnosed patients with MM and the downregulation of miR-15a/16 was correlated with International Staging System (ISS) stage. We then demonstrated miR-15a/16 inhibited myeloma cells proliferation, and increased apoptosis rate of U266 cells by suppressing the expression of anti-apoptosis protein Bcl-2. We also found miR-15a/16 could decrease VEGF-A and IL-17 levels in the supernatant of myeloma cells. These results indicate that miR-15a/16 may function as a tumor suppressor in MM through multiple regulatory mechanisms and they may be potential targets for the therapy of MM.

Bone Marrow–derived Endothelial Progenitor Cells Promote Hematopoietic Reconstitution After Hematopoietic Stem Cell Transplantation

OBJECTIVE A hematopoietic deficit is a serious complication after hematopoietic stem cell transplantation. It has been shown that fetal blood-derived endothelial progenitor cells (EPCs) can promote hematopoietic reconstitution after transplantation. This study investigated whether EPCs from bone marrow (BM) of adult mice could promote hematopoietic reconstitution. METHODS Lethally irradiated BALB/c mice were administered BM cells or BM cells plus EPCs. RESULTS The results showed that EPC-treated mice displayed accelerated recovery of peripheral blood white blood cells and reticulocytes. But the platelets were not significantly different with versus without EPCs. Accelerated recovery of BM sinusoidal vessels, promotion of stem cell implantation, and decreased adipocyte formation were associated with the mechanism. Systemic administration of anti-vascular endothelial cadherin antibody neutralized these effects significantly. CONCLUSION These data showed that BM-derived EPC infusions augmented hematopoiesis suggesting a new approach to promote hematopoiesis.

Vascular Endothelium Changes After Conditioning in Hematopoietic Stem Cell Transplantation: Role of Cyclophosphamide and Busulfan

Vascular endothelial injury, a feature of some complications of hematopoietic stem cell transplantation (HSCT), is characterized by increased endothelial cells. We investigated that classical pretreatment drugs in HSCT could result in vascular endothelial injury in mice. Six-to eight-week-old female BALB/c mice were divided into a control group, a cyclophosphamide group (60 mg/kg per day for 2 days) and a busulfan group (4 mg/kg per day for 4 days). We observed the general state of health and regularly counted the number of white blood cells. Circulating endothelial cells and their progenitors were estimated by flow cytometry. Morphologic endothelial changes were analyzed with optical and transmission electron microscopy. After conditioning with cyclophosphamide or busulfan, white blood cells fell to a low number with injuries noted on hematoxylin and eosin-stained pathology sections. Circulating endothelial cells and their progenitors peaked significantly higher than in the control group. Vascular endothelial injuries were observed in the 2 experimental groups by transmission electron microscopy. These data support the hypothesis the vascular endothelial injury occurs during conditioning with cyclophosphamide or busulfan for HSCT, with simultaneous mobilization of endothelial progenitor cells.

Piperlongumine selectively suppresses ABC-DLBCL through inhibition of NF-κB p65 subunit nuclear import

Constitutive NF-κB activation is required for survival of activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL). However, current NF-κB targeting strategies lack cancer cell specificity. Here, we identified a novel inhibitor, piperlongumine, features direct binding to NF-κB p65 subunit and suppression of p65 nuclear import. This was accompanied by NF-κB reporter activity suppression and NF-κB target gene downregulation. Moreover, mutation of Cys(38) to Ser in p65 abolished this effect of piperlongumine on inhibition of p65 nuclear import. Furthermore, we show that piperlongumine selectively inhibited proliferation and induced apoptosis of ABC-DLBCL cells. Most notably, it has been reported that piperlongumine did not affect normal cells even at high doses and was nontoxic to animals. Hence, our current study provides new insight into piperlongumines mechanism of action and novel approach to ABC-DLBCL target therapy.

Increased numbers of circulating ECs are associated with systemic GVHD.

Introduction:  Circulating endothelial cells (ECs) are known to reflect endothelial injury, and endothelial injury is associated with graft‐versus‐host disease (GVHD). We hypothesised that circulating ECs might be associated with systemic acute graft‐versus‐host disease (aGVHD).

MicroRNA-150 negatively regulates the function of CD4+ T cells through AKT3/Bim signaling pathway

Donor-derived CD4(+) T lymphocytes are the major effector cells directly involved in the development of graft-versus-host disease (GVHD). As a negative regulator of immune cell differentiation and development, microRNA-150 (miR-150) induces immunological tolerance in CD4(+) T cells after transplantation. However, the specific mechanisms have not been fully elucidated. In this study, we demonstrated that miR-150 is capable of not only inhibiting proliferation and activation of CD4(+) T cells but also promoting apoptosis. Mechanistically, miR-150 targets v-akt murine thymoma viral oncogene homolog 3 (AKT3), and subsequently downregulates B-cell lymphoma 2 (Bcl-2) interacting mediator of cell death (BIM). We have also demonstrated that re-expression of AKT3 reversed miR-150-mediated inhibition of CD4(+) T lymphocyte development. Therefore, we conclude that miR-150 negatively regulates CD4(+) T cell function by inhibiting the AKT3/BIM signaling pathway. These findings also suggest that manipulating the levels of miRNA-150 could be a valuable strategy in prevention and/or treatment of acute graft-versus-host disease.

Identification of Suitable Reference Genes for Normalization of Real-Time Quantitative Polymerase Chain Reaction in an Intestinal Graft-Versus-Host Disease Mouse Model.

BACKGROUND With the development of real-time quantitative polymerase chain reaction (RT-qPCR) and intensive research on acute graft-versus-host disease (GVHD), selecting the best reference gene for normalization of RT-qPCR analysis in a GVHD model becomes more and more important. In this study, we aimed to identify suitable reference genes for mRNA studies in an intestinal GVHD mouse model after bone marrow transplantation (BMT). METHODS BALB/c recipients received 7.5 Gy total body irradiation (TBI) followed by injection of 5 × 10(6) bone marrow cells, without infusion of spleen cells for BMT, with infusion of 5 × 10(5) or 2.5 × 10(6) spleen cells for mild or moderate GVHD, respectively. Healthy mice were chosen as normal control subjects. Duodenum, jejunum, ileum, colon, and small intestine were collected at days 7, 14, 21, and 28 after transplantation. Transcription levels of 9 candidate genes, B2M, SDHA, HPRT, ACTB, GAPDH, HMBS, TBP, YWHAZ, and RPLP0, in each tissue were measured with the use of RT-qPCR. Combined data from these tissues in each group were defined as all samples. The expression stability of these genes was analyzed with the use of Genorm, Normfinder, Bestkeeper, and ΔCt. RESULTS Our results showed that in all samples, ACTB and HMBS displayed the highest and lowest expression levels, respectively. Genorm identified HRPT and SDHA as the most stable reference genes, whereas Normfinder and ΔCt method showed HPRT as the most stably expressed gene. Bestkeeper ranked YWHAZ and HPRT as the top 2 most suitable genes. In conclusion, HPRT was recommended as the most suitable reference gene after comprehensive ranking, suggesting that it could be used as an internal control for mRNA studies in intestinal GVHD after BMT.