Keizo Minamino

Mobilization of Bone Marrow Cells by G-CSF Rescues Mice from Cisplatin-Induced Renal Failure, and M-CSF Enhances the Effects of G-CSF

Cisplatin, which is a broadly used anticancer drug, is widely known to induce acute renal failure as a result of renal tubular injury. This article examines whether G-CSF and/or M-CSF rescues mice from renal failure induced by cisplatin. BALB/c mice received intraperitoneal injections with or without G-CSF and/or M-CSF for 5 d (from day -5 to day -1). The day after the last injection of G-CSF and/or M-CSF (day 0), the mice received an intraperitoneal injection of cisplatin. When pretreated with G-CSF or G-CSF + M-CSF, the mice showed longer survival and lower serum creatinine and blood urea nitrogen levels than mice that had been received injections of M-CSF or saline. Histologically, pretreatment with G-CSF or G-CSF + M-CSF attenuated the damage to renal tubules induced by cisplatin. BALB/c mice that had received a transplant of bone marrow cells of enhanced green fluorescent protein (EGFP)-transgenic mice ([EGFP-->BALB/c] mice) were treated with or without G-CSF and/or M-CSF, followed by injection of cisplatin as well as above. [EGFP-->BALB/c] mice that were treated with G-CSF or G-CSF + M-CSF showed a significantly higher number of EGFP(+) tubular epithelial cells in the kidney than mice that were treated with only M-CSF or saline. These results suggest that bone marrow cells mobilized by G-CSF accelerate the improvement in renal functions and prevent the renal tubular injury induced by cisplatin and that M-CSF enhances the effects of G-CSF.

Macrophage colony-stimulating factor (M-CSF), as well as granulocyte colony-stimulating factor (G-CSF), accelerates neovascularization

It has been reported that bone marrow cells (BMCs) differentiate into endothelial cells of blood vessels, and that granulocyte colony‐stimulating factor (G‐CSF) mobilizes progenitors in the BMCs to the peripheral blood, while macrophage colony‐stimulating factor (M‐CSF) augments the production of monocytes. We examined whether M‐CSF augments the differentiation of BMCs into endothelial cells of blood vessels using a hindlimb‐ischemic model. Either G‐CSF or M‐CSF, or both, was administered to the hindlimb‐ischemic mice for 3 days. Both M‐CSF and G‐CSF augmented the differentiation of BMCs into endothelial cells of blood vessels through vascular endothelial cell growth factor (VEGF), resulting in early recovery of blood flow in the ischemic limbs.

Choroidal Neovascularization Is Provided by Bone Marrow Cells

Choroidal neovascularization (CNV) is a known cause of age‐related macular degeneration (ARMD). Moreover, the most common cause of blindness in the elderly in advanced countries is ARMD with CNV. It has recently been shown that bone marrow cells (BMCs) can differentiate into various cell lineages in vitro and in vivo. Adults maintain a reservoir of hematopoietic stem cells included in BMCs that can enter the circulation to reach various organs in need of regeneration. It has recently been reported that endothelial progenitor cells (EPCs) included in BMCs are associated with neovascularization. We examine the role of BMCs in CNV using a model of CNV in adult mice. Using methods consisting of fractionated irradiation (6.0 Gy × 2) followed by bone marrow transplantation (BMT), adult mice were engrafted with whole BMCs isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP). Three months after BMT, we confirmed that the hematopoietic cells in the recipients had been completely replaced with donor cells. We then carried out laser photocoagulation to induce CNV in chimeric mice (donor cells >95%). Two weeks after the laser photocoagulation, by which time CNV had occurred, immunohistochemical examination was carried out. The vascular wall cells of the CNV expressed both EGFP and CD31. These findings indicate that newly developed blood vessels in the CNV are derived from the BMCs and suggest that the inhibition of EPC mobilization from the bone marrow to the eyes could be a new approach to the fundamental treatment of CNV in ARMD.

Treatment and transfer of emphysema by a new bone marrow transplantation method from normal mice to Tsk mice and vice versa.

We have recently established a new bone marrow transplantation (BMT) method in which bone marrow cells are injected into the intrabone marrow (IBM). In the present study, we used an animal model for emphysema (tight‐skin [Tsk] mouse) to examine whether IBM‐BMT could be used to treat emphysema in Tsk mice. IBM‐BMT was carried out from C3H mice into Tsk mice (8–10 weeks old) that had already shown emphysema. Six months after transplantation, the lungs of all the Tsk mice treated with IBM‐BMT [C3H→Tsk] showed similar structures to those of normal mice, whereas the [Tsk→Tsk] mice showed emphysema, as seen in age‐matched Tsk mice. Next, we attempted to transfer emphysema from Tsk mice to C3H mice by IBM‐BMT. Six months after IBM‐BMT, the [Tsk→C3H] mice showed emphysema. These results strongly suggest that emphysema in Tsk mice originates from defects of stem cells in the bone marrow.

Hepatocyte Growth Factor Delivered by Ultrasound-Mediated Destruction of Microbubbles Induces Proliferation of Cardiomyocytes and Amelioration of Left Ventricular Contractile Function in Doxorubicin-Induced Cardiomyopathy

At present, there is no curative strategy for advanced cardiomyopathy except for cardiac transplantation, which is not easily performed, mainly due to a shortage of donors. It has been reported that myocardial progenitor cells exist even in the postnatal heart, suggesting that myocardial progenitor cells could proliferate under some situations and might improve cardiac function in cardiomyopathy‐induced hearts. In this study, recombinant human hepatocyte growth factor (rhHGF) was delivered using ultrasound‐mediated destruction of microbubbles (UMDM) into the cardiomyopathy‐induced heart by doxorubicin (20 mg/kg). Intravenous injection of rhHGF (IV‐rhHGF) alone or UMDM alone failed to improve the morphology or the function of the cardiomyopathy‐induced heart, but (IV‐rhHGF + UMDM) treatment significantly improved the heart morphologically and functionally, and repetitive treatments of (IV‐rhHGF + UMDM) enhanced the effects. The number of bromodeoxy‐uridine‐positive cardiomyocytes significantly increased in the (IV‐rhHGF + UMDM)–treated hearts compared with the untreated hearts. Moreover, Sca‐1+ myocardial progenitor cells express c‐Met, a receptor for HGF. These results suggest that (IV‐rhHGF + UMDM) treatment could morphologically and functionally improve the heart in the case of doxorubicin‐induced cardiomyopathy through the proliferation of the myocardial progenitor cells.

Simultaneous Injection of Bone Marrow Cells and Stromal Cells into Bone Marrow Accelerates Hematopoiesis In Vivo

We have previously demonstrated that stromal cells can support the proliferation and differentiation of hematopoietic cells in vitro and in vivo and that a major histocompatibility complex restriction exists between hematopoietic stem cells and stromal cells. We have also found that intra–bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) leads to more rapid reconstitution of hematopoietic cells than intravenous injection. In the present study, we examine the effect of simultaneous injection of stromal cells and BMCs into the same bone marrow on the recovery of donor hematopoietic cells and demonstrate that simultaneous IBM injection of BMCs plus stromal cells is more effective in reconstituting recipients with donor hematopoietic cells than intravenous injection of BMCs plus stromal cells or IBM injection of BMCs alone.

Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. By using a retrovirus-mediated signal sequence trap method, we identified a previously undescribed gene, termed ARIA (apoptosis regulator through modulating IAP expression), which regulates endothelial apoptosis and angiogenesis. ARIA was expressed in blood vessels during mouse embryogenesis, as well as in endothelial cells both in vitro and in vivo. ARIA is a unique protein with no homology to previously reported conserved domain structures. Knockdown of ARIA in HUVECs by using small interfering RNA significantly reduced endothelial apoptosis without affecting either cell migration or proliferation. ARIA knockdown significantly increased inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression, although their mRNA expression was not changed. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the antiapoptotic effect of ARIA knockdown. Using yeast 2-hybrid screening, we identified the interaction of ARIA with 20S proteasome subunit α-7. Thereafter, we found that cIAP-1 and cIAP-2 were degraded by proteasomes in endothelial cells under normal condition. Overexpression of ARIA significantly reduced cIAP-1 expression, and this reduction was abolished by proteasomal inhibition in BAECs. Also, knockdown of ARIA demonstrated an effect similar to proteasomal inhibition with respect to not only expression but also subcellular localization of cIAP-1 and cIAP-2. In vivo angiogenesis studied by Matrigel-plug assay, mouse ischemic retinopathy model, and tumor xenograft model was significantly enhanced by ARIA knockdown. Together, our data indicate that ARIA is a unique factor regulating endothelial apoptosis, as well as angiogenesis, presumably through modulating proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells.

A New Strategy for Treatment of Malignant Tumor: Intra‐Bone Marrow–Bone Marrow Transplantation Plus CD4− Donor Lymphocyte Infusion

Donor lymphocyte infusion (DLI) is clinically used for the treatment of malignant tumors. We have found recently that intra‐bone marrow–bone marrow transplantation (IBM‐BMT) can be used to treat various autoimmune diseases, even when radiation doses are reduced. In addition, recently we have found that IBM‐BMT can prevent not only graft failure but also graft‐versus‐host disease (GvHD). Based on these findings, we attempted to prevent and treat the progression of a tumor (Meth‐A cell line: BALB/c‐derived fibrosarcoma) by DLI plus IBM‐BMT. When the tumors had grown to approximately 10 × 10 mm, the tumor‐bearing BALB/c (H‐2d) mice were irradiated with 5 Gy, and whole spleen cells from C57BL/6J (B6) (H‐2b) mice (as DLI) were then intravenously injected into the BALB/c mice. Simultaneously, bone marrow cells (BMCs) from B6 mice were injected directly into the bone marrow cavity of the BALB/c mice (IBM‐BMT). The tumors decreased in size, but the mice died of GvHD. However, when CD4+ T‐cell–depleted spleen cells were used for DLI, the recipients showed only mild GvHD and survived longer, due to the slow growth of the tumor. In contrast, when CD8+ T‐cell–depleted spleen cells were used for DLI, the recipients showed more severe GvHD than those injected with whole spleen cells. These results suggest that IBM‐BMT plus DLI (the depletion or reduction of a certain cell population like CD4+ T cells) could be helpful to suppress both GvHD and tumor growth.

Expression of pigment epithelium-derived factor and vascular endothelial growth factor in fibrovascular membranes from patients with proliferative diabetic retinopathy.

PurposeThe expression of pigment epithelium-derived factor (PEDF), a strong inhibitor of angiogenesis, has not been examined in human ocular fibrovascular membranes, to the best of our knowledge. The purpose of this study was to determine whether PEDF is expressed in the fibrovascular membranes in eyes of patients with proliferative diabetic retinopathy (PDR), and to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF).MethodsThe expression of PEDF and VEGF in the fibrovascular membranes excised during vitreous surgery in eight cases of PDR was determined by immunohistochemistry.ResultsVEGF was strongly expressed in the endothelial cells of newly formed vessels in the fibrovascular membranes. In contrast, PEDF was weakly expressed in the endothelial cells and was prominently expressed in the extracellular matrix and fibrous tissue surrounding the new vessels.ConclusionsOur results suggest that PEDF, along with VEGF, may modulate the formation of fibrovascular membranes in patients with PDR. Jpn J Ophthalmol 2006;50:116–120

Autopsy case of primary choriocarcinoma of the urinary bladder.

Choriocarcinomas usually develop in the uterus and ovaries in the female, being extremely rare in the extragenital organs in the male. Extragenital choriocarcinomas in the male usually develop in the mediastinum or retroperitoneum. The frequency of choriocarcinoma in the urinary bladder is extremely low. The purpose of the present paper was to report an autopsy case of choriocarcinoma in the urinary bladder in the male. An 81‐year‐old male patient with macrohematuria was first diagnosed with transitional cell carcinoma (TCC). At autopsy a hemorrhagic necrotic tumor, which was found in the urinary bladder with metastatic lesions in the lungs, was diagnosed as choriocarcinoma microscopically. There was no evidence for choriocarcinoma derived from any other organs than the urinary bladder, although there were metastatic lesions in both lungs and the direct invasion into the prostate. From these findings it is concluded that the tumor was a primary choriocarcinoma in the urinary bladder in a male patient. Choriocarcinoma of the urinary bladder is very rare, but the prognosis is extremely poor in comparison with TCC even in the urinary bladder. Therefore, it is essential to clearly discriminate between choriocarcinomas and TCC.