Kazuhiko Harada

Allogeneic injection of fetal membrane-derived mesenchymal stem cells induces therapeutic angiogenesis in a rat model of hind limb ischemia.

Bone marrow‐derived mesenchymal stem cells (BM‐MSC) have been demonstrated to be an attractive therapeutic cell source for tissue regeneration and repair. However, it remains unknown whether or not allogeneic transplantation of mesenchymal stem cells (MSC) derived from fetal membranes (FM), which are generally discarded as medical waste after delivery, has therapeutic potential. FM‐MSC were obtained from Lewis rats and had surface antigen expression and multipotent potential partly similar to those of BM‐MSC. Compared with BM‐MSC, FM‐MSC secreted a comparable amount of hepatocyte growth factor despite a small amount of vascular endothelial growth factor. FM‐MSC and BM‐MSC both expressed major histocompatibility complex (MHC) class I but not MHC class II antigens and did not elicit allogeneic lymphocyte proliferation in mixed lymphocyte culture. FM‐MSC or BM‐MSC obtained from Lewis rats were injected into a MHC‐mismatched August‐Copenhagen‐Irish rat model of hind limb ischemia. Three weeks after injection, blood perfusion and capillary density were significantly higher in the FM‐MSC and BM‐MSC groups than in the phosphate‐buffered saline group, and allogeneic FM‐MSC and BM‐MSC were still observed. In nonischemic hind limb tissues, allogeneic FM‐MSC and BM‐MSC injection were associated with a comparatively small amount of T lymphocyte infiltration, compared with the injection of allogeneic splenic lymphocytes. In conclusion, allogeneic FM‐MSC injection did not elicit a lymphocyte proliferative response and provided significant improvement in a rat model of hind limb ischemia, comparable to the response to BM‐MSC. Thus, allogeneic injection of FM‐MSC may be a new therapeutic strategy for the treatment of severe peripheral vascular disease.

Comparison of Angiogenic, Cytoprotective, and Immunosuppressive Properties of Human Amnion- and Chorion-Derived Mesenchymal Stem Cells

Although mesenchymal stem cells (MSCs) can be obtained from the fetal membrane (FM), little information is available regarding biological differences in MSCs derived from different layers of the FM or their therapeutic potential. Isolated MSCs from both amnion and chorion layers of FM showed similar morphological appearance, multipotency, and cell-surface antigen expression. Conditioned media obtained from amnion- and chorion-derived MSCs inhibited cell death caused by serum starvation or hypoxia in endothelial cells and cardiomyocytes. Amnion and chorion MSCs secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Transplantation of human amnion or chorion MSCs significantly increased blood flow and capillary density in a murine hindlimb ischemia model. In addition, compared to human chorion MSCs, human amnion MSCs markedly reduced T-lymphocyte proliferation with the enhanced secretion of PGE2, and improved the pathological situation of a mouse model of acute graft-versus-host disease. Our results highlight that human amnion- and chorion-derived MSCs, which showed differences in their soluble factor secretion and angiogenic/immuno-suppressive function, could be ideal cell sources for regenerative medicine.

Adrenomedullin induces lymphangiogenesis and ameliorates secondary lymphoedema

AIMS Adrenomedullin (AM) is a multifunctional peptide hormone that plays a significant role in vasodilation and angiogenesis. Lymphoedema is a common but refractory disorder that is difficult to be treated with conventional therapy. We therefore investigated whether AM promotes lymphangiogenesis and improves lymphoedema. METHODS AND RESULTS The effects of AM on lymphatic endothelial cells (LEC) were investigated. AM promoted proliferation, migration, and network formation of cultured human lymphatic microvascular endothelial cells (HLMVEC). AM increased intracellular cyclic adenosine monophosphate (cAMP) level in HLMVEC. The cell proliferation induced by AM was inhibited by a cAMP antagonist and mitogen-activated protein kinase kinase (MEK) inhibitors. Phosphorylated extracellular signal-regulated kinase (ERK) in HLMVEC was increased by AM. Continuous administration of AM (0.05 microg/kg/min) to BALB/c mice with tail lymphoedema resulted in a decrease in lymphoedema thickness. AM treatment increased the number of lymphatic vessels and blood vessels in the injury site. CONCLUSION AM promoted LEC proliferation at least in part through the cAMP/MEK/ERK pathway, and infusion of AM induced lymphangiogenesis and improved lymphoedema in mice.

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5 × 10(5) cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.

Systemic transplantation of allogenic fetal membrane-derived mesenchymal stem cells suppresses Th1 and Th17 T cell responses in experimental autoimmune myocarditis.

We have reported that systemic administration of autologous bone marrow or allogenic fetal membrane (FM)-derived mesenchymal stem cells (MSCs) similarly attenuated myocardial injury in rats with experimental autoimmune myocarditis (EAM). Since rat EAM is a T-helper (Th) cell-mediated autoimmune disease, and recent evidence has indicated that both autologous and allogenic MSCs exert an immunosuppressive effect on Th cell activity, we focused on Th cell differentiation in allogenic FM-MSC administered EAM rats. EAM was induced in Lewis rats by injecting porcine cardiac myosin (day 0). Allogenic FM-MSCs, obtained from major histocompatibility complex mismatched ACI rats, were intravenously injected (5 × 10(5)cells/rat) on days 7, 10, or 14 (MSCd7, MSCd10, or MSCd14 groups, respectively). At day 21, echocardiography confirmed that reduced ejection fraction in the untreated EAM group (63 ± 2%) was significantly improved in the MSCd10 and MSCd14 groups (74 ± 1 and 75 ± 2%, respectively, P<0.01). CD68 immunostaining revealed that prominent macrophage infiltration in the myocardium of the EAM group (1466 ± 93 cells/mm(2)) was significantly decreased in the MSCd10 group (958 ± 139 cells/mm(2), P<0.05). To evaluate Th cell differentiation, we used flow cytometry to determine the percentage of interferon (IFN)-γ positive Th1 and interleukin (IL)-17 positive Th17 cells in peripheral CD4-positive Th cells. The percentage of Th1 cells at day 16 was significantly lower in the MSCd10 (1.3 ± 0.2%) and MSCd14 (1.6 ± 0.3%) groups compared to the EAM group (2.4 ± 0.3%, P<0.05), as was the percentage of Th17 cells in the MSCd10 group (1.9 ± 0.5%) compared to the EAM group (2.2 ± 0.9%, P<0.05). At day 21, infiltrating Th17 cells in myocardium were significantly decreased in the MSCd10 group (501 ± 132 cells/mm(2), P<0.05) compared to EAM (921 ± 109 cells/mm(2)). In addition, human CD4+ Th cells co-cultured with human FM-MSCs exhibited reduced Th1 and Th17 cell-differentiation and proliferation, with increased expression of immunosuppressive molecules including indoleamine 2,3-dioxygenase 2 and IL-6 in co-cultured FM-MSCs. These results suggest that intravenous administration of allogenic FM-MSCs ameliorates EAM via the suppression of Th1/Th17 immunity.

High-cholesterol feeding aggravates cerebral infarction via decreasing the CB1 receptor.

We examined how feeding conditions affect the CB1 receptor and cerebral infarction caused by cerebral ischemia. Mice were divided into the following three groups: normal diet (ND), caloric restriction (CR) and high-cholesterol-enriched diet (HCD), and were kept for 6 weeks. After 6 weeks, we measured both serum and brain cholesterol and the expression level of cannabinoid CB1 receptor within the brain in intact mice. In addition, middle cerebral artery (MCA) was occluded for 2 h following reperfusion. Serum cholesterol significantly increased in the HCD group in comparison with both the ND and CR groups. However, brain cholesterol decreased in the HCD group. Then, the expression level of CB1 receptor significantly decreased in the HCD group, while that of the CR group clearly increased in comparison with the ND group in intact mice. In MCA-occluded mice, The HCD group produced the most severe cerebral infarction, while cerebral infarction was significantly decreased in the CR group. These results suggest that CR prevents infarction by increasing CB1 receptor expression, while high-cholesterol feeding aggravates cerebral infarction both by hypercholesterolemia in serum and by decreasing CB1 receptor expression modulated by hypocholesterolemia within the brain.

Rehabilitative Training Tasks Improve Spatial Learning Impairment in the Water Maze Following Hypoxic-Ischemic Insult in Neonatal Rats

We recently reported that hypoxic-ischemic (HI) insult to the brain of 7-d-old rats resulted in a slowly progressive learning and memory disability, which started at around 5 wk after HI, a time frame that is representative of human adolescence. The purpose of the present study was to examine whether physical or mental exercises can prevent this late-onset, slowly progressing disability. Wistar rats were subjected to left carotid ligation followed by 2 h of hypoxic stress (8% O2 and 92% N2 at 33°C). Sham-control rats were subjected to the same procedure without ligation and hypoxic stress. Six weeks after the HI, the animals were divided into four groups: pretraining control, no training control, pretraining HI, and no training HI groups. We used the plus maze, eight-arm radial maze, and choice reaction time task as the rehabilitative training. Sixteen weeks after the HI, the water maze task was performed over 5 d to evaluate spatial learning ability; thereafter, cerebral morphology of the animals was examined. There were no differences in swimming length and latency between the pretraining control and no training control groups. Swimming length and latency in the pretraining HI group were significantly shorter and swifter than those in the no training HI group. The infarct areas on the left cerebral hemisphere were equivalent between pretraining HI and no training HI groups at each sectional slice. Rehabilitative training tasks prevented the neonatal HI-induced late-onset slowly progressive learning and memory disability.

Sustained-release adrenomedullin ointment accelerates wound healing of pressure ulcers

Pressure ulcers are one of the most common complications in elderly, incontinent or paralyzed patients. For the healing of pressure ulcers, the development of granulation tissue and reepithelialization is required. Adrenomedullin (AM), an endogenous vasodilator peptide, is reported to stimulate the proliferation and migration of various cells including endothelial cells, fibroblasts and keratinocytes. Therefore, we hypothesized that AM might accelerate the healing process of pressure ulcers in which these cells were involved. We developed a sustained-release ointment containing human recombinant AM, and applied it in a mouse model of pressure ulcer twice a day for 14 days. Human AM was efficiently absorbed in wound area, but its blood concentration was negligible. AM ointment significantly reduced the wound area on day 5 to 7 after injury. In addition, AM ointment accelerated the formation of granulation tissue and angiogenesis as well as lymphangiogenesis after 7 days of treatment. Immunological analysis revealed that Ki-67-positive proliferating cells in granulation tissue expressed AM receptors. In summary, sustained-release AM significantly improved wound healing of pressure ulcers through acceleration of granulation and induction of angiogenesis and lymphangiogenesis. Therefore, sustained-release AM ointment may be a novel therapeutic agent for pressure ulcers.