Hiroshi Shimoda

Immunohistochemical Demonstration of Lymphatic Vessels in Adult Zebrafish

Morphological profiles of lymphatic vessels in adult zebrafish trunk and ovary were studied by immunohistochemistry and electron microscopy. The present immunohistochemistry for Prox1 was successful in demonstrating lymphatic vessels in zebrafish. The zebrafish trunk revealed two types of bilateral longitudinal lymphatic trunks draining lymph centripetally along dorsal aorta and posterior cardinal veins. Large honeycomb lymphatic sinus was further shown around common cardinal veins. In the zebrafish ovary, the lymphatic vessels, comprising endothelial cells only, encompassed arterioles in their lumen. This peculiar structure appeared to be conserved in vertebrates including mammals and might serve for control of blood temperature and tissue homeostasis. The present study is first to delineate lymphatic vessels in adult zebrafish by immunohistochemistry. Our immunohistochemical results showed usefulness of immunostaining for Prox1 not only for demonstration of lymphatic vessels in zebrafish, but also for examination of their function and dynamics in pathophysiological condition.

Antitumor effects of the hyaluronan inhibitor 4-methylumbelliferone on pancreatic cancer

Hyaluronan (HA) is a major component of the extracellular matrix (ECM), and influences tumor invasion and metastasis. In a previous study, the present authors reported for the first time that 4-methylumbelliferone (MU) inhibited HA synthesis and suppressed tumor growth. However, the localization of HA and the changes in ECM morphology caused by MU in pancreatic cancer remain to be examined in detail. In the present study, the cytotoxicity of MU and its effect on cellular proliferation was evaluated in the human pancreatic cancer cell line MIA PaCa-2. The amount of HA synthesized and the retention of HA around the cells were quantitatively and immunohistochemically analyzed in vitro and in vivo. Structural changes in the ECM in the tumor tissue were investigated using an electron microscope. MU treatment led to a decrease in extracellular HA retention, as evidenced by a particle exclusion assay and immunohistochemical staining. Cell proliferation was suppressed by MU in a dose-dependent manner. The release of lactate dehydrogenase into the culture medium due to damage to the cellular membrane did not increase following MU administration. In tumor-inoculated mice, MU suppressed any increase in tumor volume and decreased the quantity of HA. Electron microscopy revealed that MU attenuated the intercellular space and caused it to be less cohesive. These data indicate that MU inhibits HA synthesis and reduces the amount of HA in the ECM while exhibiting no obvious cytotoxic effect. These findings suggest that MU has potential as a novel therapeutic agent for pancreatic cancer.

Engraftment and morphological development of vascularized human iPS cell-derived 3D-cardiomyocyte tissue after xenotransplantation

One of the major challenges in cell-based cardiac regenerative medicine is the in vitro construction of three-dimensional (3D) tissues consisting of induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and a blood vascular network supplying nutrients and oxygen throughout the tissue after implantation. We have successfully built a vascularized iPSC-CM 3D-tissue using our validated cell manipulation technique. In order to evaluate an availability of the 3D-tissue as a biomaterial, functional morphology of the tissues was examined by light and transmission electron microscopy through their implantation into the rat infarcted heart. Before implantation, the tissues showed distinctive myofibrils within iPSC-CMs and capillary-like endothelial tubes, but their profiles were still like immature. In contrast, engraftment of the tissues to the rat heart led the iPSC-CMs and endothelial tubes into organization of cell organelles and junctional apparatuses and prompt development of capillary network harboring host blood supply, respectively. A number of capillaries in the implanted tissues were derived from host vascular bed, whereas the others were likely to be composed by fusion of host and implanted endothelial cells. Thus, our vascularized iPSC-CM 3D-tissues may be a useful regenerative paradigm which will require additional expanded and long-term studies.

Transplantation of three‐dimensional artificial human vascular tissues fabricated using an extracellular matrix nanofilm‐based cell‐accumulation technique

We have established a novel three‐dimensional (3D) tissue‐constructing technique, referred to as the ‘cell‐accumulation method’, which is based on the self‐assembly of cultured human cells. In this technique, cells are coated with fibronectin and gelatin to construct extracellular matrix (ECM) nanofilms and cultured to form multi‐layers in vitro. By using this method, we have successfully fabricated artificial tissues with vascular networks constructed by co‐cultivation of human umbilical vein‐derived vascular endothelial cells between multi‐layers of normal human dermal fibroblasts. In this study, to assess these engineered vascular tissues as therapeutic implants, we transplanted the 3D human tissues with microvascular networks, fabricated based on the cell‐accumulation method, onto the back skin of nude mice. After the transplantation, we found vascular networks with perfusion of blood in the transplanted graft. At the boundary between host and implanted tissue, connectivity between murine and human vessels was found. Transmission electron microscopy of the implanted artificial vascular tubules demonstrated the ultrastructural features of blood capillaries. Moreover, maturation of the vascular tissues after transplantation was shown by the presence of pericyte‐like cells and abundant collagen fibrils in the ECM surrounding the vasculature. These results demonstrated that artificial human vascular tissues constructed by our method were engrafted and matured in animal skin. In addition, the implanted artificial human vascular networks were connected with the host circulatory system by anastomosis. This method is an attractive technique for engineering prevascularized artificial tissues for transplantation. Copyright

Construction and histological analysis of a 3D human arterial wall model containing vasa vasorum using a layer-by-layer technique

There is considerable global demand for three-dimensional (3D) functional tissues which mimic our native organs and tissues for use as in vitro drug screening systems and in regenerative medicine. In particular, there has been an increasing number of patients who suffer from arterial diseases such as arteriosclerosis. As such, in vitro 3D arterial wall models that can evaluate the effects of novel medicines and a novel artificial graft for the treatment are required. In our previous study, we reported the rapid construction of 3D tissues by employing a layer-by-layer (LbL) technique and revealed their potential applications in the pharmaceutical fields and tissue engineering. In this study, we successfully constructed a 3D arterial wall model containing vasa vasorum by employing a LbL technique for the first time. The cells were coated with extracellular matrix nanofilms and seeded into a culture insert using a cell accumulation method. This model had a three-layered hierarchical structure: a fibroblast layer, a smooth muscle layer, and an endothelial layer, which resembled the native arterial wall. Our method could introduce vasa vasorum into a fibroblast layer in vitro and the 3D arterial wall model showed barrier function which was evaluated by immunostaining and transendothelial electrical resistance measurement. Furthermore, electron microscopy observations revealed that the vasa vasorum was composed of single-layered endothelial cells, and the endothelial tubes were surrounded by the basal lamina, which are known to promote maturation and stabilization in native blood capillaries. These models should be useful for tissue engineering, regenerative medicine, and pharmaceutical applications.

Transplantation of artificial human lymphatic vascular tissues fabricated using a cell‐accumulation technique and their engraftment in mouse tissue with vascular remodeling

Transplantation of engineered tissues with microvascular structure is advancing towards therapeutic application to improve the flow of blood and/or lymphatic fluids. In lymphatic disorders, transplantation of tissue‐engineered lymphatic grafts can be an ideal treatment for draining excessive lymphatic fluid. In this study, we examined the transplantation of 3‐dimensional artificial human lymphatic network tissue (AHLT) fabricated by the cell accumulation technique into the subcutaneous tissue and fascia of mice. At 2 weeks after transplantation, the AHLT showed engraftment of artificial lymphatic vessels immunopositive for human CD31 and human podoplanin. Notably, we also observed the generation of blood vessel‐like structure comprising endothelial cells immunopositive for human CD34 and mural‐like cells immunopositive for human CD90 and αSMA, which were considered as myofibroblasts. In the fabrication of AHLT in vitro, the sporadic emergence of human CD34‐positive/Prox‐1‐negative sites was observed, followed by the formation of blood vessel‐like structure in the graft within 7 days after transplantation. The fine structure of engrafted AHLT observed by transmission electron microscopy showed that the engrafted artificial lymphatic vessels possess the specific structures of native lymphatic capillaries such as loose interendothelial connections and anchoring filaments. In contrast, blood vessel‐like structure showed tight interendothelial connections, thick basement membranes, and layers of mural‐like cells, which resemble small blood vessels. These results suggested the remodelling of artificial lymphatic network to form blood vessel‐like structure associated with mural‐like cells along with AHLT fabrication and engraftment.

4-Methylumbelliferone Decreases the Hyaluronan-rich Extracellular Matrix and Increases the Effectiveness of 5-Fluorouracil

Background/Aim: Pancreatic cancer responds poorly to most chemotherapeutic agents. Several studies have reported that hyaluronan (HA)-rich extracellular matrix (ECM) is a biological barrier against chemotherapeutic agents. 4-methylumbelliforone (MU) led to inhibition of HA synthesis and its preservation in ECM, which may enhance 5-fluorouracil (5-FU) cytotoxicity. Thus, new therapy with MU and 5-FU may be developed for pancreatic cancer. Materials and Methods: A 5-fluorouracil (5-FU) concentration and 4-methylumbelliferone (MU) dosage was analyzed by high-performance liquid chromatography (HPLC). Change in antitumor efficacy of 5-FU in combination with MU was also examined in vivo and in vitro. Results: Combined 5-FU and MU treatment inhibited cell proliferation better than 5-FU alone; 0.01 mM 5-FU alone decreased cell proliferation by 37.7 %, while 0.01 mM 5-FU with 0.5 mM MU decreased cell proliferation by 57.4%. MU enhanced the intracellular concentration of 5-FU by 47.3% compared to control. Mice tumors treated with 5-FU and MU decreased in size and animal survival was prolonged. Moreover, MU decreased cohesiveness of the intercellular space. Conclusion: Combination therapy of 5-FU with MU was effective. A novel therapy can be designed for pancreatic cancer by using ECM modulation.

Microanatomical profiles on the lymphatic system in the human ampulla of Vater (immunohistochemistry and scanning electron microscopy)

Little information is available regarding microanatomy of lymphatic system in the ampulla of Vater, though it is of critical importance for an understanding of tumor progression via the lymphatics and determination of surgical strategy. The present study, therefore, aimed to demonstrate the distribution and microanatomical profiles on the lymphatic system in the ampulla.

Fine structure of human thoracic duct as revealed by light and scanning electron micr oscopy

Little information has been available regarding microanatomy of human thoracic duct in spite of the importance for an understanding of pathophysiology in clinical medicine. The present study demonstrated a fine structure of human thoracic duct system by light and scanning electron microscopy. A number of longitudinal or spiral ridges and grooves were formed on luminal surfaces of the lymphangia and lymph sac, it likely facilitating fluent lymph flow. The endothelial cells displayed various cell shapes in compliance with their distributed regions. The lymph sac joining large vein composed a peculiar multiple valve structure presumably ensuring lymph storage and prevention of lymph backflow. The longitudinal muscle sheet in the tunica intima and circular muscle bundles in the tunica media constructed an integrated power unit probably eliciting spontaneous lymph propulsion. Furthermore, the thoracic duct was richly supplied with blood vessels not only in the tunica externa, but also just beneath the endothelium. The present findings provide a morphological basis for investigation of human thoracic duct in basic and clinical medicine.