Identification of novel yolk ferritins unique to planarians: planarians supply aluminum rather than iron to vitellaria in egg capsules.

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity.

The Cell Line-Dependent Diversity in Initial Morphological Dynamics of Pancreatic Cancer Cell Peritoneal Metastasis Visualized by an Artificial Human Peritoneal Model.

BACKGROUND: Pancreatic ductal adenocarcinoma is considered as one of the most malignant types of cancer with rapid metastasis and invasion of the cancer cells, having peritoneal metastasis (PM) as a dominant factor of poor prognosis. Although the prevention of peritoneal dissemination would result in the inhibition of the initial metastatic process and contribute in improving the poor prognosis of the pancreatic cancer, the initial dynamics of PM are still unclear because of the lack of adequate models in studying the morphological and molecular details of pancreatic cancer cells. MATERIALS AND METHODS: The artificial human peritoneal tissue (AHPT) that can be applied in studying for the spatial dynamics of cancer PM in vitro has been established previously. In this study, the initial dynamics of the three pancreatic cell lines, undifferentiated carcinoma MIA PaCa-2, poorly differentiated adenocarcinoma Panc-1, and moderately differentiated adenocarcinoma BxPC3 on AHPT are examined. RESULTS: In a morphological analysis using light and electron microscopy, MIA PaCa-2 cells spread on the mesothelial layer with disruption of the sheet structure and infiltrated into the stroma-like tissue in AHPT. On the other hand, BxPC3 cells changed shapes from round into flat ones with rapid proliferation and formed sheet structure at the surface of the tissue replacing the mesothelial layer without vertical invasion into the tissue. Panc-1 cells demonstrated the intermediate characteristics of MIA PaCa-2 and BxPC3 on AHPT. These diverse morphological characteristics were verified by the correspondence with the results in a mouse model and were reflected by the profile of secreted oncogenic proteins of the three pancreatic cell lines. CONCLUSIONS: The initial dynamics in the peritoneal dissemination of these pancreatic cancer cell lines were demonstrated by AHPT, showing the morphological and molecular diversity depending on the degree of differentiation or the properties of oncogenic protein secretion.

Inhibitory effect of carbonyl reductase 1 against peritoneal progression of ovarian cancer: evaluation by ex vivo 3D-human peritoneal model.

The current authors previously reported that a carbonyl reductase 1 (CR1) DNA-dendrimer complex could potentially be used in gene therapy for peritoneal metastasis of ovarian cancer. The aims of the current study were to observe the cellular dynamics of peritoneal metastasis of epithelial ovarian cancer cells and to ascertain changes in the dynamics of ovarian cancer cells as a result of transfection of CR1 DNA. (1) Artificial human peritoneal tissue (AHPT) was seeded with serous ovarian cancer cells, and the process leading to development of peritoneal carcinomatosis was observed over time. (2) Peritoneal carcinomatosis was produced in mice and compared to a model using AHPT to determine the appropriateness of AHPT. (3) CR1 DNA was transfected into cancer cells seeded on AHPT, and the dynamics of cancer cells were observed over time. (1) Cancer cells perforated the mesothelium, leaving normal mesothelium intact. However, the cells proliferated between the layers of the mesothelium, forming a mass. After 24 h, cancer cells had invaded the lymphatics, and after 48-72 h cancer cells had invaded deep into the mesothelium, where they formed a mass. (2) Invasion of the peritoneum by cancer cells in a murine model of peritoneal carcinomatosis resembled that in a model using AHPT, and results substantiated the reproducibility of peritoneal carcinomatosis in AHPT. (3) Proliferation of cells transfected with CR1 DNA was significantly inhibited on AHPT, and necrosis was evident. Nevertheless, cancer cell invasion deep into the mesothelium was not inhibited. Use of a new tool, AHPT, in an in vitro model of peritoneal metastasis revealed that CR1 DNA inhibited cancer cell proliferation. CR1 DNA does not play a role in inhibiting invasion of the mesothelium during peritoneal metastasis, but it does affect cancer cell proliferation. Results suggested that CR1 DNA inhibits cancer cell proliferation via necrosis.

Construction of artificial human peritoneal tissue by cell-accumulation technique and its application for visualizing morphological dynamics of cancer peritoneal metastasis.

Human peritoneum is composed of mesothelial monolayer and stromal tissue containing microvasculature. Dissemination and infiltration of cancer cells to the peritoneum result in cancer peritoneal metastasis which is an important prognostic factor of intraperitoneal or intrapelvic carcinoma. To elucidate an initial metastatic mechanism of cancer cells, in vitro human peritoneal models are demanded. In this study, we created a three-dimensional artificial human peritoneal tissue (AHPT) harboring the blood or lymphatic vascular network by cell-accumulation technique. Morphological analysis demonstrated that AHPT had mesothelial monolayer with polygonal flat cells with apical microvilli, and stroma-like structure containing fibroblasts surrounded by extracellular matrix and blood or lymphatic vascular network. To assess AHPT as a tool for cancer peritoneal metastasis model, colon and ovarian cancer cells (HT-29 and SKOV3) were seeded onto AHPT. HT-29 cells showed poor metastatic characteristics forming thick clusters in mesothelial layer without invasion into stroma-like structure. On the other hand, SKOV3 cells rapidly invaded intercellular spaces between mesothelial cells and then spread over the stroma-like structure accompanying lymphatic invasion, showing aggressive metastatic characteristics. These results demonstrated that the metastatic dynamics of cancer cells with different characteristics are able to visualized by AHPT, suggesting that this tissue can be a powerful tool for the basic research of cancer peritoneal dissemination and metastasis.

Promoting biological similarity by collagen microfibers in 3D colorectal cancer-stromal tissue: Replicating mechanical properties and cancer stem cell markers.

The extracellular matrix (ECM) of cancer tissues is rich in dense collagen, contributing to the stiffening of these tissues. Increased stiffness has been reported to promote cancer cell proliferation, invasion, metastasis, and prevent drug delivery. Replicating the structure and mechanical properties of cancer tissue in vitro is essential for developing cancer treatment drugs that target these properties. In this study, we recreated specific characteristics of cancer tissue, such as collagen density and high elastic modulus, using a colorectal cancer cell line as a model. Using our original material, collagen microfibers (CMFs), and a constructed three-dimensional (3D) cancer-stromal tissue model, we successfully reproduced an ECM highly similar to in vivo conditions. Furthermore, our research demonstrated that cancer stem cell markers expressed in the 3D cancer-stromal tissue model more closely mimic in vivo conditions than traditional two-dimensional cell cultures. We also found that CMFs might affect an impact on how cancer cells express these markers. Our 3D CMF-based model holds promise for enhancing our understanding of colorectal cancer and advancing therapeutic approaches. STATEMENT OF SIGNIFICANCE: Reproducing the collagen content and stiffness of cancer tissue is crucial in comprehending the properties of cancer and advancing anticancer drug development. Nonetheless, the use of collagen as a scaffold material has posed challenges due to its poor solubility, hindering the replication of a cancer microenvironment. In this study, we have successfully recreated cancer tissue-specific characteristics such as collagen density, stiffness, and the expression of cancer stem cell markers in three-dimensional (3D) colorectal cancer stromal tissue, utilizing a proprietary material known as collagen microfiber (CMF). CMF proves to be an ideal scaffold material for replicating cancer stromal tissue, and these 3D tissues constructed with CMFs hold promise in contributing to our understanding of cancer and the development of therapeutic drugs.

Interferon-stimulated gene 56 positively regulates Toll-like receptor 3-mediated CXCL10 expression in human renal proximal tubular epithelial cells.

Viral infections in tubular epithelial cells lead to the production of inflammatory cytokines by innate immunity, causing tubulointerstitial nephritis. TLR3 recognizes viral infections and acts via the activation of interferon (IFN)/IFN-stimulated genes (ISGs). This study investigates the role of ISG56, a representative ISG, in TLR3 signaling in cultured human renal proximal tubular epithelial cells (hRPTECs). To this end, hRPTECs were stimulated by a synthetic TLR3 ligand, polyinosinic-polycytidylic acid (poly IC), recombinant human interferon-ß [r(h)IFN-ß] or Japanese encephalitis virus (JEV) infection and assayed for inflammatory cytokine mRNA expression by RT-qPCR, and protein expression via western blotting or ELISA. ISG56 was expressed by poly IC or r(h)IFN-ß and IFN-ß knockdown reduced poly IC-induced expression of ISG56 and CXCL10. Moreover, ISG56 knockdown reduced poly IC- or r(h)IFN-ß-induced expression of CXCL10 at the same time as increasing JEV growth and reducing CXCL10 expression induced by JEV infection. Overall, TLR3 signaling induced IFN-ß-dependent expression of ISG56 and CXCL10. We show that ISG56 possibly plays a critical role in antiviral immunity of hRPTECs by positive regulation of IFN-ß-mediated CXCL10 expression downstream of TLR3.

Autolysin amidase of Listeria monocytogenes promotes efficient colonization of mouse hepatocytes and enhances host immune response.

Listeria monocytogenes is an intracellularly growing pathogen which is able to infect and to spread from cells to cells. It produces several virulence factors required for invasion and intracellular niche colonization. Endogenous peptidoglycan hydrolases which are important for survival of bacteria have been shown to be involved in pathogenesis. An autolysin amidase (Ami)-deficient mutant of L. monocytogenes (Δami) is attenuated in virulence as evidenced by a reduction in mortality of infected mice. We showed that Ami is not essential for bacterial growth and protein secretion. Histopathological analysis suggests that Ami promotes bacterial colonization of hepatocytes. By using cultured eukaryotic cells, we present evidence that a critical function of Ami in pathogenesis is to promote an efficient listerial adherence and internalization into mouse hepatocytes. Simultaneously, the peptidoglycan hydrolase activity of Ami linked to the release of immunologically active cell wall components enhances production of tumor necrosis factor (TNF)-α and interleukin 6. In the early phase of infection, interferon-γ and TNF-α production of Δami-infected mice is significantly less than that of wild-type controls, suggesting a contribution of Ami to enhance the host innate immune response to listerial infection.

Construction of transplantable artificial vascular tissue based on adipose tissue-derived mesenchymal stromal cells by a cell coating and cryopreservation technique.

Prevascularized artificial three-dimensional (3D) tissues are effective biomaterials for regenerative medicine. We have previously established a scaffold-free 3D artificial vascular tissue from normal human dermal fibroblasts (NHDFs) and umbilical vein-derived endothelial cells (HUVECs) by layer-by-layer cell coating technique. In this study, we constructed an artificial vascular tissue constructed by human adipose tissue-derived stromal cells (hASCs) and HUVECs (ASCVT) by a modified technique with cryopreservation. ASCVT showed a higher thickness with more dense vascular networks than the 3D tissue based on NHDFs. Correspondingly, 3D-cultured ASCs showed higher expression of several angiogenesis-related factors, including vascular endothelial growth factor-A and hepatic growth factor, compared to that of NHDFs. Moreover, perivascular cells in ASCVT were detected by pericyte markers, suggesting the differentiation of hASCs into pericyte-like cells. Subcutaneous transplantation of ASCVTs to nude mice resulted in an engraftment with anastomosis of host's vascular structures at 2 weeks after operation. In the engrafted tissue, the vascular network was surrounded by mural-like structure-forming hASCs, in which some parts developed to form vein-like structures at 4 weeks, suggesting the generation of functional vessel networks. These results demonstrated that cryopreserved human cells, including hASCs, could be used directly to construct the artificial transplantable tissue for regenerative medicine.

Mice with disrupted TGFbeta signaling have normal cerebella development, but exhibit facial dysmorphogenesis and strain-dependent deficits in their body wall.

The transforming growth factor betas (TGFbetas) are context-dependent regulators of neurons in vitro, but their physiological functions in the brain are unclear. Haploinsufficiency of either Tgfbeta1 or Tgfbeta2 leads to age-related deterioration of neurons, but the development of the brain is normal in the full absence of either of these genes. However, some individuals with mis-sense mutations of TGFbeta receptors are mentally retarded, suggesting that the TGFbeta isoforms can compensate for each other during brain development. This possibility was tested by generating mice (NSE x PTR) with neuron-specific expression of a dominant-negative inhibitor of TGFbeta signaling. The NSE x PTR mice with a FVBxC57Bl/6 genetic background were viable and developed normally despite strong neuronal expression of the inhibitor of TGFbeta signaling. Their cerebella were of normal size and contained normal numbers of neurons. When the genetic background of the mice was changed to C57BL/6, the phenotype of the mice became neonatal lethal, with the neonates exhibiting various malformations. The malformations correlated with sites of non-neuronal expression of the transgenes and included facial dysmorphogenesis, incomplete closure of the ventral body wall and absence of intestinal motility. The C57BL/6 Tgfbm1-3 alleles, which modulate the phenotype of Tgfbeta1(-/-) mice, were not major determinants of the NSE x PTR phenotype. The data suggest that the development of the cerebellum is insensitive to the level of TGFbeta signaling, although this may be dependent on the genetic background.

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

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.

In vitro 3D blood/lymph-vascularized human stromal tissues for preclinical assays of cancer metastasis.

Tumour models mimicking in vivo three-dimensional (3D) microenvironments are of increasing interest in drug discovery because of the limitations inherent to current models. For example, preclinical assays that rely on monolayer or spheroid cell cultures cannot easily predict 3D cancer behaviours because they have no vasculature. Furthermore, there are major differences in cancer behaviour between human and animal experiments. Here, we show the construction of 3D blood/lymph-vascularized human stromal tissues that can be combined with cancer cells to mimic dynamic metastasis for real-time throughput screening of secreted proteinases. We validated this tool using three human carcinoma cell types that are known to invade blood/lymph vessels and promote angiogenesis. These cell types exhibited characteristic haematogenous/lymphogenous metastasis and tumour angiogenesis properties. Importantly, these carcinoma cells selectively secreted different matrix metalloproteinases depending on their metastasis stage and target vasculature, suggesting the possibility of developing drugs that can target each secreted proteinase. We conclude that the 3D tissue tool will be a powerful throughput system for predicting cancer cell responses and time-dependent secretion of molecules in preclinical assays.

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 © 2015 John Wiley & Sons, Ltd.

Rhodopsin-like proteins in planarian eye and auricle: detection and functional analysis

The presence of rhodopsin-like proteins in the eyes and auricles of the freshwater planarian Dugesia japonica was confirmed using anti-frog-rhodopsin rabbit IgG. The apparent relative molecular masses of these proteins were 65x10(3) and 62x10(3), and positive reactions to IgG were localized to the microvilli of the photoreceptor cells in the eyes and to the sensory cilia, rootlets and microvilli in the auricles. Eye- or head-excised planarians showed no negative phototaxis, whereas intact or auricle-excised planarians did. During regeneration in head-excised planarians, the appearance of rhodopsin-like proteins in the regenerating eyes corresponded to the recovery of negative phototaxis behaviour. Head or auricle excision enhanced asexual fission under continuous illumination. However, eye excision had no such effect. These results suggest that the rhodopsin-like proteins in the eyes work as photoreceptors for negative phototaxis behaviour and that, in the auricles, they are involved in asexual fission originating from the circadian rhythm.

Effects of angiogenic factors and 3D-microenvironments on vascularization within sandwich cultures.

The in vitro fabrication of vascularized tissue is a key challenge in tissue engineering, but little is known about the mechanisms of blood-capillary formation. Here we investigated the mechanisms of in vitro vascularization using precisely-controlled 3D-microenvironments constructed by a sandwich culture using the cell-accumulation technique. 3D-microenvironments controlled at the single layer level showed that sandwich culture between more than 3 fibroblast-layers induced tubule formation. Moreover, the secretion of angiogenic factors increased upon increasing the number of sandwiching layers, which induced highly dense tubular networks. We found that not only angiogenic factors, but also the 3D-microenvironments of the endothelial cells, especially apical side, played crucial roles in tubule formation in vitro. Based on this knowledge, the introduction of blood and lymph capillaries into mesenchymal stem cell (MSC) tissues was accomplished. These findings would be useful for the in vitro vascularization of various types of engineered organs and studies on angiogenesis.

Suppression of starvation-induced autophagy by recombinant toxic shock syndrome toxin-1 in epithelial cells.

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced from Staphylococcus aureus, has been reported to bind directly to unknown receptor(s) and penetrate into non-immune cells but its function is unclear. In this study, we demonstrated that recombinant TSST-1 suppresses autophagosomal accumulation in the autophagic-induced HeLa 229 cells. This suppression is shared by a superantigenic-deficient mutant of TSST-1 but not by staphylococcal enterotoxins, suggesting that autophagic suppression of TSST-1 is superantigenic-independent. Furthermore, we showed that TSST-1-producing S. aureus suppresses autophagy in the response of infected cells. Our data provides a novel function of TSST-1 in autophagic suppression which may contribute in staphylococcal persistence in host cells.

Differentiation of mouse induced pluripotent stem cells into alveolar epithelial cells in vitro for use in vivo.

Alveolar epithelial cells (AECs) differentiated from induced pluripotent stem cells (iPSCs) represent new opportunities in lung tissue engineering and cell therapy. In this study, we modified a two-step protocol for embryonic stem cells that resulted in a yield of ∼9% surfactant protein C (SPC)(+) alveolar epithelial type II (AEC II) cells from mouse iPSCs in a 12-day period. The differentiated iPSCs showed morphological characteristics similar to those of AEC II cells. When differentiated iPSCs were seeded and cultured in a decellularized mouse lung scaffold, the cells reformed an alveolar structure and expressed SPC or T1α protein (markers of AEC II or AEC I cells, respectively). Finally, the differentiated iPSCs were instilled intratracheally into a bleomycin-induced mouse acute lung injury model. The transplanted cells integrated into the lung alveolar structure and expressed SPC and T1α. Significantly reduced lung inflammation and decreased collagen deposition were observed following differentiated iPSC transplantation. In conclusion, we report a simple and rapid protocol for in vitro differentiation of mouse iPSCs into AECs. Differentiated iPSCs show potential for regenerating three-dimensional alveolar lung structure and can be used to abrogate lung injury.

Ultrastructure of blood and lymphatic vascular networks in three-dimensional cultured tissues fabricated by extracellular matrix nanofilm-based cell accumulation technique.

Cell accumulation technique is an extracellular matrix (ECM) nanofilm-based tissue-constructing method that enables formation of multilayered hybrid culture tissues. In this method, ECM-nanofilm is constructed using layer-by-layer assembly of fibronectin and gelatin on culture cells. The ECM-nanofilm promotes cell-to-cell interaction; then the three-dimensional (3D) multilayered tissue can be constructed with morphological change of the cells mimicking living tissues. By using this method, we have successfully produced tubular networks of human umbilical venous endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (HDLECs) in 3D multilayered normal human dermal fibroblasts (NHDFs). This study demonstrated morphological characteristics of the vascular networks in the engineered tissues by using light and electron microscopy. In light microscopy, HUVECs and HDLECs formed luminal structures such as native blood and lymphatic capillaries, respectively. Electron microscopy showed distinct ultrastructural aspects of the vasculature of HUVECs or HDLECs with intermediated NHDFs and abundant ECM. The vasculature constructed by HUVECs exhibited structures similar to native blood capillaries, involving overlapping endothelial connections with adherens junctions, abundant vesicles in the endothelial cells and basement membrane-like structure. The detection of laminin around HUVEC-constructed vessels supported the presence of perivascular basal lamina. The vasculature constructed by HDLECs showed some ultrastructural characteristics similar to those of native lymphatic capillaries such as irregular vascular shape, loose adhesive connection and gap formation between endothelial cells. In conclusion, our novel vascular network models fabricated by the cell accumulation technique provide highly organized blood and lymphatic capillary networks mimicking the vasculatures in vivo.

Three-dimensional multilayers of smooth muscle cells as a new experimental model for vascular elastic fiber formation studies.

OBJECTIVE: Elastic fiber formation is disrupted with age and by health conditions including aneurysms and atherosclerosis. Despite considerable progress in the understanding of elastogenesis using the planar culture system and genetically modified animals, it remains difficult to restore elastic fibers in diseased vessels. To further study the molecular mechanisms, in vitro three-dimensional vascular constructs need to be established. We previously fabricated vascular smooth muscle cells (SMCs) into three-dimensional cellular multilayers (3DCMs) using a hierarchical cell manipulation technique, in which cells were coated with fibronectin-gelatin nanofilms to provide adhesive nano-scaffolds. Since fibronectin is known to assemble and activate elastic fiber-related molecules, we further optimized culture conditions. METHODS AND RESULTS: Elastica stain, immunofluorescence, and electron microscopic analysis demonstrated that 3DCMs, which consisted of seven layers of neonatal rat aortic SMCs cultured in 1% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium, exhibited layered elastic fibers within seven days of being in a static culture condition. In contrast, the application of adult SMCs, 10% FBS, ε-poly(lysine) as an alternative adhesive for fibronectin, or four-layered SMCs, failed to generate layered elastic fiber formation. Radioimmunoassay using [(3)H]valine further confirmed the greater amount of cross-linked elastic fibers in 3DCMs than in monolayered SMCs. Layered elastic fiber formation in 3DCMs was inhibited by the lysyl oxidase inhibitor ß-aminopropionitrile, or prostaglandin E2. Furthermore, infiltration of THP-1-derived macrophages decreased the surrounding elastic fiber formation in 3DCMs. CONCLUSION: 3DCMs may offer a new experimental vascular model to explore pharmacological therapeutic strategies for disordered elastic fiber homeostasis.

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry.

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.

Single axon branching analysis in rat thalamocortical projection from the anteroventral thalamus to the granular retrosplenial cortex.

The granular retrosplenial cortex (GRS) in the rat has a distinct microcolumn-type structure. The apical tufts of dendritic bundles at layer I, which are formed by layer II neurons, co-localize with patches of thalamic terminations from anteroventral (AV) thalamic nucleus. To further understand this microcolumn-type structure in the GRS, one of remaining questions is whether this structure extends into other layers, such as layers III/IV. Other than layer I, previous tracer injection study showed that AV thalamic nucleus also projects to layer III/IV in the GRS. In this study, we examined the morphology of branches in the GRS from the AV thalamus in single axon branch resolution in order to determine whether AV axon branches in layer III/IV are branches of axons with extensive branch in layer I, and, if so, whether the extent of these arborizations in layer III/IV vertically matches with that in layer I. For this purpose, we used a small volume injection of biotinylated dextran-amine into the AV thalamus and reconstructing labeled single axon branches in the GRS. We found that the AV axons consisted of heterogeneous branching types. Type 1 had extensive arborization occurring only in layer Ia. Type 2 had additional branches in III/IV. Types 1 and 2 had extensive ramifications in layer Ia, with lateral extensions within the previously reported extensions of tufts from single dendritic bundles (i.e., 30-200 µm; mean 78 µm). In type 2 branches, axon arborizations in layer III/IV were just below to layer Ia ramifications, but much wider (148-533 µm: mean, 341 µm) than that in layer Ia axon branches and dendritic bundles, suggesting that layer-specific information transmission spacing existed even from the same single axons from the AV to the GRS. Thus, microcolumn-type structure in the upper layer of the GRS was not strictly continuous from layer I to layer IV. How each layer and its components interact each other in different spatial scale should be solved future.