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Introduction: Fibroblasts contribute to the structure and function of tissue and organs; however, their properties differ in each organ given the topographic variation in gene expression among tissues. We previously reported that LYPD1, which is expressed in cardiac fibroblasts, has the capacity to inhibit sprouting of vascular endothelial cells. LYPD1 has been shown to be highly expressed in the human brain and heart, but the regulation of LYPD1 expression in cardiac fibroblasts has not been elucidated in detail. Methods: To identify the LYPD1-modulating transcription factor, motif enrichment analysis and differential expressed gene analysis using microarray data were performed. Quantitative real-time PCR was used to evaluate gene expression. Gene silencing were performed by transfection of siRNA. Western blot analyzed protein expression in NHCF-a. To assess the effect of GATA6 on the regulation of LYPD1 gene expression, dual-luciferase reporter assay was performed. Co-culture and rescue experiments were performed to evaluate endothelial network formation. Results: Motif enrichment analysis and differential expressed gene analysis using microarray data and quantitative real-time PCR revealed that CUX1, GATA6, and MAFK were candidate transcription factors. Of these, the inhibition of GATA6 expression using siRNA decreased LYPD1 gene expression and co-expression of GATA6 with a reporter vector containing the upstream sequence of the LYPD1 gene resulted in increased reporter activity. Endothelial cell network formation was attenuated when co-cultured with cardiac fibroblasts, but it was significantly restored when co-cultured with cardiac fibroblasts wherein the expression of GATA6 was knocked down with siRNA. Conclusion: GATA6 regulate the anti-angiogenic properties of cardiac fibroblasts by modulating LYPD1 expression.

Angiogenesis is regulated by a balance between promoting and inhibitory mechanisms. Although angiogenesis-promoting mechanisms have been well studied in ischemic heart diseases, angiogenesis-inhibitory mechanisms have not. Recently, we identified LYPD-1 as a novel anti-angiogenic factor derived from human heart-derived fibroblasts, which suppresses endothelial cell network formation in co-culture. However, it remains unclear whether the low angiogenicity of heart-derived fibroblasts with high expression of LYPD-1 is also observed in other mammalian species, and the properties of LYPD-1 under normal and pathological conditions remain elusive. Fibroblasts isolated from neonatal and adult rat heart also express LYPD-1 and inhibit endothelial network formation in co-culture. Moreover, immunohistochemical analysis revealed that LYPD-1 was predominantly observed in the interstitial tissues of rat heart and LYPD1 expression levels were identical from late developmental period to adult. Conversely, LYPD-1 mRNA expression was significantly downregulated temporally in myocardial infarction model rats, suggesting that angiogenesis-inhibitory mechanisms might not be sufficiently suppressed to promote angiogenesis in ischemic heart diseases. These findings suggest that heart has relatively low angiogenicity compared with other organs via the high expression of LYPD-1 by fibroblasts. Moreover, understanding the regulatory mechanisms of LYPD-1-mediated inhibition of angiogenesis might lead a novel angiogenic therapy for ischemic heart diseases and contribute to development of bioengineered cardiac tissue.

A well-organised vascular network is essential for metabolic exchange to maintain homoeostasis in the body. Therefore, for progress in regenerative medicine, it is particularly important to establish methods of vascularization in bioengineered three-dimensional (3D) functional tissues. In addition, it is necessary to develop methods to supply a large number of iPS cell-derived endothelial cells for fabricating the vascular network structure. There are already many reports on the method of inducing the differentiation of endothelial cells from iPS cells using 2D culture. However, there are few reports on methods for preparing a large number of iPS cell-derived endothelial cells. Therefore, we developed methods for inducing vascular endothelial cells from human inducible pluripotent stem (hiPS) cells using 3D suspension culture. hiPS cell-derived CD31+ cells expressed several endothelial marker genes and formed endothelial cell network structures, similar to human umbilical vein endothelial cells. These results indicate that hiPS cell-derived CD31+ cells may be a useful cell source for pre-vascularised network structures in 3D functional tissues, and it is important to develop 3D mass culture system for preparing a large number of cells to fabricate bioengineered tissues. Keywords: Human inducible pluripotent stem cells, Three-dimensional suspension culture, Endothelial cell differentiation, Microvascular network formation

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

A 26-year-old man highly suspected of having antiglomerular basement membrane (GBM) disease was treated with corticosteroid pulse therapy 9 days after initial infection-like symptoms with high procalcitonin value. The patient required hemodialysis the next day of the treatment due to oliguria. In addition to corticosteroid therapy, plasmapheresis was introduced and the patient could discontinue hemodialysis 43 days after the treatment. Kidney biopsy after initiation of hemodialysis confirmed anti-GBM disease with 86.3% crescent formation. Physician should keep in mind that active anti-GBM disease shows even high procalcitonin value in the absence of infection. To pursue recovery of renal function, the challenge of the immediate and persistent treatment with high-dose corticosteroids plus plasmapheresis for highly suspected anti-GBM disease is vitally important despite the presence of reported predictors for dialysis-dependence including oliguria and requiring hemodialysis at presentation.

Protein-protein interaction and gene regulatory networks are likely to be locked in a state corresponding to a disease by the behavior of one or more bistable circuits exhibiting switch-like behavior. Sets of genes could be over-expressed or repressed when anomalies due to disease appear, and the circuits responsible for this over- or under-expression might persist for as long as the disease state continues. This paper shows how a large-scale analysis of network bistability for various human cancers can identify genes that can potentially serve as drug targets or diagnosis biomarkers.