GATA6 regulates anti-angiogenic properties in human cardiac fibroblasts via modulating LYPD1 expression.

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.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes.

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.

Heart-derived fibroblasts express LYPD-1 and negatively regulate angiogenesis in rat.

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.

Cell sheet engineering for heart tissue repair.

Recently, myocardial tissue engineering has emerged as one of the most promising therapies for patients suffering from severe heart failure. Nevertheless, conventional methods in tissue engineering involving the seeding of cells into biodegradable scaffolds have intrinsic shortcomings, such as inflammatory reactions and fibrous tissue formation caused by scaffold degradation. On the other hand, we have developed cell sheet engineering as scaffoldless tissue engineering, and applied it for myocardial tissue engineering. Using temperature-responsive culture surfaces, cells can be harvested as intact sheets and cell-dense thick tissues are constructed by layering these cell sheets. Myocardial cell sheets non-invasively harvested from temperature-responsive culture surfaces are successfully layered, resulting in electrically communicative 3-dimensional (3-D) cardiac constructs. Transplantation of cell sheets onto damaged hearts improved heart function in several animal models. In this review, we summarize the development of myocardial tissue engineering using cell sheets harvested from temperature-responsive culture surfaces and discuss about future views.

Preparation of iPS cell-derived CD31+ endothelial cells using three-dimensional suspension culture.

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.

Inhibition of LYPD1 is critical for endothelial network formation in bioengineered tissue with human cardiac fibroblasts.

Fibroblasts not only play key roles under physiological and pathological conditions in various tissues and organs including the heart but also are indispensable for fabricating bioengineered cardiac tissues and their functions through cell-cell interactions. Because tissue functions and cells surrounding fibroblasts in vivo are different among tissues, the properties of fibroblasts might be different according to their tissue origin. Understanding the molecular mechanisms of fibroblasts may lead to fabrication of bioengineered tissues close to biological tissues. In this study, we found a unique less angiogenic property of human cardiac fibroblasts in vitro compared with human dermal fibroblasts and identified the responsible gene. Cardiac fibroblasts inhibited vascular network formation in co-cultures with various types of vascular endothelial cells. Using microarray analysis and short interfering RNA (siRNA) screening experiments, we identified Ly6/Plaur domain-containing 1 (LYPD1) as responsible for the lack of endothelial cell network formation mediated by cardiac fibroblasts. Inhibition of the LYPD1 gene by siRNA attenuated the anti-angiogenic properties of cardiac fibroblasts, whereas the functional defect was rescued by addition of recombinant LYPD1. These findings suggest that cardiac fibroblasts possess anti-angiogenic properties mediated by LYPD1 and that inhibition of LYPD1 might contribute to the fabrication of vascularized functional bioengineered tissues.

cDNA cloning and some additional peptide characterization of a single-chain vascular apoptosis-inducing protein, VAP2.

Vascular apoptosis-inducing proteins (VAPs) from hemorrhagic snake venom are apoptosis-inducing toxins targeting vascular endothelial cells. Well-characterized VAPs consist of disulfide-bridged double chains (ddVAPs). The authors previously described a single-chain VAP (scVAP), VAP2 from Crotalus atrox, which also induces apoptosis in endothelial cells (Masuda et al., 1998, European Journal of Biochemistry, 253, 36-41). The authors report here the whole cDNA sequences and some additional peptide characteristics of VAP2. In addition to the apoptosis-inducing activity of VAP2, the toxin displays a cell-detaching activity after incubation in high-salt conditions. These observations indicate that the apoptosis and cell-detaching functions can be discriminated. Analysis of the cell-detaching activity also revealed that VAP2 consists of two similar peptides, VAP2A and VAP2B, which are members of the PIII-type snake venom metalloproteases (SVMPs). The VAP2A cDNA encodes a 609-amino acid protein. In contrast, the peptide sequences of VAP2B were identical to that of catrocollastatin, an inhibitor of platelet aggregation. VAP2A and VAP2B interact with each other to form a noncovalent dimer similar to the ddVAPs, which was detected by native polyacrylamide gel electrophoresis. These data show some new characteristics of VAPs, which are important to clarify the apoptotic pathways in vascular endothelial cells.

Three-dimensional cardiac tissue fabrication based on cell sheet technology.

Cardiac tissue engineering is a promising therapeutic strategy for severe heart failure. However, conventional tissue engineering methods by seeding cells into biodegradable scaffolds have intrinsic limitations such as inflammatory responses and fibrosis arising from the degradation of scaffolds. On the other hand, we have developed cell sheet engineering as a scaffold-free approach for cardiac tissue engineering. Confluent cultured cells are harvested as an intact cell sheet using a temperature-responsive culture surface. By layering cardiac cell sheets, it is possible to form electrically communicative three-dimensional cardiac constructs. Cell sheet transplantation onto damaged hearts in several animal models has revealed improvements in heart functions. Because of the lack of vasculature, the thickness of viable cardiac cell sheet-layered tissues is limited to three layers. Pre-vascularized structure formation within cardiac tissue and multi-step transplantation methods has enabled the formation of thick vascularized tissues in vivo. Furthermore, development of original bioreactor systems with vascular beds has allowed reconstruction of three-dimensional cardiac tissues with a functional vascular structure in vitro. Large-scale culture systems to generate pluripotent stem cell-derived cardiac cells can create large numbers of cardiac cell sheets. Three-dimensional cardiac tissues fabricated by cell sheet engineering may be applied to treat heart disease and tissue model construction.

Cardiac fibroblast-derived VCAM-1 enhances cardiomyocyte proliferation for fabrication of bioengineered cardiac tissue.

BACKGROUND: Fibroblasts are indispensable for the fabrication of cell-sheet-based bioengineered cardiac tissues; however, whether cardiac fibroblasts can improve tissue properties for transplantation or in vitro models compared with other fibroblast types remains unclear. We compared the cell organization and functional properties of cardiomyocyte sheets derived from co-culture with different fibroblast types and investigated the molecular mechanisms for the observed differences. METHODS AND RESULTS: Cardiac cell sheets were fabricated by co-culturing mouse embryonic stem cell (ESC)-derived cardiomyocytes with mouse neonatal cardiac fibroblasts (NCFs), mouse adult cardiac fibroblasts (ACFs), and mouse adult dermal fibroblasts (ADFs). Cardiac cell sheets obtained from NCF or ACF co-culture showed numerous uniformly distributed and functional (beating) cardiomyocytes, while cell sheets obtained by co-culture with ADFs showed fewer and aggregated cardiomyocytes. The greater number of cardiomyocytes in the presence of NCFs was because of enhanced cardiomyocyte proliferation, as revealed by protein markers of mitosis and BrdU incorporation. Microarray analysis revealed that NCFs expressed substantially higher levels of vascular cell adhesion molecule-1 (VCAM-1) than ADFs. Treatment of ESC-derived cardiomyocytes in monoculture with soluble VCAM-1 significantly increased the number of functional cardiomyocytes, while the enhancement of cardiomyocyte number by co-culture with NCFs was abolished by anti-VCAM-1 antibodies. CONCLUSIONS: Cardiac fibroblasts enhance the proliferation of ESC-derived cardiomyocytes through VCAM-1 signaling, leading to an increase in functional myocardial cells in bioengineered tissue sheets. These sheets may be advantageous for cell-based therapy and in vitro heart research.

Formation of vascular network structures within cardiac cell sheets from mouse embryonic stem cells.

Bioengineered cardiac tissues represent a promising strategy for regenerative medicine. However, methods of vascularization and suitable cell sources for tissue engineering and regenerative medicine have not yet been established. In this study, we developed methods for the induction of vascular endothelial cells from mouse embryonic stem (ES) cells using three-dimensional (3D) suspension culture, and fabricated cardiac cell sheets with a pre-vascularized structure by co-culture of mouse ES cell-derived endothelial cells. After induction, isolated CD31+ cells expressed several endothelial cell marker genes and exhibited the ability to form vascular network structures similar to CD31+ cells from neonatal mouse heart. Co-culture of ES cell-derived CD31+ cells with ES cell-derived cardiomyocytes and dermal fibroblasts resulted in the formation of cardiac cell sheets with microvascular network formation. In contrast, microvascular network formation was reduced in co-cultures without cardiomyocytes, suggesting that cardiomyocytes within the cell sheet might enhance vascular endothelial cell sprouting. Polymerase chain reaction array analysis revealed that the expression levels of several angiogenesis-related genes, including fibroblast growth factor 1 (FGF1), were up-regulated in co-culture with cardiomyocytes compared with cultures without cardiomyocytes. The microvascular network in the cardiac sheets was attenuated by treatment with anti-FGF1 antibody. These results indicate that 3D suspension culture methods may be used to prepare functional vascular endothelial cells from mouse ES cells, and that cardiomyocyte-mediated paracrine effects might be important for fabricating pre-vascularized cardiac cell sheets.

Involvement of specific integrins in apoptosis induced by vascular apoptosis-inducing protein 1.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of vascular apoptosis-inducing protein 1 (VAP1) that specifically induces apoptosis in VEC. VAP1 belongs to the metalloprotease/disintegrin family. Yet the mechanism of inducing apoptosis by VAP1 is still not known. Since other various metalloproteases and disintegrins in snake venoms are known to influence extracellular matrix and cell adhesion, we investigated here the involvement of these adhesion molecules in VAP1-induced apoptosis. Consequently, VAP1 induced apoptosis without degrading extracellular matrix or inhibiting adhesion of VEC. However, VAP1-induced apoptosis was inhibited by antibodies for integrin alpha3, alpha6, beta1. Additionally, apoptosis was inhibited by antibody for CD9, an integrin associated protein. These results suggest that integrins are involved in VAP1-induced apoptosis by some specific role rather than that of adhesion to extracellular matrix.

Cell sheet-based cardiac tissue engineering.

Tissue engineering is indispensable for the advancement of regenerative medicine and the development of tissue models. Cell sheet-based method is one the promising strategies for cardiac tissue engineering. To date, cell sheet transplantation using wide variety of cells has been performed for the treatment of various heart diseases. These cell sheet transplantations have shown to ameliorate cardiac dysfunction and improve symptoms of heart failure. Recent progress of the technologies on the layering of cardiac cell sheets accompanied with vascularization and the large scale cultivation system of embryonic stem cell and induced pluripotent stem cell is about to turn the fabrication of thickened human cardiac tissue for transplant and tissue models into reality.

Creation of mouse embryonic stem cell-derived cardiac cell sheets.

Research on heart tissue engineering is an exciting and promising area. Although we previously developed bioengineered myocardium using cell sheet-based tissue engineering technologies, the issue of appropriate cell sources remained unresolved. In the present study, we created cell sheets of mouse embryonic stem (ES) cell-derived cardiomyocytes after expansion in three-dimensional stirred suspension cultures. Serial treatment of the suspension cultures with noggin and granulocyte colony-stimulating factor significantly increased the number of cardiomyocytes by more than fourfold compared with untreated cultures. After drug selection for ES cells expressing the neomycin-resistance gene under the control of the α-myosin heavy chain promoter, almost all of the cells showed spontaneous beating and expressed several cardiac contractive proteins in a fine striated pattern. When ES-derived cardiomyocytes alone were seeded onto temperature-responsive culture dishes, cell sheets were not created, whereas cocultures with cardiac fibroblasts promoted cell sheet formation. The cardiomyocytes in the cell sheets beat spontaneously and synchronously, and expressed connexin 43 at the edge of adjacent cardiomyocytes. Furthermore, when the extracellular action potential was recorded, unidirectional action potential propagation was observed. The present findings suggest that stirred suspension cultures with appropriate growth factors are capable of producing cardiomyocytes effectively and easily, and that ES-derived cardiac cell sheets may be a promising tool for the development of bioengineered myocardium.

Mass preparation of size-controlled mouse embryonic stem cell aggregates and induction of cardiac differentiation by cell patterning method.

Embryonic stem cells (ESCs) are promising cell sources for cell-based therapy. It has been established that the formation of ESC aggregates promotes their differentiation into the derivatives of all three germ layers. ESC aggregates are generally prepared via the formation of suspended spherical aggregates called embryoid bodies (EBs). Because the differentiation efficiency depends on the size of EBs, it becomes one of the research topics how to prepare size-controlled EBs in a scalable manner for reproducible and high-throughput experiments. Here, we have developed a novel culture method that enables simple mass preparation of size-controlled ESC aggregates on a culture surface instead of floating EBs. We developed a maskless photolithography device that enabled rapid fabrication of micropatterned surfaces. Utilizing this device, we fabricated the culture substrates the surfaces of which comprised arrays of cell-adhesive circular micro-domains (100-400 microm in diameter) and the rest of non-cell-adhesive domains. We seeded mouse ESCs on this substrate and prepared size-controlled ESC aggregates on the micro-domains. We analyzed cardiac differentiation in the ESC aggregates and found that the optimal diameter of micro-domains was 200 microm. The present method is useful for the simple and reproducible mass preparation of ESC-derived differentiated cells and high-throughput assays.

Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases.

In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.

Involvement of the V2 receptor in vasopressin-stimulated translocation of placental leucine aminopeptidase/oxytocinase in renal cells.

The placental leucine aminopeptidase (P-LAP)/oxytocinase is a membrane-bound enzyme thought to play an important role during pregnancy. In this study, we identified the presence of P-LAP protein in the renal distal tubules and collecting ducts. In rat NRK52E cells derived from renal tubules, P-LAP was localized mainly in the intracellular compartment. Upon the treatment of cells with 8-arginine-vasopressin (AVP), a significant increase in the surface level of P-LAP was observed. [deamino-Cys1, d-Arg8]-vasopressin (DDAVP), a specific V2 receptor agonist, increased the surface level of P-LAP, while [adamantaneacetyl1, O-Et-d-Tyr2, Val4, aminobutyryl6, Arg8,9]-vasopressin (AEAVP), a potent V2 receptor antagonist, blocked the AVP-stimulated enhancement. Moreover, reagents known to enhance the intracellular level of cAMP have also been shown to increase the surface level of P-LAP. When we examined the colocalization of P-LAP with the cell surface water channel aquaporin-2 (AQP-2) that is regulated by AVP, the P-LAP-containing vesicles had a relatively higher density than the AQP-2-containing vesicles, suggesting that P-LAP and AQP-2 are differently distributed in NRK52E cells. These results suggest that AVP induces the translocation of P-LAP via V2 receptor and P-LAP plays a role in the regulation of excessive AVP level in the renal collecting duct, acting as a negative feedback mechanism for the AVP action of regulating water reabsorption.