Measurement of solid size in early-stage lung adenocarcinoma by virtual 3D thin-section CT applied artificial intelligence.

An artificial intelligence (AI) system that reconstructs virtual 3D thin-section CT (TSCT) images from conventional CT images by applying deep learning was developed. The aim of this study was to investigate whether virtual and real TSCT could measure the solid size of early-stage lung adenocarcinoma. The pair of original thin-CT and simulated thick-CT from the training data with TSCT images (thickness, 0.5-1.0 mm) of 2700 pulmonary nodules were used to train the thin-CT generator in the generative adversarial network (GAN) framework and develop a virtual TSCT AI system. For validation, CT images of 93 stage 0-I lung adenocarcinomas were collected, and virtual TSCTs were reconstructed from conventional 5-mm thick-CT images using the AI system. Two radiologists measured and compared the solid size of tumors on conventional CT and virtual and real TSCT. The agreement between the two observers showed an almost perfect agreement on the virtual TSCT for solid size measurements (intraclass correlation coefficient = 0.967, P < 0.001, respectively). The virtual TSCT had a significantly stronger correlation than that of conventional CT (P = 0.003 and P = 0.001, respectively). The degree of agreement between the clinical T stage determined by virtual TSCT and the clinical T stage determined by real TSCT was excellent in both observers (k = 0.882 and k = 0.881, respectively). The AI system developed in this study was able to measure the solid size of early-stage lung adenocarcinoma on virtual TSCT as well as on real TSCT.

Microsurgery "Under the Eaves" Using ORBEYE: A Case of Dural Arteriovenous Fistula of the Anterior Cranial Fossa.

BACKGROUND: One of the merits of exoscopes, including ORBEYE, is that they are superior to a microscope in terms of ergonomic features. We report a case of dural arteriovenous fistula (dAVF) that was cured by direct surgery using the ergonomic advantages of ORBEYE. CASE DESCRIPTION: A 78-year-old man was found to have dAVF of the anterior cranial fossa incidentally. We performed direct surgery via bifrontal craniotomy. Because the frontal sinus was large, we reserved the frontal bone-like eaves in order not to open the frontal sinus. The vertex of his head was sufficiently down to shift the frontal lobe downward with gravity. During surgery, we set the angle of the operative visual axis of ORBEYE approximately horizontal to avoid the reserved frontal bone. We performed a stable operation using ORBEYE in a comfortable posture. CONCLUSIONS: ORBEYE facilitates ergonomic microsurgery, even under the eaves, with the angle of the operative visual axis approximately horizontal using gravity.

Liver, Stroke, Rhinosinusitis.

In addition to main types of fibrosis, other types of fibrosis in incredible diseases have been reported. Among them, liver fibrosis, stroke, and rhinosinusitis are described as new cases of periostin action. To understand periostin function in a new area of fibrosis linked with inflammation, periostin can be used as a new tool to elucidate the mechanisms of unknown diseases.

Periostin is a negative prognostic factor and promotes cancer cell proliferation in non-small cell lung cancer.

Periostin is a matricellular protein that is secreted by fibroblasts and interacts with various cell-surface integrin molecules. Although periostin is known to support tumor development in human malignancies, little is known about its effect on lung-cancer progression. We here demonstrate that periostin is a negative prognostic factor that increases tumor proliferation through ERK signaling in non-small cell lung carcinoma. We classified 189 clinical specimens from patients with non-small cell lung-cancer according to high or low periostin expression, and found a better prognosis for patients with low rather than high periostin, even in cases of advanced-stage cancer. In a syngenic implantation model, murine Ex3LL lung-cancer cells formed smaller tumor nodules in periostin-/- mice than in periostin+/+ mice, both at the primary site and at metastatic lung sites. An in vitro proliferation assay showed that stimulation with recombinant periostin increased Ex3LL-cell proliferation. We also found that periostin promotes ERK phosphorylation, but not Akt or FAK activation. These findings suggest that periostin represents a potential target in lung-cancer tumor progression.

Periostin attenuates tumor growth by inducing apoptosis in colitis-related colorectal cancer.

Inflammatory bowel diseases, which are multifactorial autoimmune colitis diseases, are occurring with increasing prevalence. One of the most serious complications of these diseases is colorectal cancer. Here we investigated the role of periostin (Postn), a matricellular protein that interacts with various integrin molecules on the cell surface, in colitis-induced colorectal cancer. Immunohistochemistry of mouse and human colorectal cancer samples revealed that Postn was expressed in the stroma and was upregulated in close proximity to the cancer cells. The colonic tumorigenesis in an inflammation-related colon carcinogenesis mouse model was increased in Postn knock-out (Postn-/-) mice compared to Postn+/+ mice. Although no difference was found in the degree of colitis between Postn+/+ and Postn-/- mice, Postn inhibited tumor growth and induced the apoptosis of mouse rectal cancer cells in vitro. Furthermore, fewer apoptotic colorectal cancer cells were observed in Postn-/- than in Postn+/+ mice. These data suggested that Postn has an anti-tumor effect on colitis-induced colorectal cancer.

Periostin function in communication with extracellular matrices.

Periostin is a secretory protein with a multi-domain structure, comprising an amino-terminal cysteine-rich EMI domain, four internal FAS 1 domains, and a carboxyl-terminal hydrophilic domain. These adjacent domains bind to extracellular matrix proteins (type I collagen, fibronectin, tenascin-C, and laminin γ2), and BMP-1 that catalyzes crosslinking of type I collagen, and proteoglycans, which play a role in cell adhesion. The binding sites on periostin have been demonstrated to contribute to the mechanical strength of connective tissues, enhancing intermolecular interactions in close proximity and their assembly into extracellular matrix architectures, where periostin plays further essential roles in physiological maintenance and pathological progression. Furthermore, periostin also binds to Notch 1 and CCN3, which have functions in maintenance of stemness, thus opening up a new field of periostin action.

The sp7 gene is required for maturation of osteoblast-lineage cells in medaka (Oryzias latipes) vertebral column development.

Sp7 is a zinc finger transcription factor that is essential for osteoblast differentiation in mammals. To verify the characteristic features of osteoblast-lineage cells in teleosts, we established medaka sp7 mutants using a transcription activator-like effector nuclease (TALEN) genome editing system. These mutants showed severe defects in the formation of skeletal structures. In particular, the neural and the hemal arches were not formed, although the chordal centra were formed. Analysis of the transgenic medaka revealed that sp7 mutant had normal distribution of type X collagen a1 a (col10a1a)-positive osteoblast-like cells around the centrum and at the proximal region of the vertebral arch. The sp7 mutant phenotype could be rescued by exogenous sp7 expression in col10a1a-positive cells, as well as in sp7-positive osteoblast cells. Furthermore, runx2-positive osteoblast progenitors were observed on the vertebral arches, but not on the centrum, during vertebral column development. In addition, these osteoblast progenitors differentiated into the col10a1a-positive cells. In sp7 mutant, the runx2-positive cells were normally distributed at the region of unformed vertebral arch but failed to differentiate into col10a1a-positive cells. These results indicate that osteoblast-lineage cells undergo two distinct differentiation processes during development of the vertebral arch and the centrum. Nevertheless, our results verified that sp7 gene expression in osteoblast-lineage cells is required for differentiation into mature osteoblasts to form the vertebral column and other skeletal structures.

Introductory review: periostin-gene and protein structure.

Although many studies have described the role of periostin in various diseases, the function of the periostin protein structures derived from alternative splicing and proteinase cleavage at the C-terminal remain unknown. Further experiments revealing the protein structures that are highly related to diseases are essential to understand the function of periostin in depth, which would accelerate its clinical application by establishing new approaches for curing intractable diseases. Furthermore, this understanding would enhance our knowledge of novel functions of periostin related to stemness and response to mechanical stress.

Periostin Promotes Scar Formation through the Interaction between Pericytes and Infiltrating Monocytes/Macrophages after Spinal Cord Injury.

Scar formation is a prominent pathological feature of traumatic central nervous system (CNS) injury, which has long been implicated as a major impediment to the CNS regeneration. However, the factors affecting such scar formation remain to be elucidated. We herein demonstrate that the extracellular matrix protein periostin (POSTN) is a key player in scar formation after traumatic spinal cord injury (SCI). Using high-throughput RNA sequencing data sets, we found that the genes involved in the extracellular region, such as POSTN, were significantly expressed in the injured spinal cord. The expression of POSTN peaked at 7 days after SCI, predominantly in the scar-forming pericytes. Notably, we found that genetic deletion of POSTN in mice reduced scar formation at the lesion site by suppressing the proliferation of the pericytes. Conversely, we found that recombinant POSTN promoted the migration capacity of the monocytes/macrophages and increased the expression of tumor necrosis factor-α from the monocytes/macrophages in vitro, which facilitated the proliferation of pericytes. Furthermore, we revealed that the pharmacological blockade of POSTN suppressed scar formation and improved the long-term functional outcome after SCI. Our findings suggest a potential mechanism whereby POSTN regulates the scar formation after SCI and provide significant evidence that POSTN is a promising therapeutic target for CNS injury.

Periostin is required for matricellular localization of CCN3 in periodontal ligament of mice.

CCN3 is a matricellular protein that belongs to the CCN family. CCN3 consists of 4 domains: insulin-like growth factor-binding protein-like domain (IGFBP), von Willebrand type C-like domain (VWC), thrombospondin type 1-like domain (TSP1), and the C-terminal domain (CT) having a cysteine knot motif. Periostin is a secretory protein that binds to extracellular matrix proteins such as fibronectin and collagen. In this study, we found that CCN3 interacted with periostin. Immunoprecipitation analysis revealed that the TSP1-CT interacted with the 4 repeats of the Fas 1 domain of periostin. Immunofluorescence analysis showed co-localization of CCN3 and periostin in the periodontal ligament of mice. In addition, targeted disruption of the periostin gene in mice decreased the matricellular localization of CCN3 in the periodontal ligament. Thus, these results indicate that periostin was required for the matricellular localization of CCN3 in the periodontal ligament, suggesting that periostin mediated an interaction between CCN3 and the extracellular matrix.

TGFß-2 signaling is essential for osteoblast migration and differentiation during fracture healing in medaka fish.

TGFß is known as a canonical coupling factor based on its effects on bone formation and bone resorption. There are 3 different isoforms of it related to bone metabolism in mammals. TGFß function in vivo is complicated, and each isoform shows a different function. Since TGFßs are secreted during inflammation accompanied by the release of latent TGFß from inside of the bones where they are stored in the extracellular matrix, TGFß function is potentially related to fracture healing. Although a few reports examined the TGFß expression during fracture healing, the function of TGFß in this process is poorly understood. To investigate TGFß function during fracture healing in vivo, we used the fracture healing model of the medaka fish, which enabled us to observe the behavior and function of living cells in response to a bone-specific injury. RNA in-situ hybridization analysis showed that only tgfß-2 of the 4 TGFß isoforms in medaka was expressed in the bone-forming region. To examine the TGFß-2 function for bone formation by osteoblasts, we used a medaka transgenic line, Tg (type X collagen: GFP); and the results revealed that type X collagen-positive immature osteoblasts migrated to the fracture site and differentiated to osterix-positive osteoblasts. However, only a few type X collagen-positive osteoblasts exhibited BrdU incorporation after the fracture. Then we inhibited TGFß signaling by using a chemical TGFß receptor kinase inhibitor (SB431542), and demonstrated that inhibition of TGFß strongly impaired osteoblast migration and differentiation. In addition, this TGFß inhibitor reduced the RANKL expression and caused a delay of osteoclast differentiation. Our findings thus demonstrated that TGFß-2 functioned specifically during fracture healing to stimulate the migration of osteoblasts as well as the differentiation of osteoblasts and osteoclasts.

Osteoblast and osteoclast behaviors in the turnover of attachment bones during medaka tooth replacement.

Tooth replacement in polyphyodont is a well-organized system for maintenance of homeostasis of teeth, containing the dynamic structural change in skeletal tissues such as the attachment bone, which is the supporting element of teeth. Histological analyses have revealed the character of tooth replacement, however, the cellular mechanism of how skeletal tissues are modified during tooth replacement is largely unknown. Here, we showed the important role of osteoblasts for controlling osteoclasts to modify the attachment bone during tooth replacement in medaka pharyngeal teeth, coupled with an osterix-DsRed/TRAP-GFP transgenic line to visualize osteoblasts and osteoclasts. In the turnover of the row of attachment bones, these bones were resorbed at the posterior side where most developed functional teeth were located, and generated at the anterior side where teeth were newly erupted, which caused continuous tooth replacement. In the cellular analysis, osteoclasts and osteoblasts were located at attachment bones separately, since mature osteoclasts were localized at the resorbing side and osteoblasts gathered at the generating side. To demonstrate the role of osteoclasts in tooth replacement, we established medaka made deficient in c-fms-a by TALEN. c-fms-a deficient medaka showed hyperplasia of attachment bones along with reduced bone resorption accompanied by a low number of TRAP-positive osteoclasts, indicating an important role of osteoclasts in the turnover of attachment bones. Furthermore, nitroreductase-mediated osteoblast-specific ablation induced disappearance of osteoclasts, indicating that osteoblasts were essential for maintenance of osteoclasts for the proper turnover. Taken together, our results suggested that the medaka attachment bone provides the model to understand the cellular mechanism for tooth replacement, and that osteoblasts act in the coordination of bone morphology by supporting osteoclasts.

Proliferation following tetraploidization regulates the size and number of erythrocytes in the blood flow during medaka development, as revealed by the abnormal karyotype of erythrocytes in the medaka TFDP1 mutant.

BACKGROUND: For the delivery of oxygen, the correct size/number of erythrocytes is required for proper blood flow. RESULTS: By combined analyses of wild-type (WT) medaka and the kyoho (kyo) mutant, we found proliferation-mediated adaptation for size/number of erythrocytes in the blood flow during medaka development. Before the start of heart beating in the WT medaka, the karyotype of erythrocytes was 2N-4N. After the start of blood flow, the karyotype changed to 4N-8N with tetraploidization, and the cell size became larger. After the start of intersegmental and pharyngeal blood flow, the erythrocytes became smaller. The medaka mutant kyo showed erythrocytes of large size, and positional cloning of kyo demonstrated the candidate gene TFDP1, indicating higher polyploidization due to arrest in S-phase in erythrocytes of the kyo mutant. CONCLUSIONS: From our findings, we uncovered a previously unrecognized system for the regulation of the size/number in the blood flow:proliferation of erythrocytes following tetraploidization during embryonic development.

A diffusible signal derived from hematopoietic cells supports the survival and proliferation of regenerative cells during zebrafish fin fold regeneration.

Multicellular organisms maintain body integrity by constantly regenerating tissues throughout their lives; however, the overall mechanism for regulating regeneration remains an open question. Studies of limb and fin regeneration in teleost fish and urodeles have shown the involvement of a number of locally activated signals at the wounded site during regeneration. Here, we demonstrate that a diffusible signal from a distance also play an essential role for regeneration. Among a number of zebrafish mutants, we found that the zebrafish cloche (clo) and tal1 mutants, which lack most hematopoietic tissues, displayed a unique regeneration defect accompanying apoptosis in primed regenerative tissue. Our analyses of the mutants showed that the cells in the primed regenerative tissue are susceptible to apoptosis, but their survival is normally supported by the presence of hematopoietic tissues, mainly the myeloid cells. We further showed that a diffusible factor in the wild-type body fluid mediates this signal. Thus, our study revealed a novel mechanism that the hematopoietic tissues regulate tissue regeneration through a diffusible signal.

Eda/Edar signaling guides fin ray formation with preceding osteoblast differentiation, as revealed by analyses of the medaka all-fin less mutant afl.

BACKGROUND: Ectodysplasin (Eda) signaling is essential for the morphogenesis of several ectodermal appendages. RESULTS: Here, we report a medaka mutant, all-fin less (afl), which has a nonsense mutation in its eda gene. The adult afl fish displayed various abnormalities of its dermal skeleton, such as short and twisted fin rays, missing and abnormally shaped scales and teeth, and skull deformation. Focusing on the developing fin rays in the caudal region of afl larvae, we found that the fin rays did not elongate; although the initial formation of fin rays proceeded normally. Additionally, eda expression was lost, and the expression pattern of edar, the gene for the receptor of Eda, was different from wild-type one. In vivo imaging of the double-transgenic medaka expressing enhanced green fluorescent protein under control of the edar promoter and DsRed under control of the osterix promoter revealed that edar expression preceded that of osterix and that the edar-expressing cells migrated in the direction of fin ray elongation, indicating that the Eda/Edar signaling event precedes osteoblast differentiation. CONCLUSIONS: Our findings provide evidence that Eda signaling accompanied with the binding of Eda to Edar are essential for fin ray formation guided by cell migration.

Periostin accelerates human malignant melanoma progression by modifying the melanoma microenvironment.

Given no reliable therapy for advanced malignant melanoma, it is important to elucidate the molecular mechanisms underlying the disease progression. Using a quantitative proteomics approach, the 'isobaric tags for relative and absolute quantitation (iTRAQ)' method, we identified that the extracellular matrix protein, periostin (POSTN), was highly expressed in invasive melanoma compared with normal skin. An immunohistochemical analysis showed that POSTN was expressed in all invasive melanoma (n = 20) and metastatic lymph node (n = 5) tissue samples, notably restricted in their stroma. In terms of the intercellular regulation of POSTN, we found that there was upregulation of POSTN when melanoma cells and normal human dermal fibroblasts (NHDFs) were cocultured, with restricted expression of TGF-ß1 and TGF-ß3. In a functional analyses, recombinant and NHDF-derived POSTN significantly accelerated melanoma cell proliferation via the integrin/mitogen-activated protein kinase (MAPK) signaling pathway in vitro. The size of implanted melanoma tumors was significantly suppressed in POSTN/Rag2 double knockout mice compared with Rag2 knock-out mice. These results indicate that NHDF-derived POSTN accelerates melanoma progression and might be a promising therapeutic target for malignant melanoma.

The niche component periostin is produced by cancer-associated fibroblasts, supporting growth of gastric cancer through ERK activation.

Overexpression of periostin (POSTN), an extracellular matrix protein, has been observed in several cancers. We investigated the importance of POSTN in gastric cancer. Genome-wide gene expression analysis using publicly available microarray data sets revealed significantly high POSTN expression in cancer tissues from stage II-IV gastric cancer, compared with background normal tissues. The POSTN/vimentin mRNA expression ratio was highly associated with gene groups that regulate the cell cycle and cell proliferation. IHC showed that periglandular POSTN deposition, comprising linear deposition abutting the glandular epithelial cells in normal mucosa, disappeared during intestinal gastric cancer progression. Stromal POSTN deposition was also detected at the invasive front of intestinal-type and diffuse-type cancers. In situ hybridization confirmed POSTN mRNA in cancer-associated fibroblasts, but not in tumor cells themselves. POSTN enhanced the in vitro growth of OCUM-2MLN and OCUM-12 diffuse-type gastric cancer cell lines, accompanied by the activation of ERK. Furthermore, coinoculation of gastric cancer cells with POSTN-expressing NIH3T3 mouse fibroblast cells facilitated tumor formation. The OCUM-2MLN orthotopic inoculation model demonstrated that tumors of the gastric wall in Postn(-/-) mice were significantly smaller than those in wild-type mice. Ki-67 and p-ERK positive rates were both lower in Postn(-/-) mice. These findings suggest that POSTN produced by cancer-associated fibroblasts constitutes a growth-supportive microenvironment for gastric cancer.

Periostin promotes the generation of fibrous membranes in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a severe, vision-threatening disorder characterized by the fibrous membrane formation that leads to tractional retinal detachment. There has been no effective therapeutic approach other than vitreoretinal surgery. In this study, DNA microarray analysis of the fibrous membranes revealed significant up-regulation of periostin. We also found increased periostin expression in the vitreous and retinal pigment epithelial (RPE) cells from fibrous membranes of PVR patients. In vitro, periostin increased proliferation, adhesion, migration, and collagen production in RPE cells through integrin αV-mediated FAK and AKT phosphorylation. Periostin blockade suppressed migration and adhesion induced by TGFß2 and PVR vitreous. In vivo, periostin inhibition had the inhibitory effect on progression of experimental PVR in rabbit eyes without affecting the viability of retinal cells. These results identified periostin as a pivotal molecule for fibrous membrane formation as well as a promising therapeutic target for PVR.

Altered distribution of extracellular matrix proteins in the periodontal ligament of periostin-deficient mice.

Verifying whether periostin affects the distribution of type I collagen, fibronectin and tenascin C in the periodontal ligament (PDL) is important to contribute to a more thorough understanding of that protein's functions. In this study, we have histologically examined incisor PDL of mandibles in 20 week-old male wild-type and periostin-deficient (periostin-/-) mice, by means of type I collagen, fibronectin, tenascin C, proliferating cell nuclear antigen, matrix metallo-proteinase (MMP)-1 and F4/80-positive monocyte/macrophage immunostaining, transmission electron microscopy and quantitative analysis of cell proliferation. Wild-type PDL featured well-arranged layers of collagen bundles intertwined with PDL cells, whose longitudinal axis ran parallel to the collagen fibers. However, cells in the periostin-/- PDL were irregularly distributed among collagen fibrils, which were also haphazardly arranged. Type I collagen and fibronectin reactivity was seen throughout the wild-type PDL, while in the periostin-/- PDL, only focal, uneven staining for these proteins could be seen. Similarly, tenascin C staining was evenly distributed in the wild-type PDL, but hardly seen in the periostin-/- PDL. MMP-1 immunoreactivity was uniformly distributed in the wild-type PDL, but only dotted staining could be discerned in the periostin-/- PDL. F4/80-positive monocyte/macrophages were found midway between tooth- and bone-related regions in the wild-type PDL, a pattern that could not be observed in the periostin-/- PDL. In summary, periostin deficiency may not only cause PDL collagen fibril disorganization, but could also affect the distribution of other major extracellular matrix proteins such as fibronectin and tenascin C.

The role of periostin in tissue remodeling across health and disease.

Periostin, also termed osteoblast-specific factor 2, is a matricellular protein with known functions in osteology, tissue repair, oncology, cardiovascular and respiratory systems, and in various inflammatory settings. However, most of the research to date has been conducted in divergent and circumscribed areas meaning that the overall understanding of this intriguing molecule remains fragmented. Here, we integrate the available evidence on periostin expression, its normal role in development, and whether it plays a similar function during pathologic repair, regeneration, and disease in order to bring together the different research fields in which periostin investigations are ongoing. In spite of the seemingly disparate roles of periostin in health and disease, tissue remodeling as a response to insult/injury is emerging as a common functional denominator of this matricellular molecule. Periostin is transiently upregulated during cell fate changes, either physiologic or pathologic. Combining observations from various conditions, a common pattern of events can be suggested, including periostin localization during development, insult and injury, epithelial-mesenchymal transition, extracellular matrix restructuring, and remodeling. We propose mesenchymal remodeling as an overarching role for the matricellular protein periostin, across physiology and disease. Periostin may be seen as an important structural mediator, balancing appropriate versus inappropriate tissue adaption in response to insult/injury.