KRT13 is upregulated in pancreatic cancer stem-like cells and associated with radioresistance.

Pancreatic cancer is one of the most aggressive cancers and the seventh leading cause of cancer-associated death in the world. Radiation is performed as an adjuvant therapy as well as anti-cancer drugs. Because cancer stem-like cells (CSCs) are considered to be radioresistant and cause recurrence and metastasis, understanding their properties is required for the development of novel therapeutic strategies. To investigate the CSC properties of pancreatic cancer cells, we used a pancreatic CSC model, degron (++) cells, which have low proteasome activity. Degron (++) cells displayed radioresistance in comparison with control cells. Using Ribonucleic acid (RNA) sequencing, we successfully identified KRT13 as a candidate gene responsible for radioresistance. Knockdown of KRT13 sensitized the degron (++) cells to radiation. Furthermore, a database search revealed that KRT13 is upregulated in pancreatic cancer cell lines and that high expression of KRT13 is associated with poorer prognosis. These results indicate that a combination therapy of KRT13 knockdown and radiation could hold therapeutic promise in pancreatic cancer.

Osteopontin-derived synthetic peptide SVVYGLR upregulates functional regeneration of oral and maxillofacial soft-tissue injury.

Wound healing in the oral and maxillofacial region is a complicated and interactive process. Severe mucosal or skeletal muscle injury by trauma or surgery induces worse healing conditions, including delayed wound closure and repair with excessive scar tissue. These complications lead to persistent functional impairment, such as digestive behavior or suppression of maxillofacial growth in infancy. Osteopontin (OPN), expressed in a variety of cells, is multifunctional and comprises a number of functional domains. Seven amino acids sequence, SVVYGLR (SV peptide), exposed by thrombin cleavage of OPN, has angiogenic activity and promotes fibroblast differentiation into myofibroblasts and increased expression of collagen type III. Additionally, synthetic SV peptide shows faster dermal and oral mucosal wound closure by facilitating cell motility and migratory activities in dermal- or mucosal-derived keratinocytes and fibroblasts. Moreover, cell motility and differentiation in myogenic cell populations are accelerated by SV peptide, which contributes to the facilitation of matured myofibers and scarless healing and favorable functional regeneration after skeletal muscle injury. SV peptide has high affinity with TGF-ß, with potential involvement of the TGF-ß/Smad signaling pathway. Clinical application of single-dose SV peptide could be a powerful alternative treatment option for excessive oral and maxillofacial wound care to prevent disadvantageous events.

The synthetic peptide SVVYGLR promotes myogenic cell motility via the TGFß1/Smad signaling pathway and facilitates skeletal myogenic differentiation in vitro.

In the present study, we investigated the possible involvement of the TGF-ß/Smad signaling pathway in the osteopontin-derived SVVYGLR (SV) peptide-mediated migratory activities of myogenic cells and evaluated the facilitative effects of the SV peptide on the differentiation of myogenic cells in vitro. The SV peptide-induced migration in both human-derived satellite cells and myoblasts was substantially suppressed by the TGF-ß1 receptor inhibitor SB431542 or SB505124. Besides, the expression level of the Smad3 phosphorylation was further enhanced by the addition of the SV peptide in comparison with control groups. Furthermore, an increase in the expression of myogenin-positive nuclei and a higher number of nascent myotubes with myosin heavy chain expression was confirmed in cultured myoblasts supplemented with the SV peptide. These results suggest that the involvement of the TGF-ß/Smad signaling pathway in the SV peptide-mediated migration and the facilitative effect of the SV peptide on the differentiation of myogenic cells into myotubes.

Synthetic peptide SVVYGLR upregulates cell motility and facilitates oral mucosal wound healing.

Osteopontin-derived SVVYGLR (SV) 7-amino-acid sequence is a multifunctional and synthetic SV peptide implicated in angiogenesis, production of collagen III, and fibroblast differentiation into myofibroblasts. This study investigated the effect of the SV peptide on mucosal wound healing activity. Normal human-derived gingival fibroblasts (NHGF) and human oral mucosa keratinocytes (HOMK) were used for in vitro experiments. In addition, an oral punch wound was prepared at the buccal mucosa in male rats aged 11 weeks, and we evaluated the effect of local injection of SV peptide on wound healing. The synthetic SV peptide showed no influence on the proliferation and adhesion properties of NHGF and HOMK, but it enhanced the cell motility and migration activities. TGF-ß1 receptor inhibitor, SB431542 or SB505124, substantially suppressed the SV peptide-induced migration activity, suggesting an involvement of TGF-ß1 receptor activation. Furthermore, SV peptide accelerated the healing process of an in vivo oral wound model, compared with control groups. Further immunohistological staining of wound tissue revealed that an increase in capillary growth and the greater number of fibroblasts and myofibroblasts that migrated into the wound area might contribute to the facilitation of the healing process produced by the SV peptide. The SV peptide has beneficial effects on oral wound healing through enhancement of the earlier phase consisting of angiogenesis and remodeling with granulation tissue. The synthetic SV peptide can be a useful treatment option, particularly for intractable mucosal wounds caused by trauma or surgery for progressive lesions such as oral cancer.

Osteopontin-derived synthetic peptide SVVYGLR has potent utility in the functional regeneration of oral and maxillofacial skeletal muscles.

Oral and maxillofacial skeletal muscles are critical for oral motor functions, and severe damage to these muscles by trauma or surgery may lead to persistent functional impairment. This study investigated the effects of SVVYGLR (SV) peptide, a thrombin-cleaved osteopontin-derived motif, on histopathological wound healing and functional repair after severe injury of skeletal muscles. A rat model of volumetric muscle loss bilateral masseter muscle was developed. A single dose of SV-peptide or phosphate-buffered saline (PBS) was separately injected into the injured muscle belly. Histopathological and functional analyses were performed 1-8 weeks after the treatment. Behavioral analysis during free-feeding revealed that the feeding rate markedly increased in the SV-peptide group, in contrast, the PBS group showed fewer changes after the injury. Electromyogram recordings from injured muscles demonstrated amplification of rectified burst activity over time accompanied by increased maximal amplitude and duration in the SV-peptide group, in contrast, the PBS group showed moderate changes. A lissajous figure for bilateral masseter muscle activities also revealed superior functional recovery by the SV-peptide treatment. The SV-peptide also facilitated regeneration of muscles composed of matured myofibers with a greater diameter compared to the PBS group. In addition, granulation in the earlier period and fibrosis in the later period of wound healing were significantly inhibited by the SV-peptide treatment but not by the PBS treatment. Therefore, local application of the SV-peptide could help facilitate regeneration of muscles, inhibition of fibrosis, and improvement of functional impairment of oral and maxillofacial skeletal muscles damaged by severe trauma or surgery.

Mesothelial cells facilitate cancer stem­like properties in spheroids of ovarian cancer cells.

Ovarian cancer is characterized by widespread peritoneal dissemination with ascites. Spheroids observed in the ascites of ovarian cancer patients are a mixture of cancer cells and mesothelial cells. In the present study, we evaluated whether mesothelial cells exfoliated from the peritoneum facilitate tumor spheroid formation and give rise to cancer stem­like properties in ovarian cancer cells. Spheroids from the CAOV3 and A2780 ovarian cancer cell lines grew much larger in co­culture with mesothelial cells than in monoculture under 3D conditions. The spheroids in co­culture displayed high Ki­67 expression in the peripheral zone and low expression in the central zone area. The expression of CD133 emerged in the inner portion of spheroids at later time­points (96 and 168 h), indicating that cancer cells expanded to the inner spheroid and acquired stem cell­properties. The mRNA levels of cancer stem cell markers Dclk­1, CD44 and Bmi­1 significantly increased in co­cultured CAOV3 and mesothelial cells compared to CAOV3 cells alone. Furthermore, the mesothelial cells promoted the tumorigenesis and growth of the CAOV3 cells in a mouse xenograft model compared to cancer cells alone. In conclusion, mesothelial cells promoted spheroid formation by ovarian cancer cells and facilitated cancer stem­like properties.

Overexpression of collagen type III in injured myocardium prevents cardiac systolic dysfunction by changing the balance of collagen distribution.

OBJECTIVE: Left ventricular (LV) remodeling alters the contractile and relaxation properties and induces myocardial stiffness. As LV remodeling progresses, the amount of collagen type III (Col3) is gradually decreased, being replaced by collagen type I (Col1). We evaluated whether Col3 overexpression improved cardiac function and remodeling in a rat with ischemic cardiomyopathy (ICM). We also investigated the functional motif and mechanism of thrombin-cleaved N-terminal osteopontin (N-OPN) on cardiac remodeling. METHODS: The rats with ICM were divided into 3 groups: ligation only (Control) group and groups transplanted with nontransfected fibroblast sheets (normal Fb group) or with Col3-secretory fibroblast sheets (Col3 Fb group). A gelatin hydrogel containing the N-terminal fragment (N-OPN), N-OPN lacking the SVVYGLR sequence (⊿SV), the Arg-Gly-Asp (RGD) sequence (⊿RGD), RGD and SVVYGLR sequences (⊿RGD-SV), SVVYGLR alone (SV), or a random SV peptide was implanted into an ICM model rat. RESULTS: The Col3 Fb group exhibited significantly attenuated LV systolic dysfunction. LV dilatation, myocyte hypertrophy, and LV fibrosis at the infarcted area were also attenuated by Col3 Fb implantation. Furthermore, N-OPN, ⊿RGD, and SV peptide suppressed the depression of cardiac function, LV dilatation, and myocyte hypertrophy, and also induced increased Col3 expression and reduction in the ratio of Col1 to Col3 in the infarcted and border areas. CONCLUSIONS: Overexpression of Col3 improved cardiac function by changing the balance of collagen distribution in LV remodeling. The SVVYGLR motif of the thrombin-cleaved N-OPN and SV peptide attenuated cardiac dysfunction by increasing Col3 and changing the pattern of collagen balance in the impaired area.

Evaluation of dermal wound healing activity of synthetic peptide SVVYGLR.

SVVYGLR peptide (SV peptide) is a 7-amino-acid sequence with angiogenic properties that is derived from osteopontin in the extracellular matrix and promotes differentiation of fibroblasts to myofibroblast-like cells and the production of collagen type Ⅲ by cardiac fibroblasts. However, the effects of SV peptide on dermal cells and tissue are unknown. In this study, we evaluated the effects of this peptide in a rat model of dermal wound healing. The synthetic SV peptide was added to dermal fibroblasts or keratinocytes, and their cellular motility was evaluated. In an in vivo wound healing exeriment, male rats aged 8 weeks were randomly assigned to the SV peptide treatment, non-treated control, or phosphate-buffered saline (PBS) groups. Wound healing was assessed by its repair rate and histological features. Scratch assay and cell migration assays using the Chemotaxicell method showed that SV peptide significantly promoted the cell migration in both fibroblasts and keratinocytes. In contrast the proliferation potency of these cells was not affected by SV peptide. In the rat model, wound healing progressed faster in the SV peptide-treated group than in the control and PBS groups. The histopathological analyses showed that the SV peptide treatment stimulated the migration of fibroblasts to the wound area and increased the number of myofibroblasts. Immunohistochemical staining showed a marked increase of von Willebland factor-positive neomicrovessels in the SV peptide-treated group. In conclusion, SV peptide has a beneficial function to promote wound healing by stimulating granulation via stimulating angiogenesis, cell migration, and the myofibroblastic differentiation of fibroblasts.

The integrin-binding defective FGF2 mutants potently suppress FGF2 signalling and angiogenesis.

We recently found that integrin αvß3 binds to fibroblast growth factor (FGF)-αvß31 (FGF1), and that the integrin-binding defective FGF1 mutant (Arg-50 to glutamic acid, R50E) is defective in signalling and antagonistic to FGF1 signalling. R50E suppressed angiogenesis and tumour growth, suggesting that R50E has potential as a therapeutic. However, FGF1 is unstable, and we had to express R50E in cancer cells for xenograft study, since injected R50E may rapidly disappear from circulation. We studied if we can develop antagonist of more stable FGF2. FGF2 is widely involved in important biological processes such as stem cell proliferation and angiogenesis. Previous studies found that FGF2 bound to αvß3 and antagonists to αvß3 suppressed FGF2-induced angiogenesis. However, it is unclear how FGF2 interacts with integrins. Here, we describe that substituting Lys-119/Arg-120 and Lys-125 residues in the predicted integrin-binding interface of FGF2 to glutamic acid (the K119E/R120E and K125E mutations) effectively reduced integrin binding to FGF2. These FGF2 mutants were defective in signalling functions (ERK1/2 activation and DNA synthesis) in NIH3T3 cells. Notably they suppressed, FGF2 signalling induced by WT FGF2 in endothelial cells, suggesting that the FGF2 mutants are antagonists. The FGF2 mutants effectively suppressed tube formation in vitro, sprouting in aorta ring assays ex vivo and angiogenesis in vivo The positions of amino acids critical for integrin binding are different between FGF1 and FGF2, suggesting that they do not interact with integrins in the same manner. The newly developed FGF2 mutants have potential as anti-angiogenic agents and useful tools for studying the role of integrins in FGF2 signalling.

Usefulness of measurement of heart rate variability by holter ECG in hemodialysis patients.

BACKGROUND: Major adverse cardiac and cerebrovascular event (MACCE) is one of most common complications of hemodialysis patients. Heart rate variability (HRV) is the predictor of death in heart disease patients. However, there are no studies on the role of HRV in hemodialysis patients. METHODS: From September 2009 to March 2011, 24-h electrocardiography was performed in 101 hemodialysis patients. Standard deviation of sequential 5-minute N-N interval means (SDANN) and standard deviation of the N-N interval (SDNN) was examined by a 24-h ECG analysis. Patients were observed prospectively. The primary endpoints were incidence of MACCE and MACCE-free survival. RESULTS: We studied 90 hemodialysis patients (64 males, 63.4 ± 11.8 years old). During a follow-up period of 32.0 ± 11.7 months, 33 patients developed MACCE. 24-h ECG showed mean SDNN 93.4 ± 33.4 ms and mean SDANN 83.2 ± 31.3 ms. MACCE group showed significantly lower SDNN and SDANN than event-free group. In Kaplan-Meier analysis higher SDNN and SDANN group showed significantly higher event-free survival rate than lower group. Using a Cox proportional hazards model, SDNN was independent prognostic factor while SDANN or diabetic status was not significant. In diabetic cases, there were no differences in any factors for the incidence of MACCE between higher SDNN, SDANN groups and lower groups. On the other hand in non-diabetic cases, lower SDNN or SDANN group developed significantly higher MACCE than higher groups. CONCLUSION: Measurement of HRV by Holter ECG is useful to predict MACCE in hemodialysis patients, especially non-diabetic group.

Laminin α2-secreting fibroblasts enhance the therapeutic effect of skeletal myoblast sheets.

Objectives: Skeletal myoblast sheet (SMB) transplantation, a method used for treating failing hearts, results in the secretion of cytokines that improve heart function. Enhancing the survival rate of implanted myoblasts should yield more continuous and effective therapies. We hypothesized that laminin-211 (merosin), a major component of skeletal muscle extracellular matrix (ECM), which mediates cell-to-ECM adhesion by binding to α -dystroglycan ( α DG) on muscle cells, could inhibit detachment of implanted myoblasts from host myocardia. Methods: Multilayered sheets composed of fibroblasts expressing laminin G-module (LG)4-5 of α 2 and skeletal myoblasts were transplanted into ischemic cardiomyopathy model rats. Animals were divided into four groups: the ligation only (Control) group, and those transplanted with SMB alone, with both myoblasts and control fibroblast sheets (SMB + normal Fb), or with myoblasts and laminin α 2 LG4-5-expressing fibroblast sheets (SMB + laminin Fb). Results: Quantitative estimation of nebulin mRNA levels indicated that the transplanted myoblasts in SMB + laminin Fb group exhibited significantly higher survival rates than those in the other groups. Consistent with these findings, the myoblasts in SMB + laminin Fb group exhibited elevated expression of growth factors, while SMB + laminin Fb rats also showed significant improvements in percent fractional shortening (%FS) and left ventricular remodelling, compared to the other groups. Conclusions: Laminin secreted by implanted fibroblasts inhibited the detachment of implanted myoblasts from grafted myocardia, resulting in more permanent therapeutic effects upon myoblast sheet transplantation.

Enhanced Expression of Integrin αvß3 Induced by TGF-ß Is Required for the Enhancing Effect of Fibroblast Growth Factor 1 (FGF1) in TGF-ß-Induced Epithelial-Mesenchymal Transition (EMT) in Mammary Epithelial Cells.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is regulated by growth factors such as transforming growth factor ß (TGF-ß) and fibroblast growth factors (FGF) secreted from the stromal and tumor cells. However, the role of growth factors in EMT has not been fully established. Several integrins are upregulated by TGF-ß1 during EMT. Integrins are involved in growth factor signaling through integrin-growth factor receptor crosstalk. We previously reported that FGF1 directly binds to integrin αvß3 and the interaction was required for FGF1 functions such as cell proliferation and migration. We studied the role of αvß3 induced by TGF-ß on TGF-ß-induced EMT. Here, we describe that FGF1 augmented EMT induced by TGF-ß1 in MCF10A and MCF12A mammary epithelial cells. TGF-ß1 markedly amplified integrin αvß3 and FGFR1 (but not FGFR2). We studied if the enhancing effect of FGF1 on TGF-ß1-induced EMT requires enhanced levels of both integrin αvß3 expression and FGFR1. Knockdown of ß3 suppressed the enhancement by FGF1 of TGF-ß1-induced EMT in MCF10A cells. Antagonists to FGFR suppressed the enhancing effect of FGF1 on EMT. Integrin-binding defective FGF1 mutant did not augment TGF-ß1-induced EMT in MCF10A cells. These findings suggest that enhanced integrin αvß3 expression in addition to enhanced FGFR1 expression is critical for FGF1 to augment TGF-ß1-induced EMT in mammary epithelial cells.

Improvement of cardiac function after implanting the osteopontin-derived peptide SVVYGLR in a hamster model of dilated cardiomyopathy.

OBJECTIVES: Osteopontin is a multifunctional cytokine that can modulate a variety of cellular activities, such as fibrotic response and inflammation. Osteopontin-derived peptide Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR; SV) induces angiogenesis and the expression of smooth muscle actin (SMA) in fibroblasts. In this study, we determined the effects of SV peptide on dilated cardiomyopathy (DCM). METHODS: Gels containing SV peptide (SV group), a random SV peptide (GYRVLSV) (random group) or a simple phosphate-buffered saline solution (PBS group) were transplanted on to the left ventricular (LV) anterior wall of a DCM hamster model. A control group simply underwent chest opening and closing. We used echocardiography to measure cardiac function before gel implantation (week 0) and 2, 4, 6 and 8 weeks after gel implantation. Changes in histology and myocardial remodelling were evaluated 8 weeks after the gel implantation. RESULTS: At 8 weeks post-treatment, the SV group had significantly better maintained cardiac function compared with the other groups. Histological analysis showed that LV chamber dilatation and cardiomyocyte hypertrophy were significantly attenuated, and the distribution of SMA-positive cells in the LV anterior wall area was greater in the SV group. The capillary density in the epicardial aspect of the anterior wall in the SV group was also significantly increased, indicating that the SV peptide released from the implanted gel had promoted angiogenesis. Furthermore, Western blotting and histological analyses showed that the level of expression of collagen type III at the gel-implanted anterior wall in the SV group was significantly increased, and the type III/type I collagen ratio was higher in the SV group than in the control or PBS groups. CONCLUSIONS: SV peptide treatment improved cardiac function, and inhibited the dilatation of the LV chamber and cardiomyocyte hypertrophy. By inducing the differentiation of fibroblasts to SMA-positive muscle-like cells and increasing type III collagen, SV peptide conferred a contractile property on the gel-implanted wall. We believe that the SVVYGLR peptide treatment could be used as a bridge to a left ventricular assist device and heart transplantation and for cardiac regeneration therapy without cell transplantation in the future.

SVVYGLR motif of the thrombin-cleaved N-terminal osteopontin fragment enhances the synthesis of collagen type III in myocardial fibrosis.

Osteopontin (OPN) is involved in various physiological processes such as inflammatory and wound healing. However, little is known about the effects of OPN on these tissues. OPN is cleaved by thrombin, and cleavage of the N-terminal fragment exposes a SVVYGLR sequence on its C-terminus. In this study, we examined the effects of the thrombin-cleaved OPN fragments on fibroblasts and myocardial fibrosis, particularly the role of the SVVYGLR sequence. The recombinant thrombin-cleaved OPN fragments (N-terminal fragment [N-OPN], C-terminal fragment [C-OPN], and the N-terminal fragment lacking the SVVYGLR sequence [ΔSV N-OPN]) were added to fibroblasts, and the cellular motility, signal activity, and production of collagen were evaluated. A sustained-release gel containing an OPN fragment or SVVYGLR peptide was transplanted into a rat model of ischemic cardiomyopathy and the quantities and ratio of collagen type I (COL I) and type III (COL III) were estimated. N-OPN significantly promoted fibroblast migration. Smad signal activity, expression of smooth muscle actin (SMA), and the production of COL III were enhanced by N-OPN and SVVYGLR peptide. Conversely, ΔSV N-OPN and C-OPN had no effect. In vivo, the expression level of N-OPN was associated with COL III distribution, and the COL III/COL I ratio was significantly increased by the sustained-release gel containing N-OPN or SVVYGLR peptide. The cardiac function was also significantly improved by the N-OPN- or SVVYGLR peptide-released gel treatment. The N-terminal fragment of thrombin-cleaved OPN-induced Smad signal activation, SMA expression, and COL III production, and its SVVYGLR sequence influences this function.

Tissue inhibitor of metalloproteinase-1 and -3 improves cardiac function in an ischemic cardiomyopathy model rat.

Matrix metalloproteinases (MMPs) and a family of tissue inhibitors of metalloproteinases (TIMPs) may contribute to myocardial remodeling in heart failure. TIMPs are the main inhibitors of MMPs and have other MMP-independent functions. Because little is known of the role of TIMPs in the heart, we examined the effects of TIMPs on cardiac fibroblasts (CFs) and cardiomyocytes. In vitro, TIMP-1-4 enhanced smooth muscle actin (SMA) expression in CFs, and TIMP-1 and TIMP-3 enhanced the expression of phosphorylated Smad-3 and phosphorylated transforming growth factor (TGF)-ß type 1 receptor in CFs; this effect was inhibited by TGF-ß receptor blocker SB-505124. TIMPs-1, -3, and -4 also inhibited the FAK, AKT, and ERK pathways that induce cardiac hypertrophy. TIMP-1 and TIMP-2 suppressed apoptosis in cardiomyocytes; in contrast, TIMP-4 induced apoptosis in CFs. TIMP-2 stimulated collagen synthesis. Collagen gels containing TIMP-1 or TIMP-3, which exhibit cardioprotective effects in vitro, were transplanted to the left ventricular anterior wall of a rat heart model of myocardial infarction. Gel-released TIMP-1 and TIMP-3 significantly improved cardiac function and myocardial remodeling and enhanced SMA expression in the infarcted area in ischemic cardiomyopathy model rats. Further, the transplantation of TIMP-1 or TIMP-3 gels inhibited apoptosis in the ischemic myocardium and reduced MMP-2 activity. TIMPs may be an ideal target of cardiac regeneration therapy.

Transplantation of myoblast sheets that secrete the novel peptide SVVYGLR improves cardiac function in failing hearts.

AIMS: Transplantation of myoblast sheets is a promising therapy for enhancing cardiac function after heart failure. We have previously demonstrated that a 7-amino-acid sequence (Ser-Val-Val-Tyr-Gly-Leu-Arg) derived from osteopontin (SV peptide) induces angiogenesis. In this study, we evaluated the long-term therapeutic effects of myoblast sheets secreting SV in a rat infarction model. METHODS AND RESULTS: Two weeks after ligation of the left anterior descending coronary artery, the animals were divided into the following three groups: a group transplanted with wild-type rat skeletal myoblast sheets (WT-rSkMs); a group transplanted with SV-secreting myoblast sheets (SV-rSkMs); and a control group (ligation only). We evaluated cardiac function, histological changes, and smooth muscle actin (SMA) expression through transforming growth factor-ß (TGF-ß) signalling. The ejection fraction and fractional shortening were significantly better, and the enlargement of end-systolic volume was also significantly attenuated in the SV-rSkM group. Left ventricular remodelling, including fibrosis and hypertrophy, was significantly attenuated in the SV-rSkM group, and SV secreted by the myoblast sheets promoted angiogenesis in the infarcted border area. Furthermore, many clusters of SMA-positive cells were observed in the infarcted areas in the SV-rSkM group. In vitro SMA expression was increased when SV was added to the isolated myocardial fibroblasts. Moreover, SV bound to the TGF-ß receptor, and SV treatment activated TGF-ß receptor-Smad signalling. CONCLUSION: The SV-secreting myoblast sheets facilitate a long-term improvement in cardiac function. The SV can induce differentiation of fibroblasts to myofibroblasts via TGF-ß-Smad signalling. This peptide could possibly be used as a bridge to heart transplantation or as an ideal peptide drug for cardiac regeneration therapy.

A dominant-negative FGF1 mutant (the R50E mutant) suppresses tumorigenesis and angiogenesis.

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvß3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvß3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvß3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent ("FGF1 decoy").

The preparation of PLLA/calcium phosphate hybrid composite and its evaluation of biocompatibility.

In tissue engineering, biodegradable polymer materials with both high biocompatibility and high strength are very important as scaffolds for long term use. Therefore, in this research, we tried to prepare the three types of poly(L-lactic acid) (PLLA)/calcium phosphate (CP) hybrid composite for a scaffold biomaterial. The effects of addition of different CP on both biocompatibility and mechanical properties were evaluated. CP powders and voids were three-dimensionally and uniformly distributed in the solid samples and porous composite samples. These compositions of CP and PLLA greatly improved the cellular adhesiveness, which increased as the volume fraction of CP in the composite increased. For the porous samples, cells migrated into the pores. This study demonstrated that a composite of PLLA and CP is an effective new scaffold material that results in better osteoconductivity, bone regeneration, and mineralization and has moderately high strength.

Transplantation of elastin-secreting myoblast sheets improves cardiac function in infarcted rat heart.

Myoblast sheet transplantation for cardiac failure is a promising therapy to enhance cardiac function via paracrine mechanism. However, their efficacies of treatment showed a gradual decline. The gene modification of the implanted myoblast is important in improving the long-term results of the treatment. Elastin fiber enhances the extensibility of the infarcted wall and can prevent left ventricular dilation. We therefore hypothesized that the elastin gene modification of the implanted myoblast could strengthen and maintain the long-term improvement effects of cardiac function. In this study, we evaluated long-term follow-up benefits of functional myoblast sheets that secrete elastin in an infarcted model. The animal models were divided into three groups: a group transplanted with nontransfected, wild-type, skeletal myoblast-type sheets (WT-rSkM); group transplanted with myoblast sheets that secreted elastin fragments (ELN-rSkM); and a control group (ligation only). Cardiac function was examined by echocardiography, and cardiac remodeling after infarction was evaluated by histological examination. The cardiac function was significantly improved and the left ventricle end-diastolic dimensions were significantly reduced in the ELN-rSkM group. Histological analysis showed that left ventricular remodeling was attenuated in the ELN-rSkM group and that elastic fiber was formed in the epicardial area of ELN-rSkM group. The functionalization of myoblast sheet by elastin gene transfer showed the long-term improvement of cardiac function. Expressed recombinant elastin fiber prevented the dilation of the left ventricular chamber after myocardial infarction. The functional myoblast sheet transplantation maintained the treatment effect by the paracrine effect of myoblast and the formed recombinant elastin.

Platelet-derived growth factor regulates breast cancer progression via ß-catenin expression.

OBJECTIVE: The knowledge on the association between platelet-derived growth factor (PDGF) signaling and epithelial cancers is scarce, although overexpression of PDGF and PDGF receptors has been reported in some human mesenchymal tumors. Thus, we studied the effect of PDGF on breast cancer cells in vitro and the distribution of PDGF in breast cancer tissues. METHODS: The effect of PDGF-BB on breast cancer cells was assessed by Western blotting, immunofluorescence, WST and 5-bromo-2-deoxyuridine incorporation experiments. PDGF-B and ß-catenin expression was investigated in breast cancer tissues by immunohistochemistry. RESULTS: PDGF-BB induces ß-catenin expression in breast cancer cells, and immunohistochemically the distribution of PDGF-B was similar to ß-catenin in breast cancer cells. PDGF-B-positive cancer cells were more frequent in cases of ductal carcinoma in situ (87.5%) than invasive carcinoma (61.2%). In addition, PDGF-B staining was stronger in intraductal than invasive cancer cells. PDGF-BB tended to induce nuclear translocation of ß-catenin, cell proliferation and DNA incorporation in MDA-MB231 cells, while these results were not found in MCF-7 cells. CONCLUSION: Our results suggest that PDGF-BB regulates protein expression of ß-catenin and is associated with cancer cell behavior.