High Positive End-Expiratory Pressure Renders Spontaneous Effort Noninjurious.

RATIONALE: In acute respiratory distress syndrome (ARDS), atelectatic solid-like lung tissue impairs transmission of negative swings in pleural pressure (Ppl) that result from diaphragmatic contraction. The localization of more negative Ppl proportionally increases dependent lung stretch by drawing gas either from other lung regions (e.g., nondependent lung [pendelluft]) or from the ventilator. Lowering the level of spontaneous effort and/or converting solid-like to fluid-like lung might render spontaneous effort noninjurious. OBJECTIVES: To determine whether spontaneous effort increases dependent lung injury, and whether such injury would be reduced by recruiting atelectatic solid-like lung with positive end-expiratory pressure (PEEP). METHODS: Established models of severe ARDS (rabbit, pig) were used. Regional histology (rabbit), inflammation (positron emission tomography; pig), regional inspiratory Ppl (intrabronchial balloon manometry), and stretch (electrical impedance tomography; pig) were measured. Respiratory drive was evaluated in 11 patients with ARDS. MEASUREMENTS AND MAIN RESULTS: Although injury during muscle paralysis was predominantly in nondependent and middle lung regions at low (vs. high) PEEP, strong inspiratory effort increased injury (indicated by positron emission tomography and histology) in dependent lung. Stronger effort (vs. muscle paralysis) caused local overstretch and greater tidal recruitment in dependent lung, where more negative Ppl was localized and greater stretch was generated. In contrast, high PEEP minimized lung injury by more uniformly distributing negative Ppl, and lowering the magnitude of spontaneous effort (i.e., deflection in esophageal pressure observed in rabbits, pigs, and patients). CONCLUSIONS: Strong effort increased dependent lung injury, where higher local lung stress and stretch was generated; effort-dependent lung injury was minimized by high PEEP in severe ARDS, which may offset need for paralysis.

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.

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.

In vivo differentiation of induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Induced pluripotent stem cells (iPSCs) hold promise for a new era in treating heart failure. However, the functional microstructure of iPSC-derived cardiomyocytes (iPSC-CMs) and their ability to attach to the extracellular matrix of the recipient myocardium require further elucidation. Thus, we analyzed the functional microstructure and adhesion molecules of iPSC-CM. METHODS AND RESULTS: Immunostaining analysis showed that iPSC-CMs were similar to neonatal cardiomyocytes (CMs) in expressing the cytoskeletal proteins myosin heavy chain (MHC), myosin light chain (MLC) 2a, MLC2v, and especially ß-MHC (a neonatal CM marker), as well as the adhesion molecules N-cadherin, α7-integrin, dystrophin, α-dystroglycan, α-sarcoglycan, and laminin-α2. Electron microscopy showed abundant myofibrillar bundles with transverse Z-bands and a developed mitochondrial structure in both iPSC-CMs and neonatal CMs, although the iPSC-CMs contained fewer mitochondria with lower-density cristae. When transplanted from in vitro conditions to nude rat hearts, iPSC-CMs acquired the ability to express α-MHC, a molecule specific to adult CMs. Mechanical stretch or stimulation by insulin-like growth factor-1 enhanced the α-MHC expression in iPSC-CMs in vitro. CONCLUSIONS: Our findings in vitro and in vivo indicate that CMs derived from iPSCs contain cardiac-specific organelles and adhesion systems. These results indicate that iPSC-derived CMs may be useful in new cell therapies for heart failure.

Myocardial layer-specific effect of myoblast cell-sheet implantation evaluated by tissue strain imaging.

BACKGROUND: The implantation of skeletal myoblast (SMB) cell-sheets over the damaged area of a myocardial infarction (MI) has been shown to improve global left ventricular (LV) function through a paracrine effect. However, the regeneration process has not been fully evaluated. We hypothesized that the use of tissue Doppler strain M-mode imaging to assess myocardial layer-specific strain might enable detailed visual evaluation of the regenerative ability of SMBs. METHODS AND RESULTS: SMBs were cultured on temperature-responsive culture dishes to generate cell-sheets. At 4 weeks after inducing anterior MI, the animals were divided into 2 groups: SMB cell-sheet implantation and sham operation (n=6 in each). A total of 30 cell-sheets (1.5×10(7) cells/sheet) were placed on the epicardium, covering the infarct and border regions. Subendocardial and subepicardial strain values were measured in the infarct, border, and remote regions by tissue Doppler strain analysis. SMB cell-sheet implantation produced the following major effects: progression of LV remodeling was prevented and global LV ejection fraction increased; the subendocardial strain was significantly greater than the subepicardial strain in the treated border region; vascular density in the subendocardium was significantly higher than in the subepicardium in the treated region; the expression of vascular endothelial growth factor was significantly increased. CONCLUSIONS: Tissue Doppler strain analysis allows precise evaluation of the effect of cell-sheet implantation on layer-specific myocardial function.

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.

Aberrant expression of disintegrin-metalloprotease proteins in the formation and progression of uterine cervical cancer.

OBJECTIVE: Dysregulated expression of disintegrin-metalloprotease proteins [a disintegrin and metalloproteases (ADAMs) and ADAMs with thrombospondin motif (ADAMTSs)] has been reported in many types of cancers and is believed to play an important role in cancer formation and metastasis. However, little is known about the expression of ADAMs and ADAMTSs in the development of human cervical cancer. METHODS: Reverse transcriptase polymerase chain reaction and immunoblotting were performed to assess the expression of several disintegrin-metalloproteases and tissue inhibitors of metalloproteinases (TIMPs) in squamous-type cervical cancer cells and oncogenically modified keratinocytes (immortalized human cervical keratinocytes transduced with human papilloma virus-16 E6/E7 proteins with or without oncogenes). Immunohistochemistry of ADAM-9, ADAM-10 and TIMP-3 was performed on 31 primary human cervical tissue specimens of preinvasive and invasive cervical carcinoma. RESULTS: mRNA levels of ADAM-9, ADAM-10, ADAM-12, TIMP-2 and TIMP-3 were upregulated as cervical cells progressed from dysplastic to malignant lesions compared to normal cervical cells. These results were corroborated at the protein level by Western blot analysis and immunohistochemistry. CONCLUSION: The expression of disintegrin-metalloproteases and their endogenous regulators was dysregulated during cervical carcinogenesis. The aberrant expression of ADAMs might contribute to the pathogenesis of cervical cancer formation and progression.

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.

The synthetic peptide SVVYGLR promotes cell motility of myogenic cells and facilitates differentiation in skeletal muscle regeneration.

The present study was designed to evaluate the effects of the osteopontin-derived multifunctional short peptide, SVVYGLR (SV) peptide on the biological properties of skeletal muscle-specific myogenic cells. We employed human-derived satellite cells (HSkMSC) and skeletal muscle myoblasts (HSMM) and performed a series of biochemical experiments. The synthetic SV peptide showed no influence on the proliferation and adhesion properties of HSkMSC and HSMM, while it showed a significant increase in cell motility, including migration activities upon treatment with the SV peptide. In a rat model with volumetric loss of masticatory muscle, immunohistochemical staining of regenerating muscle tissue immediately after injury demonstrated an increase of the number of both MyoD- and myogenin-positive cells in SV peptide-treated group. These results suggest that SV peptide plays a potent role in facilitating skeletal muscle regeneration by promoting the migration, and differentiation of myogenic precursor and progenitor cells.

The role of MT2-MMP in cancer progression.

The role of MT2-MMP in cancer progression remains to be elucidated in spite of many reports on MT1-MMP. Using a human fibrosarcoma cell, HT1080 and a human gastric cancer cell, TMK-1, endogenous expression of MT1-MMP or MT2-MMP was suppressed by siRNA induction to examine the influence of cancer progression in vitro and in vivo. In HT1080 cells, positive both in MT1-MMP and MT2-MMP, the migration as well as the invasion was impaired by MT1-MMP or MT2-MMP suppression. Also cell proliferation in three dimensional (3D) condition was inhibited by MT1-MMP or MT2-MMP suppression and tumor growth in the nude mice transplanted with tumor cells were reduced either MT1-MMP or MT2-MMP suppression with a prolongation of survival time in vivo. MT2-MMP suppression induces more inhibitory effects on 3D proliferation and in vivo tumor growth than MT1-MMP. On the other hand, TMK-1 cells, negative in MT1-MMP and MMP-2 but positive in MT2-MMP, all the migratory, invasive, and 3D proliferative activities in TMK-1 are decreased only by MT2-MMP suppression. These results indicate MT2-MMP might be involved in the cancer progression more than or equal to MT1-MMP independently of MMP-2 and MT1-MMP.

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.

Survivin determines cardiac function by controlling total cardiomyocyte number.

BACKGROUND: Survivin inhibits apoptosis and regulates cell division in many organs, but its function in the heart is unknown. METHODS AND RESULTS: We show that cardiac-specific deletion of survivin resulted in premature cardiac death. The underlying cause was a dramatic reduction in total cardiomyocyte numbers as determined by a stereological method for quantification of cells per organ. The resulting increased hemodynamic load per cell led to progressive heart failure as assessed by echocardiography, magnetic resonance imaging, positron emission tomography, and invasive catheterization. The reduction in total cardiomyocyte number in alpha-myosin heavy chain (MHC)-survivin(-/-) mice was due to an approximately 50% lower mitotic rate without increased apoptosis. This occurred at the expense of DNA accumulation because survivin-deficient cardiomyocytes displayed marked DNA polyploidy indicative of consecutive rounds of DNA replication without cell division. Survivin small interfering RNA knockdown in neonatal rat cardiomyocytes also led to polyploidization and cell cycle arrest without apoptosis. Adenoviral overexpression of survivin in cardiomyocytes inhibited doxorubicin-induced apoptosis, induced DNA synthesis, and promoted cell cycle progression. The phenotype of the alphaMHC-survivin(-/-) mice also allowed us to determine the minimum cardiomyocyte number sufficient for normal cardiac function. In human cardiomyopathy, survivin was potently induced in the failing heart and downregulated again after hemodynamic support by a left ventricular assist device. Its expression positively correlated with the mean cardiomyocyte DNA content. CONCLUSIONS: We suggest that the ontogenetically determined cardiomyocyte number may be an independent factor in the susceptibility to cardiac diseases. Through its profound impact on both cardiomyocyte replication and apoptosis, survivin may emerge as a promising new target for myocardial regeneration.

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.

Gene expression of colorectal cancer: preoperative genetic diagnosis using endoscopic biopsies.

In colorectal cancer, to predict the response to chemo- and/or radio-therapy or the existence of lymph node metastasis preoperatively, a more competent diagnostic system is required, in addition to conventional diagnosis based on morphology and pathology. The application of gene expression profiling to preoperative cancer diagnosis using endoscopic biopsies could enable the selection of a more appropriate therapy for patients. In this study, we evaluated the feasibility of gene expression profiling using preoperative biopsies of colorectal tumors in a clinical setting, by investigating the influence of intra-tumor heterogeneity on the profiles and testing the prediction ability of tumor malignancy. Under endoscopic examination, two biopsies were sampled from each of 10 colorectal cancers and 10 adenomas, and their gene expression data were obtained using cDNA microarrays. The intra- and inter-tumor heterogeneities of the profiles were compared with unsupervised clustering analysis. Molecular prediction of tumor malignancy using biopsies was performed with the supervised classification algorithm. In clustering analysis, almost all paired biopsies from the same tumors joined each other. Pearson's correlation coefficients of the profiles between biopsies from the same tumors (mean, 0.83) were significantly greater than those of the profiles between biopsies from other cancers (mean, 0.58) (p<0.0001). In the supervised classification method, malignancy was correctly predicted in 39 out of 40 biopsies with 8-71 informative genes. Gene expression profiling using endoscopic biopsies of colorectal tumors revealed that the intra-tumor heterogeneity was smaller than the inter-tumor heterogeneity and tumor malignancy was correctly predicted. Our findings suggest that the technique of gene expression profiling accurately represents the biological properties of colorectal cancer and could help the preoperative diagnosis of this disease.

The implication of N-acetylglucosaminyltransferase V expression in gastric cancer.

OBJECTIVE: N-acetylglucosaminyltransferase V (GnT-V) is a key enzyme that catalyzes beta(1-6) branching of N-acetylglucosamine on N-glycan of cell proteins, some of which are linked with metastasis. GnT-V expression was studied immunohistochemically in gastric cancer to compare clinicopathological parameters and evaluate the role of GnT-V in the prognosis of gastric cancer. METHODS: Immunohistochemistry was carried out to detect GnT-V expression in 50 advanced gastric cancer tissues where the depth of invasion exceeded the subserosa, and the relationship between GnT-V expression and various clinicopathological factors, including survival, was analyzed. RESULTS: GnT-V was expressed in 23 (46%) gastric cancer tissues. GnT-V expression was significantly correlated with lymph node metastases, peritoneal dissemination and liver metastases, respectively (p = 0.005, p = 0.013 and p = 0.023). Patients with GnT-V-positive gastric cancer had a significantly shorter survival than those without GnT-V expression (5-year survival rate: 31.2 and 54.4%, respectively; p = 0.045). CONCLUSION: GnT-V expression is correlated with a poor prognosis in gastric cancer patients due to metastases.

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.

Synthetic osteopontin-derived peptide SVVYGLR can induce neovascularization in artificial bone marrow scaffold biomaterials.

We have previously reported that an osteopontin-derived SVVYGLR peptide exhibited potent angiogenic activity in vitro and in vivo. In the present study, the focus points were on the in vitro effect of SVVYGLR on bone marrow stromal cell proliferation, as well as its in vivo effect on bone tissue formation when grafts made of CO3Ap-collagen sponge- as a scaffold biomaterial containing the SVVYGLR motif - were implanted. SVVYGLR peptide promoted bone marrow stromal cell proliferation. When a CO3Ap-collagen sponge containing SVVYGLR peptide was implanted as a graft into a tissue defect created in rat tibia, the migration of numerous vascular endothelial cells - as well as prominent angiogenesis - inside the graft could be detected after one week. These results thus suggested that our scaffold biomaterials including the peptide could be useful for bone tissue regeneration.

Longer preservation of cardiac performance by sheet-shaped myoblast implantation in dilated cardiomyopathic hamsters.

OBJECTIVES: Cell therapy is a promising strategy for ischemic cardiomyopathy. However, the direct injection method has limitations for generalized cell delivery, especially in dilated cardiomyopathy (DCM). We hypothesized that a sheet-shaped myoblast graft would be superior to direct injection for improving cardiac performance in DCM. METHODS: Male 27-week-old BIO TO-2 (DCM) hamsters that showed moderate cardiac remodeling were used as recipients. Myoblasts isolated from BIO F1B hamsters were cultured on dishes coated with poly(N-isopropylacrylamide), a temperature-responsive polymer, and spontaneously detached as a sheet-shaped graft at 20 degrees C without enzymatic treatment. Three different therapies were conducted: (1) sheet-shaped myoblast graft implantation (S group, n=29); (2) myoblast injection (M group, n=28); and (3) sham operation (C group, n=28). In the S group, two sheet-shaped myoblast grafts were implanted on the left ventricle (LV) wall, and in the M group, myoblasts were injected into the right ventricle (RV) and LV walls. RESULTS: After the sheet-shaped myoblast grafts were implanted, echocardiography demonstrated that the dilated LV dimension was significantly reduced, whereas the hearts in other groups showed a progression of LV dilation. The fractional shortening in the M and C groups decreased significantly while that in the S group was maintained at the preoperative level for 3 months after the operation. Histological examination demonstrated that in the S group, the LV wall thickness was increased, with viable myoblasts, and myocardial fibrosis was decreased compared with the other groups. Immunohistochemical staining demonstrated alpha-sarcoglycan and beta-sarcoglycan expression on the basement membrane of the cardiomyocytes in the S group but not in the other groups. The life expectancy was significantly prolonged in the S group. CONCLUSION: Sheet-shaped myoblast graft implantation improved cardiac performance and prolonged life expectancy, associated with a reduction in myocardial fibrosis and re-organization of the cytoskeletal proteins in DCM hamsters. Thus, sheet-shaped autologous myoblast graft implantation may induce restoration of the heart in DCM.

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.