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.

Effects of various types of anesthesia on hemodynamics, cardiac function, and glucose and lipid metabolism in rats.

Anesthesia can affect respiratory, circulatory, and endocrine systems but is necessary for certain experimental procedures such as echocardiography and blood sampling in small animals. We have now investigated the effects of four types of anesthesia [pentobarbital sodium (PENT), ketamine-xylazine (K/X), and low- or high-dose isoflurane (ISO)] on hemodynamics, cardiac function, and glucose and lipid metabolism in Sprague-Dawley rats. Aortic pressure, heart rate, and echocardiographic parameters were measured at various time points up to 45 min after the induction of anesthesia, and blood was then collected for measurement of parameters of glucose and lipid metabolism. Systolic aortic pressure remained constant in the PENT group, whereas it showed a biphasic pattern in the K/X group and a gradual decline in the ISO groups. Marked bradycardia was observed in the K/X group. The serum glucose concentration was increased and the plasma insulin level was reduced in the K/X and ISO groups compared with the PENT group. The concentrations of free fatty acids and norepinephrine in plasma were increased in the K/X group. Despite the metabolic effects of K/X and ISO, our results suggest that the marked bradycardic effect of K-X renders this combination appropriate for measurement of Doppler-derived indexes of left ventricular diastolic function, whereas the relative ease with which the depth of anesthesia can be controlled with ISO makes it suitable for manipulations or data collection over long time periods. On the other hand, PENT may be best suited for experiments that focus on measurement of cardiac function by M-mode echocardiography and metabolic parameters.

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.

[A novel diagnostic method for detection of lymph node metastases].

In order to improve the sensitivity of diagnosis of regional lymph node metastases in cancer patients, the novel molecular method called the one-step nucleic acid amplification (OSNA) assay using cytokeratin 19 mRNA as a molecular marker was developed. In this method, the supernatant of a homogenized lymph node solution is directly analyzed without the mRNA purification process that is usually required in RT-PCR. Therefore the results are available within 30 min via a simple procedure, and the results correlate well with the number of cancer cells in the lymph node. We conducted multicenter clinical trials in Japan to evaluate the OSNA assay for the detection of lymph node metastases in breast cancer, colorectal cancer, gastric cancer, and lung cancer. The results showed that the OSNA assay provides a judgment performance equivalent to that of 2-mm-interval histopathologic examinations of lymph nodes in each type of cancer. Furthermore, the OSNA assay is unlikely to yield false-positives for histopathologically negative lymph nodes. Following these results, the OSNA assay was approved as a diagnostic method for lymph node status in breast, colorectal, and gastric cancer patients by the Japanese Ministry of Health, Labor and Welfare. The OSNA assay is promising as a quick, simple, subjective, quantitative method for the detection of lymph node metastases in various cancers.

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.

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.

Effects of ramelteon on cardiac injury and adipose tissue pathology in rats with metabolic syndrome.

Melatonin regulates circadian rhythms but also has antioxidative and anti-inflammatory effects that ameliorate metabolic disorders. We investigated the effects of the selective melatonin agonist ramelteon on cardiac and adipose tissue pathology in the DahlS.Z-Leprfa /Leprfa (DS/obese) rat, a model of metabolic syndrome (MetS). Rats were treated with a low (0.3 mg/kg per day) or high (8 mg/kg per day) dose of ramelteon from 9 to 13 weeks of age. Ramelteon treatment at either dose attenuated body weight gain, left ventricular fibrosis, and diastolic dysfunction, as well as cardiac oxidative stress and inflammation, without affecting hypertension or insulin resistance. Although ramelteon did not affect visceral white adipose tissue (WAT) mass, it attenuated inflammation and downregulated insulin signaling in this tissue. In contrast, ramelteon reduced fat mass, adipocyte hypertrophy, and inflammation, and ameliorated impaired insulin signaling in subcutaneous WAT. In addition, ramelteon attenuated adipocyte hypertrophy, downregulated mitochondrial uncoupling protein 1, and upregulated 11ß-hydroxysteroid dehydrogenase type 1 expression in interscapular brown adipose tissue (BAT). In summary, ramelteon treatment attenuated obesity and cardiac injury, improved insulin signaling in visceral and subcutaneous WAT, and inhibited the whitening of BAT in rats with MetS.

Anti-inflammatory effects of heat-killed Lactobacillus plantarum L-137 on cardiac and adipose tissue in rats with metabolic syndrome.

The effects of heat-killed Lactobacillus plantarum L-137 (HK L-137) on chronic inflammation associated with metabolic disorders have remained unknown. We examined the effects of HK L-137 on cardiac and adipose tissue pathophysiology in DahlS.Z-Lepr fa /Lepr fa (DS/obese) rats as a model of metabolic syndrome. DS/obese rats were treated orally with HK L-137 (2 or 75 mg kg-1 day-1) from 9 to 13 weeks of age. HK L-137 attenuated left ventricular (LV) inflammation and fibrosis as well as adipocyte hypertrophy, inflammation, and up-regulation of sterol regulatory element-binding protein-1c (SREBP-1c) gene expression in visceral and subcutaneous adipose tissue, without affecting body weight gain or hypertension. The low dose of HK L-137 also ameliorated LV diastolic dysfunction, the increase in subcutaneous fat mass, and insulin resistance as well as attenuated the down-regulation of Akt phosphorylation in visceral and subcutaneous adipose tissue, and the elevation of the circulating interleukin-6 concentration. Furthermore, the proportion of regulatory T (Treg) cells among CD4+ T cells in the spleen was increased by HK L-137. These results suggest that the anti-inflammatory effects of HK L-137 on the heart and adipose tissue are related, at least partly, to suppression of systemic inflammation associated with an increase in splenic Treg cell.

Effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism in rats with metabolic syndrome.

The mammalian target of rapamycin (mTOR) is a regulator of metabolism and is implicated in pathological conditions such as obesity and diabetes. We aimed to investigate the role of mTOR in obesity. A new animal model of metabolic syndrome (MetS), named DahlS.Z-Leprfa /Leprfa (DS/obese) rats was established previously in our laboratory. In this study, we used this model to evaluate the effects of mTOR inhibition on cardiac and adipose tissue pathology and glucose metabolism. DS/obese rats were treated with the mTOR inhibitor, everolimus, (0.83 mg/kg per day, per os) for 4 weeks at 9 weeks of age. Age-matched homozygous lean (DahlS.Z-Lepr+ /Lepr+ or DS/lean) littermates of DS/obese rats were used as controls. Treatment with everolimus ameliorated hypertension, left ventricular (LV) hypertrophy and fibrosis, and LV diastolic dysfunction, and attenuated cardiac oxidative stress and inflammation in DS/obese rats, but had no effect on these parameters in DS/lean rats. Treatment with everolimus reduced Akt Thr308 phosphorylation in the heart of DS/obese rats. It also alleviated obesity, hyperphagia, adipocyte hypertrophy, and adipose tissue inflammation in DS/obese rats. Everolimus treatment exacerbated glucose intolerance, but did not affect Akt phosphorylation levels in the fat or liver in these rats. Pancreatic ß-cell mass was increased in DS/obese rats compared with that in DS/lean rats and this effect was attenuated by everolimus. Activation of mTOR signaling contributes to the pathophysiology of MetS and its associated complications. And mTOR inhibition with everolimus ameliorated obesity as well as cardiac and adipose tissue pathology, but exacerbated glucose metabolism in rats with MetS.

Atorvastatin reduces cardiac and adipose tissue inflammation in rats with metabolic syndrome.

BACKGROUND: Statins are strong inhibitors of cholesterol biosynthesis and help to prevent cardiovascular disease. They also exert additional pleiotropic effects that include an anti-inflammatory action and are independent of cholesterol, but the molecular mechanisms underlying these additional effects have remained unclear. We have now examined the effects of atorvastatin on cardiac and adipose tissue inflammation in DahlS.Z-Leprfa/Leprfa (DS/obese) rats, which we previously established as a model of metabolic syndrome (MetS). METHODS AND RESULTS: DS/obese rats were treated with atorvastatin (6 or 20mgkg-1day-1) from 9 to 13weeks of age. Atorvastatin ameliorated cardiac fibrosis, diastolic dysfunction, oxidative stress, and inflammation as well as adipose tissue inflammation in these animals at both doses. The high dose of atorvastatin reduced adipocyte hypertrophy to a greater extent than did the low dose. Atorvastatin inhibited the up-regulation of peroxisome proliferator-activated receptor γ gene expression in adipose tissue as well as decreased the serum adiponectin concentration in DS/obese rats. It also activated AMP-activated protein kinase (AMPK) as well as inactivated nuclear factor-κB (NF-κB) in the heart of these animals. The down-regulation of AMPK and NF-κB activities in adipose tissue of DS/obese rats was attenuated and further enhanced, respectively, by atorvastatin treatment. CONCLUSIONS: The present results suggest that the anti-inflammatory effects of atorvastatin on the heart and adipose tissue are attributable at least partly to increased AMPK activity and decreased NF-κB activity in this rat model of MetS.

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.

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.

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.

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.