In vivo characterization of a decellularized dermis-polymer complex for use in percutaneous devices.

To develop a method for making percutaneous devices that have high biocompatibility and do not induce downgrowth of epidermal cells, we prepared a partial decellularized dermis (DD)/poly(methyl methacrylate) (PMMA) complex (PDPC) with a PMMA rod firmly stabilized inside. The porcine decellularized tissue was chosen because of its high biocompatibility and mechanical properties, and MMA was used because it would adhere firmly to a polymer such as a catheter. The MMA filled the cavities in the dermis and polymerized, anchoring to the collagenous fibrils inside the porcine DD. The PDPC was cemented to the PMMA rod tightly and it was integrated with the surrounding tissue within 12 weeks of implantation. Furthermore, no downgrowth of the epidermis, which may cause clinical problems, was observed. We consider that the tissue-polymer complex may be a suitable candidate for use in percutaneous devices.

Chemoprevention of liver carcinogenesis with retinoids: Basic and clinical aspects.

The strategy to prevent liver carcinogenesis consists of: (i) antiviral modalities such as vaccination, lamivudin, and interferon; (ii) anti-inflammatory modality; and (iii) chemoprevention using such compounds as retinoid analog and vitamin K. Cancer chemoprevention is defined as an approach where natural or synthetic chemical compound works to arrest or reverse premalignant cells by using physiological pathways. As a consequence, such a clone of premalignant cells is eradicated (clonal deletion) by differentiation induction or apoptosis, and thus the process toward the development of clinically detectable cancer is disrupted. A particularly effective candidate target of chemoprevention in liver diseases is an advanced stage of chronic hepatitis, that is supposed to contain transformed hepatocyte clone(s); that is, primary prevention from liver cirrhosis and prevention of recurrent and second primary hepatocellular carcinoma following the treatment of the initial cancer. Retinoid is a collective term of vitamin A analog that binds to nuclear retinoid receptors;retinoic acid receptors (RAR) and retinoid X receptors (RXR). After ligand coupling, these receptors form homo- or heterodimers, bind to the response element (RARE or RXRE) upstream of the target gene, and regulate the gene expression as a transcriptional factor. Biological phenotypes of such transcriptional regulation by retinoid include cellular differentiation, tissue morphogenesis, and programmed cell death (apoptosis). Due to these functions, retinoid analogs are clinically tried to prevent/treat carcinoma in a wide variety of organs including head and neck cancer, uterine cervical cancer, certain leukemia and liver cancer. In this article, clinical trials of retinoid analog to inhibit second primary hepatoma, supposed molecular mechanism of the action of the compound, and aberrant metabolism of RXR and its role in liver carcinogenesis are briefly reviewed.

Decellularized dermis-polymer complex provides a platform for soft-to-hard tissue interfaces.

To develop a soft-to-hard tissue interface, we made a decellularized dermis/poly(methyl methacrylate) (PMMA) complex by soaking the decellularized dermis in methyl methacrylate (MMA) and an initiator, and then polymerizing the MMA. The decellularized tissue was chosen because of its good biocompatibility and the easiness of suturing it, and MMA because of its hard tissue compatibility and wide use in the biomedical field. The MMA filled the cavities in the dermis and polymerized within 10 min. No leaking or polymer aggregation was observed, implying that a homogenous tissue-polymer complex had formed. The cell infiltration and the integration between the tissue and the dermis occurred in vivo, whereas the cells could not infiltrate the tissue-polymer complex. This implies that the interface tissue should possess both complex and noncomplex parts, where the cells infiltrate the noncomplex part and stop when they encounter the complex part, integrating the soft and hard tissue or hard polymer.

Effect of high-pressure polymerization on mechanical properties of PMMA denture base resin.

The aim of this study was to assess the effect of high-pressure polymerization on mechanical properties of denture base resin. A heat-curing denture base resin and an experimental PMMA were polymerized under 500MPa of pressure by means of an isostatic pressurization machine at 70°C for 24h to make rectangular specimens whose dimensions were 30mm×2mm×2mm. Each specimen was deflected on a three-point flexural test until either fracture occurred or the sample was loaded up to 8mm in deflection. The molecular weight of the PMMA without filler was analyzed using the high-speed liquid chromatography system. Increased ductility without fracture was shown in the specimens subjected to high pressure, while most of the control specimens (ambient pressure) fractured. The mean toughness of the PMMA specimens polymerized under the high pressure was significantly higher than the same material polymerized under ambient pressure (p<0.01). The high pressure groups of the denture resin and the PMMA revealed a significantly lower mean 0.2% yield stress, flexural strength, and elastic modulus than control groups (p<0.01). There were certain amounts of higher molecular weight polymers in the high pressure specimens than were present in the controls. The increased toughness shown in the PMMA polymerized under the high pressure was presumably attributed to the higher molecular weight produced by the pressure. The result suggests a potential application of the high-pressure polymerization to the development of PMMA-based denture resin with improved fracture resistance.

Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway.

Human airway trypsin-like protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20-55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [3H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular Ca2+ mobilization in HBF. The HAT-induced increase in Ca2+ was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts.

Effect of TRB3 on insulin and nutrient-stimulated hepatic p70 S6 kinase activity.

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.

Injection of the insulin receptor alpha subunit increases blood glucose levels in mice.

Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged alpha subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRalpha, we purified the protein secreted from the CHO cells. The His-tagged hIRalpha was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRalpha into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRalpha showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hIRalpha in the blood stream inhibits insulin actions by binding with plasma insulin.

Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions.

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.

K(ATP) channel knockout mice crossbred with transgenic mice expressing a dominant-negative form of human insulin receptor have glucose intolerance but not diabetes.

Impaired insulin secretion and insulin resistance are thought to be two major causes of type 2 diabetes mellitus. There are two kinds of diabetic model mice: one is a K(ATP) channel knockout (Kir6.2KO) mouse which is defective in glucose-induced insulin secretion, and the other is a transgenic mouse expressing the tyrosine kinase-deficient (dominant-negative form of) human insulin receptor (hIR(KM)TG), and which has insulin resistance in muscle and fat. However, all of these mice have no evidence of overt diabetes. To determine if the double mutant Kir6.2KO/hIR(KM)TG mice would have diabetes, we generated mutant mice by crossbreeding, which would show both impaired glucose-induced insulin secretion and insulin resistance in muscle and fat. We report here that: 1) blood glucose levels of randomly fed and 6 h fasted double mutant (Kir6.2KO/hIR(KM)TG) mice were comparable with those of wild type mice; 2) in intraperitoneal glucose tolerance test (ipGTT), Kir6.2KO/hIR(KM)TG mice had an impaired glucose tolerance; and 3) during ipGTT, insulin secretion was not induced in either Kir6.2KO/hIR(KM)TG or Kir6.2KO mice, while the hIR(KM)TG mice showed a more prolonged insulin secretion than did wild type mice; 4) hyperinsulinemic euglycemic clamp test revealed that Kir6.2KO, Kir6.2KO/hIR(KM)TG and hIR(KM)TG mice, showed decreased whole-body glucose disposal compared with wild type mice; 5) Kir6.2KO, but not Kir6.2KO/hIR(KM)TG mice had some obesity and hyperleptinemia compared with wild type mice. Thus, the defects in glucose-induced insulin secretion (Kir6.2KO) and an insulin resistance in muscle and fat (hIR(KM)TG) were not sufficient to lead to overt diabetes.