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Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury.

Background: The experimental pulmonary hypertension that develops in hypobaric hypoxia is characterized by structural remodeling of the heart. The P2X4 receptor (P2X4R) controls vascular tone and vessel remodeling in several blood vessels, and it has emerged as a key factor in the enhancement of cardiovascular performance., Methods and Results: To study the possible effects of hypobaric hypoxia on the P2X4R-synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of the 5,500 m altitude level for 21 days. After 14 days' exposure to hypobaric hypoxia, pulmonary arterial pressure (PAP) was significantly increased. In the right ventricle (RV) of the heart, P2X4R expression was significantly increased on days 1 and 14 (mRNA) and on days 7 and 21 (protein) of hypobaric hypoxic exposure. Immunohistochemical staining for P2X4R protein became more intense in RV in the late phase of exposure. These changes in P2X4R synthesis in RV occurred alongside the increase in PAP. In addition, P2X1R and P2Y2R mRNA levels in the RV were significantly increased on days 1, 14, and 21, and day 5, respectively, of exposure. The level of P2X1R protein in the RV was significantly increased on day 21 of exposure., Conclusions: Conceivably, P2 receptors, including P2X4R and P2X1R, might play roles in modulating the RV hypertrophy that occurs due to pulmonary hypertension in hypobaric hypoxia., (All rights are reserved to the Japanese Circulation Society.)

Background: Gene transduction has been considered advantageous for the sustained delivery of proteins to specific target tissues. However, in the case of hard tissues, such as bone, local gene delivery remains problematic owing to anatomical accessibility limitations of the target sites., Methodology/principal Findings: Here, we evaluated the feasibility of exogenous gene transduction in the interior of bone via axonal transport following intramuscular administration of a nonviral vector. A high expression level of the transduced gene was achieved in the tibia ipsilateral to the injected tibialis anterior muscle, as well as in the ipsilateral sciatic nerve and dorsal root ganglia. In sciatic transection rats, the gene expression level was significantly lowered in bone., Conclusions/significance: These results suggest that axonal transport is critical for gene transduction. Our study may provide a basis for developing therapeutic methods for efficient gene delivery into hard tissues.

Peripheral nerve injury occasionally causes chronic neuropathic pain with hyperalgesia and allodynia. However, its treatment is difficult. Here, we used a chronic constriction injury (CCI) model in rats to investigate the effects on experimental neuropathic pain of the human hepatocyte growth factor (HGF) gene delivered into the nervous system by retrograde axonal transport following its repeated intramuscular transfer, using liposomes containing the hemagglutinating virus of Japan (HVJ). CCI (control) rats exhibited marked mechanical allodynia and thermal hyperalgesia, and decreased blood flow in sciatic nerve and hind paw. All these changes were significantly reversed by HGF gene transfer. In the sciatic nerve in HGF-treated rats, the size-frequency distributions for myelinated and unmyelinated axons each showed a rightward shift, the number of myelinated axons >5 microm in diameter was significantly increased, and the mean diameter of unmyelinated axons was significantly increased (versus CCI rats). Levels of P2X3, P2X4, and P2Y1 receptor mRNAs, and of interleukin-6 (IL-6) and activating transcription factor 3 (ATF3) mRNAs, were elevated in the ipsilateral dorsal root ganglia and/or sciatic nerve by CCI, and these levels were decreased by HGF gene transfer. These results may point toward a potential new treatment strategy for chronic neuropathic pain in this model.

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays an important role in tumor growth and metastasis by regulating energy metabolism and inducing angiogenesis. Elevated levels of HIF-1alpha, a subunit of HIF-1, are noted in various malignant tumors, but it is unclear whether this is so in esophageal carcinoma. The purpose of this study was to evaluate the implications of HIF-1alpha expression in esophageal squamous cell carcinoma. In 215 patients with esophageal carcinoma, we examined immunoreactivity for HIF-1alpha protein, vascular endothelial growth factor (VEGF) protein and p53 protein. In 38 patients, we examined the expression of HIF-1alpha messenger ribonucleic acid (mRNA) (using the semiquantitative reverse transcriptase-polymerase chain reaction [RT-PCR]). A positive HIF-1alpha protein expression was recognized in 95% of the patients, and was strongly apparent within both the nuclei and/or cytoplasm of tumor cells. The proportion of patients in the 'high score' group for HIF-1alpha protein expression increased significantly with increasing VEGF protein expression. Immunoreactivity for HIF-1alpha protein was found to have a significant effect on disease-free survival rate in our univariate analysis, but no effect on overall survival rate. In RT-PCR, HIF-1alpha mRNA scores correlated significantly with scores for HIF-1alpha protein expression, but not with any clinicopathologic factor or either of the survival rates. The detection of HIF-1alpha protein and mRNA would appear to offer limited information as to progression and prognosis in esophageal carcinoma.

Peripheral neuropathy is common and ultimately accounts for significant morbidity in diabetes. Recently, several neurotrophic factors have been used to prevent progression of diabetic neuropathy. In this study, we gave repeated intramuscular injections of the human hepatocyte growth factor (HGF) gene percutaneously, using liposomes containing the hemagglutinating virus of Japan (HVJ), to examine therapeutic efficacy of nonviral gene transfer of HGF for experimental diabetic sensorimotor neuropathy in rats. Experimental diabetes induced by intraperitoneal injection of streptozotocin resulted in a marked tactile allodynia (but not in a thermal hyperalgesia), in a reduction of both the conduction velocity and the amplitude, and in a decreased laser Doppler flux of the nerve and the muscle at 6 weeks after the induction. All these changes were significantly reversed by repeated gene transfer of HGF. Furthermore, we analyzed the density of endoneurial capillaries and morphometrical changes of the nerve. The density of endoneurial capillaries, disclosing marked reduction in diabetic rats, was also reversed significantly by repeated gene transfer of HGF; however, no considerable differences were observed morphometrically in either myelinated or unmyelinated axons. These results suggest that nonviral HVJ liposome-mediated gene transfer of human HGF has potential for the safe effective treatment of diabetic sensorimotor neuropathy.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Adrenomedullin (AM) has diuretic, natriuretic, and hypotensive effects. To study the possible effects of HHE on the AM synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of a 5,500-m altitude level for 21 days. After 14 days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased (compared with control rats). The plasma AM protein level was significantly increased on day 21 of exposure to HHE. In the right ventricle (RV), right atrium, and left atrium of the heart, the expressions of AM mRNA and protein were increased in the middle to late phase (5-21 days) of HHE, whereas in the brain and lung they were increased much earlier (0.5-5 days). In situ hybridization and immunohistochemistry showed AM mRNA and protein staining to be more intense in the RV in animals in the middle to late phase of HHE exposure than in the controls. During HHE, these changes in AM synthesis, which occurred strongly in the RV, occurred alongside the increase in PAP. Conceivably, AM may play a role in modulating pulmonary hypertension in HHE.

Hepatocyte growth factor (HGF) regulates cell growth, cell motility, and morphogenesis in various types of cells, including epithelial and endothelial cells, indicating that it probably promotes epithelial repair and neovascularization during wound healing. To better understand the effects of HGF on wound healing, we performed human HGF-gene transfer into skin wounds in rats. The rat HGF mRNA levels, and human and rat HGF protein concentrations in the wounds in HGF gene-transfer rats were significantly elevated at 3 days, 3 to 14 days, and 3 and 14 days after gene transfer, respectively. An expression of human HGF mRNA and protein was revealed in squamous cells in the epidermis, in endothelial cells and smooth muscle cells in blood vessels, and in fibroblasts in granulation tissues at 3, 7, and 14 days after gene transfer in HGF gene-transfer rats. The wound lesion area in HGF gene-transfer rats was significantly less than that in control rats from 3 to 7 days after gene transfer. The re-epithelialization rate, microvessel counts in granulation tissues, proliferating cell nuclear antigen index of fibroblasts in granulation tissues, and the proliferating cell nuclear antigen index in the epidermis of HGF gene-transfer rats were significantly increased at 3 and 7 days after gene transfer. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that the expression levels of transforming growth factor-beta1 and Colalpha2(I) mRNAs in the wounds of HGF gene-transfer rats were significantly decreased at 7 and 14 days, respectively. The hydroxyproline concentration in the wound was significantly less in HGF gene-transfer rats than in control rats at 3 days after gene transfer. These results suggest that HGF gene transfer into a skin wound may aid re-epithelialization and neovascularization in the early phase of wound healing, and that HGF may play a role in modulating cutaneous wound healing.

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations.