Nonviral gene transfer of human hepatocyte growth factor improves streptozotocin-induced diabetic neuropathy in rats.

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.

Nonviral HVJ (hemagglutinating virus of Japan) liposome-mediated retrograde gene transfer of human hepatocyte growth factor into rat nervous system promotes functional and histological recovery of the crushed nerve.

Hepatocyte growth factor (HGF) is well known to be involved in many biological functions, such as organ regeneration and angiogenesis, and to exert neurotrophic effects on motor, sensory, and parasympathetic neurons. In this study, we gave repeated intramuscular injections of the human HGF gene, using nonviral HVJ (hemagglutinating virus of Japan) liposome method, to examine whether transfection of the rat nervous system with this gene is able to exert neurotrophic effects facilitating recovery of a crushed nerve. The expression of HGF protein and HGF mRNA indicated that gene transfer into the nervous system did occur via retrograde axonal transport. At 4 weeks after crush, electrophysiological examination of the crushed nerve showed a significantly shorter mean latency and a significantly greater mean maximum M-wave amplitude with repeated injections of HGF gene. Furthermore, histological findings showed that the mean diameter of the axons, the axon number and the axon population were significantly larger in the group with repeated injections of HGF gene. The above results show that repeated human HGF gene transfer into the rat nervous system is able to promote crushed-nerve recovery, both electrophysiologically and histologically, and suggest that HGF gene transfer has potential for the treatment of crushed nerve.

Expression of survivin does not predict survival in patients with transitional cell carcinoma of the upper urinary tract.

The members of the IAP (inhibitors of apoptosis) family, which includes survivin, have recently emerged as modulators of an evolutionarily conserved step in apoptosis. Survivin is present during embryonic and fetal development, but it is downregulated in normal adult tissues. However, it becomes re-expressed in a variety of cancers. We investigated the prognostic importance of the expression of survivin in transitional cell carcinoma of the upper urinary tract (TCC-UUT). In 126 cases of TCC-UUT, we examined its expression (using immunohistochemistry), and also its relationship with the expressions of bcl-2 oncoprotein, p53 oncoprotein, and proliferating cell nuclear antigen (PCNA) immunoreactivity, clinicopathologic parameters, and clinical outcome. A positive expression of survivin was recognized in 12.7% of samples, a granular pattern being apparent within the cytoplasm of tumor cells. Survivin expression did not correlate with clinicopathologic findings, bcl-2 oncoprotein expression, p53 oncoprotein expression, PCNA index, or prognosis. In the normal urothelium, its expression was not detected. In conclusion, the expression of survivin does not predict prognosis in TCC-UUT.

Usefulness of photocrosslinkable chitosan for endoscopic cancer treatment in alimentary tract.

Endoscopic mucosal resection (EMR), an established treatment for superficial cancer in the stomach, colon, and esophagus, involves extracting the lesion with the aid of high-frequency current (as in snare polypectomy), following saline injection into the submucosal layer. Of interest in this study was the ability of photocrosslinkable chitosan, which is converted to a hydrogel (like a soft rubber) by 30-s ultraviolet irradiation, to expand the submucosal layer when used as an injection material. Photocrosslinkable-chitosan solution or normal saline (0.3 mL) was injected into the submucosal layer of the rat stomach, and the thickness of this layer was measured at various time points thereafter. In these two groups, the values obtained were 3.8 and 2.0 mm, respectively (means; n = 5 each group; p < 0.005), at 30 min after the injection. The cumulative blood loss measured at 20 min after mucosal incision was 113 mg (photocrosslinkable-chitosan solution), versus 1682 mg (saline) (means, n = 10 each group; p < 0.001). Histologic observation revealed that chitosan, which was retained within the submucosal layer, filled the ulcer base and completely surrounded the bleeding focus. Thus, the photocrosslinkable chitosan developed holds promise for use in EMR as a submucosal injection agent that avoids complications.

Peptic ulcer data from 4 years of endoscopy in 955 pilots of the Japan Air Self Defense Force.

BACKGROUND: The treatment of peptic ulcer disease has undergone profound changes due to the recognition of Helicobacter pylori as a causative factor. A survey of medical records was made to determine the prevalence of peptic ulcer among pilots of the Japan Air Self Defense Force (JASDF) and to decide on a possible change in JASDF medical policy toward an ulcer-treatment regime involving therapy to eradicate Helicobacter pylori. METHODS: The subjects were 955 male pilots, age 40 or older. Between 1996 and 1999, they underwent gastrointestinal endoscopy 2.47 times on average. Annual ulcer rates and recurrence rates were obtained from the endoscopic file data. Smoking habits and use of NSAIDs were also assessed as important risk factors for peptic ulcer. RESULTS: The detection rate of open ulcer for each year was 2.3-3.1% in the stomach and 1.9-4.4% in the duodenum. For ulcers including scarring, the corresponding figures were 7.3-9.5% and 12.7-19.9%. The recurrence rate from S1 scars in the duodenum was 34%, significantly higher than that from S2 scars (7%) (p < 0.0005). There was no significant difference in recurrence rate between S1 scars and S2 scars in the stomach. There was a significant association between gastric ulcer and smoking (p < 0.0005). None of the pilots took long-term NSAID medications. CONCLUSIONS: Peptic ulcer occurs more frequently in the JASDF pilots than in the general population. S1 scarring in the duodenum, as well as open ulcers in either stomach or duodenum, are candidates for Helicobacter pylori eradication therapy if the bacterium is detected. Other types of scars are also candidates for this therapy when pilots have symptoms related to infection with Helicobacter pylori.

In vivo targeted gene transfer in skin by the use of laser-induced stress waves.

BACKGROUND AND OBJECTIVES: Much interest has been shown in the use of lasers for nonviral targeted gene transfer, since the spatial characteristics of laser light are quite well defined. The aim of this study was to demonstrate in vivo gene transfer by the use of laser-induced stress waves (LISWs). STUDY DESIGN/MATERIALS AND METHODS: After reporter genes had been intradermally injected to rat skin in vivo, a laser target was placed on the gene-injected skin. LISWs were generated by the irradiation of an elastic laser target with 532-nm nanosecond laser pulses of a Q-switched Nd:YAG laser. RESULTS: Levels of luciferase activities for the skin exposed to LISWs were two orders of magnitude higher than those for the skin injected with naked DNA. Expressions of enhanced green fluorescent protein (EGFP) and beta-galactosidase were observed only in the area that was exposed to LISWs, and in addition, epidermal cells were selectively transfected. No major side effects were observed, and luciferase activity levels as high as 10(5) RLU per mg of protein were sustained even 5 days after gene transfer. CONCLUSION: Highly efficient and site-specific gene transfer can be achieved by applying a few pulses of nanosecond pulsed LISWs to rat skin in vivo.

Efficient gene transfer from innervated muscle into rat peripheral and central nervous systems using a non-viral haemagglutinating virus of Japan (HVJ)-liposome method.

We evaluated the feasibility of gene delivery into the peripheral and central nervous systems via retrograde axonal transport following injection of a haemagglutinating virus of Japan (HVJ)-liposome-DNA complex vector into an innervated muscle. Transfection efficiency was assessed by measuring luciferase activity, and was compared statistically with that achieved using a liposome-DNA control vector. High luciferase activity was observed in the injected muscle, the ipsilateral sciatic nerve, and the ipsilateral dorsal root ganglia on day 1 after gene transfer. The spinal cord also showed luciferase activity, although this was lower than in the other tissues. However, no activity was observed in the contralateral sciatic nerve or the contralateral dorsal root ganglia. In addition, we performed gene transfer twice, with a 1-week interval, to evaluate the feasibility of repeated therapeutic gene delivery. Again, a high transfection efficiency was observed immediately, even after the second gene transfer, and transfection efficiency was significantly higher at each defined time-point using the HVJ-liposome complex vector than using a control vector. These results indicate that this method could be used for repeated therapeutic gene delivery into muscle, nerve, dorsal root ganglia, and possibly spinal cord, without the need for a surgical approach, making it well suited to clinical applications.

Expressions of adrenomedullin mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension.

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.

Changes in myosin heavy chain and its localization in rat heart in association with hypobaric hypoxia-induced pulmonary hypertension.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodelling of the heart. In rat cardiac ventricles, pressure and volume overload are well known to be associated with changes in cardiac myosin heavy chain (MHC) isoforms. To study the effects of HHE on the MHC profile in the ventricles, 83 male Wistar rats were housed in a chamber at the equivalent of 5500 m altitude for 1-8 weeks. Pulmonary arterial pressure, right ventricular free wall (RVFW) weight, the ratio of RVFW weight over body weight (BW), the ratio of left ventricular free wall (LVFW) weight over BW, and myocyte diameter in both ventricles showed significant increases after 1 week, 2 weeks, 1 week, 6 weeks, and 4 weeks of HHE, respectively. Semi-quantitative reverse transcriptase-polymerase chain reaction revealed that beta-MHC mRNA expression was increased significantly in both ventricles at 6 and 8 weeks of HHE, whereas alpha-MHC mRNA expression was decreased significantly at 6 and 8 weeks of HHE in the right ventricle (RV) and at 6 weeks of HHE in the left ventricle (LV). The percentage of myosin containing the beta-MHC isoform was increased significantly at 4-8 weeks of HHE in RV and at 6 weeks of HHE in LV. In situ hybridization showed that the area of strong staining for beta-MHC mRNA was increased in both ventricles at 8 weeks of HHE, and showed a decrease from RVFW to cardiac septum, and from cardiac septum to LVFW. These results suggest that HHE has a significant effect on the expression of both MHC mRNA and protein in the heart, particularly in RV. These changes may reflect a role for cardiac MHC in the response to pulmonary hypertension in HHE.

Gene transfer of human hepatocyte growth factor into rat skin wounds mediated by liposomes coated with the sendai virus (hemagglutinating virus of Japan).

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.