Effective healing of diabetic skin wounds by using nonviral gene therapy based on minicircle vascular endothelial growth factor DNA and a cationic dendrimer.

BACKGROUND: The development of an efficient method to improve the wound healing process is urgently required for diabetic patients suffering a threat of limb amputations. Various growth factors have been proposed for treatment; however, more research still has to be carried out to maintain their curative effect. In the present study, we describe a simple nonviral gene therapy method for improving wound healing. METHODS: Minicircle plasmid DNA encoding vascular endothelial growth factor (VEGF) was combined with an arginine-grafted cationic dendrimer, PAM-RG4. The formed complexes were injected subcutaneously into the skin wounds of diabetic mice. RESULTS: Actively proliferating cells in wound tissue were efficiently transfected, resulting in a high level of VEGF expression. Within 6 days after injection, skin wounds in the diabetic mice were generally healed and displayed a well-ordered dermal structure, which was confirmed by histological staining. CONCLUSIONS: This simple and effective gene therapy method may represent a powerful tool for the treatment of diabetic foot ulcers and other diseases that are refractory to treatment.

EGCG and quercetin protected INS-1 cells in oxidative stress via different mechanisms.

EGCG and quercetin are known as beneficial dietary flavonoid for various diseases including diabetes mellitus. But it is not certain whether they could protect pancreatic beta cell directly. We performed this study to test both EGCG and quercetin could directly protect beta cell line under oxidative stress, and verify the action mechanisms. The protective effect of quercetin on INS-1 cells against oxidative stress was concentration dependent, but EGCG showed specific concentration zone for the protection. The protective effect of EGCG was more pronounced in pre-treatment before oxidative stress, while quercetin showed dramatic improvement of viability in simultaneous incubation with H2O2. In EGCG pre-treatment, antioxidant enzymes and activity were decreased, but the phosphorylated PI3K and Akt were significantly increased. PI3K inhibitor significantly reduced cell viability in EGCG pre-treatment. In conclusion, EGCG and quercetin have protective effect on INS-1 cells against oxidative stress through both antioxidant effect and anti-apoptosis signaling. In EGCG, pre-treatment make its effect better by the enhancement of anti-apoptosis signaling. Quercetin protected INS-1 cells more in simultaneous incubation via strong antioxidant defense.

Polymyxin B, scavenger of endotoxin, enhances isolation yield and in vivo function of islets.

Collagenase purified from bacteria has been used to isolate islets for transplantation. However, collagenase is contaminated with small amounts of endotoxin, which induces dysfunction or apoptosis of islets. In this study, we investigated the effects of polymyxin B, endotoxin scavenger, on the yield and quality of isolated islets. It is revealed that polymyxin B neutralized endotoxin in vitro and inhibited endotoxin-mediated decreases of the glucose stimulation index. Additionally, adenosine triphosphate (ATP) quantitation, islet regression assay, and caspase-3 activation assay demonstrated that polymyxin B efficiently blocked the toxic effects induced by endotoxin. Thereafter, we isolated mouse islets both with and without polymyxin B and compared total islet equivalents (IEQs), glucose-stimulated insulin release, and ATP content. Polymyxin B enhanced islet recovery, and ATP content of islets, and glucose stimulation index, and reduced TNF-alpha expression of islets. Marginal transplantation (200 IEQs/mouse) under the kidney capsule of diabetic mice induced normoglycemia in 30% of the polymyxin B group, but not in any mouse of control group. This result suggests that islets isolated with polymyxin B more effectively lower blood glucose levels as compared with control islets. Thus, polymyxin B could serve as a useful agent in the protection of islets from endotoxin-induced inflammation and apoptosis.

Comparison of the efficiency and toxicity of sonoporation with branched polyethylenimine-mediated gene transfection in various cultured cell lines.

OBJECTIVE: The objective of this study is to evaluate transfection efficiency and safety for gene delivery by sonoporation in comparison with cationic polymer gene carrier branched polyethylenimine (BPEI). METHODS: The cDNA expressing VEGF(165) was cloned under chicken beta-actin promoter. The plasmid DNA was transfected into the CHO, HEK293, and NIH3T3 cells using microbubble-based sonoporation and BPEI (25 kDa) under various conditions. Enzyme-linked immunosorbent assay (ELISA) was used to determine the expressed protein level. Cytotoxicities of transfection methods were compared by Cell Counting Kit-8. RESULTS: At 1 MHz intensity, transfection efficiency of sonoporation was enhanced by microbubble concentration with no detrimental effects. By contrast, BPEI exacerbated cell viability, despite its high transgene expression efficiency. CONCLUSION: Sonoporation gene therapy might be the safest technique to be used in actual clinical practice.