Delivery of hypoxia-inducible VEGF gene to rat islets using polyethylenimine

Abstract

Islet transplantation is a promising strategy for treatment of diabetes. However, islets are exposed to hypoxia in the process of isolation and transplantation and prone to apoptosis. Vascular endothelial growth factor (VEGF) gene transfer is one of the promising strategies to address this problem. However, VEGF expression in the cells under normoxia is undesirable since it may induce pathological angiogenesis. Therefore, VEGF expression should be regulated to avoid this problem. In this study, hypoxia-inducible VEGF gene was transferred to islets using a non-viral carrier. Rat islets were transfected with high molecular weight PEI (25 kDa, PEI25K), low molecular weight PEI (2 kDa, PEI2K), and polyamidoamine dendrimer (PAMAM). PEI25K had higher transfection efficiency to rat islets than PAMAM or PEI2K. The hypoxia-inducible gene expression vector, pRTP801-Luc or pRTP801-VEGF was transferred to rat islets using PEI25K. Transfection assay with pRTP801-Luc showed that luciferase expression was induced in rat islets under hypoxia. In addition, transfer of pRTP801-VEGF showed that VEGF gene expression was higher under hypoxia than normoxia in rat islets. In conclusion, delivery of pRTP801-VEGF using PEI25K induces VEGF level specifically under hypoxia and may be useful for the development of anti-apoptotic strategies for islet transplantation.

A comparison of non-viral vectors for gene delivery to pancreatic β-cells: Delivering a hypoxia-inducible vascular endothelial growth factor gene to rat islets

“…This effect was not observed in islets transfected with pSV40-VEGF, however, suggesting that the RTP801 promoter increased VEGF expression specifically in islets under hypoxia. This finding was in accordance with our previous report using PEI as a gene carrier (14).…”

“…PEI has different transfection efficiencies, depending on its molecular weight and the form of PEI. In this study, branched 25-kDa PEI was compared with other non-viral vectors for transfection into the pancreatic ß-cells based on a previous report that the efficiency of 25 kDa branched PEI was significantly higher than that of a PAMAM dendrimer or 2 kDa linear PEI (14). Hemagglutinating virus of Japan envelope (HVJ-E, an inactivated Sendai virus envelope) was reported to be useful and efficient as a non-viral vector for gene delivery (23).…”

“…After 4 h, the transfection mixtures were removed and 100 μl of fresh Medium 199 containing 10% FBS was added. Cells were incubated at the desired concentration of oxygen (normoxia, 20% oxygen; hypoxia, 1% oxygen) for 12 h. ELISA was performed using a Biosource Human Vascular Endothelial Growth Factor ELISA kit (Biosource International Inc., Camarillo, CA) (14).…”

Oxygenation of islets and its role in transplantation

“…3), including overexpression of vascular endothelial growth factor-A (VEGF-A) [40]. Recently, a strategy to induce VEGF selectively during hypoxia was developed and included the use of a nonviral vector, polyethylenimine [41 ] and, with even higher efficiency, Effectene (Qiagen GmbH, Hilden, Germany) [42 ]. In addition, mobilization of angioblasts from bone marrow [43,44 ] improves islet graft revascularization and function.…”

Hemoglobin Conjugates with Antioxidant Enzymes (Hemoglobin-Superoxide Dismutase-Catalase) via Poly(Ethylene Glycol) Crosslinker for Protection of Pancreatic Beta RINm5F Cells in Hypoxia

“…41 These conjugated Hbs may also be used for enhancing the survival time of different cell or tissue types for temporary storage after isolation or during transplantation of beta cells, cardiac cells, and in ischemic conditions or related complications. [42][43][44] For isolated beta cell transplantation in the low partial oxygen sites such as peritoneal cavity, exogenous low p50 Hb conjugated with antioxidants may help prevent oxygen transport issues and reduce cell death from hypoxic and free radicals stresses. Conjugated Hbs with low p50 in such situations may be more beneficial than high p50 systems or other oxygen carriers as the systems may be capable of unloading more oxygen into the cellular environment more efficiently in hypoxic conditions and thus improve the overall metabolic function.…”