Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets

Abstract

Islet transplantation is limited by islet graft failure due to poor revascularization, host immune rejection and nonspecific inflammatory response. Delivery of human vascular endothelial growth factor (hVEGF) gene to the islets is likely to promote islet revascularization and survival. We used a bicistronic adenoviral vector encoding hVEGF and CpG-free allele of green fluorescent protein (Adv-GFP-hVEGF) and introduced into human pancreatic islets by transfection. We found that transfection efficiency and apoptosis were dependent on the multiplicity of infection (MOI). Compared to Adv-GFP transfected and nontransfected islets, the levels of hVEGF secreted from Adv-GFP-hVEGF transfected islets were higher and exhibit a linear relationship between hVEGF expression and MOI (10-5000). Persistent, but low level expression of hVEGF from nontransfected islets was also observed. This may be due to expression of the endogenous hVEGF gene under hypoxic conditions. The levels of DNA fragmentation determined by ELISA of islet lysates were dependent on the MOI of Adv-GFP-hVEGF. On glucose challenge, insulin release from transfected islets was comparable to nontransfected islets. Immunohistochemical staining for hVEGF was very high in Adv-GFP-hVEGF transfected islets. Weak staining was also observed for hCD31 in both transfected and nontransfected islets. These findings suggest that Adv-GFP-hVEGF is a potential candidate for promoting islet revascularization.

Biological and Biomaterial Approaches for Improved Islet Transplantation

“…Although it may be difficult to prevent a hypoxic condition of the inner islet cell mass during in vitro culture, genetic modulation of islets to express genes that promote rapid revascularization upon transplantation and reduced culture time could play an important role in preventing hypoxic damage to the islets (Mahato et al, Cheng et al, 2004;Narang et al, 2004). These approaches are discussed in section V.…”

“…Other considerations are the safety and toxicity to the islets. The latter include effects on islet function in terms of glucose-stimulated insulin secretion as well as islet viability as assessed by apoptotic and necrotic cell death upon such treatment (Mahato et al, 2003;Cheng et al, 2004;Narang et al, 2004). Transfection efficiency and toxicity of vectors are usually inversely related to each other.…”

“…Higher and dose-dependent gene expression is observed from adenoviral vectors. Redrawn from Narang et al (2004), with kind permission of Springer Science and Business Media, and Cheng et al (2004). (Narang et al, 2005).…”

“…Ex vivo gene transfer to isolated islets has been done using several gene candidates including VEGF (Mahato et al, 2003;Cheng et al, 2004;Narang et al, 2004) for revascularization and interleukin-1 receptor antagonist (Sandberg et al, 1993;Gysemans et al, 2003), TNF-␣ antagonist (Dobson et al, 2000), Bcl-2 (Rabinovitch et al, 1999;Contreras et al, 2001), A20 (Grey et al, 1999), heme oxygenase I (Pileggi et al, 2001;Tobiasch et al, 2001), IL-10 (Takayama et al, 1998), TGF-␤ , CTLA-4 Ig (Lu et al, 1999;O'Rourke et al, 2000), IL-4 (Chang and Prud'homme, 1999;Ko et al, 2001), IL-10 (Benhamou et al, 1996;Deng et al, 1997;, IL-12 (Yasuda et al, 1998), IFN-␥ receptor (Chang and Prud'homme, 1999), FasL (Kang et al, 1997;Judge et al, 1998), Adv E3 (von Herrath et al, 1997, insulin-like growth factor-I (Giannoukakis et al, 2000a;George et al, 2002a), dominant negative protein kinase C␦ (Carpenter et al, 2001(Carpenter et al, , 2002, dominant negative MyD88 (Dupraz et al, 2000), nuclear factor B (Wei and Zheng, 2003), inhibitor of B repressor (Giannoukakis et al, 2000b), heat shock protein 70 (Burkart et al, 2000), manganese superoxide dismutase (Hohmeier et al, 1998), and catalase (Benhamou et al, 1998;Xu et al, 1999) for immune, inflammation, and apoptosis protection. The therapeutic utility and mechanism of intervention of these gene candidates has bee...…”

Transfection of Rat Pancreatic Islet Tissue by Polymeric Gene Vectors

“…This prompted investigators to develop longacting islets through various means. As one strategy, genetic modification of islet cells following isolation from a pancreas has been attempted with various genes for immunomodulation (e.g., CTLA-4Ig 1 ), b-cell proliferation (e.g., hepatocyte growth factor 2 ), anti-hypoxia (e.g., myoglobin 3 ), neovascularization (e.g., vascular endothelial growth factor 4,5 ), anti-apoptosis (e.g., bcl-2 6,7 ), and cytoprotection (e.g., interleukin-4 8 ). It was hypothesized that such genetic modification would improve the viability and functionality of transplanted islets.…”