Date of Award

12-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Ram I. Mahato, Ph.D.

Committee

John K. Buolamwini, Ph.D. Ivan C. Gerling, Ph.D. Ramareddy V. Guntaka, Ph.D. Yi Lu, Ph.D.

Keywords

Adenoviral Vectors, Diabetes, Gene/Drug Delivery, Islet Transplantation, Liver Fibrosis, Triplex Forming Oligonucleotide

Abstract

Ex vivo gene transfer can improve the outcome of islet transplantation for treating Type I diabetes. Earlier we have shown co‑expression of human vascular endothelial growth factor (hVEGF) and human interleukin‑1 receptor antagonist (hI‑1Ra) after transfection of plasmid DNA encoding these two genes. Due to poor transfection efficiency of plasmid DNA and the better known islet transduction efficiency of adenoviral (Adv) vectors, in this study, we constructed Adv‑hVEGF‑hIL‑1Ra by cloning hVEGF and hIL‑1Ra coding sequences and polyA signal under separate cytomegalovirus (CMV) promoters in Adenoquick plasmid (Ad 13.1). There was dose and time dependent expression of these genes after transduction of Adv‑hVEGF‑hIL‑1Ra into human islets. The mRNA expression of hVEGF and hIL‑1Ra was more than 100 times higher than that of the non‑transduced and bicistronic plasmid transfected control islets. Transduced islets were viable as evidenced by insulin release upon glucose challenge. Co‑expression of hVEGF and hIL‑1Ra by islets showed decrease in caspase‑3 activity and apoptosis induced by a cocktail of inflammatory cytokines such as TNF‑α IL‑1βand IFN‑γ. Compared to non‑treated or Adv‑LacZ transduced islets, transduction of islets with Adv‑hVEGF‑hIL‑1Ra prior to transplantation under the kidney capsules of diabetic NOD‑SCID mice reduced the blood glucose levels, and increased serum insulin and c‑peptide levels. Immunohistochemical staining of the islet bearing kidney sections at day 30 after transplantation was positive for human insulin, hVEGF and von Willebrand factor.

Further, we decided to replace VEGF with hepatocyte growth factor (HGF) as it not only can promote revascularization of islets but also increases β ‑cell proliferation. It can also protect the islets from apoptosis. Adv‑hHGF‑hIL‑1Ra showed dose and time dependent expression of these genes after transduction into human islets and further showed significant decrease in caspase‑3 induced by the cytokines. Compared to un‑transduced islets, transduction of islets with Adv‑hHGF‑hIL‑1Ra at 1000 MOI prior to transplantation under the kidney capsules of streptozotocin‑induced‑diabetic NOD‑SCID mice reduced blood glucose levels, and increased serum insulin and c‑peptide levels.

These results indicate that the bicistronic Adv vector efficiently expresses both growth factor and antiapoptotic genes, decreases apoptosis and improves the outcome of islet transplantation.

Liver fibrosis is a consequence of chronic liver disorders which lead to accumulation of extra cellular matrix (ECM). Particularly, there is an increased accumulation of collagen in the fibrotic liver. We have therefore used a triplex forming oligonucleotide (TFO) against the type α1 (I) collagen and evaluated, whether it can attenuate liver fibrosis induced by common bile duct ligation (CBDL). There was a significant decrease in hydroxyproline levels in TFO‑treated groups compared to non‑treated CBDL group. Masson’s trichrome staining showed weak staining for collagen in TFO‑treated liver tissues and there was a predominantly high staining in non‑treated CBDL group. There was over expression of α ‑smooth muscle actin (α ‑SMA), marker for myofibroblasts following CBDL. Western blotting analysis also revealed an increase in TGF‑β1 expression, which is a potential marker for fibrosis in the CBDL group compared to TFO‑treated group. These results suggest that TFO can be used to downregulate Type I collagen gene expression and can alleviate liver fibrosis induced by common bile duct ligation.

DOI

10.21007/etd.cghs.2010.0236

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