Work

Hepatocyte Growth Factor Enhances Engraftment and Function of Nonhuman Primate Islets

Year: 2008

Type: article

Abstract: OBJECTIVE-Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control.Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets.RESEARCH DESIGN AND METHODS-NHP islets were transduced with murine (Ad.mHGF) or human (Ad.hHGF) adenoviral HGF (Ad.HGF) at low multiplicity of infection and studied in vitro.To study the function of Ad.HGF-transduced NHP islets in vivo, a renal subcapsular marginal mass islet transplant model was developed in streptozotocin-induced diabetic NOD-SCID mice.RESULTS-Baseline glucose values were 454.7 Ϯ 11.3 mg/dl (n ϭ 7).Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 Ϯ 9.7 mg/dl, n ϭ 5).In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0Ϯ 6.2 mg/dl, n ϭ 7) for the 6-week duration of the experiment.Unilateral nephrectomy resulted in an immediate return of glucose to baseline diabetic levels.Interestingly, adenoviral DNA, as well as mouse HGF (mHGF) mRNA derived from the adenovirus, were present for 42 days posttransplantation. Surprisingly, transplant of 500 IE with Ad.hHGF, as compared with Ad.mHGF, resulted in only marginal correction of blood glucose, suggesting that human HGF is less efficient than mHGF in this system.CONCLUSIONS-These studies demonstrate that mHGF markedly improves islet transplant outcomes in the highest preclinical species examined to date.HGF has promise as an agent that can improve islet mass and function in transplant models and likely in other models of types 1 and 2 diabetes.Diabetes 57: 2745-2754, 2008 H epatocyte growth factor (HGF) is a mesenchyme-derived protein originally identified as a circulating factor involved in liver regeneration (1-7).It is now recognized as a widely expressed mitogen, morphogen, and motogen with actions on many organs, including the liver, kidney, brain, and islets (1-7).Mature HGF is a heterodimeric protein composed of a 69-kDa ␣-chain and a 34-kDa ␤-chain.This mature HGF is generated by cleavage by extracellular proteases of the inactive pro-HGF precursor to the active HGF heterodimer.Hayek and colleagues (1,2) have shown that HGF is a potent mitogen for fetal and adult human ␤-cells in vitro.We and others have shown that HGF is a ␤-cell mitogen in vivo and is also a ␤-cell survival factor (3-7).Transgenic or viral overexpression of HGF in murine or rat ␤-cells results in an increased quantity of engrafted ␤-cells and ␤-cell proliferation.Moreover, HGF upregulates GLUT-2, glucokinase, and insulin gene expression in ␤-cells in vivo (4).As a result of these events, HGF-overexpressing islets sense glucose and secrete insulin in a fashion superior to normal islets (3,4), and ␤-cell-targeted HGF transgenic mice develop mild hypoglycemia (3,4).Moreover, using two different preclinical models, the SCID mouse and the "Edmonton rat," we have shown that adenoviral delivery of HGF to the mouse and rat islets markedly enhances transplant performance and reduces the number of islets required for successful islet transplantation (5,6).Finally, HGF decreases streptozotocin (STZ)-induced ␤-cell death in RIP-HGF mice (4), and adenoviral-mediated HGF transfer into normal mouse islets improves graft survival (5).In light of the promise of HGF as a therapeutic agent, we have now examined the efficacy and safety of HGF in a higher-level preclinical model, using nonhuman primate (NHP) islets transplanted into NOD-SCID mice.The results clearly indicate that NHP islets overexpressing mouse HGF (mHGF) function more effectively than normal islets.These anticipated outcomes were accompanied by three unexpected observations: 1) HGF overexpression and enhanced ␤-cell engraftment were not associated with increases in ␤-cell proliferation, 2) abundant adenovirus expression persisted the full 6 weeks of the experiment, and 3) murine HGF was superior to human HGF (hHGF) in this system.These results extend the efficacy of HGF from rodent islets to the primate level and clearly demonstrate that HGF should be explored as a therapeutic agent in human diabetes. RESEARCH DESIGN AND METHODSGeneration of recombinant adenovirus.Adenoviruses encoding green fluorescent protein (GFP), ␤-galactosidase (LacZ), and mHGF and hHGF were prepared as described previously (5,6,8,9).Multiplicity of infection (MOI) was determined using optical density (OD 260 ) and plaque assay.MOI calculations assumed 1,000 cells per islet equivalent (IE; 1 IE ϭ 125-m-diameter islet).NHP islets were maintained in culture for 24 -48 h before they were transduced for 1 h with 250 -500 MOI of the adenovirus, as previously described in detail (5,6,8,9) and in Fig. 2. Uninfected and infected islets were used 24 -48 h after infection, as indicated in the Figures.To determine the efficiency of adenoviral transduction, relative semiquantitative PCR analysis and immunoblots were performed using standard methods as described previously (5,6,8,9).NHP islet isolation.NHP islets from cynomolgus donors (Macaca fascicularis, aged 3-10 years) were isolated at the University of Miami using modifications (10) of the automated method for human islet isolation (11).

Source: Diabetes

DOI: 10.2337/db07-1085