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Hepatocyte growth factor enhances engraftment and function of nonhuman primate islets.

Fiaschi-Taesch NM, Berman DM, Sicari BM, Takane KK, Garcia-Ocaña A, Ricordi C, Kenyon NS, Stewart AF - Diabetes (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. taeschn@dom.pitt.edu

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 +/- 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 +/- 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 +/- 6.2 mg/dl, n = 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.

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Glucose-stimulated insulin secretion and HGF expression in nontransduced and transduced NHP islets. A: NHP islets were exposed to 250–500 MOI for 1 h. At 24 h after transduction, groups of 15 IE of similar size were incubated with 5 or 22 mmol/l glucose for 30 min, and insulin secreted into the media was measured by radioimmunoassay. Results are the means ± SE of 5–13 experiments performed in triplicate. The data are presented as the percentage above the insulin secreted by uninfected islets (100%). B and C: HGF expression as assessed by RT-PCR (B) and Western blot (C) analysis of RNA extracts and protein extracts obtained from nontransduced NHP islets and islets transduced with 250 MOI of Ad.LacZ, Ad.GFP, Ad.mHGF, or Ad.hHGF. At 24 h after transduction, islets were harvested and mRNA and protein were extracted. For the RT-PCR, primers were specifically designed to amplify either mHGF or hHGF cDNA, as appropriate. HGF was detected using an antibody recognizing both mHGF and hHGF. Actin was used as an internal control for loading. Note that both Ad.mHGF and Ad.hHGF were able to transduce NHP islets equally as assessed by RT-PCR and Western blot.
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f2: Glucose-stimulated insulin secretion and HGF expression in nontransduced and transduced NHP islets. A: NHP islets were exposed to 250–500 MOI for 1 h. At 24 h after transduction, groups of 15 IE of similar size were incubated with 5 or 22 mmol/l glucose for 30 min, and insulin secreted into the media was measured by radioimmunoassay. Results are the means ± SE of 5–13 experiments performed in triplicate. The data are presented as the percentage above the insulin secreted by uninfected islets (100%). B and C: HGF expression as assessed by RT-PCR (B) and Western blot (C) analysis of RNA extracts and protein extracts obtained from nontransduced NHP islets and islets transduced with 250 MOI of Ad.LacZ, Ad.GFP, Ad.mHGF, or Ad.hHGF. At 24 h after transduction, islets were harvested and mRNA and protein were extracted. For the RT-PCR, primers were specifically designed to amplify either mHGF or hHGF cDNA, as appropriate. HGF was detected using an antibody recognizing both mHGF and hHGF. Actin was used as an internal control for loading. Note that both Ad.mHGF and Ad.hHGF were able to transduce NHP islets equally as assessed by RT-PCR and Western blot.

Mentions: Adenoviruses encoding green fluorescent protein (GFP), β-galactosidase (LacZ), and mHGF and hHGF were prepared as described previously (5,6,8,9). Multiplicity of infection (MOI) was determined using optical density (OD260) and plaque assay. MOI calculations assumed 1,000 cells per islet equivalent (IE; 1 IE = 125-μm-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).


Hepatocyte growth factor enhances engraftment and function of nonhuman primate islets.

Fiaschi-Taesch NM, Berman DM, Sicari BM, Takane KK, Garcia-Ocaña A, Ricordi C, Kenyon NS, Stewart AF - Diabetes (2008)

Glucose-stimulated insulin secretion and HGF expression in nontransduced and transduced NHP islets. A: NHP islets were exposed to 250–500 MOI for 1 h. At 24 h after transduction, groups of 15 IE of similar size were incubated with 5 or 22 mmol/l glucose for 30 min, and insulin secreted into the media was measured by radioimmunoassay. Results are the means ± SE of 5–13 experiments performed in triplicate. The data are presented as the percentage above the insulin secreted by uninfected islets (100%). B and C: HGF expression as assessed by RT-PCR (B) and Western blot (C) analysis of RNA extracts and protein extracts obtained from nontransduced NHP islets and islets transduced with 250 MOI of Ad.LacZ, Ad.GFP, Ad.mHGF, or Ad.hHGF. At 24 h after transduction, islets were harvested and mRNA and protein were extracted. For the RT-PCR, primers were specifically designed to amplify either mHGF or hHGF cDNA, as appropriate. HGF was detected using an antibody recognizing both mHGF and hHGF. Actin was used as an internal control for loading. Note that both Ad.mHGF and Ad.hHGF were able to transduce NHP islets equally as assessed by RT-PCR and Western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551685&req=5

f2: Glucose-stimulated insulin secretion and HGF expression in nontransduced and transduced NHP islets. A: NHP islets were exposed to 250–500 MOI for 1 h. At 24 h after transduction, groups of 15 IE of similar size were incubated with 5 or 22 mmol/l glucose for 30 min, and insulin secreted into the media was measured by radioimmunoassay. Results are the means ± SE of 5–13 experiments performed in triplicate. The data are presented as the percentage above the insulin secreted by uninfected islets (100%). B and C: HGF expression as assessed by RT-PCR (B) and Western blot (C) analysis of RNA extracts and protein extracts obtained from nontransduced NHP islets and islets transduced with 250 MOI of Ad.LacZ, Ad.GFP, Ad.mHGF, or Ad.hHGF. At 24 h after transduction, islets were harvested and mRNA and protein were extracted. For the RT-PCR, primers were specifically designed to amplify either mHGF or hHGF cDNA, as appropriate. HGF was detected using an antibody recognizing both mHGF and hHGF. Actin was used as an internal control for loading. Note that both Ad.mHGF and Ad.hHGF were able to transduce NHP islets equally as assessed by RT-PCR and Western blot.
Mentions: Adenoviruses encoding green fluorescent protein (GFP), β-galactosidase (LacZ), and mHGF and hHGF were prepared as described previously (5,6,8,9). Multiplicity of infection (MOI) was determined using optical density (OD260) and plaque assay. MOI calculations assumed 1,000 cells per islet equivalent (IE; 1 IE = 125-μm-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).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. taeschn@dom.pitt.edu

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 +/- 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 +/- 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 +/- 6.2 mg/dl, n = 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.

Show MeSH
Related in: MedlinePlus