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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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Related in: MedlinePlus

Adenoviral persistence in islet grafts after transplantation. A: Viral constructs and primers. The primers were designed to specifically amplify only mHGF DNA derived from the adenovirus. Primers used were 5′-GCC ATG CCA AAT CGT CCT GG-3′ and reverse 3′-GTA GTT TGT CCA ATT ATG TCA CAC CAC-5′ for Ad.mHGF and 5′-CAT TCC AAA TAT GAG ATG CAT TG-3′ and reverse 5′-TAA AAA AGT ATT AAG GCG AAG ATT A-3′ for NHP actin. B: Persistence of viral DNA in vitro. NHP islets transduced with Ad.mHGF at 250 MOI were harvested 24 h after transduction. INS1 cells were transduced with Ad.mHGF at 1,000 MOI for 1 h and were used as a positive control. Note that NHP actin is only present in NHP islets, but not in INS1 cells. C: Assessment of viral DNA in vivo. Kidneys bearing the NHP islet graft transduced with Ad.mHGF, and contralateral kidneys containing no islet graft were harvested 24 h and 7, 14, 28, and 42 days after transplant. DNA and RNA were extracted. INS1 cells infected with Ad.mHGF were used as a positive control. Note that the adenoviral DNA was still abundant 42 days after transplant in kidneys bearing the graft. The presence of the graft was confirmed by the presence of NHP actin. Kidneys with no islet grafts were negative for mouse HGF and NHP actin but were positive for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). D: Assessment of viral mRNA in vivo. The presence of viral mouse HGF and NHP actin mRNAs was confirmed in kidneys bearing the NHP grafts, whereas contralateral kidneys were negative. INS1 cells transduced with Ad.mHGF were used as positive controls. E: Control PCR from mRNA samples used in D with no reverse transcriptase (RT). To confirm that no genomic DNA contaminated the samples used in D, PCR was performed on the same samples used in D but with no reverse transcriptase. INS1 cells transduced with Ad.mHGF were used as a positive control. RT was performed before the PCR. Note that only the INS1 cells were positive, confirming that samples used in D were free of contaminating DNA.
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f5: Adenoviral persistence in islet grafts after transplantation. A: Viral constructs and primers. The primers were designed to specifically amplify only mHGF DNA derived from the adenovirus. Primers used were 5′-GCC ATG CCA AAT CGT CCT GG-3′ and reverse 3′-GTA GTT TGT CCA ATT ATG TCA CAC CAC-5′ for Ad.mHGF and 5′-CAT TCC AAA TAT GAG ATG CAT TG-3′ and reverse 5′-TAA AAA AGT ATT AAG GCG AAG ATT A-3′ for NHP actin. B: Persistence of viral DNA in vitro. NHP islets transduced with Ad.mHGF at 250 MOI were harvested 24 h after transduction. INS1 cells were transduced with Ad.mHGF at 1,000 MOI for 1 h and were used as a positive control. Note that NHP actin is only present in NHP islets, but not in INS1 cells. C: Assessment of viral DNA in vivo. Kidneys bearing the NHP islet graft transduced with Ad.mHGF, and contralateral kidneys containing no islet graft were harvested 24 h and 7, 14, 28, and 42 days after transplant. DNA and RNA were extracted. INS1 cells infected with Ad.mHGF were used as a positive control. Note that the adenoviral DNA was still abundant 42 days after transplant in kidneys bearing the graft. The presence of the graft was confirmed by the presence of NHP actin. Kidneys with no islet grafts were negative for mouse HGF and NHP actin but were positive for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). D: Assessment of viral mRNA in vivo. The presence of viral mouse HGF and NHP actin mRNAs was confirmed in kidneys bearing the NHP grafts, whereas contralateral kidneys were negative. INS1 cells transduced with Ad.mHGF were used as positive controls. E: Control PCR from mRNA samples used in D with no reverse transcriptase (RT). To confirm that no genomic DNA contaminated the samples used in D, PCR was performed on the same samples used in D but with no reverse transcriptase. INS1 cells transduced with Ad.mHGF were used as a positive control. RT was performed before the PCR. Note that only the INS1 cells were positive, confirming that samples used in D were free of contaminating DNA.

Mentions: For the in vitro studies, uninfected and infected islets were harvested 24 and 48 h after infection and were frozen at −80°C until extraction. For the in vivo studies, the left kidneys containing the NHP islet graft and the right (nontransplanted) kidneys were harvested on days 1, 7, 14, 28, and 42 and were frozen at −80°C until extraction. RNA and DNA extraction were performed using Trizol (Invitrogen, Carlsbad, CA). RNA samples were treated with DNase to prevent DNA contamination. PCR for adenoviral mouse HGF was performed with the oligonucleotides described in Fig. 5. As a control to insure that no contaminated DNA was amplified, PCR was performed on RNA samples treated with DNase with no previous reverse transcription.


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)

Adenoviral persistence in islet grafts after transplantation. A: Viral constructs and primers. The primers were designed to specifically amplify only mHGF DNA derived from the adenovirus. Primers used were 5′-GCC ATG CCA AAT CGT CCT GG-3′ and reverse 3′-GTA GTT TGT CCA ATT ATG TCA CAC CAC-5′ for Ad.mHGF and 5′-CAT TCC AAA TAT GAG ATG CAT TG-3′ and reverse 5′-TAA AAA AGT ATT AAG GCG AAG ATT A-3′ for NHP actin. B: Persistence of viral DNA in vitro. NHP islets transduced with Ad.mHGF at 250 MOI were harvested 24 h after transduction. INS1 cells were transduced with Ad.mHGF at 1,000 MOI for 1 h and were used as a positive control. Note that NHP actin is only present in NHP islets, but not in INS1 cells. C: Assessment of viral DNA in vivo. Kidneys bearing the NHP islet graft transduced with Ad.mHGF, and contralateral kidneys containing no islet graft were harvested 24 h and 7, 14, 28, and 42 days after transplant. DNA and RNA were extracted. INS1 cells infected with Ad.mHGF were used as a positive control. Note that the adenoviral DNA was still abundant 42 days after transplant in kidneys bearing the graft. The presence of the graft was confirmed by the presence of NHP actin. Kidneys with no islet grafts were negative for mouse HGF and NHP actin but were positive for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). D: Assessment of viral mRNA in vivo. The presence of viral mouse HGF and NHP actin mRNAs was confirmed in kidneys bearing the NHP grafts, whereas contralateral kidneys were negative. INS1 cells transduced with Ad.mHGF were used as positive controls. E: Control PCR from mRNA samples used in D with no reverse transcriptase (RT). To confirm that no genomic DNA contaminated the samples used in D, PCR was performed on the same samples used in D but with no reverse transcriptase. INS1 cells transduced with Ad.mHGF were used as a positive control. RT was performed before the PCR. Note that only the INS1 cells were positive, confirming that samples used in D were free of contaminating DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Adenoviral persistence in islet grafts after transplantation. A: Viral constructs and primers. The primers were designed to specifically amplify only mHGF DNA derived from the adenovirus. Primers used were 5′-GCC ATG CCA AAT CGT CCT GG-3′ and reverse 3′-GTA GTT TGT CCA ATT ATG TCA CAC CAC-5′ for Ad.mHGF and 5′-CAT TCC AAA TAT GAG ATG CAT TG-3′ and reverse 5′-TAA AAA AGT ATT AAG GCG AAG ATT A-3′ for NHP actin. B: Persistence of viral DNA in vitro. NHP islets transduced with Ad.mHGF at 250 MOI were harvested 24 h after transduction. INS1 cells were transduced with Ad.mHGF at 1,000 MOI for 1 h and were used as a positive control. Note that NHP actin is only present in NHP islets, but not in INS1 cells. C: Assessment of viral DNA in vivo. Kidneys bearing the NHP islet graft transduced with Ad.mHGF, and contralateral kidneys containing no islet graft were harvested 24 h and 7, 14, 28, and 42 days after transplant. DNA and RNA were extracted. INS1 cells infected with Ad.mHGF were used as a positive control. Note that the adenoviral DNA was still abundant 42 days after transplant in kidneys bearing the graft. The presence of the graft was confirmed by the presence of NHP actin. Kidneys with no islet grafts were negative for mouse HGF and NHP actin but were positive for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). D: Assessment of viral mRNA in vivo. The presence of viral mouse HGF and NHP actin mRNAs was confirmed in kidneys bearing the NHP grafts, whereas contralateral kidneys were negative. INS1 cells transduced with Ad.mHGF were used as positive controls. E: Control PCR from mRNA samples used in D with no reverse transcriptase (RT). To confirm that no genomic DNA contaminated the samples used in D, PCR was performed on the same samples used in D but with no reverse transcriptase. INS1 cells transduced with Ad.mHGF were used as a positive control. RT was performed before the PCR. Note that only the INS1 cells were positive, confirming that samples used in D were free of contaminating DNA.
Mentions: For the in vitro studies, uninfected and infected islets were harvested 24 and 48 h after infection and were frozen at −80°C until extraction. For the in vivo studies, the left kidneys containing the NHP islet graft and the right (nontransplanted) kidneys were harvested on days 1, 7, 14, 28, and 42 and were frozen at −80°C until extraction. RNA and DNA extraction were performed using Trizol (Invitrogen, Carlsbad, CA). RNA samples were treated with DNase to prevent DNA contamination. PCR for adenoviral mouse HGF was performed with the oligonucleotides described in Fig. 5. As a control to insure that no contaminated DNA was amplified, PCR was performed on RNA samples treated with DNase with no previous reverse transcription.

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