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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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Cell death rates in Ad.mHGF-treated islets in vitro and in vivo. A: Representative photomicrographs of TUNEL staining in vitro in isolated control islets or those transduced with Ad.mHGF 48 h previously. Insulin is shown in red and TUNEL staining in green. Arrows indicate examples of TUNEL-positive β-cells. B: Quantification of TUNEL-positive, insulin-positive cells. The numbers indicate the numbers of cells counted as in Fig. 6. Bars indicate SE C: Examples of TUNEL-positive β-cells in islet grafts 3 days after transduction and transplantation. Arrows indicate examples of TUNEL-positive β-cells. D: Quantitation of TUNEL-positive/insulin-positive cells in islet grafts in vivo. These observations demonstrate that Ad.mHGF treatment reduces cell death in vitro and in vivo. hHGF, human HGF; mHGF, mouse HGF. (Please see http://dx.doi.org/10.2337/db08-1085 for a high-quality representation of this figure.)
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f7: Cell death rates in Ad.mHGF-treated islets in vitro and in vivo. A: Representative photomicrographs of TUNEL staining in vitro in isolated control islets or those transduced with Ad.mHGF 48 h previously. Insulin is shown in red and TUNEL staining in green. Arrows indicate examples of TUNEL-positive β-cells. B: Quantification of TUNEL-positive, insulin-positive cells. The numbers indicate the numbers of cells counted as in Fig. 6. Bars indicate SE C: Examples of TUNEL-positive β-cells in islet grafts 3 days after transduction and transplantation. Arrows indicate examples of TUNEL-positive β-cells. D: Quantitation of TUNEL-positive/insulin-positive cells in islet grafts in vivo. These observations demonstrate that Ad.mHGF treatment reduces cell death in vitro and in vivo. hHGF, human HGF; mHGF, mouse HGF. (Please see http://dx.doi.org/10.2337/db08-1085 for a high-quality representation of this figure.)

Mentions: To assess NHP β-cell proliferation in vivo, animals were injected with bromodeoxyuridine (BrdU) (3–6,9,12,13), and graft-containing kidneys were harvested on the 5th day after transplantation at 6 h after BrdU injection, fixed in 4% paraformaldehyde overnight at 4°C, paraffin embedded, and sectioned. In the graft-containing kidneys, three serial sections separated by 25 μm were deparaffinized, rehydrated, treated in prewarmed 1 mmol/l HCl for 1 h at 37°C, blocked in 2% BSA/PBS for 1 h, and then incubated overnight with anti-BrdU (Abcam, Cambridge, MA) and anti-insulin antibodies (Zymed). After serial washing in PBS containing Tween, slides were incubated for 1 h with secondary antibodies, gel mounted, and coverslipped. Two sections per graft were analyzed. Three fields per section were quantified for BrdU- and insulin-positive cells. To assess β-cell survival, identical procedures were followed, and sections from the same paraffin blocks were costained for insulin and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The percent of TUNEL- and insulin-positive nuclei were quantified as described previously (14) and as described in the legend to Fig. 7.


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)

Cell death rates in Ad.mHGF-treated islets in vitro and in vivo. A: Representative photomicrographs of TUNEL staining in vitro in isolated control islets or those transduced with Ad.mHGF 48 h previously. Insulin is shown in red and TUNEL staining in green. Arrows indicate examples of TUNEL-positive β-cells. B: Quantification of TUNEL-positive, insulin-positive cells. The numbers indicate the numbers of cells counted as in Fig. 6. Bars indicate SE C: Examples of TUNEL-positive β-cells in islet grafts 3 days after transduction and transplantation. Arrows indicate examples of TUNEL-positive β-cells. D: Quantitation of TUNEL-positive/insulin-positive cells in islet grafts in vivo. These observations demonstrate that Ad.mHGF treatment reduces cell death in vitro and in vivo. hHGF, human HGF; mHGF, mouse HGF. (Please see http://dx.doi.org/10.2337/db08-1085 for a high-quality representation of this figure.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Cell death rates in Ad.mHGF-treated islets in vitro and in vivo. A: Representative photomicrographs of TUNEL staining in vitro in isolated control islets or those transduced with Ad.mHGF 48 h previously. Insulin is shown in red and TUNEL staining in green. Arrows indicate examples of TUNEL-positive β-cells. B: Quantification of TUNEL-positive, insulin-positive cells. The numbers indicate the numbers of cells counted as in Fig. 6. Bars indicate SE C: Examples of TUNEL-positive β-cells in islet grafts 3 days after transduction and transplantation. Arrows indicate examples of TUNEL-positive β-cells. D: Quantitation of TUNEL-positive/insulin-positive cells in islet grafts in vivo. These observations demonstrate that Ad.mHGF treatment reduces cell death in vitro and in vivo. hHGF, human HGF; mHGF, mouse HGF. (Please see http://dx.doi.org/10.2337/db08-1085 for a high-quality representation of this figure.)
Mentions: To assess NHP β-cell proliferation in vivo, animals were injected with bromodeoxyuridine (BrdU) (3–6,9,12,13), and graft-containing kidneys were harvested on the 5th day after transplantation at 6 h after BrdU injection, fixed in 4% paraformaldehyde overnight at 4°C, paraffin embedded, and sectioned. In the graft-containing kidneys, three serial sections separated by 25 μm were deparaffinized, rehydrated, treated in prewarmed 1 mmol/l HCl for 1 h at 37°C, blocked in 2% BSA/PBS for 1 h, and then incubated overnight with anti-BrdU (Abcam, Cambridge, MA) and anti-insulin antibodies (Zymed). After serial washing in PBS containing Tween, slides were incubated for 1 h with secondary antibodies, gel mounted, and coverslipped. Two sections per graft were analyzed. Three fields per section were quantified for BrdU- and insulin-positive cells. To assess β-cell survival, identical procedures were followed, and sections from the same paraffin blocks were costained for insulin and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The percent of TUNEL- and insulin-positive nuclei were quantified as described previously (14) and as described in the legend to Fig. 7.

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