Abstract
BackgroundOptimizing the collagenase G (ColG):collagenase H (ColH) ratio is a key strategy for achieving tailored donor-tissue specific islet isolation. Collagen V (Col V) and collagen III (Col III) are crucial target matrices of ColG and ColH, respectively. We herein investigated the relevance between the expression of target matrices in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome.MethodsIslet isolation was performed in Lewis and SD rats using different ColG:ColH ratios (5:1, 1:1 and 1:5; n = 7/group). The composition of Col III and Col V was examined using immunohistochemical staining, real-time polymerase chain reaction (PCR), Western blotting and mass spectrometry. Chain types in collagen I (Col I) were also assessed using mass spectrometry.ResultsNo beneficial effects were observed by increasing the ColG amount, irrespective of the rat strain. In contrast, the islet yield in Lewis rats was considerably increased by high amounts of ColH but decreased in SD rats, suggesting that Lewis pancreas contains more Col III than SD pancreas. Neither immunohistochemical nor real-time PCR showed correlation with isolation outcome. However, Western blotting revealed that Lewis contained considerably higher amount of Col III than SD (p = 0.10). Likewise, Col-I(α1)/Col-III(α1) and Col-I(α2)/Col-III(α1) were significantly lower in Lewis than in SD rats (p = 0.007, respectively). Furthermore, the isolation outcome was considerably correlated with the composition of homotrimeric Col I.ConclusionsThe Col III expression and the composition of homotrimeric Col I in pancreatic tissues determined using mass analyses appeared useful for optimizing the ColG:ColH ratio in islet isolation.
Highlights
Pancreatic islet transplantation is a promising and safe therapy for type 1 diabetic patients[1, 2], many issues remain to be solved
The collagen III (Col III) expression and the composition of homotrimeric collagen I (Col I) in pancreatic tissues determined using mass analyses appeared useful for optimizing the collagenase G (ColG):collagenase H (ColH) ratio in islet isolation
We reported that collagen III (Col III) is a key target matrix of ColH using highly purified recombinant collagenases [13]
Summary
Pancreatic islet transplantation is a promising and safe therapy for type 1 diabetic patients[1, 2], many issues remain to be solved. Pancreata from two or more donors are required for one diabetic patient to achieve insulin independence in many cases, despite improvements in human pancreatic islet isolation procedures over the past three decades [3,4,5,6]. A more efficient islet isolation procedure may increase the islet yield from one donor pancreas and solve such problems, helping relieve the organ shortage issue. Optimizing the collagenase G (ColG):collagenase H (ColH) ratio is a key strategy for achieving tailored donor-tissue specific islet isolation. We investigated the relevance between the expression of target matrices in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome
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