Date Published: May 10, 2019
Publisher: Public Library of Science
Author(s): Yuki Miyazaki, Kazutaka Murayama, Ibrahim Fathi, Takehiro Imura, Youhei Yamagata, Kimiko Watanabe, Hiroshi Maeda, Akiko Inagaki, Yasuhiro Igarashi, Shigehito Miyagi, Hiroki Shima, Kazuhiko Igarashi, Takashi Kamei, Michiaki Unno, Masafumi Goto, Atsushi Asakura.
Optimizing 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.
Islet 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.
No 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.
The 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.
Although pancreatic islet transplantation is a promising and safe therapy for type 1 diabetic patients[1, 2], many issues remain to be solved. For example, 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 [3–6]. Furthermore, the successful islet isolation rate has increased through appropriate donor selection, but the successful islet isolation rate of whole donor pancreata is still poor [7, 8]. 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.
To establish a tailor-made donor tissue-specific islet isolation protocol, two factors appear to be essential. The first is a highly purified tissue dissociation enzyme. If poorly characterized unknown proteases derived from C. histolyticum are included in the dissociation enzyme, the equation between the dissociation enzyme and target matrix will not work efficiently. The second crucial factor is identifying the target extracellular matrix of each enzyme component. We previously reported that Col III is a key target matrix of ColH , and Col V is a key substrate for ColG using highly purified recombinant collagenase of each subtype . Based on these novel findings, we investigated the relationship between Col III and Col V expression in pancreatic tissues and influence of ColG:ColH ratio on islet isolation outcome in different rat strain. Our data suggested that the Col III expression and/or the composition of homotrimeric Col I in pancreatic tissues may be useful for optimizing the ColG:ColH ratio in islet isolation.