Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming

Omori, Keiko; Kobayashi, Eiji; Komatsu, Hirotake; Rawson, Jeffrey; Agrawal, Garima; Parimi, Mounika; Oancea, Alina R.; Valiente, Luis; Ferreri, Kevin; Al-Abdullah, Ismail H.; Kandeel, Fouad; Takahashi, Masafumi; Mullen, Yoko

doi:10.1152/ajpendo.00441.2015

Cited by 6 publications

(3 citation statements)

References 47 publications

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“…Inflammation-related gene expressions ( IL1B and TNF ) ( 27 , 28 ) showed the trend of lower inflammation in microwells compared to flat dishes though not statistically significant ( Supplementary Figure 5 ). Expressions of apoptosis-related genes ( BBC3 and FASLG ) ( 29 , 30 ) and necrosis-related genes ( HMGB1 ) ( 31 ) in flat dishes and microwells did not demonstrate the primary cause of cell death; rather, they were donor-dependent. Quantification of Live/Dead stained cultured islets demonstrated that the microwell dish significantly improved the viability compared to the flat dish (60.8% vs. 86.2%, P = 0.0196; Figure 4F ).…”

Section: Resultsmentioning

confidence: 99%

Microwell culture platform maintains viability and mass of human pancreatic islets

Kato

Miwa²,

Quijano

et al. 2022

Front. Endocrinol.

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BackgroundTransplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures.MethodHuman islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for <160 µm-islets and 200 µm × 370 µm-microwells for >160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested.ResultsThe microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions.ConclusionMicrowell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

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Section: Resultsmentioning

confidence: 99%

Microwell culture platform maintains viability and mass of human pancreatic islets

Kato

Miwa²,

Quijano

et al. 2022

Front. Endocrinol.

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“…The viability index of rat islets after 72 h of cold preservation in UW solution supplemented with 500 µg/mL DAFP-1 (1.17 ± 0.08) was significantly higher than islets preserved in UW solution alone control ( Figure 5 B). It is known that the cold preservation-mediated islet injury is manifested during the rewarming of the islets [ 34 , 43 ]. Therefore, we further assessed the recovery and viability of islets after cold preservation followed by rewarming.…”

Section: Resultsmentioning

confidence: 99%

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival

et al. 2022

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Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic β-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary β-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation.

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“…PUMA activation is known to be involved in p53-mediated apoptosis 36,37 and, interestingly, we observed an increase in PUMA (BBC3) expression in rat islets incubated in vitro under hypoxia and transplanted to immunodeficient mice. Other studies reported that PUMA is involved in cell death in human and rodent islets exposed to various insults [38][39][40][41][42][43] . A correlation has also been reported between PUMA gene expression and dysfunction of the grafted human islets in immunodeficient mice 38 .…”

Section: Discussionmentioning

confidence: 98%

NLRP3 Inflammasome is Activated in Rat Pancreatic Islets by Transplantation and Hypoxia

Lavallard

Cottet‐Dumoulin

Wassmer

et al. 2020

Sci Rep

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Hypoxia, IL-1β production and oxidative stress are involved in islet graft dysfunction and destruction. However, the link between these events has not yet been determined in transplanted islets. The goal of this study was to determine whether NLRP3 inflammasome is responsible for IL-1β production and if it is activated by hypoxia-induced oxidative stress in transplanted islets. Rat islets were transplanted under the kidney capsule of immunodeficient mice. At different times post-transplantation, blood samples were collected and islet grafts harvested. Rat islets were also incubated in vitro either under normoxia or hypoxia for 24 h, in the absence or presence of inhibitors of NLRP3 inflammasome (CASP1 inhibitor) or oxidative stress (NAC). NLRP3, CASP1, IL1B, BBC3 pro-apoptotic and BCL2 antiapoptotic genes in transplanted and in vitro incubated islets were then studied using real time PCR. IL-1β released in the blood and in the supernatant was quantified by ELISA. Cell death was analysed by propidium iodide and Annexin-V staining. NLRP3, CASP1 and BBC3 in transplanted rat islets and IL-1β in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1β released in supernatant increased compared to islets incubated under normoxia. These effects were prevented by the inhibition of NLRP3 inflammasome by CASP1 or oxidative stress by NAC. However, these inhibitors did not prevent hypoxia-induced rat islet death. These data show that NLRP3 inflammasome in rat islets is transiently activated after their transplantation and induced through oxidative stress in vitro. However, NRLP3 inflammasome inhibition does not protect islet cells against hypoxia.

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Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming

Cited by 6 publications

References 47 publications

Microwell culture platform maintains viability and mass of human pancreatic islets

Microwell culture platform maintains viability and mass of human pancreatic islets

Submilligram Level of Beetle Antifreeze Proteins Minimize Cold-Induced Cell Swelling and Promote Cell Survival

NLRP3 Inflammasome is Activated in Rat Pancreatic Islets by Transplantation and Hypoxia

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