Islet Vascularization Research Articles

Adipose tissue–derived mesenchymal stem cells promote the vascularization of pancreatic islets transplanted into decellularized pancreatic skeletons

We have recently developed a model of pancreatic islet transplantation into a decellularized pancreatic tail in rats. As the pancreatic skeletons completely lack endothelial cells, we investigated the effect of co-transplantation of mesenchymal stem cells and endothelial cells to promote revascularization.Decellularized matrix of the pancreatic tail was prepared by perfusion with Triton X-100, sodium dodecyl sulfate and DNase solution. Isolated pancreatic islets were infused into the skeletons via the splenic vein either alone, together with adipose tissue–derived mesenchymal stem cells (adMSCs), or with a combination of adMSCs and rat endothelial cells (rat ECs). Repopulated skeletons were transplanted into the subcutaneous tissue and explanted 9 days later for histological examination. Possible immunomodulatory effects of rat adMSCs on the survival of highly immunogenic green protein–expressing human ECs were also tested after their transplantation beneath the renal capsule. The immunomodulatory effects of adMSCs were also tested in vitro using the Invitrogen Click-iT EdU system.In the presence of adMSCs, the proliferation of splenocytes as a response to phytohaemagglutinin A was reduced by 47% (the stimulation index decreased from 1.7 to 0.9, P = 0.008) and the reaction to human ECs was reduced by 58% (the stimulation index decreased from 1.6 to 0.7, P = 0.03). Histological examination of the explanted skeletons seeded only with the islets showed their partial disintegration and only a rare presence of CD31-positive cells. However, skeletons seeded with a combination of islets and adMSCs showed preserved islet morphology and rich vascularity. In contrast, the addition of syngeneic rat ECs resulted in islet-cell necrosis with only few endothelial cells present. Live green fluorescence–positive endothelial cells transplanted either alone or with adMSCs were not detected beneath the renal capsule.Though the adMSCs significantly reduced in vitro proliferation stimulated by either phytohaemagglutinin A or by xenogeneic human ECs, in vivo co-transplanted adMSCs did not suppress the post-transplant immune response to xenogeneic ECs. Even in the syngeneic model, ECs co-transplantation did not lead to sufficient vascularization in the transplant area. In contrast, islet co-transplantation together with adMSCs successfully promoted the revascularization of extracellular matrix in the subcutaneous tissue.

Antihyperlipidemic and Histopathological Pancreas Analysis of Muntingia calabura L. Fruit Extract on Alloxan-Induced Diabetic Mice

Hyperlipidemia is clinically manifested by high levels of Total Cholesterol (TC), Triglycerides (TG), Low Density Lipoprotein (LDL) and low levels of High Density Lipoprotein (HDL) in the blood. Antihyperlipidemic drugs available currently have adverse effects. One of the medicine plants with antihyperlipidemic properties with no side effects is Muntingia calabura fruit. The sample used for this research were 30 male mice were divided into 5 groups, i.e., a negative control, a diabetic control, a positive control group, and two treatment groups which were given 100 and 300mg/kg of M. calabura fruit extract every day for 14 days. Examination of TC, TG, HDL, LDL and histopathological pancreas were determined after administration of the extract orally for 14 days. The results demonstrated an administration of glibenclamide and M. calabura fruit could effectively reduce TC, TG and LDL and increase HDL compared to the diabetes control group (P<0.05). In the diabetes control group, the mice given alloxan 150mg/kg showed a decrease in Langerhans islet density, vascularity, and islet injury compared to the normal control group. Mice given M. calabura fruit extract showed a significant increase in pancreatic Langerhans cells granulation and cell density. The conclusion of this research is M. calabura fruit extract showed improvements in lipid profile and pancreatic Langerhans cell regeneration. Therefore, the fruit extract of M. calabura is a potential antihyperlipidemic drug.

Dapagliflozin improves pancreatic islet function by attenuating microvascular endothelial dysfunction in type 2 diabetes.

Sodium-glucose co-transporter 2 inhibitors, including dapagliflozin, improve ß cell function in type 2 diabetic individuals. Whether dapagliflozin can protect islet microvascular endothelial cells (IMECs) and thus contribute to the improvement of ß cell function remains unknown. The db/db mice were treated with dapagliflozin or vehicle for 6weeks. ß cell function, islet capillaries and the levels of inflammatory chemokines in IMECs were detected. The mouse IMEC cell line MS-1 cells were incubated with palmitate and/or dapagliflozin for 24h. Angiogenesis and inflammatory chemokine levels were evaluated, and the involved signalling pathways were analysed. The mouse ß cell line MIN6 cells, in the presence or absence of co-culture with MS-1 cells, were treated with palmitate and/or dapagliflozin for 24h. The expression of ß cell specific markers and insulin secretion in MIN6 cells were determined. Dapagliflozin significantly improved ß cell function, increased islet capillaries and decreased the levels of inflammatory chemokines of IMECs in db/db mice. In the palmitate-treated MS-1 cells, angiogenesis was enhanced and the levels of inflammatory chemokines were downregulated by dapagliflozin. Either a PI3K inhibitor or mTOR inhibitor eliminated the dapagliflozin-mediated effects. Importantly, dapagliflozin attenuated the palmitate-induced downregulation of ß cell function-related gene expression and insulin secretion in MIN6 cells co-cultured with MS-1 cells but not in those on mono-culture. Dapagliflozin restores islet vascularisation and attenuates the inflammation of IMECs in type 2 diabetic mice. The dapagliflozin-induced improvement of ß cell function is at least partially accounted for by its beneficial effects on IMECs in a PI3K/Akt-mTOR-dependent manner.

13-OR: Cystic Fibrosis Donors Exhibit Striking Decrease in Pancreatic Islet and Exocrine Capillaries

Glucose intolerance and insulin deficiency are common features of cystic fibrosis (CF) , with nearly half of adult CF patients developing CF-related diabetes (CFRD) , but the underlying pathophysiology remains poorly understood. Extensive destruction of the exocrine pancreas occurs in most CF patients. However, islets are still readily detectable, suggesting β cell loss is not the major driver of impaired insulin release. Islets are normally highly vascularized, and islet capillaries are the source of key growth factors that maintain β cell health and function. Islet capillary morphology is altered in type 1 and type 2 diabetes, with increased capillary density and/or vessel thickening being observed. In the present study we aimed to determine whether islet capillaries are also disrupted in CF/CFRD. Pancreas sections (8 control and 8 CF/CFRD, age-matched) were stained for CD31, a marker of vascular endothelial cells. Islets in microscopy images were manually identified (17.8±2.8[SEM] per sample) and CD31-positive area within islet and exocrine was quantified. Mean islet cross-sectional area did not significantly differ between CF and non-CF pancreas specimens. CD31 positive area, expressed as a fraction of islet area, was significantly decreased in CF pancreas specimens (2.8±0.9% vs. 6.1±1.2%; p=0.05) . Islet capillary density (the number of CD31+ objects normalized to islet area) was also decreased (0.88±0.17x103/μm2 vs. 3.8±0.57x103/μm2; p=0.0004) , as was exocrine tissue CD31 positive area (0.17±0.03% vs. 1.5±0.23%; p<0.0001) . Our data demonstrate a significant deficit in islet and pancreatic vascularity in CF/CFRD. This contrasts markedly with the increase in islet capillary density seen in both T1D and T2D and underscores the presence of distinct features of islet morphology that characterize CF. Given that islet capillaries support β-cell function and survival, their loss likely contributes to impaired β-cell function. Disclosure J.J.Castillo: None. A.Aplin: None. R.Akter: None. R.L.Hull-meichle: Research Support; Casma Therapeutics. Funding Cystic Fibrosis Foundation Fellowship (CASTIL20F0)

Systematic evaluation of clinically used biomaterials to determine their suitability for fabrication of beta cell delivery devices

As a treatment for type I diabetes, clinical islet transplantation (CIT) in which donor islets of Langerhans are transplanted intrahepatically has become a viable option for patients. However, the success of this procedure is limited by factors including ischemia, host immunological factors, and delayed vascularization of the hypoxia-sensitive islets. One solution would be to use a synthetic polymer scaffold as a carrier for the transplanted islets, as it would allow for their transplantation into a more favorable environment and could protect the cells from host immune reactions. To realize this potential solution, it is important that the synthetic polymer used does not interfere with the functionality and survival of the islets. In order to determine which synthetic polymers best meet this requirement, we examined the interactions of human islets from six donors with four clinically approved materials: polyetheretherketone (PEEK), polyvinylidene fluoride (PVDF), polyphenylsulfone (PPSU) and polysulfone (PSU) in vitro. Human islet morphology, viability, insulin secretion, functionality and gene expression were investigated to assess the suitability of these synthetic polymers as a carrier for transplanted islets. We found three of the synthetic synthetic polymers (PEEK, PPSU and PVDF) showed promise based on their overall performance, while the glucose responsiveness of islets cultured on PSU resulted in significantly reduced insulin secretion from five of six donors. Our findings demonstrate that close examination of human islets and their interaction with synthetic polymers is an important factor to consider when selecting synthetic polymers for engineering islet replacement devices.

Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient β cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

KRP-203 Is a Desirable Immunomodulator for Islet Allotransplantation.

Background.The current standard immunosuppressive regimens, calcineurin inhibitors, have diabetogenic and anti-vascularization effects on islet grafts. KRP-203, a sphingosine-1-phosphate functional antagonist, exerts its immunomodulatory function through lymphocyte sequestration. However, the effect of this antagonist on islets is unclear. We examined the effect of KRP-203 on the islet function and vascularization and sought a calcineurin-free regimen for islet allotransplantation.Methods.KRP-203 was administered for 14 d to mice, then diabetogenic effect was evaluated by blood glucose levels and a glucose tolerance test. Static glucose stimulation, the breathing index, and insulin/DNA were examined using isolated islets. Islet neovascularization was evaluated using a multiphoton laser scanning microscope. After islet allotransplantation with either KRP-203 alone, sirolimus alone, or both in combination, the graft survival was evaluated by blood glucose levels and immunohistochemical analyses. A mixed lymphocyte reaction was also performed to investigate the immunologic characteristics of KRP-203 and sirolimus.Results.No significant differences in the blood glucose levels or glucose tolerance were observed between the control and KRP-203 groups. Functional assays after islet isolation were also comparable. The multiphoton laser scanning microscope showed no inhibitory effect of KRP-203 on islet neovascularization. Although allogeneic rejection was effectively inhibited by KRP-203 monotherapy (44%), combination therapy prevented rejection in most transplanted mice (83%).Conclusions.KRP-203 is a desirable immunomodulator for islet transplantation because of the preservation of the endocrine function and lack of interference with islet neovascularization. The combination of KRP-203 with low-dose sirolimus may be promising as a calcineurin-free regimen for islet allotransplantation.

Chronic Fetal Leucine Infusion Does Not Potentiate Glucose-Stimulated Insulin Secretion or Affect Pancreatic Islet Development in Late-Gestation Growth-Restricted Fetal Sheep

Growth-restricted fetuses have attenuated glucose-stimulated insulin secretion (GSIS), smaller pancreatic islets, less pancreatic β-cells, and less pancreatic vascularization compared with normally growing fetuses. Infusion of leucine into normal late-gestation fetal sheep potentiates GSIS, as well as increases pancreatic islet size, the proportion of the pancreas and islet comprising β-cells, and pancreatic and islet vascularity. In addition, leucine stimulates hepatocyte growth factor (HGF ) mRNA expression in islet endothelial cells isolated from normal fetal sheep. We hypothesized that a 9-d leucine infusion would potentiate GSIS and increase pancreatic islet size, β-cells, and vascularity in intrauterine fetal growth restriction (IUGR) fetal sheep. We also hypothesized that leucine would stimulate HGF mRNA in islet endothelial cells isolated from IUGR fetal sheep. Late-gestation Columbia-Rambouillet IUGR fetal sheep (singleton or twin) underwent surgeries to place vascular sampling and infusion catheters. Fetuses were randomly allocated to receive a 9-d leucine infusion to achieve a 50-100% increase in leucine concentrations or a control saline infusion. GSIS was measured and pancreas tissue was processed for histologic analysis. Pancreatic islet endothelial cells were isolated from IUGR fetal sheep and incubated with supplemental leucine. Data were analyzed by mixed-models ANOVA; Student, Mann-Whitney, or a paired t test; or a test of equality of proportions. Chronic leucine infusion in IUGR fetuses did not affect GSIS, islet size, the proportion of the pancreas comprising β-cells, or pancreatic or pancreatic islet vascularity. In isolated islet endothelial cells from IUGR fetuses, HGF mRNA expression was not affected by supplemental leucine. IUGR fetal sheep islets are not responsive to a 9-d leucine infusion with respect to insulin secretion or any histologic features measured. This is in contrast to the response in normally growing fetuses. These results are important when considering nutritional strategies to prevent the adverse islet and β-cell consequences in IUGR fetuses.

Islet vascularization is regulated by primary endothelial cilia via VEGF-A-dependent signaling

Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Bbs4-/-islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted Bbs4-/- islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to β-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human BBS4 silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.

Grafting Islets to a Dissected Peritoneal Pouch to Improve Transplant Survival and Function.

Although the liver is the primary site for clinical islet transplantation, it poses several restrictions, especially limited tissue volume due to portal vein pressure. We evaluated the preperitoneal space as an extrahepatic islet transplant site to deliver high tissue volumes and sustain long-term graft function. A peritoneal pouch was formed by dissecting the parietal peritoneum from the transversalis fascia of mice. Syngeneic C57BL/6 donor islets were transplanted into the peritoneal pouch of diabetic mouse recipients. Blood glucose was monitored for islet function, and miR-375 was analyzed for islet damage. Islet graft morphology and vascularization were evaluated by immunohistochemistry. [F] fluoro-D-glucose positron emission tomography/computed tomography was used to image islet grafts. Transplantation of 300 syngeneic islets into the peritoneal pouch of recipients reversed hyperglycemia for >60 days. Serum miR-375 was significantly lower in the peritoneal pouch group than in the peritoneal cavity group. Peritoneal pouch islet grafts showed high neovascularization and sustained insulin and glucagon expression up to 80 days posttransplantation. A peritoneal pouch graft with high tissue volume (1000 islets) could be visualized by positron emission tomography/computed tomography imaging. Human islets transplanted into the peritoneal pouch of diabetic nude mice also reversed hyperglycemia successfully. Islets transplanted into a dissected peritoneal pouch show high efficiency to reverse diabetes and sustain islet graft function. The preperitoneal site has the advantages of capacity for high tissue volume, enriched revascularization and minimal inflammatory damage. It can also serve as an extrahepatic site for transplanting large volume of islets necessitated in islet autotransplantation.

A Chronic Fetal Leucine Infusion Potentiates Fetal Insulin Secretion and Increases Pancreatic Islet Size, Vascularity, and β Cells in Late-Gestation Sheep

Background:Infusion of a complete amino acid mixture into normal late-gestation fetal sheep potentiates glucose-stimulated insulin secretion (GSIS). Leucine acutely stimulates insulin secretion in late-gestation fetal sheep and isolated fetal sheep islets in vitro. Objectives:We hypothesized that a 9-d leucine infusion would potentiate GSIS in fetal sheep. Methods:Columbia-Rambouillet fetal sheep at 126 days of gestation received a 9-d leucine infusion to achieve a 50%–100% increase in leucine concentrations or a control infusion. At the end of the infusion we measured GSIS, pancreatic morphology, and expression of pancreatic mRNAs. Pancreatic islet endothelial cells (ECs) were isolated from fetal sheep and incubated with supplemental leucine or vascular endothelial growth factor A (VEGFA) followed by collection of mRNA. Data measured at multiple time points were compared with a repeated-measures 2-factor ANOVA. Data measured at 1 time point were compared using Student's t test or the Mann–Whitney test. Results:Glucose-stimulated insulin concentrations were 80% higher in leucine-infused (LEU) fetuses than in controls (P < 0.05). In the pancreas, LEU fetuses had a higher proportion of islets >5000 μm2 than controls (75% more islets >5000 μm2; P < 0.05) and a larger proportion of the pancreas that stained for β cells (12% greater; P < 0.05). Pancreatic and pancreatic islet vascularity were both 25% greater in LEU fetuses (P < 0.05). PancreaticVEGFAand hepatocyte growth factor (HGF) mRNA expressions were 38% and 200% greater in LEU fetuses than in controls (P < 0.05), respectively. In isolated islet ECs,HGFmRNA was 20% and 50% higher after incubation in ce:supplemental leucine (P < 0.05) or VEGFA (P < 0.01), respectively. Conclusions:A 9-d leucine infusion potentiates fetal GSIS, demonstrating that glucose and leucine act synergistically to stimulate insulin secretion in fetal sheep. A greater proportion of the pancreas being comprised of β cells and higher pancreatic vascularity contributed to the higher GSIS.

Beta-cell β1 integrin deficiency affects in utero development of islet growth and vascularization.

The β1 integrin subunit contributes to pancreatic beta cell growth and function through communication with the extracellular matrix (ECM). The effects of in vitro and in vivo β1 integrin knockout have been extensively studied in mature islets, yet no study to date has examined how the loss of β1 integrin during specific stages of pancreatic development impacts beta cell maturation. Beta-cell-specific tamoxifen-inducible Cre recombinase (MIP-CreERT) mice were crossed with mice containing floxed Itgb1 (β1 integrin) to create an inducible mouse model (MIPβ1KO) at the second transition stage (e13.5)of pancreas development. By e19.5-20.5, the expression of beta-cell β1 integrin in fetal MIPβ1KO mice was significantly reduced and these mice displayed decreased beta cell mass, density and proliferation. Morphologically, fetal MIPβ1KO pancreata exhibited reduced islet vascularization and nascent endocrine cells in the ductal region. In addition, decreased ERK phosphorylation was observed in fetal MIPβ1KO pancreata. The expression of transcription factors needed for beta-cell development was unchanged in fetal MIPβ1KO pancreata. The findings from this study demonstrate that β1 integrin signaling is required during a transition-specific window in the developing beta-cell to maintain islet mass and vascularization.

A better reception for islets

Transplantation Pancreatic islet transplantation can stabilize blood glucose concentrations in individuals with type 1 diabetes, but transplant function decreases over time. Forbes et al. investigated the supportive effects of cotransplanting islets and human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs). HUCPVCs inhibited T cells, expressed proregenerative and immune-regulatory markers in vitro, and increased islet vascularization in vivo. Cotransplanting human islets and HUCPVCs under the kidney capsule or via the hepatic portal vein improved control of blood glucose in diabetic immunodeficient and immunocompetent mouse models for up to 16 weeks. Sci. Transl. Med. 12 , eaan5907 (2020).

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization.

Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes.

Using Recombinant Human Collagen With Basic Fibroblast Growth Factor to Provide a Simulated Extracellular Matrix Microenvironment for the Revascularization and Attachment of Islets to the Transplantation Region.

Islet transplantation is considered a potential therapeutic option to reverse diabetes. The pancreatic basement membrane contains a variety of extracellular matrix (ECM) proteins. The abundant ECM is essential for the survival of transplanted islets. However, the ECM proteins necessary for maintaining islet vascularization and innervation are impaired by enzymatic digestion in the isolation process before islet transplantation, leading to destruction of islet microvessels. These are the primary concern and major barrier for long-term islet survival and function. Thus, it is crucial to create an appropriate microenvironment for improving revascularization and islet function to achieve better transplantation outcome. Given the importance of the presence of ECM proteins for islets, we introduce recombinant human collagen (RHC) to construct a simulated ECM microenvironment. To accelerate revascularization and reduce islet injury, we add basic fibroblast growth factor (bFGF) to RHC, a growth factor that has been shown to promote angiogenesis. In order to verify the outcome, islets were treated with RHC combination containing bFGF and then implanted into kidney capsule in type 1 diabetic mouse models. After transplantation, 30-day-long monitoring displayed that 16 mg–60 ng RHC-bFGF group could serve as superior transplantation outcome. It reversed the hyperglycemia condition in host rapidly, and the OGTT (oral glucose tolerance test) showed a similar pattern with the control group. Histological assessment showed that 16 mg–60 ng RHC-bFGF group attenuated apoptosis, promoted cellular proliferation, triggered vascularization, and inhibited inflammation reaction. In summary, this work demonstrates that application of 16 mg–60 ng RHC-bFGF and islets composite enhance the islet survival, function, and long-term transplantation efficiency.

Islet Architecture Controls Synchronous β Cell Response to Glucose in the Intact Mouse Pancreas &lt;i&gt;in vivo&lt;/i&gt;

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets lose synchronized intra-islet Ca2+ oscillations between β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results will have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Hypoxia induction in cultured pancreatic islets enhances endothelial cell morphology and survival while maintaining beta-cell function.

Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue. We now aim to determine the impact of hypoxia on islet vascularization. We induced hypoxia in cultured mouse islets using the hypoxia mimetic cobalt chloride (100 μM CoCl2). We measured the impact on islet metabolism (two-photon NAD(P)H and Rh123 imaging) and function (insulin secretion and survival). We also measured the impact on hypoxia related transcripts (HIF-1α, VEGF-A, PDK-1, LDHA, COX4) and confirmed increased VEGF-A expression and secretion. Finally, we measured the vascularization of islets in static and flowing culture using PECAM-1 immunofluorescence. CoCl2 did not induce significant changes in beta cell metabolism (NAD(P)H and Rh123), insulin secretion, and survival. Consistent with hypoxia induction, CoCl2 stimulated HIF-1α, PDK-1, and LDHA transcripts and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2) and this switch was noted in the glucose-stimulated cytoplasmic NAD(P)H responses. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static (dish) and microfluidic flow culture. Hypoxia induction using CoCl2 had a positive effect on islet EC morphology and survival with limited impact on beta-cell metabolism, function, and survival. The EC response appears to be due to endogenous production and secretion of angiogenic factors (e.g. VEGF-A), and mechanistically independent from survival induced by serum albumin.

Islet macrophages are associated with islet vascular remodeling and compensatory hyperinsulinemia during diabetes.

β-Cells respond to peripheral insulin resistance by first increasing circulating insulin during diabetes. Islet remodeling supports this compensation, but its drivers remain poorly understood. Infiltrating macrophages have been implicated in late-stage type 2 diabetes, but relatively little is known on islet resident macrophages, especially during compensatory hyperinsulinemia. We hypothesized that islet resident macrophages would contribute to islet vascular remodeling and hyperinsulinemia during diabetes, the failure of which results in a rapid progression to frank diabetes. We used chemical (clodronate), genetics (CD169-diphtheria toxin receptor mice), or antibody-mediated (colony-stimulating factor 1 receptor α) macrophage ablation methods in diabetic (db/db) and diet-induced models of compensatory hyperinsulinemia to investigate the role of macrophages in islet remodeling. We transplanted islets devoid of macrophages into naïve diabetic mice and assessed the impact on islet vascularization. With the use of the above methods, we showed that macrophage depletion significantly and consistently compromised islet remodeling in terms of size, vascular density, and insulin secretion capacity. Depletion of islet macrophages reduced VEGF-A secretion in both human and mouse islets ex vivo, and this functionally translated to delayed revascularization upon transplantation in vivo. We revealed that islet resident macrophages were associated with islet remodeling and increased insulin secretion during diabetes. This suggests utility in harnessing islet macrophages during this phase to promote islet vascularization, remodeling, and insulin secretion.

Revascularizing eye-lets

Islet Transplantation Pancreatic islet transplantation is a potentially promising therapy for type 1 diabetes, but poor revascularization hinders islet long-term viability. Figueiredo et al. studied the role of protein tyrosine phosphatase 1B (PTP1B) in regulating islet vascularization. Islets from PTP1B−/− mice retained more endothelial cells during in vitro culture and restored normoglycemia when transplanted into the anterior chamber of the eye in diabetic mice. Future work is needed to determine whether targeting PTP1B can improve islet transplantation in human patients. Sci. Transl. Med. 11 , eaar6294 (2019).

Long-term c-Kit overexpression in beta cells compromises their function in ageing mice.

c-Kit signalling regulates intracellular pathways that enhance beta cell proliferation, insulin secretion and islet vascularisation in mice up to 28weeks of age and on short-term high-fat diet. However, long-term c-Kit activation in ageing mouse islets has yet to be examined. This study utilises beta cell-specific c-Kit-overexpressing transgenic (c-KitβTg) ageing mice (~60weeks) to determine the effect of its activation on beta cell dysfunction and insulin secretion. Wild-type and c-KitβTg mice, aged 60weeks, were examined using metabolic tests to determine glucose tolerance and insulin secretion. Pancreas histology and proteins in isolated islets were examined to determine the expression of beta cell transcription factors, proliferation and intracellular signalling. To determine the role of insulin receptor signalling in ageing c-KitβTg mice, we generated beta cell-specific inducible insulin receptor knockout in ageing c-KitβTg mice (c-KitβTg;βIRKO mice) and examined the ageing mice for glucose tolerance and islet histology. Ageing c-KitβTg mice progressively developed glucose intolerance, compared with age-matched wild-type littermates, due to impaired insulin secretion. Increased beta cell mass, proliferation and nuclear forkhead box transcription factor O1 (FOXO1) expression and reduced exocytotic protein levels were detected in ageing c-KitβTg mouse islets. Protein analyses of isolated islets showed increased insulin receptor, phosphorylated IRS-1Ser612 and cleaved poly(ADP-ribose) polymerase levels in ageing c-KitβTg mice. Ageing c-KitβTg mouse islets treated ex vivo with insulin demonstrated reduced Akt phosphorylation, indicating that prolonged c-Kit induced beta cell insulin insensitivity. Ageing c-KitβTg;βIRKO mice displayed improved glucose tolerance and beta cell function compared with ageing c-KitβTg mice. These findings indicate that long-term c-Kit overexpression in beta cells has a negative impact on insulin exocytosis and that temporally dependent regulation of c-Kit-insulin receptor signalling is important for optimal beta cell function.