Angiopoetin-2 signals do not mediate the hypervascularization of islets in type 2 diabetes

Aims Changes in the islet vasculature have been implicated in the regulation of β-cell survival and function during the progression to type 2 diabetes (T2D). Failure of the β-cell to compensate for the increased insulin demand in obesity eventually leads to diabetes; as a result of the complex interplay of genetic and environmental factors (e.g. ongoing inflammation within the islets) and impaired vascular function. The Angiopoietin/Tie (Ang/Tie) angiogenic system maintains vasculature and is closely related to organ inflammation and angiogenesis. In this study we aimed to identify whether the vessel area within the islets changes in diabetes and whether such changes would be triggered by the Tie-antagonist Ang-2. Methods Immunohistochemical and qPCR analyses to follow islet vascularization and Ang/Tie levels were performed in human pancreatic autopsies and isolated human and mouse islets. The effect of Ang-2 was assessed in β-cell-specific Ang-2 overexpressing mice during high fat diet (HFD) feeding. Results Islet vessel area was increased in autopsy pancreases from patients with T2D. The vessel markers Tie-1, Tie-2 and CD31 were upregulated in mouse islets upon HFD feeding from 8 to 24 weeks. Ang-2 was transiently upregulated in mouse islets at 8 weeks of HFD and under glucolipotoxic conditions (22.2 mM glucose/ 0.5 mM palmitate) in vitro in human and mouse islets, in contrast to its downregulation by cytokines (IL-1β, IFN- and TNF-α). Ang-1 on the other hand was oppositely regulated, with a significant loss under glucolipotoxic condition, a trend to reduce in islets from patients with T2D and an upregulation by cytokines. Modulation of such changes in Ang-2 by its overexpression or the inhibition of its receptor Tie-2 impaired β-cell function at basal conditions but protected islets from cytokine induced apoptosis. In vivo, β-cell-specific Ang-2 overexpression in mice induced hypervascularization under normal diet but contrastingly led to hypovascularized islets in response to HFD together with increased apoptosis and reduced β-cell mass. Conclusions Islet hypervascularization occurs in T2D. A balanced expression of the Ang1/Ang2 system is important for islet physiology. Ang-2 prevents β-cell mass and islet vascular adaptation in response to HFD feeding with no major influence on glucose homeostasis

Angiopoetin-2 Signals Do Not Mediate the Hypervascularization of Islets in Type 2 Diabetes.

Changes in the islet vasculature have been implicated in the regulation of β-cell survival and function during the progression to type 2 diabetes (T2D). Failure of the β-cell to compensate for the increased insulin demand in obesity eventually leads to diabetes; as a result of the complex interplay of genetic and environmental factors (e.g. ongoing inflammation within the islets) and impaired vascular function. The Angiopoietin/Tie (Ang/Tie) angiogenic system maintains vasculature and is closely related to organ inflammation and angiogenesis. In this study we aimed to identify whether the vessel area within the islets changes in diabetes and whether such changes would be triggered by the Tie-antagonist Ang-2.Immunohistochemical and qPCR analyses to follow islet vascularization and Ang/Tie levels were performed in human pancreatic autopsies and isolated human and mouse islets. The effect of Ang-2 was assessed in β-cell-specific Ang-2 overexpressing mice during high fat diet (HFD) feeding.Islet vessel area was increased in autopsy pancreases from patients with T2D. The vessel markers Tie-1, Tie-2 and CD31 were upregulated in mouse islets upon HFD feeding from 8 to 24 weeks. Ang-2 was transiently upregulated in mouse islets at 8 weeks of HFD and under glucolipotoxic conditions (22.2 mM glucose/ 0.5 mM palmitate) in vitro in human and mouse islets, in contrast to its downregulation by cytokines (IL-1β, IFN-ɣ and TNF-α). Ang-1 on the other hand was oppositely regulated, with a significant loss under glucolipotoxic condition, a trend to reduce in islets from patients with T2D and an upregulation by cytokines. Modulation of such changes in Ang-2 by its overexpression or the inhibition of its receptor Tie-2 impaired β-cell function at basal conditions but protected islets from cytokine induced apoptosis. In vivo, β-cell-specific Ang-2 overexpression in mice induced hypervascularization under normal diet but contrastingly led to hypovascularized islets in response to HFD together with increased apoptosis and reduced β-cell mass.Islet hypervascularization occurs in T2D. A balanced expression of the Ang1/Ang2 system is important for islet physiology. Ang-2 prevents β-cell mass and islet vascular adaptation in response to HFD feeding with no major influence on glucose homeostasis

Islet hypervascularization in T2D and effects of Ang-2 overexpression.

<p>A healthy islet is surrounded by intact capillaries, maintained by the Ang/Tie system and extracellular matrix supporting the function and survival of the islet. An increased insulin demand leads to more islet blood flow, β-cell mass and vascular expansion and consequent compensation. A transient Ang-2 upregulation promotes angiogenesis, leading islet endothelium to a non-quiescent state inhibiting Tie-2 signaling. Towards human T2D progression, β-cell failure and apoptosis occurs together with increased islet and endothelial inflammation and islet hypervascularization. Ang-2 overexpression on the other hand prevents β-cell mass and vascular expansion in response to HFD with persistent islet and endothelial inflammation.</p

Ang/Tie expression in isolated islets correlates with changes in vessel area.

<p>Isolated WT mouse and human islets were cultured for 3 days in control condition (11.1 mM glucose for mouse or 5.5 mM for human) or treated with diabetic conditions of 22.2 mM glucose + 0.5 mM palmitic acid or mixture of cytokines: 2 ng/mL IL-1β, 1000 U/ml IFN-ɣ and TNF-α (cyto). <b>(A,D)</b> GSIS is shown by the stimulatory index assessed by 16.7/2.8 mM glucose stimulation. <b>(B,C,E,F)</b> Graph shows ratio of vessel area to islet area for mouse <b>(B,C)</b> and human <b>(E,F)</b> islets, fixed and immune-labelled for vessel (CD-31,red) and islet (insulin, green). <b>(G-L)</b> qPCR analysis of treated mouse and human islets for mouse CD-31 <b>(G,J)</b>, Ang-1,-2 <b>(H,K)</b>, Tie-1,-2 <b>(I,L)</b>. All genes have been normalized to PPIA or 18s as housekeeping control. *p<0.05, treated vs. control 11.1 mM (mouse) or 5.5 mM (human). <b>(M)</b> Representative western blot from treated human islet lysates (left panel) and densitometric analyses of Ang-2 (right panel). Data are means +/-SE from 3–5 independent experiments from 3–5 different organ donors (human islets) or 3–5 independent mouse islet isolations.</p

Islet vessel area increases in T2DM.

<p><b>(A)</b> Representative images of pancreatic sections from non-diabetic controls and patients with T2D, immune-labelled for CD31 (red) and insulin (green). <b>(B)</b> Graphs show ratio of vessel area to islet area (control: n = 6; T2D: n = 10). <b>(C)</b> Plot shows no correlation of vessel density with BMI. <b>(D-H)</b> qPCR analysis of Ang-2, Tie-1, Tie-2, CD-31 from isolated mouse islets from C57BL/6 WT mice kept on normal diet (ND) or high-fat high-sucrose diet (HFD) for <b>(D)</b> 8 weeks (n = 4/group), <b>(E)</b> 16 weeks (n = 9/group) and <b>(F)</b> 24 weeks (n = 7/group), <b>(G) of</b> eNOS and <b>(H) of</b> ICAM-1. <b>(I)</b> qPCR analysis of Ang-1, Ang-2, Tie-1 and Tie-2 of isolated islets from non-diabetic (control, n = 8) and from patients with T2D (n = 7). *p<0.05, HFD vs ND or T2D vs. control</p

Ang-2 over-expression impairs islet function but protects from cytokine treatment in isolated islets.

<p>Isolated islets from RIP-rtTA;tet-O-Ang-2 (Ang2-rtTA) and RIP-rtTA control (rtTA) mice were cultured for 3 days in presence of 10 μg/ml doxycycline to achieve Ang-2 overexpression. Mouse or human islets were cultured in 11.1 (mouse) or 5.5 mM glucose (human) or treated with diabetic conditions of 22.2 mM glucose + 0.5mM palmitic acid or mixture of cytokines: 2 ng/mL IL-1β, 1000 U/ml IFN-ɣ and TNF-α (cyto). <b>(A)</b> Western blot from treated mouse islets shows Ang-2 overexpression in islets by myc-Ang-2. <b>(B)</b> GSIS is shown by the stimulatory index assessed by 16.7/2.8 mM glucose stimulation and normalized to control. <b>(C,D)</b> Treated mouse islets fixed post-GSIS and apoptotic cells detected by double staining for TUNEL and insulin. Representative images from different treatments. <b>(E,F)</b> qPCR analysis for CD31 <b>(E)</b> and ICAM <b>(F)</b> from mouse islets overexpressing Ang-2. <b>(G,H)</b> Representative western blots (upper panel) and densitometric analyses of proteins (lower panels) showing myc-Ang-2, ICAM-1, cleaved caspase 3 and actin/tubulin as housekeeping control, in human islets overexpressing Ang-2 by Ad-Ang-2 or control Ad-GFP <b>(G;</b> MOI = 50) or treated with 100 nM Tie-2 inhibitor for 72h (<b>H)</b>. Data are means +/-SE from 3–5 independent experiments from 3–5 different organ donors (human islets) or 3–5 independent mouse islet isolations. *p<0.05, treated vs. 11.1 mM glucose control, #p<0.05, Ang2-rtTA vs. rtTA.</p