Islet vascularity in wild-type and thrombospondin-1 deficient animals.

<p>Micrographs of blood vessel-stained (Bandeiraea simplicifolia; red) pancreas form one-year-old wild-type and thrombospondin-1 deficient mice (A–B). Vascular density in 10–12 week-old, 16-week-old and one-year-old wild-type and thrombospondin-1 deficient mice (C). Islet blood flow in 10–12-week-old wild-type and thrombospondin-1 deficient mice (D). All values are means±SEM for 6–10 animals in each group. * denotes P<0.05 when compared to age-matched wild-type animals. Scale bars in A–B are 50 µm.</p

Beta-cell functional tests in wild-type and thrombospondin-1 deficient animals.

<p>Intravenous glucose tolerance tests (2.5 g/kg) in 10–12 week-old (A), 16-week-old (B) and one-year-old animals (C). Perfusion of islet grafts composed of wild-type or thrombospondin-1 deficient islets implanted to wild-type or thrombospondin-1 deficient animals (D) with calculations of total insulin release (area under the curve) for the high glucose period (15–45 min; E). All values are means±SEM for 5–14 animals in each group. * denotes P<0.05 when compared to age-matched wild-type animals.</p

Age-dependent changes in islet and beta-cell mass of wild-type and thrombospondin-1 deficient mice.

<p>Micrographs of insulin-stained (red) pancreas from 10–12-week-old wild-type (A) and thrombospondin-1 deficient mice (B), 16-week-old wild-type (C) and thrombospondin-1 deficient mice (D), and one-year-old wild-type (E) and thrombospondin-1 deficient mice (F). Islet, beta- and alpha-cell mass in 10–12 week-old, 16-week-old and one-year-old wild-type and thrombospondin-1 deficient mice (G–I). Number of pancreatic islets in the same animals (J). All values are given as means±SEM for 5–8 animals in each group. * denotes P<0.05 when compared to age-matched wild-type animals. Scale bars in A–F are 400 µm.</p

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

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

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