Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation - PubMed (original) (raw)

. 2012 Jul 1;367(1):40-54.

doi: 10.1016/j.ydbio.2012.04.022. Epub 2012 Apr 24.

Marcela Brissova, Rachel B Reinert, Fong Cheng Pan, Priyanka Brahmachary, Marie Jeansson, Alena Shostak, Aramandla Radhika, Greg Poffenberger, Susan E Quaggin, W Gray Jerome, Daniel J Dumont, Alvin C Powers

Affiliations

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

Qing Cai et al. Dev Biol. 2012.

Abstract

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Overexpression of VEGF-A in early development affects islet morphology and vascularization whereas overexpression of Ang1 and Ang2 has little effect

(A) Mice were treated with Dox to induce angiogenic factor expression for the time periods shown by the arrows. (B–I) Immunostained sections of P1 stage pancreatic tissue from mice treated with Dox from E5.5 to P1. (B) Rip-rtTA; tet-O-Ang1 mice, insulin (Ins, blue) and Ang1 (red); (C) Rip-rtTA; tet-O-Ang1 mice, insulin (Ins, blue) and PECAM1 (green). (D) Rip-rtTA; tet-O-Ang2 mice, insulin (Ins, blue) and c-Myc (red; the Ang2 transgene has a Myc tag). (E) Rip-rtTA; tet-O-Ang2 mice, insulin (Ins, blue) and PECAM1 (green). (F) Rip-rtTA; tet-O-VEGF-A mice, insulin (Ins, blue) and VEGF-A (red); (G) Rip-rtTA; tet-O-VEGF-A, insulin (blue) and PECAM1 (green). (H) Rip-rtTA mice, insulin (Ins, blue) and PECAM1 (green). (I) Pancreatic insulin content at P1 in Rip-rtTA mice and Rip-rtTA;tet-O-VEGF-A mice treated with Dox from E5.5 to P1. Insulin content: Rip-rtTA, 528.5±8.1 ng/pancreas (n=5 mice); Rip-rtTA; tet-O-VEGF-A, 372.1± 23.9 ng/pancreas (n=8 mice); ***; p<0.001. (J) Pancreatic glucagon content at P1 stage from mice treated with Dox from E5.5 to P1; Glucagon content: Rip-rtTA, 22.1±1.7 ng/pancreas (n=5 mice); Rip-rtTA; tet-O-VEGF-A, 15.4±2.1 ng/pancreas (n=8 mice); *; p<0.05. (K) Percentage of β cell area per total pancreatic area; Rip-rtTA, 1.22±0.14% (n=3 mice); Rip-rtTA;tet-O-VEGF-A, 0.70±0.10% (n=3 mice); **; p<0.05. Scale bar in panel B represents 50 μm and also corresponds to panels D–H.

Figure 2

Figure 2. β cell-specific overexpression of VEGF-A increases endothelial cell proliferation but decreases number of Ngn3+ endocrine progenitors and β cell proliferation

(A and B) Pancreatic sections of Rip-rtTA and RIP-rtTA;tet-O-VEGF-A mice at E14.5 (Dox treatment from E5.5 to E14.5) were immunostained for E-cadherin (green), and Ngn3 (red). (C) Percentage of Ngn3+ cells/E-cadherin+ cells at E14.5: Control Rip-rtTA, 16.4±1.0 (n=3 mice); Rip-rtTA; tet-O-VEGF-A, 12.1±1.0 (n=3 mice); *; p<0.05. (D and E) Pancreatic sections of Rip-rtTA and RiP-rtTA;tet-O-VEGF-A mice at E14.5 (Dox treatment from E5.5 to E14.5) were immunostained for Cpa1 (green), and Sox9 (red). (F and G) Pancreatic sections of Rip-rtTA and RIP-rtTA;tet-O-VEGF-A mice at E16.5 stage (Dox treatment from E5.5 to E16.5) were immunostained for insulin (Ins, green), TUNEL positive cells (red) and counterstained with DAPI (blue). (H and I) Pancreatic sections of Rip-rtTA and RIP-rtTA;tet-O-VEGF mice at P1 (Dox treatment from E5.5 to P1) were immunostained for insulin (Ins, blue), Ki67 (red) and PECAM1 (green). (J) Rate of β-cell proliferation at E16.5 and P1 (Dox treatment from E5.5 to E16.5 or P1). Rip-rtTA (E16.5), 4.0±1.0% (n=3 mice); Rip-rtTA;tet-O-VEGF-A (E16.5), 0.08±0.05% (n=3 mice); Rip-rtTA (P1), 11.3±1.7% (n=4 mice); Rip-rtTA;tet-O-VEGF-A (P1), 2.4±0.5% (n=4 mice); **; p<0.01 (K) Rate of endothelial cell proliferation at P1 (Dox treatment from E5.5 to P1). Rip-rtTA, 14.4±3.8% (n=4 mice); Rip-rtTA;tet-O-VEGF-A, 88.2±6.1% (n=4 mice); ***; p<0.001. Insets in A–I show magnification of boxed area in corresponding panels. Scale bars in panels A–E represent 50 μm. Scale bar in panel F represents 50 μm and also corresponds to panels G–I.

Figure 3

Figure 3. Overexpression of VEGF-A during islet development dramatically increases islet vascularization, but decreases β cell number

Pancreatic sections of Rip-rtTA and Rip-rtTA;tet-O-VEGF-A mice were immunostained at different developmental stages (Dox treatment from E5.5 to E16.5, P1, or P7). (A–F) Pancreatic sections of Rip-rtTA and Rip-rtTA;tet-O-VEGF-A mice at E16.5, P1 and P7 labeled for insulin (Ins, blue), glucagon (Glu, green), and DBA ductal marker (red). (D–F) Pancreatic sections of Rip-rtTA;tet-O-VEGF-A at E16.5, P1 and P7 stages: Insulin (Ins, blue), Glucagon (Glu, green), and DBA ductal marker (red). (G–L) Pancreatic sections of Rip-rtTA and Rip-rtTA;tet-O-VEGF-A mice at E16.5, P1 and P7 were labeled for insulin (Ins, blue), PECAM1 (green), and DBA ductal marker (red). Arrows in panel B, D and F point to β cell associated with DBA+ ducts. Scale bar in panel L represents 50 μm and also corresponds to panels A–J.

Figure 4

Figure 4. Continuous and short-term overexpression of VEGF-A in β cells during postnatal period leads to altered islet morphology

(A, B) Pancreatic sections of Rip-rtTA and Rip-rtTA;tet-O-VEGF mice treated with Dox from E5.5 to P28; insulin (Ins, blue); PECAM1 (green); glucagon (Glu, red). (C) Pancreatic sections of Rip-rtTA;tet-O-VEGF mice treated with Dox from P1 to P7 were labeled for insulin (Ins, blue), PECAM1 (green), and glucagon (Glu, red) (D) Pancreatic sections of Rip-rtTA;tet-O-VEGF mice treated with Dox from P21 to P28 were labeled for insulin (Ins, blue), PECAM1 (green), and glucagon (Glu, red). (E–J) Pancreatic sections of Rip-rtTA and Rip-rtTA;tet-O-VEGF mice treated with Dox from E5.5 to P28. (E, F) Insulin (Ins, green), Pdx1 (red), and VEGF-A (blue); (G, H) Insulin (Ins, green), Nkx6.1 (red), and VEGF-A (blue); (I, J) Insulin (Ins, green), MafA (red), and VEGF-A (blue). Insets in E–J show magnification of boxed area in corresponding panels. In panels F, H, and J, the left and right insets represent β cells with basal VEGF-A expression and VEGF-A overexpressing β cells, respectively. Scale bar in panel A represents 50 μm and corresponds to panels B–J.

Figure 5

Figure 5. β cell-specific overexpression of VEGF-A alters the ultrastructure of endothelial cells

Transmission electron microscopy images show endocrine cells with adjacent endothelial cells. (A) Rip-rtTA mice; scale bar, 2 μm. (B) Rip-rtTA;tet-O-VEGF-A mice; scale bar, 2 μm. Boxed areas in panel A and B are magnified in C and D, respectively. (C) Rip-rtTA mice; scale bar, 500 nm. (D) Rip-rtTA;tet-O-VEGF-A mice; scale bar, 500 nm. Open arrowheads in C and D point to caveolae. BM, basement membrane; LM, vessel lumen; EC, endothelial cell; RBC, red blood cell. α, α cell; β, β cell; δ, δ cell.

Figure 6

Figure 6. Overexpression of Ang1 in the adult pancreas has little effect on islet morphology, vessel area, and vessel density

(A, B) Pancreatic sections of Rip-rtTA;tet-O-Ang1 mice were immunostained with anti-insulin (Ins, green) and anti-β-galactosidase (β-gal, red). The Ang1 transgene has a bicistronic cassette that encodes Ang1 and the β-gal reporter. (A) Mice without Dox. (B) Mice treated with Dox from E5.5 to 8 weeks. (C) The percentage of β-gal+ β cells in No Dox, 10±2% (n=3 mice) and Dox group, 72±2% (n=3 mice); ***; p<0.001. **(D)** Islets from No Dox and Dox group were isolated and cultured for 48 hours. The amount of secreted Ang1 in the culture media was quantified by ELISA: No Dox, 0.67±0.03 pg/islet/48h (n=3 mice); Dox, 4.8±1.0 pg/islet/48h (n=3 mice); *; p<0.05. (**E**, **F**) Immunostaining of pancreatic sections using anti-insulin (Ins, green) and anti-PECAM1 (red) in Rip-rtTA;tet-O-Ang1 mice in the absence or presence of Dox treatment. (**G**) Islet vessel density was calculated by dividing the total number of vessels over the total islet area: Control Rip-rtTA, 1,682±71 vessels/mm2 (n=3 mice); Rip-rtTA; tet-O-Ang1, 1,570±43 vessels/mm2 (n=3 mice); p>0.05. (H) Area/Vessel in islets was calculated by dividing the total area of islet vessels over the number of islet vessels: Control Rip-rtTA, 65.5±3.8 μm2 (n=3 mice); Rip-rtTA; tet-O-Ang1, 66.6±3.0 μm2 (n=3 mice), p>0.05. (I, J) Pancreatic sections of Rip-rtTA and Rip-rtTA;tet-O-hAng1 mice were immunostained with anti-insulin (Ins, green) and anti-PECAM1 (red). Both were treated with Dox from 8 weeks to 12 weeks of age. (K) Islet vessel density in Rip-rtTA, 1,626±34 vessels/mm2 (n=3 mice) and Rip-rtTA;tet-O-Ang-1, 1,643±38 vessels/mm2 (n=3 mice); p>0.05. (L)Area/Vessel in Rip-rtTA, 77±3 μm2 (n=3 mice) and Rip-rtTA;tet-O-Ang-1, 78±3 μm2 (n=3 mice); p>0.05.

Figure 7

Figure 7. Overexpression of Ang-2 in the adult pancreas has little effect on islet morphology but slightly enhances vessel area and density

(A and B) Pancreatic sections of Rip-rtTA;tet-O-Ang2 mice were immunostained with anti-insulin (Ins, green) and anti-myc (red) as Ang2 has a c-myc tag. (A) Mice without Dox; (B) Mice treated with Dox from E5.5 to 8 weeks of age. (C) The percentage of myc positive β cells in No Dox, 35±1% (n= 3 mice) and Dox, 80±1% (n=3 mice); ***; p<0.001. (**D**) Islets from both groups were isolated and cultured for 48 hours. The amount of secreted Ang2 in the culture media was quantified by ELISA in No Dox, 116±20 pg/islet/48h (n=3 mice) and Dox, 1,580±582 pg/islet/48h (n=3 mice); *; p< 0.05. (**E**, **F**) Immunostaining of pancreatic sections using anti-insulin (Ins, green) and anti-PECAM-1 (red) in Rip-rtTA;tet-O-Ang2 mice in the absence or presence of Dox treatment. (**G**) Islet vessel density: No Dox, 1,526±24 vessels/mm2 (n=3 mice); Dox, 1,660±60 vessels/mm2 (n=3 mice); *; p<0.05. (**H**) Area/vessel in No Dox, 119±4 μm2 (n=3 mice) and Dox, 140±7 μm2 (n=3 mice); **; p<0.01. (**I**, **J**). Pancreatic sections of Rip-rtTA and Rip-rtTA;tet-O-Ang2 mice were immunostained with anti-insulin (Ins, green) and anti-PECAM1 (red). Both were treated with Dox from 8 weeks to 12 weeks of age. (**K**) Islet vessel density: Rip-rtTA, 1,283±55 vessels/mm2 (n=3 mice); Rip-rtTA;tet-O-Ang2, 1,353±42 vessels/mm2 (n=3 mice); p>0.05. (L) Area/vessel: Rip-rtTA, 116±8 μm2 (n=3 mice); Rip-rtTA;tet-O-Ang2, 102±6 μm2 (n=3 mice); p>0.05.

Figure 8

Figure 8. β cell-specific overexpression of Ang1 or Ang2 alters endothelial cell ultrastructure

Transmission electron microscopy images of mice treated with Dox from E5.5 to 8 weeks. (A) Rip-rtTA mice, scale bar, 500 μm. (B) Rip-rtTA;tet-O-Ang-1 mice, scale bar, 500 μm. (C) Rip-rtTA;tet-O-Ang-2 mice, scale bar, 500 μm. Closed arrowheads in A point to fenestrations. Open arrowheads in B and C point to caveolae. LM, vessel lumen; RBC, red blood cell.

Figure 9

Figure 9. Model of pancreatic islet vascularization in VEGF-A overexpressing mice

(A) In the normal pancreas, around E14.5, endothelial cells form a single layer close to the trunk with endocrine cells. Around E16.5, endocrine cells begin to cluster near the ductal epithelium that is surrounded with single layer vessels in the normal pancreas. By P7, in normal pancreas, endocrine cell clusters migrate away from ducts to form highly vascularized islets. (B) In the pancreas with VEGF-A overexpressing β cells, around E14.5, an increased number of endothelial cells is attracted to β cells along trunk domain. Around E16.5, endocrine cells form smaller clusters near the ductal epithelium but are surrounded with multiple layers of endothelial cells. By P7, in pancreas with VEGF-A overexpressing β cells, endocrine cell clusters and ducts are surrounded with multiple layers of endothelial cells that limit proper islet formation. Endocrine cells are adjacent to the ducts with some endocrine cells remaining in the ductal epithelium.

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