Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis

Abstract

Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the underpinning cellular and molecular mechanisms are unknown. Comparing mice with whole-body insulin receptor haploinsufficiency (Insr+/-) against littermate controls, we found impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia; this was in spite of increased expression of the proangiogenic growth factor Vegfa. Insr+/- neonatal retinas exhibited reduced tip cell number and branching complexity during developmental angiogenesis, which was also found in separate studies of mice with endothelium-restricted Insr haploinsufficiency. Functional responses to vascular endothelial growth factor A (VEGF-A), including in vitro angiogenesis, were also impaired in aortic rings and pulmonary endothelial cells from Insr+/- mice. Human umbilical vein endothelial cells with shRNA-mediated knockdown of Insr also demonstrated impaired functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and endothelial nitric oxide synthase was intact, but downstream signaling to extracellular signal-reduced kinase 1/2 (ERK1/2) was impaired, as was VEGF receptor-2 (VEGFR-2) internalization, which is required specifically for signaling to ERK1/2. Hence, endothelial insulin receptors facilitate the functional response to VEGF-A during angiogenic sprouting and are required for appropriate signal transduction from VEGFR-2 to ERK1/2.

Keywords: ERK; VEGF; angiogenesis; endothelial; insulin; vascular.

Figure 1.

Angiogenesis is impaired in Insr^+/- mice with hindlimb ischemia. (A) Ischemic to nonischemic limb perfusion defined by laser Doppler imaging, with representative day 21 images, showing impaired recovery from hindlimb ischemia in Insr^+/- vs WT (n = 14, 13). (B) Confocal immunofluorescence of ischemic and nonischemic gastrocnemius muscle reveals reduced capillary density in ischemic Insr^+/- vs WT muscle. Representative images of ischemic muscle show isolectin B4 stained capillaries in red and nuclei in blue. Scale bars denote 50 μm. (n = 8, 7). (C) Vegfa mRNA normalized to 18S mRNA is higher in the ischemic limb adductor muscle of Insr^+/- vs WT (n = 4, 4). D) Vegfa mRNA normalized to 18S mRNA is higher in the nonischemic limb adductor muscle of Insr^+/- vs WT (n = 4, 4). *P < 0.05. Insr, insulin receptor; WT, wild-type.

Figure 2.

Insr^+/- exhibits impaired in vitro functional responses to VEGF. Capillary sprouting from aortic rings embedded in a collagen matrix with VEGF-A₁₆₅ is reduced in Insr^+/- vs WT (A), as is mean sprout length (B); representative images show isolectin B4 staining of endothelium in green, with scale bars denoting 1000 μm (n = 5, 5). (C) Insulin receptor (Insr) mRNA normalized to 18S mRNA is reduced in Insr^+/- vs WT PEC (n = 5, 5). (D) In vitro angiogenesis in Matrigel is impaired in Insr^+/- vs WT PEC (n = 7, 4). (E) Scratch wound closure is impaired in Insr^+/- vs WT PEC (n = 5, 6). (F) Migration toward VEGF-A₁₆₅ in Boyden chamber apparatus is impaired in Insr^+/- vs WT PEC (n = 5, 4). (G) Proliferation defined by nuclear EdU incorporation is similar in Insr^+/- and WT PEC (n = 11, 7). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; PEC, pulmonary endothelial cell; VEGF, vascular endothelial growth factor; WT, wild-type.

Figure 3.

Developmental angiogenesis is impaired in the neonatal P5 retina of Insr^+/- mice. (A) Radial outgrowth of the developing retinal vasculature is comparable in Insr^+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm (n = 7, 13). (B) Vascular endothelial area is reduced in the peripheral half of the retinal vasculature in Insr^+/- vs WT (n = 7, 13). (C) Vascular branching is reduced in the peripheral and central zones of the retinal vasculature in Insr^+/- vs WT (n = 7, 13). (D) Emerging tip cells per millimeter of vascular front perimeter are reduced in Insr^+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 50 μm (n = 7, 13). (E) The number of filopodia per tip cell is similar in Insr^+/- vs WT (n = 7, 13). (F) The number of regressed vessels, defined as Collagen IV sleeves (red) without overlying isolectin B4 (green) in representative images, is lower in Insr^+/- than WT (n = 11, 10). (G) The number of proliferating endothelial cells, defined as EdU⁺ nuclei (red) overlying isolectin B4 (white) in representative images, is similar in Insr^+/- and WT (n = 13, 5). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; WT, wild-type.

Figure 4.

Developmental angiogenesis is impaired in the neonatal P5 retina of ECInsr^+/- mice. (A) Radial outgrowth of the developing retinal vasculature is comparable in ECInsr^+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm (n = 7, 6). (B) Vascular endothelial area is reduced in the peripheral and central zones of the retinal vasculature in ECInsr^+/- vs WT (n = 7, 6). (C) Vascular branching is reduced in the peripheral and central zones of the retinal vasculature in ECInsr^+/- vs WT (n = 7, 6). (D) Emerging tip cells per millimeter of vascular front perimeter are reduced in ECInsr^+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 50 μm (n = 7, 6). (E) The number of filopodia per tip cell is similar in ECInsr^+/- vs WT (n = 7, 6). ECInsr, endothelial cell insulin receptor; WT, wild-type.

Figure 5.

Insr knockdown in HUVECs impairs functional responses to VEGF. (A) Insulin receptor protein knockdown of 40% was achieved in Insr shRNA HUVECs vs control shRNA HUVECs, with representative gel (n = 7, 7). (B) Angiogenic sprout numbers were reduced from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs, with representative microscopy images (n = 3, 3). (C) Angiogenic sprout length was similar from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs (n = 3, 3). (D) Scratch wound closure was impaired in Insr shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). (E) Adhesion to gelatin was impaired in Insr shRNA HUVECs vs control shRNA HUVECs, especially in context of media supplemented with VEGF-A₁₆₅; representative microscopy images show DAPI-defined nuclei in blue and phalloidin-defined filamentous actin in red, with scale bars denoting 250 μm (n = 4, 4). *P < 0.05. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; VEGF, vascular endothelial growth factor.

Figure 6.

Insr knockdown in HUVECs impairs signaling responses to VEGF. (A) Representative blots illustrating major VEGF signaling nodes in Insr shRNA HUVECs and control shRNA HUVECs grown on gelatin at baseline, 5 minutes, and 15 minutes after stimulation with VEGF-A₁₆₅ 50 ng/mL, (B) with quantification of VEGF-induced phosphorylation of ERK1/2 at 5 and 15 minutes (n = 4, 4). (C) Representative blot illustrating impaired insulin-stimulated Akt phosphorylation in Insr shRNA HUVECs vs control shRNA HUVECs after 15 minutes’ exposure to 10 and 100 nm insulin. (D) Representative blots and (E) quantification from surface biotinylation experiment illustrating impaired internalization of VEGFR2 in Insr shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). (D) Detected biotin-labelled VEGFR2 in the presence or absence of TE and so is not directly represented in €, which presents derived internalization data. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; TE, trypsin exposure; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.

Figure 7.

Schematic illustration of the proposed role of Insr during sprouting angiogenesis. Insr expression is known to be enriched in tip ECs, which migrate along VEGF gradients, leading emerging sprouts during angiogenesis. Knockdown of Insr in ECs impairs VEGF signaling to ERK1/2 as a result of impaired VEGFR2 internalization, which manifests as diminished sprout formation and EC migration. EC, endothelial cell; ERK, extracellular signal-regulated kinase; Insr, insulin receptor; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2.