Vascular adaptation to a dysfunctional endothelium as a consequence of Shb deficiency

Abstract

Vascular endothelial growth factor (VEGF)-A regulates angiogenesis, vascular morphology and permeability by signaling through its receptor VEGFR-2. The Shb adapter protein has previously been found to relay certain VEGFR-2 dependent signals and consequently vascular physiology and structure was assessed in Shb knockout mice. X-ray computed tomography of vessels larger than 24 μm diameter (micro-CT) after contrast injection revealed an increased frequency of 48-96 μm arterioles in the hindlimb calf muscle in Shb knockout mice. Intravital microscopy of the cremaster muscle demonstrated a less regular vasculature with fewer branch points and increased vessel tortuosity, changes that led to an increased blood flow velocity. Reduced in vivo angiogenesis was observed in Shb knockout Matrigel™ plugs. Unlike the wild-type situation, VEGF-A did not provoke a dissociation of VE-cadherin from adherens junctions in Shb knockout venules. The reduced angiogenesis and altered properties of junctions had consequences for two patho-physiological responses to arterial occlusion: vascular permeability was reduced in the Shb knockout cremaster muscle after ligation of one supplying artery and heat-induced blood flow determined by Laser-Doppler measurements was decreased in the hindlimb after ligation of the femoral artery. Consequently, the Shb knockout mouse exhibited structural and functional (angiogenesis and vascular permeability) vascular abnormalities that have implications for understanding the function of VEGF-A under physiological conditions.

Fig. 1

Micro-CT analysis of hind-limb arteries in wild-type and Shb knockout mice. The figure shows the visualization of arterial tree (a) and quantitation of the calf vasculature (b). The number of vessels in the different size ranges are shown. Means ± SEM are given. p < 0.05 when comparing the wild-type and Shb knockout vessel numbers. n = 6 mice for each genotype

Fig. 2

Analysis of cremaster vasculature in wild-type and Shb knockout mice. a Panoramic overviews of cremaster vasculature showing primarily arteries/arterioles after FITC-dextran injection. Scale bar 100 μm. Microscope used was DM5000B, ×5/0.12 or ×10/0.25 objectives (Leica Microsystems, Germany). b Whole-mount staining of cremaster microvasculature for CD31 to visualize smaller vessels. Scale bar 200 μm. Microscope used was a laser scanning confocal miscroscope C-1 with Plan Fluor ELWD ×20/0.45 and ×40/0.60 objectives; EZ-C1 software, (Nikon, Japan). (C) Quantitation of vessel length (μm) from a larger artery (A shows examples at top in the panels) to a corresponding vein and dye velocity (mm/s) are given as means ± SEM. * and ** indicate p < 0.05 and 0.01, respectively when compared with corresponding wild-type controls using a Students’ t test. N = 13–16 observations on three mice of each genotype. Quantitation of microvascular density and branchpoints. Values are in arbitrary units as means ± SEM for n = 5–7 observations on 3 mice of each genotype. ** indicates p < 0.01 when compared against wild-type with a Students’ t test

Fig. 3

Matrigel™ angiogenesis in wild-type and Shb knockout mice. a The vasculature was stained with CD31 (red) and VE-cadherin (green) and angiogenesis was estimated by confocal microscopy of zeta-stacks. Scale bar 50 μm. b Quantification of angiogenesis by counting vessel tips, vessel branches and vessel density. Values (counts per mm²) are means ± SEM, n = 3 mice each genotype. * and ** indicate p < 0.05 and 0.01, respectively, when compared with wild-type by Students’ t test

Fig. 4

Staining of wild-type and Shb knockout venules with VE-cadherin (a, b) and their ultrastructure visualized by scanning electron microscopy (SEM) (c, d). a Confocal microscopy of cremaster venules with or without treatment with VEGF-A after staining for VE-cadherin. Cremaster vasculature was injected with 20 ng/ml VEGF-A or PBS and fixated 5 min later. Microscope used was a laser scanning confocal miscroscope C-1 with Plan Fluor ELWD ×40/0.60 and ×60/1.40 objectives; EZ-C1 software, (Nikon, Japan). Original magnification 9400. Scale bars 30 μm. b Quantitation of the relative venular surface devoid of VE-cadherin staining in percent. Means ± SEM for 3–5 observations on 3 mice in each genotype are given. *** indicates p < 0.001 when compared with unstimulated wild-type. c The muscles had been exposed for 5 min to 20 ng/ml VEGF-A or not. Arrows indicate examples of junctions. A LEO 1530 instrument was used. Magnification ×60,000. Arrows indicate examples of closed junctions (unstimulated wild-type, VEGF-A stimulated Shb knockout), loose junctions (unstimulated Shb knockout) and porous junctions (VEGF-A stimulated wild-type). Scale bar 1 μm. d Quantitation of the percentages loose or porous adherens junctions in the corresponding groups for 6–12 determinations on 3 mice in each genotype. *** indicates p < 0.001 when compared with unstimulated wild-type

Fig. 5

Vascular leakage in response to arterial ligation in wild-type or Shb knockout cremaster muscles. a Rhodamine-dextran leakage at 24 h after ischemia at sites proximal and distal to the site of ligation. Scale bar 100 μm. Microscope used was DM5000B, ×5/0.12 or ×10/ 0.25 objectives (Leica Microsystems, Germany). b Rhodamine-dextran fluorescence was determined in several microscopic fields proximal or distal to the site of ligation prior to ligation, 1 h after ligation and 24 h after ligation. Values are absolute fluorescence distal to ligation or fluorescence distal to ligation relative the corresponding value proximal to ligation (basal). Means ± SEM for 3–5 mice of each genotype at each point are given. * indicates p < 0.05 when compared with control using a Students’ t test. Asterisks show examples of extravascular leakage of the fluorescent dye. Note differences in signal

Fig. 6

Analysis of blood flow changes in response to heat in wild-type and Shb knockout mice subjected to unilateral femoral artery ligation. Perfusion changes in response to heat (30 °C) in contralateral non-occluded (a) and occluded (b) wild-type and Shb knockout mice. Limb perfusion changes in response to heat challenge were measured on day 1, 3 and 7 after arterial occlusion after base line registration. Data from the contralateral non-occluded leg were compiled (a). Values are expressed as delta perfusion units and means ± SEM are given. n = 5 mice for each genotype. * indicates p < 0.05 when compared with corresponding wild-type control