1. Mice Lacking the Vascular Endothelial Growth Factor-B Gene (Vegfb) Have Smaller Hearts, Dysfunctional Coronary Vasculature, and Impaired Recovery From Cardiac Ischemia
by Daniela Bellomo, John P. Headrick, Ginters U. Silins, Carol A. Paterson, Penny S. Thomas, Michael Gartside, Arne Mould, Marian M. Cahill, Ian D. Tonks, Sean M. Grimmond, Steve Townson, Christine Wells, Melissa Little, Margaret C. Cummings, Nicholas K. Hayward, and Graham F. Kay
Circulation Research
Volume 86(2):e29-e35
February 4, 2000
Copyright © American Heart Association, Inc. All rights reserved.

2. a, Diagram of the murine Vegfb gene (top), the targeting construct used to generate a Vegfb+/− mouse (middle), and the final targeted locus (bottom).
a, Diagram of the murine Vegfb gene (top), the targeting construct used to generate a Vegfb+/− mouse (middle), and the final targeted locus (bottom). The exons of the Vegfb gene are shown as numbered boxes with the open reading frame as open boxes. The location and orientation of the PCR primers used to genotype mice are shown as PCR1, PCR2, and PCR3. b, Genotyping PCR on Vegfb+/+, Vegfb+/−, and Vegfb−/− DNA, using the primers shown in panel a.
Daniela Bellomo et al. Circ Res. 2000;86:e29-e35
Copyright © American Heart Association, Inc. All rights reserved.

3. Vegf-B/β-Gal localization in the heart during development and after birth. a, Whole-mount E12.5 Vegf-B/β-Gal–stained Vegfb+/− (right) and Vegfb+/− (left) hearts showing higher expression in the RV. b through d, Whole mount and sections of Vegf-B/β-Gal–stained E15.5 hearts. b, Vegfb+/− whole mount.
Vegf-B/β-Gal localization in the heart during development and after birth. a, Whole-mount E12.5 Vegf-B/β-Gal–stained Vegfb+/− (right) and Vegfb+/− (left) hearts showing higher expression in the RV. b through d, Whole mount and sections of Vegf-B/β-Gal–stained E15.5 hearts. b, Vegfb+/− whole mount. Vegf-B/β-Gal levels are very low in the aorta and pulmonary trunk. c, Longitudinal section of the Vegfb+/− heart shown in panel b. Note higher levels of Vegf-B/β-Gal expression (staining blue) in the RV and right side of the interventricular septum (*) than the LV. d, Sagittal section through the RV. Note expression in the trabeculations of the ventricle and absence in the atria. e through h, E17.5 hearts. e, Longitudinal heart section stained for PECAM-1 (staining brown). The rectangles represent the regions in panels f and g. f and g, Vegf-B/β-Gal and anti–PECAM-1 double labeling. f, Atrial region. The endocardium (arrows) is stained with anti–PECAM-1, but no capillaries have formed in this region. g, RV. Anti–PECAM-1 stains the endocardium (arrow) and the endothelium of numerous capillaries (arrowhead) surrounded by Vegf-B/β-Gal–expressing cardiomyocytes in the myocardium and subepicardium. h, Higher magnification of the subepicardial region stained for Vegf-B/β-Gal expression showing several Vegf-B/β-Gal–positive cells. i through k, P3 Vegfb+/− mouse hearts. i and j, Vegf-B/β-Gal staining of transverse heart sections. j, Section through the atrioventricular transition. Note prominent staining in the LV. j, Section through the ventricular region. Note the absence of staining around the coronary vessels (arrowheads). k and l, Contiguous sections through a coronary arteriole stained for PECAM-1 (j) and Vegf-B/β-Gal (k). Vegf-B/β-Gal–expressing cardiomyocytes surround the capillaries, but Vegf-B/β-Gal is undetectable in the endothelium and smooth muscle layers surrounding the artery. rv indicates right ventricle; lv, left ventricle; *, interventricular septum; ra, right atrium; aa, atrial appendage; m, myocardium; se, subepicardium; and p, pulmonary trunk. Bar=500 μm (a, b, d, e, i, and j) and 100 μm (c, f, g, h, k, and l).
Daniela Bellomo et al. Circ Res. 2000;86:e29-e35
Copyright © American Heart Association, Inc. All rights reserved.

4. Reduced postnatal heart size in Vegfb−/− hearts
Reduced postnatal heart size in Vegfb−/− hearts. a, Appearance of P25 Vegfb−/−, Vegfb+/−, and Vegfb+/+ hearts from same-sex littermates, illustrating a slightly reduced Vegfb−/− heart size. b, Percentage of body/weight ratio is significantly reduced in the juvenile (>P25) Vegfb−/− mice but not in early postnatal mice (P3-9), indicating the impaired growth of the Vegfb−/− hearts in the first few weeks after birth.
Reduced postnatal heart size in Vegfb−/− hearts. a, Appearance of P25 Vegfb−/−, Vegfb+/−, and Vegfb+/+ hearts from same-sex littermates, illustrating a slightly reduced Vegfb−/− heart size. b, Percentage of body/weight ratio is significantly reduced in the juvenile (>P25) Vegfb−/− mice but not in early postnatal mice (P3-9), indicating the impaired growth of the Vegfb−/− hearts in the first few weeks after birth. Values are mean±SEM. *P<0.05 vs Vegfb+/+ mice; P<0.01 vs Vegfb+/− mice.
Daniela Bellomo et al. Circ Res. 2000;86:e29-e35
Copyright © American Heart Association, Inc. All rights reserved.

5. Reactive hyperemic responses to 20-second coronary occlusion in Langendorff-perfused Vegfb+/+, Vegfb+/−, and Vegfb−/− hearts. a, Baseline and peak hyperemic flows. b, Flow-debt repayments over the initial 60 seconds of reperfusion.
Reactive hyperemic responses to 20-second coronary occlusion in Langendorff-perfused Vegfb+/+, Vegfb+/−, and Vegfb−/− hearts. a, Baseline and peak hyperemic flows. b, Flow-debt repayments over the initial 60 seconds of reperfusion. Values are mean±SEM. *P<0.05 vs Vegfb+/+ hearts.
Daniela Bellomo et al. Circ Res. 2000;86:e29-e35
Copyright © American Heart Association, Inc. All rights reserved.

6. Functional responses of Langendorff-perfused Vegfb+/+, Vegfb+/−, and Vegfb−/− hearts to 20 minutes of global normothermic ischemia and 30 minutes of reperfusion. a, Responses for left ventricular diastolic pressure.
Functional responses of Langendorff-perfused Vegfb+/+, Vegfb+/−, and Vegfb−/− hearts to 20 minutes of global normothermic ischemia and 30 minutes of reperfusion. a, Responses for left ventricular diastolic pressure. Rate-pressure product (heart rate×left ventricular developed pressure) (b) and coronary flow (c) are shown. Values are mean±SEM. *P<0.05 vs Vegfb+/+ hearts.
Daniela Bellomo et al. Circ Res. 2000;86:e29-e35
Copyright © American Heart Association, Inc. All rights reserved.