1. Margreet de Vries1,2, Laura Parma1,2, Erna Peters1,2,
Jaap Hamming2, Marie-José Goumans3,2, Paul Quax1,2
1 Einthoven Laboratory for Experimental Vascular Medicine, 2 Dept of Surgery,
3 Dept of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands
VEGFR2 blockade reduces intraplaque haemorrhage and enhances plaque stability by augmentation of plaque neovessel maturation
Introduction
Immature plaque neovessels contribute to atherosclerotic plaque instability and intraplaque haemorrhage by leaking erythrocytes and leukocytes in the plaque. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), together with the angiopoietin-Tie2 system, regulates the maturation of neovessels..
We have previously shown that murine vein graft lesions exhibit massive plaque neovascularization and that leaky vessels and intraplaque haemorrhage contribute to lesion growth.
Hypothesis
VEGFR2 blockade induces plaque neovessel maturation
VEGFR2 blockade reduces vein graft
thickening and augments plaque stability C * D * E * A
control
control
DC101
250 µm B *
DC101
collagen
SMC actin
macrophages
Figure 2. A. Representative cross-sections of vein grafts 28 days after surgery (Hematoxilin-Phloxine-Saffron staining). B. Quantitative measurements of lesion area. C. Collagen content (sirius red staining) in vein grafts expressed as relative % collagen. D. smooth muscle cell actin (SMC) content. E. macrophage content.
Conclusions
Blockade of VEGFR2 results in reduced intraplaque hemorrhage, decreased vein graft lesion area and increased plaque stability. Blockade of VEGFR2 induces a more mature neovessel type due to local decreased VEGF and ANG2 expression resulting in more stable neovessels.
This study identifies plaque neovessel maturation as an attractive target for the treatment of unstable atherosclerotic diseases.
VEGFR-2 blocking antibodies inhibit intraplaque haemorrhage and erythrocyte extravasation B C * A D
control
DC101 *
Figure 1. A. Representative images of lesions with neovessels and extravasated erythrocytes. Green; erythrocytes, Red; endothelial cells (CD31). B. Quantification of the length of the vein graft segment that displayed intraplaque hemorrhage (IPH). C. Quantification of the density of neovessels in vein graft sections. D. Quantification of the area of the extravasated erythrocytes.
Angiopoietin expression in vein grafts
Decreased local
angiogenic gene
expression upon
VEGFR2 blockade
Figure 3. A. In stable region of lesions, CD31 (red) positive neovessels are covered by SMC positive pericytes (blue). Erythrocytes (green). B. In regions with intraplaque haemorrhage (demonstrated by extravasated erythrocytes (green)) neovessels (red) show absence of pericytes (blue). C. Regions with intraplaque haemorrhage are positive for stabilizing factor angiopoietin 1 (green). Blue; SMCA, Red; erythrocytes. D. Regions with intraplaque hemorrhage are positive for destabilizing factor angiopoietin 2 (green). Blue; SMC, Red; erythrocytes. * A B *
Figure 4. Blockade of VEGFR2 results in decreased expression of VEGF, VEGF Receptor1 (VEGFR1) and Angiopoietin 2 (ANG2), locally in the vein graft whereas VEGFR2 and TIE2 are not regulated. Connexin 40, important in endothelial gap junction formation, is increased expressed after DC101 treatment. ICAM1 and IL6 are not differently regulated between the groups. * C D *
VEGFR2 blockade inhibits sprout formation
Materials and Methods
Donor caval veins were engrafted in the common carotid arteries of hypercholesterolemic male ApoE*3 Leiden mice. Mice were treated at day 14, 17, 21 and 25 with 10 mg/kg VEGFR2 blocking antibodies (DC101) or control IgG antibodies (GL113) and were sacrificed at day 28 (n= 12-14/group). Immunohistochemistry was used to quantify specific protein expression. qPCR was used to determine expression of genes locally in the vein grafts (n=6/group). Results are expressed as mean±SEM. An aortic ring assay was used to quantify the angiogenic effects of DC101 and effects on pericyte coverage. B
Figure 5. A. In an aortic ring assay DC101 treatment (30 µg/ml) results in less angiogenic sprouts in comparison to the control. B. Confocal imaging of sprouts stained for CD31 (red), SMC (green ) and DAPI (blue) seem to show more SMC+ pericytes in the DC101 group in comparison to the control. A *
control
DC101