Orchestration of angiogenesis and arteriovenous contribution by angiopoietins and vascular endothelial growth factor (VEGF)

Abstract

Multiple classes of factors contribute to angiogenesis. In past years, the primary focus has been to understand the functions of individual classes of angiogenic factors. However, few studies have focused on the combinatorial roles of multiple classes of factors in angiogenesis. In this report, we have investigated the in vivo angiogenic processes regulated by two major classes of angiogenic factors, the angiopoietins and vascular endothelial growth factor (VEGF). Here we show that angiopoietin-1, a factor previously considered to be proangiogenic, can offset VEGF-induced angiogenesis in vivo. We also provide direct in vivo evidence for the synergistic effect of angiopoietin-2 and VEGF on the induction of angiogenesis. Furthermore, we show that these two classes of factors control the ratio of arterial and venous blood vessel types during angiogenesis. We believe that our study is a step toward understanding how multiple classes of factors harmonize angiogenesis and blood vessel types.

Figure 1

Transgenic constructs and the cardiac-specific expression of the transgenes. (a) Schematic diagram of the transgenic constructs. cDNA encoding Ang1, Ang2, and VEGF was inserted downstream of the αMHC promoter/exons. (b) RT-PCR expression analysis of the transgenes in adult organs. The transgenes are expressed specifically in the heart. Integrity of each RNA sample was confirmed by amplifying for Tie2, which is expressed in all tissues (data not shown). WT, wild type; H, heart; B, brain; K, kidney; S, spleen.

Figure 2

Combinatorial regulation of angiogenesis by VEGF and angiopoietins. (a) Anti-CD31/PECAM-1-antibody staining of the adult heart from each transgenic line and wild-type control. The genotype of each line is indicated at the bottom left (Ang1, αMHC∷Ang1; Ang2, αMHC∷Ang2; VEGF, αMHC∷VEGF; VEGF/Ang1, αMHC∷VEGF;αMHC∷Ang1 double-transgenic line; VEGF/Ang2, αMHC∷VEGF;αMHC∷Ang2 double-transgenic line; and VEGF/Ang1/Ang2, αMHC∷VEGF;αMHC∷Ang1;αMHC∷Ang2 triple-transgenic line). An example of the aberrant clusters of capillaries in the VEGF/Ang2 double-transgenic heart is outlined by the dotted line. (Scale bar: 25 μm.) (b) Quantitation of capillary density. Capillary lumens in each transgenic line and wild-type control hearts were counted (per 10⁴-μm² field) from 6–13 randomly selected fields. (c) Flk1∷lacZ staining of the wild-type control and VEGF/Ang2 transgenic lines.

Figure 3

Severe edema and fibrosis caused by VEGF/Ang2 is corrected by Ang1. (a) The αMHC∷VEGF;αMHC∷Ang2 double-transgenic heart stained with Masson's trichrome. This histology is consistent with the widely distributed severe edema. Furthermore, many clustered extracellular matrix (ECM) fibers were detected by the trichrome method, indicating fibrosis. (b) The αMHC∷VEGF;αMHC∷Ang1;αMHC∷Ang2 triple-transgenic heart stained with trichrome. The histology is consistent with the significant improvement of severe edema. Furthermore, the decreased incidence of ECM fibers identified by the trichrome staining indicates improvement of this pathological condition.

Figure 4

Regulation of the dominance of vascular types by VEGF and VEGF/Ang2. (a) CD31/PECAM-1 (Top) and LacZ (Middle) immunolabeling of each transgenic line, αMHC∷VEGF (VEGF), αMHC∷VEGF;αMHC∷Ang2 (VEGF/Ang2), and wild type control (WT) crossed to the arterial-endothelial reporter line, ephrinB2^taulacZ/+. The merged images are shown at Bottom. (b) CD31/PECAM-1- (Top) and LacZ (Middle) immunolabeling of each transgenic line, αMHC∷VEGF (VEGF), αMHC∷VEGF;αMHC∷Ang2 (VEGF/Ang2), and wild type (WT) crossed to the venous endothelial reporter line, ephB4^taulacZ/+. The merged images are shown at Bottom. The percentages of either ephrinB2⁺ or ephB4⁺ capillaries were calculated by comparing number of CD31/PECAM-1-positive and the LacZ-immunopositive capillary lumens in the same section. Percentages of ephrinB2⁺ (i.e., arterial-type) capillaries: WT 50 ± 3.1%; and VEGF 96 ± 6 4%; VEGF/Ang2, 55 ± 17. Percentages of ephB4⁺ (i.e., venous-type) capillaries: WT 36 ± 6.4%; VEGF 10 ± 2.0%; and VEGF/Ang2, 12 ± 4%. (Scale bars: 25 μm.)