AIP1 functions as an endogenous inhibitor of VEGFR2-mediated signaling and inflammatory angiogenesis in mice
Haifeng Zhang, Yun He, Shengchuan Dai, Zhe Xu, Yan Luo, Ting Wan, Dianhong Luo, Dennis Jones, Shibo Tang, Hong Chen, William C. Sessa, Wang Min
Haifeng Zhang, Yun He, Shengchuan Dai, Zhe Xu, Yan Luo, Ting Wan, Dianhong Luo, Dennis Jones, Shibo Tang, Hong Chen, William C. Sessa, Wang Min
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Research Article Vascular biology

AIP1 functions as an endogenous inhibitor of VEGFR2-mediated signaling and inflammatory angiogenesis in mice

Abstract

ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice.

Authors

Haifeng Zhang, Yun He, Shengchuan Dai, Zhe Xu, Yan Luo, Ting Wan, Dianhong Luo, Dennis Jones, Shibo Tang, Hong Chen, William C. Sessa, Wang Min

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Figure 1

Generation of AIP1-flox and knockout mice.

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Generation of AIP1-flox and knockout mice. (A) Schematic diagram for str...
(A) Schematic diagram for strategy to generate AIP1-flox mice. The targeting vector contains exons 3/4 at 5′ arm, exons 5/6 in the targeting region, and exon 7 at 3′ arm. PCR primers P1–P6 are shown. The 5′ probe with HindIII digest, and 3′ probe with ScaI digest are indicated. (B and C) Screening of ES clones. Genomic DNA from positive clones was extracted and used for Southern blot, using 5′ probe after HindIII digest (B) and 3′ probe after ScaI digest (C). Clone 51 was positive and used for blastocyst injection to generate chimera mice. (D) Generation of AIP1+/lox and AIP1+l– mice. The chimeras were mated with C57BL/6 for germline transmission to obtain AIP1+/lox. AIP1+/lox mice were further mated with β-actin Cre mice to delete both the targeting region (exons 5/6) and the Neo gene (AIP1+/– mice). KO mice were obtained by mating between the heterozygous (AIP1+/–) male and female. (E and F) AIP1 deletion in tissues. Muscle and aorta from WT and KO mice were homogenized and AIP1 expression was detected by Western blot with anti-AIP1 antibody (E). AIP1 expression in paraffin section of muscle and aorta was determined by immunohistochemistry with anti-AIP1 antibody (F). For muscle tissue, arrows indicate capillaries and the arrowhead indicates a large vessel. For aorta tissue, the arrow indicates endothelium and the arrowhead indicates smooth muscle layer.