Haploinsufficiency of Klippel-Trenaunay syndrome geneAggf1inhibits developmental and pathological angiogenesis by inactivating PI3K and AKT and disrupts vascular integrity by activating VE-cadherin

Abstract

Aggf1 is the first gene identified for Klippel-Trenaunay syndrome (KTS), and encodes an angiogenic factor. However, the in vivo roles of Aggf1 are incompletely defined. Here we demonstrate that Aggf1 is essential for both physiological angiogenesis and pathological tumour angiogenesis in vivo. Two lines of Aggf1 knockout (KO) mice showed a particularly severe phenotype as no homozygous embryos were observed and heterozygous mice also showed embryonic lethality (haploinsufficient lethality) observed only for Vegfa and Dll4. Aggf1+/- KO caused defective angiogenesis in yolk sacs and embryos. Survived adult heterozygous mice exhibit frequent haemorrhages and increased vascular permeability due to increased phosphorylation and reduced membrane localization of VE-cadherin. AGGF1 inhibits VE-cadherin phosphorylation, increases plasma membrane VE-cadherin in ECs and in mice, blocks vascular permeability induced by ischaemia-reperfusion (IR), restores depressed cardiac function and contraction, reduces infarct sizes, cardiac fibrosis and necrosis, haemorrhages, edema, and macrophage density associated with IR. Mechanistically, AGGF1 promotes angiogenesis by activating catalytic p110α subunit and p85α regulatory subunit of PI3K, leading to activation of AKT, GSK3β and p70S6K. AKT activation is significantly reduced in heterozygous KO mice and isolated KO ECs, which can be rescued by exogenous AGGF1. ECs from KO mice show reduced capillary angiogenesis, which is rescued by AGGF1 and AKT. Tumour growth/angiogenesis is reduced in heterozygous mice, which was associated with reduced activation of p110α, p85α and AKT. Together with recent identification of somatic mutations in p110α (encoded by PIK3CA), our data establish a potential mechanistic link between AGGF1 and PIK3CA, the two genes identified for KTS.

Targeting AGGF1 (angiogenic factor with G patch and FHA domains 1) for Blocking Neointimal Formation After Vascular Injury

“…AGGF1 is required for specific differentiation of veins and development of intersegmental vessels during zebrafish embryogenesis,35 which was confirmed by Kashiwada et al 36. Our recent studies using AGGF1 +/− knockout (KO) mice showed that AGGF1 is essential for early embryogenesis and vascular development, and blocks vascular permeability 37. We have found that AGGF1 is an important signaling factor that can induce autophagy by activating JNK,33 regulate angiogenesis and vascular development by activating PI3K, AKT, GSK3β, and S6K and inhibiting ERK1/2 (also reported by Hu et al38), and maintain vascular integrity by inhibiting VE‐cadherin phosphorylation 35, 37.…”

“…Based on our extensive analyses of reported data in the literature and our preliminary data, we concluded that the AGGF1‐mediated ERK signaling pathway is the most relevant and logical signaling pathway for AGGF1 in VSMCs. Therefore, our research plans focus on the AGGF1‐ERK signaling, which has been well documented in the literature 37, 38. It should be interesting to explore other AGGF1‐mediated signaling pathways, such as AKT, JNK, and other pathways, in VSMCs functions and neointimal formation in the future.…”

“…Recombinant platelet‐derived growth factor‐BB (PDGF‐BB) was from R&D. The 6xHis‐tagged AGGF1 protein was purified as described by us previously 27, 33, 37…”

“…The AGGF1 +/− knockout (KO) mice with a gene‐trap allele were created by us and described previously 33, 37. Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Cleveland Clinic and the Ethics Committee on Animal Research of College of Life Science and Technology at Huazhong University of Science and Technology and performed in accordance with institutional guidelines.…”

“…Our recent studies using AGGF1 +/− knockout (KO) mice showed that AGGF1 is essential for early embryogenesis and vascular development, and blocks vascular permeability 37. We have found that AGGF1 is an important signaling factor that can induce autophagy by activating JNK,33 regulate angiogenesis and vascular development by activating PI3K, AKT, GSK3β, and S6K and inhibiting ERK1/2 (also reported by Hu et al38), and maintain vascular integrity by inhibiting VE‐cadherin phosphorylation 35, 37. We also showed that AGGF1 protein therapy can robustly treat CAD and MI by inducing autophagy and therapeutic angiogenesis 33.…”