ADAMTS18 Metalloproteinase Regulates Angiogenesis In Vivo: Evidence from ADAMTS18 Deficient Mice and Zebrafish

Background: ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) members play crucial role in development, inflammation, cancer, and vascular biology. ADAMTS18 is an orphan ADAMTS for which substrates have yet to be discovered. Using ADAMTS18 knock-out (KO) mice, we demonstrated that ADAMTS18 deficiency in mice generated abnormal vascular phenotypes in particular in common carotid artery that is associated with aggravated thrombosis and ischemic cerebral stroke (Blood 122: 31, 2013). Methods and Results: Here, we further investigated the role of ADAMTS18 in angiogenesis. In an established in vivo angiogenesis model (Matrigel plug assay), ADAMTS18 KO mice showed significantly reduced neovessel formation compared to WT mice (Hemoglobin: WT vs. KO, 76.25 ± 28.8 vs. 50.5 ± 17.4, P = 0.048). To systematically evaluate the effect of ADAMTS18 on embryo angiogenesis, we developed zebrafish ADAMTS18 morpholino (MO) knock-down model. Images of trunk regions were taken at 48-hpf (hours post fertilization) with the vascular structures visualized by eGFP fluorescence. Labeled ISV (intersegmental vessel) and DLAV (dorsal longitudinal anastomotic vessel) showed regular development in the embryo injected with control MO. However, embryos injected with ADAMTS18-e3i3-MO presented a lower number of incomplete and thinner ISVs and ectopic sprouts of dorsal aorta. Additionally, ADAMTS18 knock down impaired formation of the CVP (caudal vein plexus) in zebrafish. In control embryos, CVP were formed honeycomb-like structures at the tail around 48-hpf. In contrast, ADAMTS18 knock down resulted in specific defects in CVP formation. Notably, poor blood circulation in the CV (caudal vein) and CCV (common cardinal vein) was observed in ADAMTS18-e3i3-MO injected zebrafish compared to that of control zebrafish. To disclose the potential signaling molecules involved in vascular abnormities, the proteins extracted from common carotid artery (CCA) of WT and ADAMTS18 KO mice were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. A total of 510 proteins in CCA were identified, and 77 of these proteins (~15%) of ADAMTS18 KO mice were differentially expressed compared to WT mice. 22 of the 77 (~29%) proteins are responsible for the regulation of vascular morphology and angiogenesis. Ingenuity® Pathway Analysis (IPA) predicated that TGFβ signaling (z-score = 2.358) was significantly activated. The key molecules involved in TGFβ signaling were then examined by Western blotting. Compared to WT mice, the expression of ALK-1 (a type of TGFβ receptor I, TβRI), smad-1, -5, phosphorylated smad-1, -5 were significantly decreased in the carotid artery of ADAMTS18 KO mice, while the expression of ALK-5 (another type of TGF-β receptor I) smad-2, -3, phosphorylated smad-2, -3 were significantly increased, which may result in the reduced endothelial cell proliferation and migration in ADAMTS18 KO mice. Conclusions: These data imply crucial roles of ADAMTS18 in the regulation of angiogenesis in vivo.