Pathological Neovascularization Is Reduced by Inactivation of ADAM17 in Endothelial Cells but Not in Pericytes

Abstract

Key Words: ADAMs Ⅲ metalloproteinase-disintegrins Ⅲ TNF␣-convertase Ⅲ proliferative retinopathy Ⅲ pathological neovascularization P athological neovascularization has a critical role in diseases such as cancer, 1,2 rheumatoid arthritis 3 and proliferative retinopathies, including retinopathy of prematurity, diabetic retinopathy and the wet form of macular degeneration. 4,5 Therefore molecules with roles in pathological neovascularization are considered potential targets for treatment of these conditions. Previous studies have identified a role for the cell surface metalloproteinase ADAM17 (a disintegrin and metalloproteinase 17, also referred to as TACE [tumor necrosis factor ␣-converting enzyme]) in crosstalk between the VEGFR2 and extracellular signal-regulated kinase 1/2 in endothelial cells, and in processing several receptors with key functions in angiogenesis, including the VEGFR2 and Tie2. 6 The goal of the present study was to determine whether ADAM17 has a role in angiogenesis or pathological neovascularization in vivo by subjecting conditional knockout mice carrying floxed alleles of ADAM17 7 and a Cre-recombinase expressed either in endothelial cells (Tie2-Cre) or in smooth muscle cells and pericytes (␣-smooth muscle actin [␣sma] Cre) to mouse models of pathological neovascularization.ADAM17 was first discovered as the converting enzyme for tumor necrosis factor (TNF)␣, 8,9 a potent proinflammatory cytokine that is a causative factor in autoimmune diseases such as rheumatoid arthritis and Crohn's disease as well as in septic shock in mice. 10 Once mice lacking ADAM17 were generated, it became clear that ADAM17 is Original received August 14, 2009; revision received January 13, 2010; accepted January 19, 2010 The ability of ADAM17 to release endothelial cell membrane proteins on stimulation with VEGF-A raised questions about what role ADAM17 has during developmental angiogenesis and in pathological neovascularization in adult animals. Although mice lacking ADAM17 die perinatally, most likely as a consequence of their severe heart valve defects, 11,12 there have been no reports of defects in developmental angiogenesis in these animals. To address whether ADAM17 has a role in angiogenesis or pathological neovascularization or both, we conditionally inactivated ADAM17 in endothelial cells or in smooth muscle cells such as pericytes, and then determined how lack of ADAM17 affects two mouse models for pathological neovascularization, the oxygen induced retinopathy model for retinopathy of prematurity, and growth of heterotopically injected tumor cells. Moreover, we assessed proliferation and chord formation of endothelial cells lacking ADAM17, and evaluated the role of ADAM17 in the proteolytic release of membrane proteins with known roles in angiogenesis and pathological neovascularization. Methods Reagents and Cell LinesPorcine aortic endothelial cells expressing VEGFR2/KDR (PAE/ KDR cells) and mouse embryonic fibroblasts (mEFs) lacking ADAM17 have been described previously. 6,15 Reagents were from...

iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding

“…We found that PMA-stimulated shedding of the Eph receptor B4 (EphB4), KitL2, and Tie2 (12,14) was strongly reduced in iRhom2 −/− mEFs compared with WT control mEFs ( Fig.1 J-L; KitL2 shedding in primary mEFs is shown in Fig. S1C), whereas the PMA-stimulated shedding of the ADAM17 substrates CD62 ligand [cluster of differentiation 62L (CD62L)] and intercellular adhesion molecule-1 (ICAM-1) (14, 28) was not affected ( Fig.1 M and N).…”

“…The expression vectors for iRhom2, mitotic arrest deficient 2 (MAD2), and plasmids encoding alkaline phosphatase (AP)-tagged EGFR ligands, CD62L, ICAM-1, EphB4, and KitL2, have been described previously (4,12,14,20,28). The chimeras between TGFα and HB-EGF were generated by overlap extension PCR (34) using human TGFα-AP and HB-EGF-AP cDNAs as a template and cloned into pRc/CMV (Invitrogen) (see Fig.…”

“…Moreover, mice lacking ADAM17 resemble mice lacking the EGFR in that they have open eyes at birth, skin barrier defects, enlarged heart valves, abnormal mammary ductal morphogenesis, and lung defects (3,(8)(9)(10)(11). ADAM17 is also responsible for the proteolytic release, or "ectodomain shedding" of a variety of other membrane proteins from the cell surface, such as Kit ligand (KitL) 1 and 2 (12), the vascular endothelial growth factor receptor 2 and many others (13,14). However, most phenotypes caused by inactivation of ADAM17 can be explained through loss of shedding of TNFα or of EGFR ligands (3,(6)(7)(8)(9)(10)(11), so these can be considered the functionally dominant substrates of ADAM17.…”

Sjögren’s syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB

“…5,6 The metalloproteinase TACE (tumor necrosis factor a (TNF-a)-converting enzyme) has been shown to be of critical importance in angiogenesis, proposing that TACE is involved in pro-angiogenic pathways leading to vessel formation, promoting proliferation and differentiation of endothelial progenitor cells. [8][9][10] TACE is responsible for the release of the soluble form of TNF-a, and its production is closely linked to Sjö gren's syndrome (SS), an inflammatory autoimmune disorder that affects secretory organs such as the salivary and lacrimal glands [11][12][13][14] but, a literature search yielded no data on the angiogenic role of TACE-dependent VEGF-A/VEGFR2 activation pathway in this disease. 15,16 Based on these findings, in the current study we explore the involvement of TACE in VEGF/VEGFR-2-mediated angiogenesis in SS patients.…”

Ectodomain shedding and ADAMs in development

“…Although in most cases ADAM10-deficient mice phenocopy Notch signaling-deficient mice (Weber et al, 2011; Glomski et al, 2011), and ADAM17 knockout mice resemble EGFR or EGFR-ligand knockout mice (Franzke et al, 2012), there might be pathological situations under which both ADAM10 and ADAM17 share substrates in common signaling pathways, as indicated by previous cell-based experiments Bozkulak and Weinmaster, 2009). ADAM17 might also be involved in pathological neovascularization, implicating another functional importance of this protease in epithelial and endothelial maturation (Weskamp et al, 2010). …”