Angiopoietin-Like 7 Is an Anti-Angiogenic Protein Required to Prevent Vascularization of the Cornea

Abstract

PurposeWe sought to identify the anti-angiogenic molecule expressed in corneal keratocytes that is responsible for maintaining the avascularity of the cornea.MethodsHuman umbilical vein endothelial cells (HUVECs) were cultured with either human dermal fibroblasts or with human corneal keratocytes under serum-free conditions. The areas that exhibited blood vessel formation were estimated by immunostaining the cultures with an antitibody against CD31, a blood vessel marker. We also performed microarray gene-expression analysis and selected one molecule, angiopoietin-like 7 (ANGPTL7) for further functional studies conducted with the keratocytes and in vivo in mice.ResultsAreas showing blood vessel formation in normal serum-free medium were conditions were markedly smaller when HUVECs were co-cultured with corneal keratocytes than when they were co-cultured with the dermal fibroblasts under the same conditions. Microarray analysis revealed that ANGPTL7 expression was higher in keratocytes than in dermal fibroblasts. In vitro, inhibiting ANGPTL7 expression by using a specific siRNA led to greater tube formation than did the transfection of cells with a control siRNA, and this increase in tube formation was abolished when recombinant ANGPTL7 protein was added to the cultures. In vivo, intrastromal injections of an ANGPTL7 PshRNA into the avascular corneal stroma of mice resulted in the growth of blood vessels.Conclusions ANGPTL7, which is abundantly expressed in keratocytes, plays a major role in maintaining corneal avascularity and transparency.

Molecular Therapy for Degenerative Disc Disease: Clues from Secretome Analysis of the Notochordal Cell-Rich Nucleus Pulposus

“…Angiopoeitin like 7 (ANGPTL7) has been proposed as an anti-angiogeneic protein abundantly expressed in keratocytes and plays a major role in maintaining corneal avascularity and transparency45. Semaphorin 3E identified in NCCM belongs to class 3 semaphorin family of proteins, known as potent inhibitors of both pathological nerve innervation and vascular proliferation4647.…”

PAX6 regulates human corneal epithelium cell identity

“…Angiopoietin-Like 7 (ANGPTL7), which reportedly prevents neovascularization on the ocular surface (Toyono et al, 2015), indicating that PAX6 may contribute to corneal avascularity. In addition, promoter analysis revealed that PAX6 directly binds to the promoter of KRT12 (Shiraishi et al, 1998) and to ALDH3A1, activating its gene expression (Davis et al, 2008).…”

Evolution of the angiopoietin-like gene family in teleosts and their role in skin regeneration

“…In human Angptl7 is an anti-angiogenic factor and in vitro studies using cornea keratocyte cells demonstrated that this gene may be responsible for maintaining tissue avascularity in the eye [36, 37]. The decreased expression of angptl7 during sea bream skin regeneration and its negative correlation with blood vessel number suggests it may have a similar role to that in mammals (Fig.…”

“…In mouse, overexpression of ANGPTL6 in skin promotes epidermal hyperplasia and enlargement of dermal lymphatic and blood vessels to favour wound healing [34, 35]. ANGPTL7 regulates extracellular matrix (ECM) formation [36] and is highly expressed in keratinocytes and is a potent anti-angiogenic factor in the cornea [37]. This protein is also described to inhibit tumour growth in a mouse xenograph model [38] and is required for the regeneration of human hematopoietic stem and progenitor cells (HSPCs) [39, 40].…”