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Inhibition of the Notch Pathway Promotes Flap Survival by Inducing Functional Neoangiogenesis

Abbas, Ozan Luay MD; Borman, Hüseyin MD; Terzi, Yunus K. PhD; Terzi, Ayşen MD; Bayraktar, Nilüfer MD; Özkan, Burak MD; Yazici, Ayşe C. PhD

doi: 10.1097/SAP.0000000000000197

Objective The Notch pathway seems to function as an antiangiogenic factor, negatively regulating the sprouting effect of vascular endothelial growth factor (VEGF). This function is well defined in embryonic and tumor vasculature. However, little is known about its function in ischemia-induced angiogenesis. In the first part of this study, we investigated the role of Notch in reparative angiogenesis after ischemia. In the second part, we hypothesized that anti-Notch therapy will result in increased angiogenic sprouting. We analyzed the effect of Notch inhibition in the induction of angiogenic sprouting.

Methods In the first part, we investigated the effect of ischemia on the Notch ligand delta-like ligand 4 (DLL4). Twenty rats were divided equally into 2 groups. In the surgery group, dorsal skin flap was used as model of ischemia. In the control group, no surgical procedure was performed. DLL4 and VEGF gene expressions were assessed. Immunohistochemical staining was used for detection of DLL4 in tissue materials. Plasma levels of VEGF and DLL4 were measured. In the second part, we investigated the effect of Notch inhibition using a gamma-secretase inhibitor (GSI) on inducing neoangiogenesis. Twenty rats were assigned to 2 equal groups. In all animals, dorsal skin flap was raised and sutured back into its bed. Animals in the GSI-treated group received GSI intravenously after surgery for 3 days. Saline was administered in the control group. Necrotic area measurements, microangiography, and histologic evaluations were performed to compare groups.

Results In the first part, VEGF and DLL expressions increased in ischemic tissues (P < 0.01). Immunohistochemical analysis revealed that DLL4 expression was upregulated in capillary endothelial cells after ischemia. Plasma levels for VEGF and DLL4 were higher in the animals that underwent surgery (P < 0.01). In the second part, GSI treatment resulted in higher flap survival rates (P < 0.05). Microscopic analysis exhibited increase in the number of microvascular structures after GSI treatment (P < 0.05). Microangiographic evaluation showed that neovascularization increased in the GSI-applied flaps.

Conclusions We present an evidence for the importance of the Notch pathway in the regulation of ischemia-induced angiogenesis. Notch inhibition promotes flap survival by creating a neovasculature that has an increase in vascular density.

From the *Department of Plastic and Reconstructive Surgery, Ahi Evran University, Kirsehir, Turkey; and Departments of †Plastic and Reconstructive Surgery, ‡Medical Genetics, §Pathology, ∥Biochemistry, and ¶Biostatistics, Baskent University, Ankara, Turkey.

Received December 29, 2013, and accepted for publication, after revision, February 12, 2014.

Conflicts of interest and sources of funding: none declared.

Presented at the 2nd EURAPS Research Council Meeting, Antalya, Turkey, May 22 to 23, 2013, and the 34th Annual Congress of Turkish Plastic Reconstructive and Aesthetic Surgery Association, Antalya, Turkey, October 31 to November 4, 2012.

Reprints: Ozan Luay Abbas, MD, Department of Plastic and Reconstructive Surgery, Ahi Evran University Hospital, Kervansaray Mah.2019.Sk. No:1 Merkez, Kirşehir, Turkey. E-mail:

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