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Supplementary Figure 1 from Expression of Terminally Glycosylated Calcitonin Receptor–Like Receptor in Uterine Leiomyoma: Endothelial Phenotype and Association with Microvascular Density

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posted on 2023-03-31, 16:05 authored by Leonid L. Nikitenko, Tanya Cross, Leticia Campo, Helen Turley, Russell Leek, Sanjiv Manek, Roy Bicknell, Margaret C.P. Rees
Supplementary Figure 1 from Expression of Terminally Glycosylated Calcitonin Receptor–Like Receptor in Uterine Leiomyoma: Endothelial Phenotype and Association with Microvascular Density

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ARTICLE ABSTRACT

Purpose: The role for the hypoxia-inducible angiogenic factor adrenomedullin (AM) in tumor growth and progression has been suggested. Calcitonin receptor–like receptor (CL) is a G protein–coupled receptor (GPCR) that mediates effects of AM, but little information is available on its expression and functional state in human tumors. The present study attempted to determine CL potential for antiangiogenic therapy of uterine leiomyoma.Experimental Design and Results: GPCR CL is transported to the cell surface and recognized by AM only when terminally/mature glycosylated. The presence and localization of this form of the receptor in tumor and surrounding myometrial tissues obtained from leiomyoma-bearing uteri were examined using deglycosylation, immunoblotting, and immunofluorescence analysis. The mature CL glycoprotein was expressed in both tissues and localized exclusively in normal and tumor endothelium within leiomyoma-bearing uteri. The functionality of the receptor expressed in myometrial microvascular endothelial cells (MMVEC) was examined in vitro using receptor internalization and angiogenic assays. The mature CL glycoprotein expressed by primary MMVECs was functional because AM interacted with this GPCR and induced its internalization as well as angiogenic effects (proliferation and migration) in MMVECs in vitro. Finally, the levels of tissue-expressed mature CL glycoprotein as a functional form of this GPCR were analyzed by immunoblotting. The expression of this functional form of the receptor in vivo was significantly decreased (P = 0.01) in leiomyoma tissue, and this was concurrent with the decrease in microvascular density (measured by Chalkley counting) in tumor compared with surrounding myometrium (P = 0.031).Conclusions: Our findings suggest that GPCR CL mediates angiogenic effects of AM in myometrium and that further evaluation of the properties of the CL expressed in both normal and tumor endothelium in vivo may be essential before targeting this endothelial GPCR for antiangiogenic therapies.

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