Altered expression of the tachykinins substance P/neurokinin A/hemokinin-1 and their preferred neurokinin 1/neurokinin 2 receptors in uterine leiomyomata

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Altered expression of the tachykinins substance P/neurokinin A/hemokinin-1 and their preferred neurokinin 1/neurokinin 2 receptors in uterine leiomyomata Ayoze González-Santana, M.Sc., Sara Marrero-Hernández, M.Sc., Idaira Dorta, Ph.D., Mariano Hernández, Ph.D., Francisco María Pinto, Ph.D., Delia Báez, M.D., Ph.D., Aixa R. Bello, Ph.D., Luz Candenas, Ph.D., Teresa A. Almeida, Ph.D. Fertility and Sterility Volume 106, Issue 6, Pages 1521-1529 (November 2016) DOI: 10.1016/j.fertnstert.2016.07.007 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Relative quantification of TACR1-Fl, TACR1-Tr, TACR2, and TAC1 mRNA in 20 leiomyomas (LM) and their matched myometrium (MY). Statistical analyses were performed using the Mann-Whitney test. Bars show the mean fold change, and error bars indicate SEM. *P<.05, ** P<.01, *** P<.001. Fertility and Sterility 2016 106, 1521-1529DOI: (10.1016/j.fertnstert.2016.07.007) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Substance P, NKA, and HK-1 immunoreactivity in myometrium (A–C) and matched leiomyoma (D–F) tissues. (A) Substance P immunoreactivity in cytoplasm (arrowheads) and a few nuclei (black arrow) in SMCs, connective (black arrow with solid circle at base), and endothelial cells of myometrium (red arrow with solid circle at base. (D) Stronger immunoreaction in leiomyoma SMCs (black arrows) and connective cells (black arrows with solid circle at base). Blue nuclei (A-D) correspond to hematoxylin staining. (B) Neurokinin A immunoreactive nuclei in SMCs (black arrows), VSMCs (red arrows), endothelial (red arrows with solid circle at base), and connective cells (black arrows with solid circle at base) of myometrium. (E) Stronger immunoreaction in leiomyoma SMC (black arrows) and connective cells (black arrows with solid circle at base). Endothelial cells (red arrow with solid circle at base) are also shown. (C, F) Hemokinin-1 immunoreactive cytoplasm in SMCs (arrowheads) and endothelial cells (red arrows with solid circle at base) of myometrium and leiomyoma. In leiomyoma, connective HK-1 immunoreactive cells are present compared with myometrium (black arrows with solid circle at base). Asterisks denote the vessel lumen. No hematoxylin counterstain was applied for NKA and HK-1. Scale bars = 500 μm. Fertility and Sterility 2016 106, 1521-1529DOI: (10.1016/j.fertnstert.2016.07.007) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 NK1R and NK2R immunoreactivity in myometrium (A, B) and matched leiomyoma (C, D) tissues. (A) Neurokinin 1R-ir in cytoplasm of SMCs (arrowheads), VSMCs (red arrow), and weaker in connective cells (arrows with solid circle at base) of myometrium. (C) Intense NK1R-ir connective cells (arrows with solid circle at base) in leiomyoma. Leiomyoma VSMCs (red arrows) and endothelial cells (red arrow with solid circle at base) are also shown. (B, D) NK2R-ir in SMCs (black arrows), with predominantly nuclear localization, in myometrium and leiomyoma. NK2R-ir in leiomyoma connective cells (black arrow with solid circle at base (d). Asterisks denote the vessel lumen. Scale bars = 500 μm. Fertility and Sterility 2016 106, 1521-1529DOI: (10.1016/j.fertnstert.2016.07.007) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Comparative expression of tachykinin NK1R isoforms and NK2R proteins, in leiomyomas and their matched myometrium. Lanes 1–2, 3–4, 5–6, and 7–8 represent bands detected in myometrium-matched leiomyoma of four patients, respectively. (A) Western blot analysis with a specific N-terminal antibody against human NK1R showed two bands corresponding to NK1R-Fl and NK1R-Tr. Relative expression of NK1R-Tr was higher in leiomyomas compared with myometrium, but no differences were observed for NK1R-Fl. (B) Western blot analysis with a specific anti-human NK2R antibody shows similar expression levels in both tissues. Each bar represents the mean with SEM of eight different samples. *P<.05. Fertility and Sterility 2016 106, 1521-1529DOI: (10.1016/j.fertnstert.2016.07.007) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions