Slit2/Robo4 Signaling: Potential Role of a VEGF-Antagonist Pathway to Regulate Luteal Permeability

Abstract

Introduction The corpus luteum (CL) is dependent on luteal vascular permeability, which is controlled by human chorionic gonadotropin (hCG) via vascular endothelial growth factor (VEGF). In this study we investigated the role of a potential VEGF antagonist pathway – Slit2/Robo4 – and its influence on endothelial cell adhesion.

Materials and Methods Luteinized granulosa cells (LGCs) were stimulated with hCG in the absence or presence of a VEGF inhibitor. The expression of VEGF and Slit2 were measured. Human umbilical vein endothelial cells (HUVECs) were stimulated with Slit2 or VEGF, and gene expressions of cadherin 5 (CDH5) and claudin 5 (CLDN5) were measured. Following Robo4 knockdown, CDH5, CLDN5 and endothelial permeability were measured.

Results Stimulation of human LGCs with hCG significantly increased VEGF while Slit2 expression was significantly suppressed. Inhibition of VEGF action after hCG stimulation did not change Slit2 suppression. Slit2 knockdown did not affect VEGF expression. While VEGF stimulation of HUVECs significantly suppressed CDH5 and CLDN5 gene expression, stimulation of HUVECs with Slit2 resulted in a significant increase in CDH5 and CLDN5. Robo4 knockdown was done, leading to downregulation of CDH5 and CLDN5 which resulted in significantly increased permeability.

Conclusions Our results indicate the existence of a VEGF-antagonist pathway in the CL that decreases vascular permeability. During the functional life of the CL the pathway is suppressed by hCG. It is possible that stimulation of this pathway could be used to treat ovarian hyperstimulation syndrome.

Zusammenfassung

Einleitung Voraussetzung für die regelrechte Funktion des Corpus luteum ist ein durchlässiges Gefäßsystem. Diese vaskuläre Durchlässigkeit wird durch die Einwirkung des humanen Choriogonadotropins (hCG) auf den Vascular Endothelial Growth Factor (VEGF) kontrolliert. In dieser Studie untersuchten wir ein potenzielles VEGF-antagonistisches System – das Slit2/Robo4-System – und dessen Auswirkung auf die endotheliale Zelladhäsion.

Material und Methoden Luteinisierte Granulosazellen (LGC) wurden mit hCG stimuliert mit oder ohne Beigabe eines VEGF-Hemmers. Es wurde die VEGF- und Slit2-Expression gemessen. Aus der menschlichen Nabelschnur gewonnene venöse Endothelzellen (HUVECs) wurden mit Slit2 oder VEGF stimuliert. Danach wurde die Genexpression von Cadherin 5 (CDH5) und Claudin 5 (CLDN5) gemessen. Es wurden ein Robo4-Knockdown durchgeführt und die nachfolgende CDH5- und CLDN5-Expression sowie die endotheliale Durchlässigkeit gemessen.

Ergebnisse Die Stimulation von menschlichen LGCs mit dem hCG führte zu einer wesentlichen Steigerung von VEGF, während die Slit2-Expression signifikant unterdrückt wurde. Die Hemmung der VEGF-Aktivität nach der hCG-Stimulation wirkte sich nicht auf die Unterdrückung der Slit2-Expression aus. Der Slit2-Knockdown hatte keine Auswirkungen auf die VEGF-Expression. Während die VEGF-Stimulation von HUVECs die Genexpression von CDH5 und CLDN5 signifikant unterdrückte, führte die Slit2-Stimulation von HUVECs zu einer signifikanten Steigerung der CDH5- und CLDN5-Expression. Es wurde ein Robo4-Knockdown durchgeführt, was zu einer Herabregulation von CDH5 und CLDN5 führte und die Durchlässigkeit signifikant steigerte.

Schlussfolgerung Unsere Ergebnisse weisen auf das Vorhandensein eines VEGF-antagonistischen Systems im Corpus luteum hin, das die vaskuläre Gefäßdurchlässigkeit mindert. Dieses System wird während der Funktionsdauer des Corpus luteum durch das hCG unterdrückt. Es kann daher angenommen werden, dass eine Stimulation dieses Systems zur Behandlung beispielsweise des ovariellen Hyperstimulationssyndroms eingesetzt werden könnte.

Key words

VEGF - hCG - Slit2 - luteal permeability - OHSS

Schlüsselwörter

VEGF - hCG - Slit2 - luteale Durchlässigkeit - OHSS

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