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. 2017 Jan;77(1):73-80.
doi: 10.1055/s-0042-113461.

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

I Bekes  1 , V Haunerdinger  2 , R Sauter  1 , I Holzheu  1 , W Janni  1 , A Wöckel  3 , C Wulff  3
Affiliations

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

I Bekes et al. Geburtshilfe Frauenheilkd. 2017 Jan.

Abstract
in English, German

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.

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.

Keywords: OHSS; Slit2; VEGF; hCG; luteal permeability.

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Conflict of interest statement

Conflict of Interest The authors declare that they have no conflict of interests.

Figures

Fig. 1
Fig. 1
Slit2 and VEGF gene expression in LGCs after knockdown, stimulation and inhibition. Slit2 knockdown resulted in marked reduction of Slit2 gene expression (orange column) but did not result in any change in VEGF (red column) (a). Slit2 (b) and VEGF (c) gene expression after hCG stimulation and VEGF inhibition (Flt-1 Fc) in LGCs. The first column shows the control (normalized to 1), the second column shows gene expression after hCG treatment, the third column shows gene expression after VEGF inhibition (Flt-1 Fc) and the fourth column shows gene expression after simultaneous stimulation with hCG and VEGF inhibition (Flt-1 Fc). There was a significant decrease in Slit2 after hCG treatment which was not reversed when Flt-Fc was administered simultaneously. VEGF inhibition alone had no effect on Slit2 expression (b). VEGF gene expression was significantly elevated after hCG treatment and not affected by additional Flt-1 Fc administration (c).
Fig. 2
Fig. 2
Endothelial cell morphology of HUVECs after VEGF, Flt-1 Fc or Slit2 stimulation. Cell culture morphology of HUVECs (control) (a), HUVECS + VEGF (b), HUVECs + VEGF inhibition with Flt-1 Fc (c) and HUVECs + Slit2 (d) at 96 h of incubation (bar = 100 µm). VEGF stimulation resulted in an arrangement of endothelial cells (b), which was reversed after inhibition with Flt-1 Fc (c). Slit2 stimulation was comparable to controls; Slit2 did not affect the cells and did not lead to any kind of arrangement of cells (d).
Fig. 3
Fig. 3
Gene expression of CLDN5 and CDH5 in HUVECs after Slit2 or VEGF stimulation. Gene expression of CLDN5 (orange column) and CDH5 (red column) after exogenous stimulation of HUVECs with Slit2 and VEGF at 96 h. Both adhesion proteins (CLDN5 and CDH5) were found to be significantly increased after Slit2 stimulation. In contrast, VEGF stimulation resulted in a significant decrease in adhesion protein expression.
Fig. 4
Fig. 4
Gene expression of CLDN5 and CDH5 and permeability measurement in HUVECs after Robo4 knockdown. Gene expression of Robo4 (a), CLDN5 and CDH5 (b) and permeability measurement (c) at 48 h, 72 h and 96 h of incubation after Robo4 knockdown in HUVECs. Gene silencing of Robo4 was significant at all time-points (a). CLDN5 was significantly downregulated at 48 h, 72 h and 96 h of incubation (orange column), while CDH5 was first significantly reduced after 72 h (red column) (b). Endothelial permeability subsequently increased and was significant at 72 h and 96 h (c).
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