Immunohistochemical detection of vascular endothelial growth factor (VEGF) and VEGF receptors Flt-1 and KDR/Flk-1 in the cochlea of guinea pigs

doi:10.1016/S0378-5955(01)00262-3

Hearing Research

Volume 155, Issues 1–2, May 2001, Pages 175-180

https://doi.org/10.1016/S0378-5955(01)00262-3 Get rights and content

Abstract

Vascular endothelial growth factor (VEGF) is known as an endothelial cell-specific mitogen. There are no reports concerning the presence of VEGF in the inner ear. To gain information, immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF receptors Flt-1 and KDR/Flk-1 was performed on paraffin-sectioned temporal bones from five guinea pigs. Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in spiral ganglion cells. VEGF could also be found in the endothelium of blood vessels, in the spiral ligament and in the organ of Corti. Flt-1 was found in the limbus epithelium, in all supporting cells of the organ of Corti, in Claudius cells, cells of the sulcus and in the spiral ligament. Flk-1 could be detected in some supporting cells of the organ of Corti (inner pillar cells and Deiters’ cells). Immunoreactivity to Flk-1 was also found in endothelium of blood vessels and in the spiral ligament. Hair cells showed VEGF immunostaining, but did not contain staining to Flt-1 nor Flk-1. In the stria vascularis any immunoreactivity to all used VEGF and VEGF receptor antibodies could not be detected. The findings were supported by Western blot analysis on inner ear tissues and ovaries from guinea pigs. We may conclude that the growth factor VEGF and both receptors participate in cochlear physiology.

Introduction

The nitric oxide/cGMP pathway is a well accepted signal cascade integrated in the physiology of the inner ear (Fessenden and Schacht, 1998, Hess et al., 1998, Michel et al., 1999). In other organs interactions between the NO/cGMP pathway and vascular endothelial growth factor (VEGF) have been found (Morbidelli et al., 1996, Kroll and Waltenberger, 1998, Shen et al., 1999). VEGF, known as an endothelial cell mitogen, also has effects on vascular permeability, migration, vessel formation and relaxation (Ferrara et al., 1992, Keck et al., 1989). Recently, neurotrophic and mitogenic activity on neurons has been reported in addition (Sondell et al., 1999). The effects of VEGF are thought to be mediated by two high-affinity receptors, Flt-1 (fms-like tyrosine kinase) or VEGF receptor-1 (VEGFR-1) and KDR (kinase-insert-domain-containing receptor)/Flk-1 or VEGF receptor-2 (VEGFR-2). For sensory organs, constitutive expression of VEGF and receptors has been reported for the eye (Kim et al., 1999) and the vestibular system (Hess et al., 2000a, Hess et al., 2000b), but for the cochlea data are not available yet.

Section snippets

Materials and methods

Three-month-old female guinea pigs (n=5) were deeply anesthetized with Nembutal (20 mg/kg body weight), followed by cardiac perfusion with fixative (4% paraformaldehyde, buffered at pH 7.4) after flushing out the blood cells with phosphate-buffered saline (PBS, 0.1 M at pH 7.4). Both temporal bones of each animal were removed, the bulla opened and immersed in the same fixative for 12 h. Decalcification was performed with EDTA solution 10%, buffered with Tris at pH 7.0 over 7 days. The tissue

VEGF immunostaining

VEGF was detectable in spiral ganglion cells and generally in vascular endothelium (Fig. 1a,e). Also cells of the spiral ligament, possibly fibrocytes and endothelial cells (Fig. 1a,f), showed immunoreactivity to VEGF. No staining could be detected in the stria vascularis (Fig. 1f). Within the organ of Corti, staining was detected in supporting cells and sensory cells such as inner and outer hair cells (Fig. 1c,d). A significant difference between the two different VEGF antibodies could not be

Discussion

Our findings give evidence that VEGF and VEGF receptors are constitutively expressed in the cochlea of guinea pigs. It has been demonstrated that VEGF induces expression of endothelial constitutive NO synthase (ecNOS) e.g. in blood vessels (Bouloumie et al., 1999, Hood et al., 1998, Tilton et al., 1999). Although there are contradictory statements concerning the differential function of VEGF receptors, recently published data allocate the VEGF receptor 2 KDR/Flk-1 to expression (Kroll and

Acknowledgements

The financial support of the Jean-Uhrmacher-Stiftung is appreciated gratefully. The authors thank Mr. S. Basnaoglu for excellent technical support.

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Immunohistochemical detection of vascular endothelial growth factor (VEGF) and VEGF receptors Flt-1 and KDR/Flk-1 in the cochlea of guinea pigs

Abstract

Introduction

Section snippets

Materials and methods

VEGF immunostaining

Discussion

Acknowledgements

Anal. Biochem.

Biochem. Biophys. Res. Commun.

Hear. Res.

Brain Res.

Brain Res.

Neurosci. Lett.

Neurosci. Lett.

Biochem. Biophys. Res. Commun.

Hear. Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.