The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel
Aleksanteri Aspelund, … , Ilkka Immonen, Kari Alitalo
Aleksanteri Aspelund, … , Ilkka Immonen, Kari Alitalo
Published July 25, 2014
Citation Information: J Clin Invest. 2014;124(9):3975-3986. https://doi.org/10.1172/JCI75395.
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Research Article

The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel

Abstract

In glaucoma, aqueous outflow into the Schlemm’s canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

Authors

Aleksanteri Aspelund, Tuomas Tammela, Salli Antila, Harri Nurmi, Veli-Matti Leppänen, Georgia Zarkada, Lukas Stanczuk, Mathias Francois, Taija Mäkinen, Pipsa Saharinen, Ilkka Immonen, Kari Alitalo

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Figure 1

SC ECs display molecular features of lymphatic endothelium.

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SC ECs display molecular features of lymphatic endothelium. (A–M) Whole-...
(A–M) Whole-mount immunofluorescence staining of the adult murine eye using antibodies against PECAM-1, PROX1, and VEGFR-3. The entire thickness of the limbus was visualized by confocal imaging into 1 z stack. Subsets with the SC (A–D), the AV (E–H), and the ES vasculature (I–L) are shown. The joining point of the AV into the SC (arrow) and the joining point of the AV into the ES vein (arrowhead) are indicated. Dashed outlines denote the SC and ES lymphatic vessels. c, capillary; pcv, postcapillary venule; v, vein; a, artery. (M) AH drainage route in a 90° y-axis projection of the confocal stack in A–L. xyz axes are shown for orientation between A–L and M. (N) The SC (dashed outline) and ES lymphatic vessels (asterisks) in vivo in Prox1-CreERT2 LSL-tdTomato lineage tracer mice after 4-OHT administration. (O and P) Immunohistochemical PROX1 staining and negative control staining of the SC in a human eye. Arrows indicate PROX1 expression in SC ECs. (Q) Visualization of zebrafish SC by staining with antibodies against human and mouse PROX1. (R–T) Immunofluorescence staining of murine SC and ES lymphatic vessels using antibodies against CCL21 (R) and LYVE-1 (S and T). Scale bars: 100 μm (A–L, S, and T); 50 μm (M); 200 μm (N–R).