Differential expression of endothelial nutrient transporters (MCT1 and GLUT1) in the developing eyes of mice

Ayuko Kishimoto, Hiromi Takashi-Iwanaga, Masahiko Watanbe M, Toshihiko Iwanaga

The paper can be accessed here.

 Introduction

Endothelial cells at the blood brain express glucose transporter (GLUT)-1 for uptake of glucose, but also express monocarboxylate transporters (MCT), which are required because neonates depend on monocarboxylates derived form milk as a source of energy. Within adults, all blood tissue barriers, including the blood retina barrier, express both GLUT1 and MCT1.

During development, a special hyaloid vascular system develops as part of the ocular vascular system, which degenerates during later stages of development of the eye. The hyaloid artery branches into tunica vasculosa lentis (TVL) at the lens and the vasa hyaloidea propria (VHP) along the internal surface of the retina. In later stages of development, the hyaloid artery, along with its branches regress, as the central artery and its six branches develop to form the permanent vasculature of the eye supplying blood to the inner two thirds of the retina. As within the central retinal artery, tight junctions between the endothelial cells of the hyaloid artery limit diffusion into the perivascular space. This results in the need of transporters such as GLUT1 for transport of nutrient across the blood-retina barrier.

This study investigates the morphology of ocular vessels expressing GLUT1/MCT1 to characterize the new development of blood vessels differentially expressing GLUT1 and MCT1 in the eye.

Methods

Eye samples were obtained from embryos, neonates of postnatal day 0-3, 5, 7, 9, 11, 13, 15, 20 and 25 as well as adult mice. The retina and lens were isolated from these eyeballs. Immunohistorchemistry was performed on samples, and to obtain double immunoflourescnce, the samples were incubated with anti-MCT1 antibody, ant-GLUT1 antibody and anti-CD31 antibody.

Silver intensified immunogold staining was used for electron microscopy, where frozen sections were incubated with an anti-MCT1 antibody followed by a secondary antibody covalently linked with gold particles. Subsequently, the samples underwent silver enrichment and were stained with uranyl acetate and lead citrate for observation under an electron microscope.

For scanning electron microscopy, posterior sections of the eyeballs and sections of the cerebral cortex were obtained, prepared and coated with osmium in a plasma osmium coater. The samples were then examined with and SEM at an acceleration voltage of 5kV.

Results

Hyaloid Vascular system

In fetuses, the VHP (on the surface of the retina) and VTL (on the lens) of the hyaloid vascular system both possess GLUT1 transporters. The VHP had GLUT1 transporters running along the entire length, with a few MCT1 expressing cells dispersed within the endothelium f neonates. The frequency of MCT1 expressing cells was higher at the periphery of the VHP.  In the VTL, GLUT1 transporters were present throughout, but MCT1 transporters were dispersed at the posterior and lateral surfaces of the lens. The hyaloid vascular system, present only in early stages of development, expressed GLUT1 along its entire length, whereas retinal and brain endothelia predominantly express MCT1 in early stages of development. 

Fig. 6. An equatorial view of a lens stained for MCT1 and GLUT1. Some MCT1-expressing vessels intermingle with GLUT1-expressing vessels of the TVL. Bar 200 μm.

Vascular systems in the developing retina

Vascularisation of the retina begins at birth. Up to day 3, growing capillaries do not have either GLUT1 or MCT1 transporters. Around postnatal day 5, MCT1 transporters begin to appear in the retina near the optical disc and spread into the superficial layer of the retina by till day 7. Neither GLUT1 nor MCT1 is observed in the central retinal artery or any of its six branches. However, the veins and capillaries both expressed MCT1. A dense sprouting, dense around the vein but scarce around the artery, did not express MCT1 or GLUT1. The sprouting began near the optic disc at day 0 and extended to t hora serata by day 9. During angiogenesis, desmin (a component of muscle specific intermediate filaments) was expressed in pericytes and the smooth muscles cells of the vessels. The pericytes were found around MCT1 expressing central plexus. GLUT1 transporters increased slowly in the ocular vessels after day 11, and at day 20 were comparable to the frequency of MCT1 transporters. At day 25, the GLUT1 transporters appeared to be more dominant than MCT1 transporters.

Radicular Projections

MCT1 expressing capillaries displayed long projections of uniform thickness that did not branch dendritically. These were not filopodia as the were around 5um in thickness, whereas filopodia are thicker. These projections were not associated with pericytes and were seen singly or in small groups all along the capillaries. These projections were also seen around veins expressing MCT1 but not in arteries, which had no MCT1. These projections were most dense on days 5 through 11, the mid-stage for suckling, and were absent by day 20. These projections were also found in capillaries of the neonatal brain.

Figure 12. Scanning electron micrographs of capillary vessels in the retina at neonatal 7 days. The extracellular matrix has been thoroughly removed by alkaline maceration to expose the surface of endothelial cells. Many radicles project from the outer surface of endothelial cells (E). Bars 10 μm (a), 1 μm (b).

Discussion

The hyaloid vascular system initially only expressed GLUT1, whereas retinal capillaries and veins expressed MCT1. This may be because the hyaloid vasculature is a temporary vascular system. Also, MCT1 expressing endothelial cells were present amidst GLUT1 expressing endothelial cells in hyaloid vessels, indicating specific regulation of MCT1 and GLUT1 during angiogenesis, a patter not observed in the brain.

Capillaries and veins of the developing retina predominantly express MCT1, likely in response to the presence of monocarboxylates from milk as a source of energy. Also, in the developing eye, the shift from MCT1 to GLUT1 expression occurs after weaning. During angiogenesis, arteries did not express any MCT1 or GLUT1.

The radicular projections observed in the SEM were a surprising feature in this research. These were found in MCT1 expressing cells in both the retina and the brain. These projections may indicate a novel feature in vessel growth present in both retinal and brain endothelium and may be related to the use of monocarboxyaltes as an energy source.

The overall findings showed that differential expression of MCT1 and GLUT1 occurs in the ocular vasculature during development. Some of these features observed in the blood-retinal barriers can be seen in the blood-brain barrier as well. The presence of radicular projections indicate a novel feature present in angiogenesis.

Critique

This paper was an easy read, with the introduction covering all the information required to understand the research. The layout was also easy to follow, and the text of the body as well as the figures were large enough to read. The figures were extremely clear with straightforward explanations. There was no unnecessary, excessive information in the paper, making it a simple read.

The research itself was very straightforward, with three procedures. The paper explains development of vaculature in the eye in an excellent way, describing all the vessels and arteries  present during different developmental stages. It took into consideration previous research on vasculature in the eye as well as energy transporters present within the vasculature of both the eye and the brain. Incorporating previous research into their paper, the authors were also able to correct errors that arose in previous studies. A great component of the paper is that the researchers came across a novel feature of the developing vessels in the eye and brain whilst trying to understand development of blood vessels in terms of expression of endothelial transporters. This novel feature gives the paper an edge in terms of possible future research. 

The overall quality of this paper and research is excellent. However, there were some issues in the paper. The discussion mostly reiterates the results and does not expand on possible reasons of why certain results were obtained or try to provide causative information on their results. In addition, I think that the research should have been conducted on the eyes at the first 30 postnatal days in order to ensure complete understanding of developmental stages. Often times, the researchers found that a process appeared sometime between to study dates, but were not able to state the exact time of development. I believe that in a developmental study it is important to know exact timing of each developmental stage.

Whilst some aspects of the experimental design and the material of the discussion may be lacking, the paper, in general, does a wonderful job at investigating differential expression of nutrient transporters in developing ocular vasculature and opens the door to further research regarding endothelial development in the eye as well as the brain.

References

Kishimoto A., Takashi-Iwanaga H., Watanabe M., Iwanaga T., 2016. Differential expression of endothelial nutrient transporters (MCT1 and GLUT1) in teh deloping eyes of mice. Experimental Eye Research. 153:170-177.