Spatial Differences in Blood vs. Lymphatic Growth into the Cornea

Spatial Differences in Blood vs. Lymphatic Spatial Differences in Blood vs. Lymphatic Growth into the CorneaGrowth into the Cornea

Amir Reza Hajrasouliha MD, Zahra Sadrai MD, Reza Dana MD, MPH, MSc.

Schepens Eye Research Institute, Massachusetts Eye & Ear Infirmary,

Department of Ophthalmology, Harvard Medical School, Boston, MA

Financial Disclosure: The authors have no financial interest in the subject matter of this poster.

Funding Sources: NIH/NEI RO1-EY12963

Spatial Differences in Blood vs. Lymphatic Growth into the Cornea

Introduction

Angiogenesis contributes to both physiological and pathological processes. The process of lymphatic and blood vessel growth is tightly regulated by several key angiogenic factors including the fibroblast growth factors (FGF) and vascular endothelial growth factors (VEGF).

FGFs are pleiotropic molecules capable of acting on a variety of cell types

of endodermal, mesenchymal and neuroectodermal origin.

The complex biologic responses of lymph and blood vessel growth are controlled with multiple overlapping functions of these. It is not well clear how these factors selectively activate separate pathways of lymphatic or blood vessel growth when both type of vessel form simultaneously.

Purpose

To study the effect of b-FGF and its contribution to the spatial pattern of VEGF ligand expression and angiogenesis.


Either whole cornea or different zones of the cornea based on the proximity to the pellet were isolated on day 1,3 ,7 post- implantation.

Zones were selected as illustrated in the figure on right: zone 1, the temporal side (pellet side)zone 2, the superior and inferior quadrant zone 3, the nasal side (opposite side of the pellet)

Methods

Male 6-8 week old BALB/c mice were used in the experiments. Eighty-ng of b-FGF (R&D Systems, Minneapolis, MN, USA) pellets were positioned into the pocket 1.0mm apart from the limbal vascular arcade. Mice were sacrificed on day 1, 3 and 7 post-implantation and Immunohistochemical staining and real time PCR were performed. All animals were treated in accordance with the ARVO statement for the use of Animals in Ophthalmic and Vision Research. In addition, all experiments described herein were conducted under Institutional ACUC approval.

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Method:

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Figure 1. Corneal flat mounts showing spatial differences in hem- and lymph-angiogenesis stimulated by b-FGF pellet (80ng) implanted into the corneal stroma (upper panels )and stained on day 1, 3 and 7 post implantation. Blood vessel (green) grow to the central cornea from the pellet side while lymphatic vessel (red) grow on the opposite side of the pellet (lower panels, higher magnification).

Results

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Results

Figure 2. Time course of VEGF ligands’ mRNA expression was determined by real time PCR in corneas of normal and b-FGF micropellet implanted mice. The expression levels of all VEGF ligands increased in corneas after b-FGF micropellet implantation.


Figure 3. Spatial differences in mRNA levels of VEGF ligands in normal and b-FGF micropellet implanted mice were observed. The expression levels of VEGF- A increases most at the pellet side over time, while VEGF-D increases mostly on the opposite side of the pellet after b-FGF micropellet implantation. VEGF-C increased similarly in all three zones over time.

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• b-FGF stimulates VEGF-A in areas closest to the higher concentration b-FGF.• VEGF-C over-expression occurs equally in all zones, suggesting its role in both lymphatic and blood vessel formation.• Selective VEGF-D over expression in the opposite side of the pellet may indicate VEGF-D as the mediator of lymphangiogenesis induced by b-FGF.

Summary

VA: VEGF-A

VC:VEGF-C

VD: VEGF-D


Conclusions:

Angiogenesis is an intricate process that involves many cell types playing different roles. FGF is capable of regulating other growth factor signaling, and may be upstream to VEGFs.

The discrete patterns of VEGF ligands expressed in different areas of the cornea induced by FGF suggest that these spatial differences regulate blood vs. lymphatic vessel growth.

Contact [email protected]@schepens.harvard.edu

Spatial Differences in Blood vs. Lymphatic Growth into the Cornea

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