Orbital Angiogenesis and Lymphangiogenesis in Thyroid Eye Disease: An Analysis of Vascular Growth Factors with Clinical Correlation

Abstract

Purpose: The human orbit is an environment that is vulnerable to inflammation and edema in the setting of autoimmune thyroid disease. Our study investigated the tenet that orbital adipose tissue lacks lymphatic vessels and analyzed the clinicopathologic differences between patients with acute and chronic thyroid eye disease (TED). The underlying molecular mediators of blood and lymphatic vessel formation within the orbital fat also were evaluated.

Design: Retrospective cohort study.

Participants: The study included fat specimens from 26 orbits of 15 patients with TED undergoing orbital decompression. Orbital fat specimens from patients without TED as well as cadaveric orbital fat served as controls.

Methods: Tissue specimens were processed as formalin-fixed, paraffin-embedded sections or frozen cryosections for immunohistochemistry. Total RNA was extracted and analyzed via quantitative (real-time) reverse-transcription polymerase chain reaction. Clinicopathologic correlation was made by determining the clinical activity score (CAS) of each patient with TED.

Main outcome measures: Samples were examined for vascular and lymphatic markers including podoplanin, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and cluster of differentiation 31 (CD31) by immunohistochemistry, as well as for mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptors, semaphorin 3F, neuropilin 1, neuropilin 2, podoplanin, and LYVE-1 by quantitative (real-time) reverse-transcription polymerase chain reaction.

Results: Clinicopathologic correlation revealed increased staining of CD31-positive blood vessels in patients with acute TED with a CAS more than 4, as well as rare staining of podoplanin-positive lymphatic vessels within acutely inflamed orbital fat tissue. Additionally, quantitative (real-time) reverse-transcription polymerase chain reaction analysis demonstrated increased expression of VEGF receptor (VEGFR) 2 as well as VEGF signaling molecules VEGF-A, VEGF-C, and VEGF-D.

Conclusions: In acute TED, compared with chronic TED and control orbital fat, there is increased blood vessel density, suggesting neovascularization and rare lymphatic vessels suggestive of limited lymphangiogenesis. This proangiogenic and prolymphangiogenic microenvironment is likely the result of the increased expression of VEGFR-2, VEGF-A, VEGF-C, and VEGF-D. These findings imply that orbital edema in acute TED may be mediated, in part, by both the formation of new, immature blood vessels and the formation of lymphatic capillaries that are functionally incapable of draining interstitial fluid.

Figure 1. Immunohistological characterization of control specimens, eyelid skin and subcutaneous fat obtained from the neck

Localization of podoplanin and LYVE-1 (red) confirm the presence of lymphatic vessels in these samples, as expected, and proves the utility of these markers. Samples are counterstained with hematoxylin (blue). Scale bar = 100 µm

Figure 2. Immunohistological characterization of orbital fat obtained from control patients without TED

Staining for podoplanin and LYVE-1 (red) show no positive staining, indicating the absence of lymphatic vessels, which is consistent with previous reports. Samples are counterstained with hematoxylin (blue). Scale bar = 100 µm

Figure 3. Immunohistological characterization of orbital fat from patients with TED

Patients G1, G2 and G3 are in the acute, inflammatory phase of the disease and all exhibit podoplanin-positive vessel-like structures (red, arrows). Patients G4, G5, and G6 are in the chronic stage of disease and do not exhibit any podoplanin-positive cells. Middle and rightmost panels shows LYVE-1 and CD31 staining for patients G1 – G6 respectively. Samples are counterstained with hematoxylin (blue). Scale bar = 100 µm

Figure 4. Increased vasculature in tissue from patients with acute TED

Patients with TED have increased staining of CD31⁺ cells with dilated blood vessels, when compared against control fat specimens and orbital fat from patients with chronic TED. Quantification of staining revealed a statistically significant increase in CD31⁺ staining in the acute TED patients when compared against the controls. Samples are counterstained with hematoxylin (blue). Scale bar = 100 µm

Figure 5. Changes in gene expression associated with TED

Orbit specimens of five patients with acute TED (CAS > 4) and five patients with chronic TED (CAS < 4) were collected and relative mRNA expression was evaluated. The values were averaged within the chronic and acute groups. qRT-PCR analysis of genes revealed a significant increase in expression of VEGFR-2, VEGF-A, VEGF-C, and VEGF-D in acute TED when compared to chronic disease and control specimens.