Background: Corneal neovascularization increases the risk of T cell–mediated allograft rejection. Here, we investigate whether T cells promote angiogenesis in transplantation.
Methods: Conventional effector T cells were collected from draining lymph nodes of allogeneic or syngeneic corneal transplanted BALB/c mice. T cells were either cocultured with vascular endothelial cells (VECs) to assess VEC proliferation or used in a mixed lymphocyte reaction assay. Messenger RNA (mRNA) expression of vascular endothelial growth factor (VEGF)-A, -C, and VEGF receptor 2 (VEGF-R2) in VECs was assessed by real-time PCR. VEGF-A protein expression was determined by enzyme-linked immunosorbent assay. Flow cytometry was used to analyze VEGF-R2 expression in corneal CD31+ cells, and VEGF-A and IFNγ expression in corneal CD4+ T cells.
Results: Allogeneic T cells from high-risk (HR) grafted mice induced more VEC proliferation than those from syngeneic transplant recipients (P = 0.03). Vascular endothelial growth factor-A mRNA and protein expression were higher in T cells from draining lymph nodes (P = 0.03 and P = 0.04, respectively) and cornea (protein; P = 0.04) of HR compared with low-risk (LR) grafted hosts. Vascular endothelial growth factor-A, VEGF-C, and VEGF-R2 mRNA expression were increased in VECs when cocultured with T cells from HR transplants compared with LR transplants and naive mice. In addition, IFNγ blockade in T cell/VEC coculture increased VEC proliferation and VEGF-A protein expression, whereas blocking VEGF-A significantly reduced VEC proliferation (P = 0.04).
Conclusions: Allogeneic T cells from corneal transplant hosts promote VEC proliferation, probably via VEGF-A signaling, whereas IFNγ shows an antiangiogenic effect. Our data suggest that T cells are critical mediators of angiogenesis in transplantation.
ECs express more when VEGF-A, VEGF-C and VEGF-R2 mRNA when cocultured with T cells from high-risk inflamed transplants compared to low-risk transplants and naı¨ve mice. ECs proliferate in response to VEGF-A when cultured with allogeneic T cells from corneal transplant while IFNg shows an antiangiogenic effect.
1 Schepens Eye Research Institute, Massachusetts Eye & Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA.
Received 27 September 2015. Revision received 31 May 2016.
Accepted 3 June 2016.
This study was funded by the National Institutes of Health (NIH R01 EY12963) and the NIH National Eye Institute core grant P30EY003790.
The authors declare no conflicts of interest.
A.D.Z. and M.T. contributed equally to this work.
A.D.Z. participated in the performance of the research, data analysis, and writing the article. M.T. participated in the performance of the research and data analysis. B.S. participated in the performance of the research and data analysis. J.Y. participated in the performance of the research. T.H.D. participated in the performance of the research. T.I. participated in the performance of the research. A.M. participated in the performance of the research and writing the article. S.K.C. and R.D. participated in the research design and writing the article.
Correspondence: Reza Dana, MD, MPH, MSc, Schepens Eye Research Institute, Massachusetts Eye & Ear Infirmary, 20 Staniford Street, Boston, MA, 02114. (Reza_Dana@meei.harvard.edu).
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