Author: Alwin Chun Rong Tan
Tan, Alwin Chun Rong, 2021 Manipulation of the transcription of intercellular adhesion molecule 1 in human retinal endothelial cells, Flinders University, College of Medicine and Public Health
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Uveitis is the term used to describe intraocular inflammation. The disease is defined according to its location inside the eye, and can be infectious or non-infectious in nature. Patients may present with eye redness, floaters and/or blurred sight, and the vision is often impaired. Non-infectious uveitis is associated with dysregulation of the immune response, leading to autoinflammation or autoimmunity. Current treatment of non-infectious uveitis includes the use of corticosteroids and immunomodulatory drugs, such as methotrexate and adalimumab. These drugs can be effective in managing the inflammation, but they often have adverse effects. The work described in this thesis centres around a novel therapeutic approach to treat non-infectious posterior uveitis.
During non-infectious posterior uveitis, leucocytes migrate across the retinal endothelium into the posterior segment of the eye. This process is tightly regulated by adhesion molecules, which are expressed on the surface of the endothelium. In particular, intercellular adhesion molecule 1 (ICAM-1), being the most well-studied member of the immunoglobulin superfamily cell adhesion molecules, is known to interact with integrins on leucocytes to direct leucocyte trafficking. ICAM-1 expression is primarily controlled at the level of gene transcription. Its expression increases following stimulation by inflammatory cytokines that include tumour necrosis factor alpha (TNF-⍺) and interleukin-1 beta (IL-1b).
This thesis presents experimental work that explores ICAM-1 expression by human retinal endothelial cells, and investigates the manipulation of ICAM1 gene transcription in these cells as the basis of a new treatment for non-infectious posterior uveitis. In addition to the full-length ICAM-1 transcript, a novel ICAM-1 transcript variant was identified in human retinal endothelial cells. Inflammatory cytokines, TNF-⍺ and IL-1b, upregulated full-length and novel ICAM-1 transcript expression in primary human retinal endothelial cell isolates. A significant increase in ICAM-1 protein expression on cell isolates was also observed following TNF-⍺ and IL-1b stimulation.
A range of transcription factors (TFs) were predicted to regulate ICAM1 gene transcription by in silico methods, and Ets-1 was chosen as the candidate TF for targeting in human retinal endothelial cells. Ets-1 is encoded by a proto-oncogene. It is the best studied of the Ets TF family, and most often acts as a transcriptional activator. Thus, it was hypothesised that Ets-1 was a transcriptional activator of the ICAM1 gene in human retinal endothelial cells, and it was expected that ICAM-1 expression by these cells would be reduced when Ets-1 activity was inhibited.
To investigate the effect of Ets-1 blockade on ICAM-1 expression in human retinal endothelial cells, Ets-1 transcript was targeted with small interfering (si)RNA in a cell line derived from primary cells. The Ets-1 transcript was expressed in the cell line, as in primary human retinal endothelial cells, and there was a significant reduction in transcript expression following siRNA transfection. Unexpectedly, ICAM-1 transcript and protein expression increased following Ets-1 siRNA transfection and treatment with cytokines, TNF-⍺ and IL-1b, compared to the fresh medium control. These findings show for the first time that blocking Ets-1 can increase the expression of ICAM-1.
The overall goal of this thesis was to progress a new therapeutic approach for non-infectious posterior uveitis, involving manipulation of the transcription of the ICAM1 gene in retinal endothelial cells. The work has demonstrated that ICAM-1 protein expression is increased in the face of Ets-1 blockade. However, delivering an Ets-1 activator as a treatment for non-infectious posterior uveitis is unlikely to gain traction, given the potential for Ets-1 to drive tumour development. This study highlights Ets-1 as an important regulator of ICAM-1 expression in retinal endothelial cells.
Keywords: ICAM-1, uveitis, retinal endothelial cells, gene transcription, Ets-1, siRNA, TFs
Subject: Medical Science thesis
Thesis type: Masters
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Professor Justine Smith