Investigating the Role of Inter-Alpha-Trypsin Inhibitor Heavy Chain 3 in Age-Related Macular Degeneration

Abstract

Introduction: Age-related macular degeneration (AMD) is a leading cause of vision loss in those over 50, characterised by progressive damage to the macula, the central part of the retina. The Bruch's membrane, a key component of the outer blood-retinal barrier that separates the retina from the choroidal blood supply, plays a crucial role in homeostasis of the eye; disruptions in the structure and function of this extracellular matrix (ECM) contribute to AMD development. A previous proteomics study has shown that Inter-Alpha-Trypsin Inhibitor Heavy Chain 3 (ITIH3), a ‘Heavy Chain’ of the inter--inhibitor family, is present in Bruch's membrane at an increased level in early AMD. This thesis aimed to investigate the ligand interactions, expression pattern and localisation of ITIH3 in the human macula to gain insights into its potential role in AMD pathogenesis. Methods: Recombinant human ITIH3 (WT and mutants) was expressed in E. coli and purified for in vitro studies. Self-association of ITIH3 was assessed using size-exclusion chromatography and analytical ultracentrifugation. Ligand interactions were evaluated using biolayer interferometry with various glycosaminoglycans (GAGs), including chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate (HS), heparin, hyaluronan (HA), keratan sulphate (KS), and the proteins complement C3b, complement factor H, the Latency-Associated Peptide (LAP) of TGF-β1, Tumour Necrosis Factor-Stimulated Gene-6 (TSG-6) and vitronectin. ITIH3 expression and localisation were examined in human macula using in situ hybridisation (by RNAscope) and immunofluorescence staining, respectively, using tissues from AMD patients and from individuals without AMD. Results: ITIH3 was shown to form stable oligomers corresponding to dimers, trimers and higher order species. Two distinct mechanisms were identified, both requiring cations. One involved the Metal Ion Dependent Adhesion Site (MIDAS) within ITIH3’s von Willebrand Factor A (vWFA) domain along with Mn²⁺ or Zn²⁺ ions, while the other was MIDAS-independent e.g., (based on results from a mutant lacking the vWFA domain). ITIH3 bound to heparin/HS in a pH-dependent manner (under mildly acidic conditions) but did not bind the other GAGs tested. ITIH3 also interacted with TSG-6 (a regulator of ECM remodelling) and with TGF-1 LAP (which is known to bind to the related ITIH1). There was no detectable binding to vitronectin or the complement proteins, which have been implicated in AMD pathology. In situ hybridisation revealed ITIH3 mRNA expression in the choroid and in the lining cells of the suprachoroidal space. Immunofluorescence staining identified that ITIH3 protein was localised to these regions as well as to the Bruch’s membrane. Notably, there was much more intense immunostaining for ITIH3 in the Bruch's membrane of AMD patients compared to non-AMD controls. Discussion: The observed expression and localisation patterns of ITIH3 in macular tissues provide evidence that this protein accumulates in the Bruch’s membrane in the context of AMD through increased local production in the choroid. It is possible that ITIH3's oligomerisation and ligand-binding properties may protect the structural integrity of the Bruch’s membrane in AMD and ageing. The metal-ion-dependent self-association of ITIH3 could influence its ligand interactions within the macula and provides the potential for cross-linking of matrix components, such as HS proteoglycans in the Bruch’s membrane (e.g. collagen XVIII and perlecan). The pH-dependent binding of ITIH3 to HS may regulate crosslinking in the context of inflammation, where pH is known to decrease. Moreover, the interaction with TSG-6 suggests a potential role in ECM remodelling and tissue repair, although it is not known whether TSG-6 catalyses the formation of covalent complexes of ITIH3 with HA in Bruch’s membrane. Overall, the findings of this thesis provide the basis for further exploration of the mechanisms underlying ITIH3's multifaceted involvement in AMD.

Date of Award	14 Feb 2025
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Tony Day (Main Supervisor), Caroline Milner (Co Supervisor) & Simon Clark (Co Supervisor)