Assessing inter-tissue vulnerability of brain and retinal endothelium to systemic stressors in neuromyelitis optica spectrum disorder
Neuromyelitis optica spectrum disorders (NMOSD) are rare autoimmune diseases characterized by the presence of autoantibodies (AQP4-IgG) against the water channel protein aquaporin-4 (AQP4). The primary clinical manifestations are recurrent episodes of transverse myelitis and optic neuritis driven by...
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| Format: | Doctoral Thesis |
| Language: | English |
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Philipps-Universität Marburg
2024
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| Online Access: | PDF Full Text |
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| Summary: | Neuromyelitis optica spectrum disorders (NMOSD) are rare autoimmune diseases characterized by the presence of autoantibodies (AQP4-IgG) against the water channel protein aquaporin-4 (AQP4). The primary clinical manifestations are recurrent episodes of transverse myelitis and optic neuritis driven by local, AQP4-IgG-mediated inflammation. While AQP4-IgG primarily target astrocytic AQP4 in the brain, spinal cord and optic nerve, AQP4 is also expressed in retinal Müller glial cells, suggesting a potential primary retinopathy in NMOSD. To investigate whether the brain and retina are equally targeted by AQP4-IgG, it is essential to examine the initial barrier these autoantibodies must overcome to infiltrate target tissues: the endothelium. The microenvironment-induced heterogeneity in endothelial properties across different tissues necessitates research into how these endothelial cells respond to systemic, barrier-disrupting stressors, thereby determining their varying levels of protection against AQP4-IgG extravasation.
The first part of this dissertation examines the impact of systemic hydrogen peroxide (H2O2)-induced oxidative stress on the integrity of the endothelial barrier in the brain and retina, as well as its influence on the endothelial complement secretome. Real-time cell analysis (RTCA), an impedance-based method for assessing paracellular barrier integrity, revealed cell type-specific in vitro growth phases, with primary human brain microvascular endothelial cells (HBMEC) exhibiting a more stable cultivation compared to primary human retinal endothelial cells (HREC). Following exposure to 500 µM H2O2, HBMEC displayed hyperpermeability, whereas HREC maintained a functional barrier under the same conditions. These RTCA findings were confirmed by immunocytochemistry of adherens junction marker vascular endothelial cadherin, which showed a stronger signal reduction in HBMEC compared to HREC following H2O2 treatment. Investigation of the endothelial complement secretome using a bead-based multiplex enzyme-linked immunosorbent assay revealed that both endothelial cell types secrete the complement regulators factor H (FH) and factor I (FI). FH secretion remained stable under oxidative stress in both cell types, while FI secretion decreased at higher H2O2 concentrations.
The second part of this dissertation explores the effects of proinflammatory complement activation products C3a and C5a on the para- and transcellular endothelial barrier integrity in the brain and retina, and the extent to which these complement activation products regulate gene expression of CDH5, C3, C5, C3AR1 and C5AR1. RTCA indicated that C3a decreases paracellular endothelial barrier integrity in HBMEC, but not in HREC, an effect that was mitigated when C5a was present at a 1:10 ratio to C3a. Both C3a and C5a increased transcellular IgG transport in HBMEC, but not in HREC. Gene expression analysis showed an upregulation of C3 and C5 expression in HREC, but not in HBMEC.
These findings suggest that both oxidative stress and proinflammatory anaphylatoxins C3a and C5a disrupt the brain endothelial barrier more significantly than the retinal barrier. This indicates a higher susceptibility of the brain to pathological IgG extravasation compared to the retina in NMOSD, highlighting the retina’s potential protective mechanisms against autoimmune damage. |
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| Physical Description: | 78 Pages |
| DOI: | 10.17192/z2024.0391 |