Caporali, Andrea

Hardingham, Giles

Mameli, Eleonora

2023-11-23T13:51:35Z

2023-11-23T13:51:35Z

2023-11-23

Age-related cerebrovascular dysfunction plays a critical role in the pathogenesis of cerebrovascular disease, vascular dementia and Alzheimer’s disease, but therapeutic development has been largely unsuccessful until now. Endothelial cells (ECs) are a fundamental component of the neurovascular unit. Their dysfunction has been established as an early event in the pathogenesis of cerebrovascular disease and vascular dementia, leading to dysregulation of cerebral blood flow and blood-brain barrier damage. In this context, identifying novel genes associated with endothelial dysfunction will help understand the role of ECs in blood-brain barrier integrity and address new therapeutic targets. Trichoplein (TCHP) was initially characterised as a ubiquitously expressed keratin filament-binding protein associated with cell division and cilia formation. Moreover, TCHP has been reported to regulate ER-mitochondria tethering and promote mitophagy, a specialised form of autophagy necessary for the turnover/remodelling of mitochondria. In the lab, we previously demonstrated a pivotal role for the centriolar protein TCHP in linking endothelial cell function with the control of autophagy, showing that the depletion of TCHP in ECs impairs migration and sprouting and triggers cellular inflammation. In line with this, the endothelial-specific deletion of Tchp (TchpEC) in mice decreased the blood flow recovery and vascularisation following hind-limb ischaemia. Protein aggregates were detected in ECs from TchpEC mice and ECs from patients with coronary artery disease. However, the presented preliminary data regarding the role of TCHP in brain vasculature has not been explored yet. My PhD project aims to characterise the role of TCHP in brain microvascular ECs in vitro and in vivo and to reveal its role in blood-brain barrier integrity. For this study, I generated mice with endothelial selective Tchp knock-out (TchpEC ) by breeding the conditional knock-out mice with mice carrying Cre recombinase under the VE-cadherin promoter. RNA sequencing demonstrated the up-regulation of matrix-metalloproteinases and chemokine signalling pathways in the brain ECs isolated from TchpEC mice. The analysis of the in vitro permeability by Electric Cell-substrate Impedance Sensing (ECIS®) revealed an impaired barrier function in ECs lacking TCHP. Furthermore, TchpEC mice administered with the fluorescently labelled tracer dextran presented a higher tracer accumulation in the brain than WT mice, showing a loss of blood-brain barrier integrity. In addition, the presence of protein aggregates was confirmed in the cytoplasm of brain microvascular ECs lacking TCHP. The proteomic characterisation of the insoluble- protein fraction revealed RNA-binding and proteasome-associated proteins, suggesting the toxicity of these aggregates for the cells. Finally, a pharmacological screening identified an FDA-approved compound activating autophagy and, thus, restoring EC function and reducing expression of inflammatory genes in EC lacking TCHP. Collectively, this study presents the novel role played by TCHP in the cerebrovascular endothelium and identifies a new mechanism by which the silencing of TCHP could link endothelial dysfunction to impaired blood-brain barrier integrity.

https://hdl.handle.net/1842/41230

http://dx.doi.org/10.7488/era/3966

en

The University of Edinburgh

Mameli, E., Martello, A. & Caporali, A. (2022) Autophagy at the interface of endothelial cell homeostasis and vascular disease, Febs j. 289, 2976-2991.

Starikova, E., Mammedova, J., Ozhiganova, A., Lebedeva, A., Malashicheva, A., Semenova, D., Khokhlova, E., Mameli, E., Caporali, A., Wills, J. & Sokolov, A. (2021) Protective Role of Mytilus edulis Hydrolysate in Lipopolysaccharide-Galactosamine Acute Liver Injury, Frontiers in Pharmacology. 12, 1018.

Autophagy

Blood-brain barrier

Trichoplein

Cerebrovascular disease

Contribution of the centriolar protein Trichoplein to endothelial cell function in brain vasculature

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy