Cerebral small vessel disease (cSVD) is a common type of vascular dementia marked by dysfunction in brain endothelial cells (BECs), which leads to changes in white matter, an early sign of the disease. Endothelial dysfunction occurs before the blood-brain barrier (BBB) breaks down [1]. Identifying new genes linked to this dysfunction can improve our understanding of BECs in BBB integrity. A key factor in BBB disruption is the overproduction of matrix metalloproteinases due to neurovascular inflammation. While autophagy is associated with neurodegenerative diseases and inflammation, its role in endothelial barrier function in cSVD is still unclear. Increasing evidence suggests that proper autophagic flux in ECs is crucial for maintaining vascular function, while defective autophagy can lead to a pro-inflammatory phenotype in these cells [2]. We previously demonstrated that the centriolar protein trichoplein (TCHP) plays a critical role in connecting EC function with autophagy regulation. The loss of TCHP is associated with impaired autophagy and increased inflammation in ECs [3].
Here, we propose to investigate the role of TCHP in the mechanisms underlying defective autophagy, EC inflammation, and increased BBB permeability.
We then generated endothelial-specific Tchp knock-out mice (TchpEC). Using a labelled dextran injection and magnetic resonance imaging to map and quantify BBB permeability in TchpEC mice revealed a significant increase in hippocampal vascular permeability compared to wild type (WT) mice (Gd DTPA mM: 0.83±0.16 WT vs 1.13±0.25 TchpEC; n=8 unpaired t-test 0.037). TchpEC mice showed a decrease in pericytes coverage (CD13%: 21.26±6.10 WT vs 13.96±5.09 TchpEC; n=7 unpaired t-test 0.032) and capillary diameters (µm: 2.99±0.21 WT vs 3.34±0.31 TchpEC; n=7 unpaired t-test 0.029). Transcriptomics analysis of BEC sorted from WT and TchpEC mice showed that the Gene Ontology terms cytokine-mediated signalling pathway, extracellular matrix organisation, and chemokine-mediated signalling pathway are enriched in BECs from TchpEC mice. Gelatin zymography further confirmed increased Mmp9 production in TchpEC BECs (1.02±0.21 WT vs 1.44±0.26 TchpEC; n=7 unpaired t-test 0.007). Furthermore, we used a co-culture cellular model using WT pericytes and TchpEC BECs to identify key pathways affected in pericytes by the loss of Tchp in BECs. Transcriptomics analysis revealed that the Notch pathway is significantly affected in pericytes when co-cultured with TchpEC BECs. Finally, treatment of TchpEC mice with a MMP9 inhibitor (GM6001, i.p. 50mg/Kg every second day for 15 days) restored pericyte coverage (CD13%: 19.46±1.53 WT; 12.38±2.77 TchpEC ; 16.28±2.67 TchpEC+GM; n=7 two-way Anova p<0.01) and reduced BBB permeability(Permeability index; relative fluorescent unit/gr tissue 106.73±46.63 WT; 270.23±22.80 TchpEC ; 173.14±33.18 TchpEC+GM; n=7 two-way Anova p<0.01).
In conclusion, our data indicate that TCHP-induced defective autophagy in endothelial cells may result in inflammation and compromised blood-brain barrier integrity.
All experiments involving mice were performed following the guidance and operation of the Animals (Scientific Procedures) Act 1986 and the prior approval of the UK Home Office and the University of Edinburgh Animal Welfare and Ethical Review Board.