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Background: Chronic cerebral hypoperfusion (CCH) is the leading cause of cerebral small vessel disease (CSVD). CCH is strongly associated with blood–brain barrier (BBB) dysfunction and white matter lesions (WMLs) in CSVD. However, the effects of CCH on BBB integrity and components and the cellular and molecular mechanisms underlying the effects of BBB dysfunction remain elusive. Whether maintaining BBB integrity can reverse CCH-induced brain damage has also not been explored. Methods: In this study, we established a rat model of CSVD via permanent bilateral common carotid artery occlusion (2VO) to mimic the chronic hypoperfusive state of CSVD. The progression of BBB dysfunction and components of the BBB were assessed using immunostaining, Western blotting, transmission electron microscopy (TEM) and RNA sequencing. We also observed the protective role of imatinib, a tyrosine kinase inhibitor, on BBB integrity and neuroprotective function following CCH. The data were analyzed using one-way or two-way ANOVA. Results: We noted transient yet severe breakdown of the BBB in the corpus callosum (CC) following CCH. The BBB was severely impaired as early as 1 day postoperation and most severely impaired 3 days postoperation. BBB breakdown preceded neuroinflammatory responses and the formation of WMLs. Moreover, pericyte loss was associated with BBB impairment, and the accumulation of serum protein was mediated by increased endothelial transcytosis in the CC. RNA sequencing also revealed increased transcytosis genes expression. BBB dysfunction led to brain damage through regulation of TGF-β/Smad2 signaling. Furthermore, imatinib treatment ameliorated serum protein leakage, oligodendrocyte progenitor cell (OPC) activation, endothelial transcytosis, microglial activation, and aberrant TGF-β/Smad2 signaling activation. Conclusions: Our results indicate that reduced pericyte coverage leads to increased BBB permeability via endothelial transcytosis. Imatinib executes a protective role on the BBB integrity via inhibition of endothelial transcytosis. Maintenance of BBB integrity ameliorates brain damage through regulation of TGF-β/Smad2 signaling following CCH; therefore, reversal of BBB dysfunction may be a promising strategy for CSVD treatment. [ABSTRACT FROM AUTHOR]

Blood-brain barrier (BBB) dysfunction plays a pivotal role in the pathology of chronic cerebral hypoperfusion (CCH)-related neurodegenerative diseases. Continuous endothelial cells (EC) that line the blood vessels of the brain are important components of the BBB to strictly control the flow of substances and maintain the homeostatic environment of the brain. However, the molecular mechanisms from the perspective of EC-induced BBB dysfunction after CCH are largely unknown. In this study, the BBB function was assessed using immunostaining and transmission electron microscopy. The EC dysfunction profile was screened by using EC enrichment followed by RNA sequencing. After identified the key EC dysfunction factor, C-kit, we used the C-kit inhibition drug (imatinib) and C-kit down-regulation method (AAV-BR1-C-kit shRNA) to verify the role of C-kit on BBB integrity and EC transcytosis after CCH. Furthermore, we also activated C-kit with stem cell factor (SCF) to observe the effects of C-kit on BBB following CCH. We explored that macromolecular proteins entered the brain mainly through EC transcytosis after CCH and caused neuronal loss. Additionally, we identified receptor tyrosine kinase C-kit as a key EC dysfunction molecule. Furthermore, the pharmacological inhibition of C-kit with imatinib counteracted BBB leakage by reducing caveolae-mediated transcytosis. Moreover, treatment with AAV-BR1-C-kit shRNA, which targets brain EC to inhibit C-kit expression, also ameliorated BBB leakage by reducing caveolae-mediated transcytosis. Furthermore, the SCF increased the permeability of the BBB by actively increasing caveolae-mediated transcytosis. This study provides evidence that C-kit is a key BBB permeability regulator through caveolae-mediated transcytosis in EC after CCH. [Display omitted] [ABSTRACT FROM AUTHOR]