Ya, Jingyuan
(2025)
The impacts of endothelial cell senescence on ageing-related blood-brain barrier dysfunction and the potential therapeutic strategies.
PhD thesis, University of Nottingham.
Abstract
Background: Disruption of the blood-brain barrier (BBB) contributes to various agerelated neurological disorders. Brain microvascular endothelial cells (BMECs) are the most critical components of the BBB, forming a tightly sealed yet selectively permeable monolayer to maintain central nervous system (CNS) homeostasis. The accumulation of senescent cells in the CNS is thought to play a significant role in age related BBB dysfunction. A large number of recent pre-clinical and clinical studies have focused on the development of senotherapeutic interventions to prevent or delay BBB ageing and extend health span. However, the understanding of BMEC senescence and age-related BBB dysfunction remains limited, and studies illustrating the effects of potential interventions are urgently needed.
Methods: Replicative senescence (RS) in human brain microvascular endothelial cells (HBMECs) was induced by repetitive passage and confirmed to exist by a panel of senescent markers, including senescence-associated-β galactosidase staining (SA-βgal), γH2AX staining, telomere length measurement, WST-1 proliferation assay, and cyclin-dependent kinase inhibitor p16 expression. BMECs were treated with a cocktail of senolytics (dasatinib and quercetin, D+Q), or senomorphics targeting p38MAPK (BIRB796) or NF-κB (QNZ) signalling pathways from passage 16 until passage 20 to explore the effects of senotherapeutics on cellular senescence. Premature senescence was induced by exposing HBMECs (passages 6-8) to 400 μM H2O2 for 48 hours, followed by 12 days of incubation under normal complete media for the development of senescence. BIRB796 or QNZ were added to the culture media of HBMECs along with H2O2 to inhibit the activation of p38MAPK or NF-κB, respectively. D+Q was added to the culture media of HBMECs on day 10 after the H2O2 exposure for 24 hours to eliminate senescent cells. Similar phenotypic assays as the replicative senescence experiments were conducted to evaluate the senescence-associated changes evoked by oxidative stress. Using an in vitro model of BBB, composed of BMECs, astrocytes and pericytes, this study explored the correlation between BMEC senescence and BBB dysfunction. Crucial factors of BBB function were analysed in young, RS and SIPS HBMECs in the presence or absence of senotherapeutics to explore the mechanism of age-related BBB dysfunction, including expression and localisation of tight junction proteins, levels and activity of MMPs, and the expressions of angiogenic factors and pro-inflammatory cytokines.
Results: Replicative senescence was deemed present at passage ≥ 19, where HBMECs displayed enlarged morphology, shortened telomere length, reduced proliferative capacity and increased SA-β-gal activity, γH2AX staining, actin stress fibers and cyclin-dependent kinase inhibitor p16 expression. The angiogenesis capacity and migration capacity revealed a significant decline in RS BMECs, evidenced by diminished tubulogenic potentials and wound repair rate. Significant impairments observed in the integrity and function of BBB established with RS BMECs, ascertained successively by decreases in transendothelial electrical resistance and increases in paracellular flux, revealed a close correlation between BMEC senescence and BBB dysfunction. Disruptions in the localisation and decreased expression of tight junction proteins, zonula occluden-1 (ZO-1), occludin, and claudin-5, and increased activity of matrix metalloproteinase 2 (MMP2) in RS BMECs may elucidate the underlying mechanisms of the BBB dysfunction. Stress-induced premature senescence (SIPS) HBMECs exhibited a set of similar phenotypic changes and functional impairment to those induced by RS. However, in contrast to the 70% senescent rate induced by RS, oxidative stress induced approximately 30% of SA-βgal and γH2AX positively stained cells, yet it still caused significant disruption of the BBB. In addition, RS HBMECs exhibited markedly reduced overall expressions of tight junction proteins, including ZO-1, occludin, and claudin-5. Meanwhile, ZO-1 and occludin levels remained stable, whereas claudin-5 levels showed further elevation in SIPS HBMECs. Selectively elimination of senescent cells by D+Q, as well as the inhibition of the p38MAPK signalling pathway, attenuated the effects of both replicative stress and oxidative stress on senescent markers in HBMECs and preserved BBB function by restoring subcellular localisation of ZO-1 and inhibiting the activation of MMP2. NF-κB inhibitor (QNZ) delayed SIPS and protected BBB from oxidative stress, yet accelerating cellular senescence and induced cell death with RS HBMECs. Furthermore, inhibition of the p38MAPK/NF-κB pathway significantly suppressed the oxidative stress-evoked expression of senescence-associated secretory phenotypes (SASPs), namely interleukin-8, monocyte chemoattractant protein-1 and intercellular adhesion molecule-1.
Conclusion: This study supports the concept that BMECs enter senescence under replicative stress and enhanced oxidative stress during the ageing process, which ultimately damages the integrity of the BBB. The selective elimination of senescent cells by senolytics and the inhibition of p38MAPK effectively mitigate the accumulation of senescent BMECs in the cerebrovasculature and preserve BBB functions.
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