Cavβ3 Contributes to the Maintenance of the Blood-Brain Barrier and Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis

• Published in: Arteriosclerosis, thrombosis, and vascular biology Vol. 44; no. 8; pp. 1833 - 1851
• Main Authors: Martus, Damian, Williams, Sarah K, Pichi, Kira, Mannebach-Götz, Stefanie, Kaiser, Nicolas, Wardas, Barbara, Fecher-Trost, Claudia, Meyer, Markus R, Schmitz, Frank, Beck, Andreas, Fairless, Richard, Diem, Ricarda, Flockerzi, Veit, Belkacemi, Anouar
• Format: Journal Article
• Language: English
• Published: United States 01.08.2024
• Subjects: Animals; Blood-Brain Barrier - metabolism; Calcium - metabolism; Calcium Channels - genetics; Calcium Channels - metabolism; Calcium Signaling; Capillary Permeability; Cells, Cultured; Encephalomyelitis, Autoimmune, Experimental - genetics; Encephalomyelitis, Autoimmune, Experimental - metabolism; Endothelial Cells - metabolism; Female; Inositol 1,4,5-Trisphosphate Receptors - genetics; Inositol 1,4,5-Trisphosphate Receptors - metabolism; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myosin Light Chains - metabolism; Myosin-Light-Chain Kinase - genetics; Myosin-Light-Chain Kinase - metabolism; Phosphorylation; blood-brain barrier; endothelial cells; encephalomyelitis, autoimmune, experimental; calcium signaling; microvascular permeability; mice; myosin light chain kinase
• ISSN: 1079-5642; 1524-4636; 1524-4636
• DOI: 10.1161/ATVBAHA.124.321141

Tight control of cytoplasmic Ca concentration in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca (Cav) channels, in modulating Ca signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier. We investigated the function of Cavβ3 in BMECs by Ca imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3 (Cavβ3-deficient) mice as controls. We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3 mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3 BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca -dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3 than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of cDNA in Cavβ3 BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins. Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca concentration and Ca -dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.