VEGF-mediated disruption of endothelial CLN-5 promotes blood-brain barrier breakdown

Abstract

Breakdown of the blood-brain barrier (BBB) is an early and significant event in CNS inflammation. Astrocyte-derived VEGF-A has been implicated in this response, but the underlying mechanisms remain unresolved. Here, we identify the endothelial transmembrane tight junction proteins claudin-5 (CLN-5) and occludin (OCLN) as targets of VEGF-A action. Down-regulation of CLN-5 and OCLN accompanied up-regulation of VEGF-A and correlated with BBB breakdown in experimental autoimmune encephalomyelitis, an animal model of CNS inflammatory disease. In cultures of brain microvascular endothelial cells, VEGF-A specifically down-regulated CLN-5 and OCLN protein and mRNA. In mouse cerebral cortex, microinjection of VEGF-A disrupted CLN-5 and OCLN and induced loss of barrier function. Importantly, functional studies revealed that expression of recombinant CLN-5 protected brain microvascular endothelial cell cultures from a VEGF-induced increase in paracellular permeability, whereas recombinant OCLN expressed under the same promoter was not protective. Previous studies have shown CLN-5 to be a key determinant of trans-endothelial resistance at the BBB. Our findings suggest that its down-regulation by VEGF-A constitutes a significant mechanism in BBB breakdown.

Fig. 1.

In EAE, BBB disruption increases as endothelial CLN-5 and OCLN decrease. (A) Confocal Z-series projections of cerebral cortex from adult mice (C57BL/6, 11 weeks) immunostained for OCLN (Left) or CLN-5 plus GFAP (Right). CLN-5 and OCLN localize to linear profiles, confirmed as vessels using Factor VIII-related antigen (Right, Inset). Staining for GFAP illustrates close proximity of astrocytic end-feet to vessels (Right, asterisk). (B and C) Hematoxylin and eosin-stained section of lumbar spinal cord from 10-week C57BL/6 mouse 16 days after induction of EAE with MOG_35–55. Perivascular inflammation is observed in white matter, illustrated at higher magnification in C. (D and F) Confocal Z-series projections of dorsolateral lumbar spinal cord from 10-week C57BL/6 mice with EAE (tail paralysis and paraparesis, Right) 15–18 days after induction, or age-and sex-matched controls (Left), immunostained for CLN-5 and fibrinogen (D) or OCLN and albumin (F). EAE sections display disruption of the BBB as assessed by parenchymal immunoreactivity for fibrinogen or albumin. Immunoreactivity for CLN-5 and OCLN appears reduced in these areas. Individual channels are presented below each image for clarity. (E and G) Morphometric analysis (see Materials and Methods) confirms a significant correlation between BBB disruption and decreased immunoreactivity for CLN-5 and OCLN in EAE sections (E, P < 0.0001; G, P < 0.0001, Spearman rank correlation). Results shown are from individual animals with EAE (the same illustrated in D and F), and are representative of findings from 3 experiments with 5 animals per condition per experiment. (Scale bars: A, 50 μm; B, 1.5 mm; D and F, 100 μm.)

Fig. 2.

VEGF down-regulates CLN-5 and OCLN in human BMVECs. (A) Immunoblots of protein extracts from human BMVEC treated for 24 h with cytokines shown (VEGF-A, 100 ng/mL; other cytokines, 10 ng/mL). (B) Results in blots quantified by densitometry (see Materials and Methods). Note that CLN-5 and OCLN are both strongly down-regulated by VEGF-A. OCLN is also down-regulated by IL-1β. Other tight junction-associated adaptor and regulatory proteins shown include JAM-1, CASK, ZO-1, and cingulin, which are unchanged in response to inflammatory stimuli. (C) Results of real-time PCR performed on cDNA from human BMVEC treated as described earlier. Results for CLN-5 and OCLN are illustrated. Note that CLN-5 and OCLN mRNA are both strongly down-regulated by VEGF-A. OCLN mRNA is also down-regulated by IL-1β and up-regulated by TGFβ1. ***P < 0.001, ANOVA followed by Bonferroni post-test. (D) Confocal imaging of human BMVEC treated with 100 ng/mL VEGF-A for 24 h and immunostained for CLN-5, OCLN, cingulin and ZO-1. In controls, all four proteins localize to the plasma membrane in areas of cell-cell contact. Note that CLN-5 and OCLN are both down-regulated by VEGF-A. (Scale bars: D, 20 μm.) Data in all panels are representative of at least 3 separate experiments on 3 distinct cultures.

Fig. 3.

VEGF-A disrupts endothelial CLN-5 and OCLN, and induces BBB permeability. (A) Confocal Z-series projection of dorsolateral lumbar spinal cord from 10-week C57BL/6 mice with EAE (as described earlier) at 16 d after induction (Right), and age-and sex-matched controls (Left), immunostained for VEGF-A and GFAP. VEGF-A immunoreactivity localizes to reactive GFAP+ astrocytes in the EAE sample, and is not detected in normal control. (B–E) Sections of cerebral cortex from 12-week adult C57BL/6 mice 24 h following stereotactic microinjection of murine VEGF₁₆₅ (60 ng in 3 μL PBS/BSA, Right) or vehicle control (Left). Sections are immunostained for CLN-5 and OCLN (red channel), plus fibrinogen or albumin (green channel). The red channel from outlined areas in B and D is shown at higher magnification in C and E. Note that BBB permeability is observed around vessels in VEGF-injected areas (B and D, Right), and that immunoreactivity for CLN-5 and OCLN appears patchy and discontinuous in these areas (C and E, Right). (Scale bars: A, 20 μm; B and D, 75 μm; C and E, 20 μm.) Data shown are representative of findings from at least 4 mice per time point per condition.

Fig. 4.

VEGF-induced down-regulation of CLN-5 is a significant determinant of increased paracellular permeability in BMVEC. (A and B) Sandwich ELISA data showing secretion of VEGF-A by human astrocytes. (A) VEGF-A concentrations in astrocyte media after 24 h treatment with cytokines shown, followed by washout and culture for an additional 24 h. IL-1β is a potent inducer of VEGF-A in these cells, and its effect is potentiated by IFNγ (I+G). ***P < 0.001, **P < 0.01, ANOVA plus Bonferroni post-test. (B) Conditioned media generated in a from IL-1β/IFNγ-treated astrocyte cultures (I+G) and controls were diluted 1:1 with CSC medium, then applied for 16 h to confluent human BMVEC monolayers on 0.4-μm culture inserts. BMVECs exposed to conditioned medium from IL-1β/IFNγ-treated astrocytes (ACM/I+G) show a significant increase in paracellular permeability, as assessed using trans-endothelial passage of 40-kD dextran-FITC. Co-treatment with 40 ng/mL Flt-1-Fc fusion protein significantly inhibits this increase in permeability (**P < 0.01, *P < 0.05, ANOVA plus Bonferroni post-test, data from 60 min following addition of 40 kD dextran). (C) Recombinant CLN-5 (pDCLN5) or OCLN (pSOCLN) or empty vector controls were nucleofected into otherwise negative cell lines (CLN-5, HEK cells; OCLN, Hs578T cells). Note expression of recombinant CLN-5 and OCLN in negative cell lines nucleofected with relevant expression construct, and localization to sites of cell-cell contact (Upper). Note also that expression of recombinant CLN-5 is refractory to 100 ng/mL VEGF-A treatment in BMVEC nucleofected with pDCLN5 (Bottom). (D and E) Bovine BMVECs were nucleofected with pDCLN5 (D) or pSOCLN (E) or empty vector controls (pDEST, pS6), then left to grow to confluence for 10–14 d on 0.4-μm inserts. Confluent monolayers were then treated with 100 ng/mL VEGF-A or vehicle control for 24 h. Note that VEGF-A-induces a significant increase in permeability in BMVEC monolayers nucleofected with empty vector, and that this effect is rescued in pDCLN5-nucleofected cultures (D). In contrast, the effect of OCLN expressed under the same promoter is not significant (E). ***P < 0.001, **P < 0.01, ANOVA plus Bonferroni post-test, data shown in both panels from 60 min following addition of 40 kD dextran-FITC. (Scale bar: C, 12 μm.) Data in all panels representative of at least 5 separate experiments on 5 distinct cultures.