American Society of Hematology

Figure 2.

$tPA/PDGF-CCa signaling increases occludin phosphorylation and permeability. (A) Gene expression heatmap of the top differentially expressed genes in isolated vascular fragments from WT mice 4 hours after ICV injection of either PBS or PDGF-CCa. (B) The vascular fragments from WT mice were isolated 4 hours after ICV injection of PBS, PDGF-CCa, or tPA and were analyzed by using quantitative polymerase chain reaction for differential expression of Angptl4. (C) The isolated vascular fraction from WT mice, either control without MCAO or isolated 24 hours after MCAO, was analyzed by using quantitative polymerase chain reaction for the expression of Angptl4. (D) WT mice were given an ICV injection with PBS, tPA, or tPA in combination with imatinib, or ANGPTL4, or VEGFR2 inhibitor (VEGFR2i), or PKCβ inhibitor (PKCβi). At 5 hours, Evans blue was injected intravenously; at 6 hours, animals were perfused with PBS. One brain hemisphere was blotted to determine pS490 and total occludin protein levels. (E) Quantification of blot S490 phosphorylation. (F) The other brain hemisphere was processed for BBB permeability by measuring Evans blue dye extravasation. (G) BBB permeability was assessed after 6 hours of either saline or tPA ICV injection in PDGFiCre+ mice and PDGFiCre+; S490AOCC+/+ mice by measuring Evans blue dye extravasation. One-way analysis of variance followed by a Holm-Šídák post hoc test was used for comparison of ≥3 groups; a t test was used for comparison between 2 groups. *P < .05, **P < .01, ***P < .001, ****P < .0001. ns, nonsignificant; Veh, vehicle.$

tPA/PDGF-CCa signaling increases occludin phosphorylation and permeability. (A) Gene expression heatmap of the top differentially expressed genes in isolated vascular fragments from WT mice 4 hours after ICV injection of either PBS or PDGF-CCa. (B) The vascular fragments from WT mice were isolated 4 hours after ICV injection of PBS, PDGF-CCa, or tPA and were analyzed by using quantitative polymerase chain reaction for differential expression of Angptl4. (C) The isolated vascular fraction from WT mice, either control without MCAO or isolated 24 hours after MCAO, was analyzed by using quantitative polymerase chain reaction for the expression of Angptl4. (D) WT mice were given an ICV injection with PBS, tPA, or tPA in combination with imatinib, or ANGPTL4, or VEGFR2 inhibitor (VEGFR2i), or PKCβ inhibitor (PKCβi). At 5 hours, Evans blue was injected intravenously; at 6 hours, animals were perfused with PBS. One brain hemisphere was blotted to determine pS490 and total occludin protein levels. (E) Quantification of blot S490 phosphorylation. (F) The other brain hemisphere was processed for BBB permeability by measuring Evans blue dye extravasation. (G) BBB permeability was assessed after 6 hours of either saline or tPA ICV injection in PDGFiCre⁺ mice and PDGFiCre⁺; S490AOCC^+/+ mice by measuring Evans blue dye extravasation. One-way analysis of variance followed by a Holm-Šídák post hoc test was used for comparison of ≥3 groups; a t test was used for comparison between 2 groups. *P < .05, **P < .01, ***P < .001, ****P < .0001. ns, nonsignificant; Veh, vehicle.

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