Fig 2: CLDN3 and CLDN5 protein expression in endothelial cell monocultures following a transfer of conditioned.Endothelial Monoculture Conditioned Media, (ECM+), Astrocyte Monoculture Conditioned Media, (ACM+), Pericyte Monoculture Conditioned Media, (PCM+), and Triculture Conditioned Media, (TCM+), were prepared by either a 1 hour incubation with 5% severely injured patient plasma (CM+TP) or left as is with no plasma additions made (CM-TP). Final working concentration of severely injured patient plasma and conditioned media in endothelial monocultures receiving conditioned media was 5% and 10% respectively. Each condition was compared to its Naïve control (CM+TP—and CM-TP -) receiving no conditioned media and no severely injured patient plasma to produce fold changes. CM+TP were compared against one another to determine the differential effect amongst the conditioned media types. Both 4 and 6 hour incubation times were assessed. * Denotes p<0.05, ** Denotes p<0.01, *** Denotes p<0.001 (9) CLDN3 protein expression: Endothelial monocultures did not present with any significant changes in CLDN3 protein expression for all conditions. (10) CLDN5 protein expression: All conditions tested resulted in a significant increase in the fold change of CLDN5 expression compared to the naïve control (not shown). The Endothelial monoculture receiving 6Hr ECM+/CM+TP showed a significant increase in fold change vs. control compared to 6Hr ACM+/CM+TP (p<0.01), and 6Hr PCM+/CM+TP (p<0.05) n = 2; The endothelial monoculture receiving 6Hr TCM+/CM+TP showed a significant increase in fold change compared to 6Hr ACM+/CM+TP (p<0.001), and 6Hr PCM+/CM+TP (p<0.001) n = 2.

Fig 3: CLDN3, CLDN5, and ZO-1 mRNA expression in endothelial cell monocultures following a 4 hour incubation with cell type specific conditioned media.Endothelial Monoculture Conditioned Media, (ECM+), Astrocyte Monoculture Conditioned Media, (ACM+), Pericyte Monoculture Conditioned Media, (PCM+), and Triculture Conditioned Media, (TCM+), were prepared by either a 1-hour incubation with 5% severely injured patient plasma (CM+TP) or left as is with no plasma addition (CM-TP). All conditioned media was added to endothelial cell monocultures activated with severely injured patient plasma to a working concentration of 5%. Final working concentration of severely injured patient plasma and conditioned media in endothelial monocultures receiving conditioned media was 5% and 10% respectively. Each condition was compared to an endothelial cell monoculture naïve control (CM+TP—and CM-TP -). CM+TP wells were compared against one another to determine the differential effect each cell type’s conditioned media had. (A) CLDN3 mRNA expression: The endothelial monoculture receiving ECM+/CM+TP showed a 4.96-fold change increase vs. control, p<0.01, n = 2; ECM+/CM+TP produced a significant fold change increase in CLDN3 mRNA expression compared to ACM+/ CM+TP, (p<0.01), PCM+/CM+TP, (p<0.01), and TCM+/CM+TP, (p<0.01). Endothelial monocultures receiving ACM+, PCM+, or TCM+ did not show significant fold change vs. control for either CM-TP or CM+TP. Additionally, endothelial monocultures receiving ECM+/CM-TP showed no significant change in transcription. (B) CLDN5 mRNA expression: The Endothelial monoculture ECM+/CM+TP showed a 2.94-fold change increase vs. control, p<0.0001, n = 2; ECM+/CM+TP produced a significant fold change increase in CLDN5 mRNA expression compared to ACM+/CM+TP, (p<0.0001), PCM+/CM+TP, (p<0.0001), and TCM+/CM+TP (p<0.0001). Endothelial monocultures receiving ACM+, PCM+, or TCM+ did not show significant fold change vs. control for either CM+TP or CM-TP. Endothelial monocultures receiving ECM+/CM-TP showed no significant change in transcription. (C) ZO-1 mRNA expression: Endothelial monocultures receiving ACM+, ECM+, PCM+, or TCM+ did not show significant fold change vs. control for either CM+TP or CM-TP. Two experimental replicates of the conditioned media experiment were performed, n = 2. ** Denotes p<0.01. **** Denotes p<0.0001.

Fig 4: CLDN5, and CLDN3 protein expression following a 4- or 6-hour incubation with 5% plasma from severely injured patients.(A) CLDN3 protein expression: We observed no significant change in fold change of CLDN3 expression was observed in either the endothelial monoculture or triculture, n = 2 (B) CLDN5 protein expression: Endothelial monoculture showed a 5.12- and 2.58-fold change increase vs. control following 4 and 6 hours of incubation respectively with 5% severely injured plasma. p<0.001, p<0.05, n = 2; There is a significant difference between the fold change at 4 hours of incubation compared to 6 hours of incubation following 5% severely injured patient plasma in the endothelial monoculture, p<0.01. The triculture did not show any significant fold change vs. control. One ELISA was performed with the combined whole cell lysate of three experimental replicates, n = 2. * Denotes p<0.05, ** Denotes p<0.01, *** Denotes p<0.001.