Fig 2: 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 3: CLDN5, CLDN3, and ZO-1 mRNA expression following a 4 hour incubation with a working concentration of 5% plasma from healthy patients (HP) or 5% Trauma Plasma from severely injured patients (TP).(A) CLDN3 mRNA expression: The Endothelial monoculture incubated with TP showed a 3.98-fold increase vs. control, p<0.001, n = 6; Astrocyte monocultures, Pericyte monocultures, and the triculture did not show any significant change vs. control. All cell cultures incubated with HP showed no significant change vs. control. (B) CLDN5 mRNA expression: Endothelial monoculture showed a 3.51-fold increase vs. control, p<0.001, n = 6; Astrocyte monocultures, Pericyte monocultures, and the triculture did not show any significant fold change vs. control. All cell cultures incubated with HP showed no significant change vs. control. (C) ZO-1 mRNA expression: ZO-1 mRNA expression did not change significantly vs. control for either the monoculture or tricultures, n = 5. All cell cultures incubated with HP showed no significant change vs. control. Six experimental replicates were performed n = 6. ** Denotes p<0.01 *** Denotes p<0.001.

Fig 4: 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.