Inflammatory mediators reduce surface PrPc on human BMVEC resulting in decreased barrier integrity

Abstract

The cellular prion protein (PrP^c) is a surface adhesion molecule expressed at junctions of various cell types including brain microvascular endothelial cells (BMVEC) that are important components of the blood-brain barrier (BBB). PrP^c is involved in several physiological processes including regulation of epithelial cell barrier function and monocyte migration across BMVEC. BBB dysfunction and disruption are significant events in central nervous system (CNS) inflammatory processes including HIV neuropathogenesis. Tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) are two inflammatory factors that have been implicated in the processes that affect BBB integrity. To examine the effect of inflammation on PrP^c expression in BMVEC, we used these mediators and found that TNF-α and VEGF decrease surface PrP^c on primary human BMVEC. We also showed that these factors decrease total PrP^c protein as well as mRNA, indicating that they regulate expression of this protein by de novo synthesis. To determine the effect of PrP^c loss from the surface of BMVEC on barrier integrity, we used small hairpin RNAs to knockdown PrP^c. We found that the absence of PrP^c from BMVEC causes increased permeability as determined by a fluorescein isothiocyanate (FITC)-dextran permeability assay. This suggests that cell surface PrP^c is essential for endothelial monolayer integrity. To determine the mechanism by which PrP^c downregulation leads to increased permeability of an endothelial monolayer, we examined changes in expression and localization of tight junction proteins, occludin and claudin-5, and found that decreased PrP^c leads to decreased total and membrane-associated occludin and claudin-5. We propose that an additional mechanism by which inflammatory factors affect endothelial monolayer permeability is by decreasing cell-associated PrP^c. This increase in permeability may have subsequent consequences that lead to CNS damage.

Figure 1

TNF-α and VEGF decrease PrP^c surface expression on endothelial cells. Surface PrP^c was analyzed by flow cytometry after treatment with TNF-α (10ng/ml) or VEGF (100ng/ml) (A) Representative plot showing the change in surface PrP^c determined by FACS analysis after treatment with TNF-α for 18h and 24h. Three independent experiments were quantified (B) The fold change in mean fluorescence intensity (MFI) of PrP^c on BMVEC after TNF-α treatment as compared to control was calculated after subtracting the contribution of the isotype matched negative control antibody. Surface PrP^c after 18h and 24h of TNF-α treatment decreased by 0.5 fold (50%) and 0.4 fold (40%), respectively. (C) Representative plot showing the change in surface PrP^c determined by FACS analysis after treatment with VEGF for 18h and 24h (D). The fold change in MFI of PrP^c on BMVEC after VEGF treatment as compared to control was calculated after subtracting the contribution of the isotype matched negative control antibody. Surface PrP^c after 18h and 24h of VEGF treatment decreased by 0.4 fold (40%) and 0.5 fold (50%), respectively. Significance was determined using a two-tailed paired Student t test. *p < 0.05, **p < 0.01.

Figure 2

TNF-α and VEGF decrease total PrP^c in endothelial cells. (A). Representative blot showing PrP^c decrease in BMVEC treated with TNF-α (10ng/ml) for either 18h or 24h and lysates prepared to examine total PrP^c by Western blotting. GAPDH was used as a loading control (B) Four independent experiments were quantified. TNF-α decreased total PrP^c by 0.4-fold (40%) at 18h with PrP^c returning to baseline at 24h. (C) Representative blot showing PrP^c decrease in BMVEC treated with VEGF (100 ng/ml) for either 18h or 24h and lysates prepared to examine total PrP^c by Western blotting. GAPDH was used as a loading control (D) Three independent experiments were quantified. VEGF decreased total PrP^c by 0.2 fold (20%) at 18h and by 0.6 fold (60%) at 24h. Significance was determined using a two-tailed paired Student’s t test. *p < 0.05, **p < 0.01.

Figure 3

TNF-α and VEGF decrease PrP^c mRNA in endothelial cells. (A) BMVEC were treated with TNF-α (10ng/ml) for 12h, 18h, or 24h and mRNA levels of PrP^c were evaluated using qRT-PCR. TNF-α decreased PrP^c mRNA at 12h and 18h by 0.2 fold (20%) and 0.4 fold (40%), respectively, and PrP^c mRNA returned to baseline at 24h, n=5. (B) BMVEC were treated with VEGF (100ng/ml) for 12h, 18h, or 24h and mRNA levels of PrP^c were evaluated using qRT-PCR. VEGF decreased PrP^c mRNA by 0.4 fold (40%), 0.5 fold (50%), and 0.6 fold (60%) at 12h, 18h, and 24h respectively, n=3. Significance was determined using a two-tailed paired Student’s t test. *p < 0.05, **p < 0.01, ***p< 0.0005.

Figure 4

Significant knockdown of PrP^c in BMVEC with shRNA. (A) mRNA levels of PrP^c were analyzed by qRT-PCR in control cells and 3 lines infected with PRNP shRNA. One cell line (PrP^c KD1) showed 90% decrease in PrP^c mRNA. The second cell line (PrP^c KD2) and the third cell line (PrP^c KD3) showed 25% and 65% fold decrease in PrP^c mRNA, respectively (B) Surface PrP^c was analyzed by flow cytometry in control cell lines and 3 cell lines that were infected with lentivirus containing PRNP shRNA. (C) Change in the mean fluorescence intensity (MFI) of PrP^c on BMVEC cell lines infected with PRNP shRNA as compared to control was calculated after subtracting the contribution of the isotype matched negative control antibody. PrP^c KD1 showed a 90% decrease in surface PrP^c while PrP^c KD2 showed a 40% decrease in surface PrP^c and PrP^c KD3 showed a 70% decrease in PrP^c.

Figure 5

PrP^c knock down results in increased BMVEC monolayer permeability. Permeability was measured by the passage of dextran-FITC (70kDA) across BMVEC monolayer. Transfected BMVECs were cultured on tissue culture inserts with 3 μm pores placed in 24-well tissue culture plates. Permeability of the barrier was measured by passage of dextran-FITC (70kDA) (125ug/ml) as described in the Materials and Methods section. The cell line with a 90% knock down of PrP^c (PrP^c KD1) showed a 2.0-fold increase in permeability as compared to control cell lines. PrP^c KD2, which has a 40% knock down of PrP^c, showed no increase in permeability while PrP^c KD3, which has a 70% knock down of PrP^c, showed a 1.6 fold increase in permeability. Significance was determined using a two-tailed paired Student’s t test. *p < 0.01, ***p< 0.0005.

Figure 6

PrP^c knock down downregulates total and membrane associated expression of occludin and claudin-5. Occludin and claudin-5 expression was measured by confocal microscopy and analyzed. Control and PrP^c knock down cells were plated on ibdi dishes and fixed with PFA. Cells were stained using occludin and claudin-5 antibodies and analyzed for changes in expression and localization. (A) Cells were stained with DAPI to identify cell nuclei and labeled with phalloidin to identify the shape of the cells. PrP^c KD1 and PrP^c KD3 show reduced total occludin and cell membrane occludin. (B) To compare conditions, similar number of cells (300 to 800 cells) and total area was analyzed. Control cells had 43% of their total occludin as membrane protein. PrP^c KD1 had reduced membrane occludin (28%) while PrP^c KD3 had 27% of their total occludin as membrane occludin. (C) Cells were stained with DAPI to identify cell nuclei and labeled with phalloidin to identify the shape of the cells. PrP^c KD1 and PrP^c KD3 show reduced total claudin-5 and cell membrane claudin-5. (D) To compare conditions, similar number of cells (300 to 800 cells) and total area was analyzed. Control cells had 32.64% of their total claudin-5 as membrane protein. PrP^c KD1 had reduced membrane claudin-5 (25%) while PrP^c KD3 had 22.65% of their total claudin-5 as membrane claudin-5. Significance was determined using ANOVA test. * p≤0.001, # p≤0.0002.