Fig 1.
Cholangiocyte migration assay.
Cholangiocyte migration was measured in an invasion chamber using a collagen gel matrix to detect cells with invasive and migratory behavior after exposure to DCD conditions (panel A). Cholangiocytes migrated through the matrix at increased rates depending on how long they were allowed to recover after exposure to DCD conditions. Specifically, a progressively larger percentage of cells reached the bottom of the chamber after 48 hours as the recovery time increased from 1–7 days (panel B). Results are expressed as mean +/- SD, n = 5.
Fig 2.
Cholangiocyte morphology changes.
Light microscopic appearance of cultured cholangiocytes before or after cold storage (24 hours) and warm ischemia (1 hour). Panel 1 shows Naive cholangiocytes: Normal cuboidal epithelial cell appearance; Panel 2 shows 24-hour cold storage cells: Mixed cell shapes from cuboidal towards spindle-shaped cell; Panel 3 shows cells exposed to both cold storage and 1 hour of warm ischemia: Mostly spindle mesenchymal cell appearance with loss of cuboidal architecture in any cells. The normal cuboidal epithelial appearance has been replaced by a spindle shaped mesenchymal appearing cell morphology in response to DCD ischemic conditions. Magnification = 400x.
Fig 3.
Immunocytochemical (ICC) staining of the epithelial cell markers.
Human cholangiocyte-specific protein staining (upper panels) includes both CK7 and E-cadherin from groups of cells that include Naïve cells without DCD ischemia, from cells with 24 hour exposure to cold storage (CS), and from cells exposed to both cold storage and 60 minutes of warm ischemia (WI). Each antibody (Ab) specific stain is paired with the same cells stained with the nuclear stain DAPI (blue images). For the epithelial markers, the magnitude of the specific staining DECREASES as the degree of DCD ischemia increases (Naïve to CS to CS+WI). The bottom groups of cells are similar to the top except these cells were stained for the mesenchymal specific markers SNAIL, N-cadherin, and Vimentin. Opposite to the pattern seen with epithelial markers, the mesenchymal specific markers are shown to INCREASE in staining intensity as the level of DCD ischemic stress increases (Naïve to CS to CS+WI). These protein expression patterns are consistent with cells undergoing an epithelial-to-mesenchymal transition (EMT). Magnification is 200x.
Fig 4.
Epithelial protein expression profiles on cholangiocytes.
Immunocytochemical expression of cholangiocyte CK7 and E-Cadherin (epithelial cell markers, panel A) and SNAIL, E-Cadherin, and Vimentin (mesenchymal markers, panel B) 24 hours after sham ischemia (Naive controls), after 24 hours after cold storage (CS), and after both warm ischemia (60 minutes) and CS (WI+CS). The kinetics of expression of E-Cadherin (Panel C) and Vimentin (Panel D) are also shown for all three groups of cholangiocytes. * P<0.05, data expressed as the sample mean +/- SD from 5 independent experiments.
Fig 5.
Mesenchymal protein expression profiles on cholangiocytes.
Cholangiocyte expression (via western blot) of Vimentin (panel A), SNAIL (panel B), and N-Cadherin (panel-C) in Naive cholangiocytes, from those cold stored for 24 hours (CS), and from those with both 60 minutes of warm ischemia and 24 hours of cold storage (WI+CS). Expression is shown for 24 hours, 1, 2, and 3 weeks from ischemia exposure to staining. Panel D shows an actual chemiluminescent western blot analysis of these proteins and the GAPDH loading control. All samples were loaded at 30 μg protein per lane. *P<0.05, values are expressed as mean +/- SD from 5 independent experiments.
Fig 6.
Cholangiocyte TGFβ gene expression and migratory behavior.
qPCR results for TGF-β gene expression from DCD exposed cholangiocytes that either migrate (bottom) or do not migrate (top). The top and bottom are the respective top and bottom layer of cholangiocytes at the three time points (days 1, 5, and 7). The day 0 cells were cholangiocytes immediately after exposure to DCD ischemic conditions. Expression values are measured relative to the internal control GAPDH. * P< 0.05 relative to the corresponding top values, n = 4.
Fig 7.
TGFβ signaling and cholangiocyte morphology changes.
Cell morphology results from the grading of images taken with light microscopy. Data are the mean percent of cells out of total cells. There are three main groups, the fresh Naive cholangiocytes (Naive), vehicle DMSO control cholangiocytes, and cholangiocytes pretreated with the selective TGFβ receptor-1 antagonist Galunisertib (1 μM, 1 hour before DCD ischemia). Cells were examined for morphometric changes immediately after induction of DCD ischemia (D0), 1 day after (D1), 5 days after (D5), or 7 days after (D7). The bar indicates the groups exposed to DCD ischemic conditions of 1 hour of warm ischemia and 24 hours of cold storage followed by the indicated time in days of return to normoxia and 37C temperatures. G1 refers to normal cuboidal appearance, GII to mostly cuboidal appearance, GIII refers to mostly spindle appearance, and GIV refers to spindle appearance. All images were graded in a blinded manner. * P <0.05 relative to the corresponding bar in the control or treated (Rx) groups at D0. N = 4.
Fig 8.
Rat liver ischemic cholangiopathy model.
Immunohistochemical analysis of expression of SNAIL and CK-7 from rat liver tissue 7 days after transplantation into syngeneic rats. Sections were prepared from fresh non-ischemic livers, from livers with 24 hours of cold storage time in UW solution, and from livers with both 24 hours of cold storage and 30 minutes of warm ischemia prior to liver recovery from the donor (panel A). Arrows show individual cholangiocytes staining for the selected markers. Quantitation of stain intensity shown in panel A was done for each of the 5 livers from each group using ImageJ analysis. These results are graphically shown in Panel B, * P<0.05. Panel C shows the serum ALT and Total Bilirubin values measured in liver isograft recipients from the time of transplantation until 7 days after transplantation. Values are mean +/- SD from 5 rats, *P<0.05 relative to the control rats (24 hours of cold storage only).