Differential increases in the expression of intermediate filament proteins and concomitant morphological changes of transdifferentiating rat hepatic stellate cells observed in vitro

Abstract

The primary function of hepatic stellate cells (HSCs) is the storage of vitamin A. However, they are also responsible for liver fibrosis and are therapeutic targets for treatment of liver cirrhosis. Among the many molecular markers that define quiescent or activated states of HSCs, the characteristics of type III intermediate filaments are of particular interest. Whereas vimentin and desmin are upregulated in activated HSCs, glial fibrillary acidic protein is downregulated in activated HSCs. The functional differences between vimentin and desmin are poorly understood. By time-course quantifications of several molecular markers for HSC activation, we observed that the expression of vimentin preceded that of desmin during the transdifferentiation of HSCs. The immunoreactivity of vimentin in transdifferentiated HSCs was more intense in perinuclear regions compared to that of desmin. We propose that the delayed expression of desmin following the expression of vimentin and the peripheral localization of desmin compared to vimentin are both related to the more extended phenotype of transdifferentiating HSCs observed in vitro.

Keywords: desmin; hepatic stellate cell; intermediate filament; myofibroblast; vimentin.

Fig. 1

Morphological changes of rat hepatic stellate cells (HSCs) cultured in vitro. (A–F) Male Wistar rats at the age of eight weeks were sacrificed for isolation of HSCs as described in Materials and Methods. Cells were cultured on uncoated polystyrene dishes and observed daily by phase contrast microscopy. Bar in panel F represents 50 µm and applies to all the panels in this figure. (G) The area occupied by a cell was measured. Ten cells were used to calculate the occupied area for each day. Asterisks indicate significant (P<0.05) differences compared to day seven by ANOVA.

Fig. 2

Expression of vimentin and desmin proteins in transdifferentiating rat HSCs. (A) HSCs were collected after the indicated number of days in culture and ten µg of RIPA-soluble cell lysates were separated by SDS-PAGE. Protein expression was assessed by Western blotting. Approximate molecular weights are indicated on the left of the panels. (B) The same experiments shown in panel A were done at least three times using other lots of HSCs. The intensities of chemiluminescent signals are indicated by means±standard deviations. The signal intensities on day seven were set to one for each protein analyzed. Asterisks indicate significant (P<0.05) differences compared to proteins on day seven by ANOVA.

Fig. 3

Immunofluorescent staining of desmin and vimentin in transdifferentiated rat HSCs. HSCs, plated on uncoated polystyrene dishes, were then subcultured on poly-L-lysine-coated glass-bottom dishes and cultured for two additional days. Cells were fixed and stained with mouse anti-desmin and goat anti-vimentin antibodies. Fluorescently-labeled secondary antibodies were used to visualize the locations of desmin (A, shown in red in panels C and E) and that of vimentin (B, shown in green in panels C and E). A differential interference contrast (DIC) image (D) is also shown. Bar in panel D represents 50 µm and applies to panels A–D; the bar in E represents 100 µm. Arrows in panel E indicate HSCs with prominent perinuclear staining for vimentin in contrast to desmin and arrowheads in panel E indicate HSCs with uniform distributions of vimentin and desmin. (F) Quantification of cytoplasmic distributions of vimentin and desmin. (G) Schema for quantification of distributions of cellular components in a cell. Lines extending from the nuclear membrane to the plasma membrane were drawn. Each line was divided evenly into ten segments from the perinuclear (region 1) to the peripheral (region 10) cytoplasmic regions and staining intensities in each segment were added together and expressed as percentages. Ten lines per cell were drawn and ten cells were used to calculate the distributions.

Fig. 4

Distribution of organelles and intermediate filaments. The HSCs plated on uncoated polystyrene dishes were subcultured on poly-L-lysine-coated glass-bottom dishes and cultured for four additional days. Cells were fixed and stained with mouse anti-desmin and goat anti-vimentin antibodies. Fluorescently-labeled secondary antibodies were used to visualize the locations of desmin (A and G, shown in red in panels D and J) and that of vimentin (B and H, shown in green in panels D and J). Prior to fixation, mitochondria were visualized by using MitoTracker Deep Red 633 (C) and shown in blue (D). Immediately before observation, lipid droplets were visualized by using BODIPY 493/503 (I) and shown in blue (J). DIC images (E and K) are also shown. Bar in panel K represents 50 µm and applies to all the panels. (F and L) Quantification of cytoplasmic distributions of mitochondria and lipid droplets, respectively.

Fig. 5

Effect of stress fiber disruption on desmin and vimentin locations in transdifferentiated rat HSCs. HSCs plated on uncoated polystyrene dishes were subcultured on poly-L-lysine-coated glass-bottom dishes and cultured for three additional days. Activated HSCs were treated with either DMSO (A–E) or two µM cytochalasin D (F–J) for one h. Cells were then fixed and stained with mouse anti-desmin (B and G, shown in red in panels D and I) and goat anti-vimentin (C and H, shown in green in panels D and I) antibodies. F-actin was stained by Alexa Fluor 546 phalloidin (A and F) and shown in blue (D and I). DIC images (E and J) are also shown. Bar in panel J represents 50 µm and applies to all the panels.