Synaptophysin: A novel marker for human and rat hepatic stellate cells

Abstract

Synaptophysin is a protein involved in neurotransmitter exocytosis and is a neuroendocrine marker. We studied synaptophysin immunohistochemical expression in 35 human liver specimens (normal and different pathological conditions), in rat models of galactosamine hepatitis and carbon tetrachloride-induced cirrhosis, and in freshly isolated rat stellate cells. Synaptophysin reactivity was present in perisinusoidal stellate cells in both human and rat normal liver biopsies. The number of synaptophysin-reactive perisinusoidal cells increased in pathological conditions. Double staining for alpha-smooth muscle actin and synaptophysin, detected by confocal laser scanning microscopy, unequivocally demonstrated colocalization of both markers in lobular stellate cells. In addition, freshly isolated rat stellate cells expressed synaptophysin mRNA (detected by polymerase chain reaction) and protein. Finally, electron microscopy showed the presence of small electron translucent vesicles, comparable to the synaptophysin-reactive synaptic vesicles in neurons, in stellate cell projections. We conclude that synaptophysin is a novel marker for quiescent as well as activated hepatic stellate cells. Together with the stellate cell's expression of neural cell adhesion molecule, glial fibrillary acidic protein, and nestin, this finding raises questions about its embryonic origin and its differentiation. In addition, the presence of synaptic vesicles in stellate cell processes suggests a hitherto unknown mechanism of interaction with neighboring cells.

Figure 1.

a: Liver biopsy of patient with chronic biliary disease showing synaptophysin reactivity in perisinusoidal cells with stellate projections. Original magnification, ×250. b: Liver biopsy of patient with chronic hepatitis C showing α-smooth muscle actin-reactivity in lobular perisinusoidal stellate cells, in septal myofibroblasts (left upper side of figure) and in smooth vessel cells of an arterial wall (arrow) Original magnification, ×250. c: Liver biopsy of patient with chronic hepatitis C showing synaptophysin-immunoreactivity in lobular perisinusoidal cells with stellate projections. In the fibrotic septum (upper left side of figure), no reactivity is present. Arterial wall, arrow. Original magnification, ×250. d-f: Liver biopsy of patient with chronic hepatitis C: double staining for synaptophysin and α-smooth muscle antigen, detected by the confocal laser scanning microscope: Lobular stellate cell showing reactivity for synaptophysin (green labeling, d), α-smooth muscle actin (red labeling, f) and colocalization for synaptophysin and α-smooth muscle actin (yellow double-labeling, e). Original magnification, ×1000.

Figure 2.

a: Electron micrograph of normal human liver biopsy showing a stellate cell in the Disse space, flanked by part of two hepatocytes (H). The stellate cell contains fat droplets. Collagen bundles (C). Original magnification, ×14,500. Inset: Detail of a cytoplasmic processus containing many small clear vesicles, comparable to the synaptic vesicles found in neurons. Original magnification, ×46,000. b: Electron micrograph of human liver biopsy with chronic subobstruction. Disse space (*) and sinusoidal recess between two hepatocytes (H). Part of a cytoplasmic process of a stellate cell (arrow) containing many small clear vesicles (small arrows). Collagen bundles (C). Original magnification, ×36,800.

Figure 3.

a: Normal rat biopsy immunostained for synaptophysin, showing reactivity in perisinusoidal stellate cells. Fat droplets can be recognized in the cytoplasm of some immunoreactive cells (arrow; original magnification, ×400). b: Rat liver biopsy 36 hours after galactosamine intoxication. Synaptophysin reactivity is seen in a higher number of stellate cells, compared with the normal rat liver shown in a. Stellate cells are clustered around necrotic areas. Original magnification, ×100. c: Rat liver biopsy 48 hours after galactosamine intoxication. Synaptophysin reactivity is seen in stellate cells. The number of stellate cells reaches its maximum at 48 hours. Stellate cells are clustered around necrotic areas. P, portal tract; *, necrotic areas. Original magnification, ×100. d-f: Rat liver biopsy 36 hours after galactosamine intoxication. Double staining for synaptophysin and α-smooth muscle antigen, detected by the confocal laser scanning microscope: lobular stellate cell showing reactivity for synaptophysin (green labeling, d), α-smooth muscle actin (red labeling, f) and colocalization for synaptophysin and α-smooth muscle actin (yellow double-labeling, e). Original magnification, ×1000.

Figure 4.

Transcription of the SYN gene in freshly isolated rat liver stellate cells was demonstrated by RT-PCR. The PCR product was calculated to a length of 448 bp, as was confirmed on agarose gel electrophoresis stained with ethidium bromide. Rat hepatic stellate cells (lane 1) show transcription; FTO-2B rat hepatocyte cell line (lane 2) showed no transcription; rat brain (lane 3) showed abundant transcription. Lane M: 100-bp ladder. Lane 4: negative control (no sample). Identification of the PRC product as rat synaptophysin cDNA was achieved by sequencing.