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Regulation of Hepatic Stellate Cell Proliferation

Melissa Potts

In today’s society, there is an increase number of individuals who suffer from liver damage. Chronic injury of the liver leads to fibrosis, an overgrowth of scar or connective tissue, which is a response to viral hepatitis, heavy alcohol consumption, metabolic disorders, and autoimmune diseases. Although there have been continuous and steady advances in basic research that has explored the possible mechanisms that initiate and perpetuate the fibrogenic response, the mechanisms remain unclear. It is clear, however, that after liver injury, hepatic stellate cells and hepatocytes play a crucial and facilitating role in fibrogenesis. Specifically, hepatic stellate cells undergo a response known as “activation” which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts (Friedman 2000).

            Hepatic stellate cells (HSC) are located in the subendothelial space of Disse (Olaso and Friedman 1998). The space of Disse is a cavity that is enclosed by plates of hepatocytes and sinusoids which are wide, leaky capillaries (Marieb and Mallatt 487). HSC have a stellate or star shape and compromise 15% of the total number of resident liver cells (Friedman 2000). HSC constitute a heterogeneous population of cells that differ in their expression of cytoskeletal filaments, retinoid content, and potential for extracellular matrix production (Friedman 2000). In a normal, healthy liver, hepatic stellate cells store vitamin A and fat and show minimal proliferation and collagen synthesis (Gaca et al. 2002). However, after the liver has sustained injury or damage and the HSC are “activated;” they metabolize vitamin A, synthesize extracellular matrix materials such as collagen, proteoglycans, and glycoproteins, and secrete growth factors that stimulate hepatocyte proliferation (Uyama et al. 2002).

            Hepatic stellate cell activation is an organized and defined sequence of cellular events. The process of HSC activation is actually defined by a two-stage process including initiation and perpetuation (Figure 1). The initiation stage, also referred to as the pre-inflammatory stage, refers

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to early changes in gene expression and phenotype which render the cells responsive to other local stimuli (Friedman 2000). The perpetuation stage is comprised of a series of events that includes proliferation, contractility, fibrogenesis, matrix degradation, HSC chemotaxis, retinoid loss, and leukocyte chemotaxis (Friedman 2003). These events ultimately enhance degradation of normal matrix and accumulation of fibrillar, or scar matrix (Friedman 2003).

Figure 1 A schematic diagram of the stellate cell activation pathway

            As shown in figure 1, the hepatic stellate cell activation pathway begins with proliferation in the perpetuation stage. Proliferation is characterized by an increase in the number of stellate cells in an injured liver that arise in part from local proliferation in response to polypeptide growth factors (Friedman 2000). Growth factors are large proteins that cause cells to divide and, in some cases, differentiate (Nelson and Cox 474). Polypeptide growth factors stimulate division

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of HSC because they specifically interact with tyrosine kinase receptors (Nelson and Cox 474). Most of the polypeptide growth factors, or mitogenic factors, signal through receptor tyrosine kinases (Olaso and Friedman 1998). Amplified stellate cell and hepatocyte proliferation specifically reflects increased secretion of active mitogens, extracellular heparan sulfate (HS) and HS proteoglycan, as well as agonists for proteinase-activated receptors-1 and -2 (Olaso and Friedman 1998).

            In one study, primary culture models were used to study and reveal the stellate cell-derived factors that regulate hepatocyte proliferation. Specifically, culture models were used to reveal that stellate cell-derived factors such as extracellular heparan sulfate (HS) and HS proteoglycan regulate and stimulate liver cell proliferation. In this study, isolation of HSC and hepatocytes were initially performed as well as a mono-culture of hepatocytes in the initial phase (Figure 2). Hepatic stellate cells were isolated from Wistar rats and cell purity was determined by the typical star-like shape and vitamin A autofluorescence. The isolated HSC were then suspended in Williams-E medium, which contained fetal bovine serum (FBS), penicillin, and streptomycin, and plated on collagen-coated culture dishes to be used in co-cultures. Hepatocytes were also isolated from Wistar rats and suspended in Williams-E medium that did not contain FBS, penicillin, or streptomycin. To prepare a mono-culture, hepatocytes were plated on collagen-coated culture dishes and the culture continued up to 4 days (Uyama et al. 2002).

            The second phase of the experiment consisted of a mixed co-culture and a separated co-culture of hepatocytes and HSC (Figure 2). To prepare a mixed co-culture, the freshly isolated hepatocytes from the initial phase of the experiment were plated onto culture dishes where isolated HSC had already been cultured and the culture was continued up to 4 days. In this type of co-culture, the hepatocytes and HSC had cell-to-cell contact. To prepare a separated co-culture, hepatocytes from the initial phase were plated onto other culture dishes where isolated

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HSC had already been cultured and the culture also continued up to 4 days. In this type of co-culture, the hepatocytes and HSC were separated by a culture insert to avoid any cell-to-cell contact (Uyama et al. 2002).

Figure 2 Culture systems used in this experiment. a). Hepatocyte mono-culture. b). Hepatocyte and HSC mixed co-culture. c). Hepatocyte and HSC separated co-culture.

 

In the final phase of the study, hepatocytes in each type of culture were treated with 5-Bromo-2′-deoxyuridine (BrdU). After 24 hours, incorporated BrdU was immunocytochemically evaluated. This evaluation included counting the number of cells with brown-colored nuclei in four randomly selected microscopic fields. From this data, the BrdU labeling index (BrdU L.I.) was calculated at 48 h and 72 h after plating. The BrdU L.I. was calculated as the number of BrdU-positive cells divided by the number of cells in the identical area multiplied by

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100 to yield a percentage. The BrdU labeling index is a measure of the DNA synthesis of hepatocytes (Uyama et al. 2002).

            The three different types of cultures used in this study revealed that active growth factors, extracellular heparan sulfate (HS) and HS proteoglycan, secreted from HSC, stimulated and regulated hepatocyte proliferation. In the mono-culture of hepatocytes, the number of hepatocytes decreased to 49% of the original number. This result is expected because without the presence of activated HSC, hepatocytes are not stimulated to divide. The decreased percentage of hepatocytes reflects the fact that hepatocyte proliferation is stimulated in the presence of HSC which secrete extracellular HS and HS proteoglycan. In the mixed co-culture of hepatocytes and HSC, the number of hepatocytes were roughly maintained at 106% initially, then decreased to 50% of the original number. When the hepatocytes were co-cultured with HSC, they came into contact with the processes of HSC, and thereafter, they were surrounded by activated HSC which secreted the growth factors. Unlike the mono-culture of hepatocytes, the number of hepatocytes did not decrease initially and as greatly because they were in the presence of HSC. In the separated co-culture of hepatocytes and HSC, the number of hepatocytes significantly increased to 135% initially, but then decreased to 76% of the original cell number. The drastic increase in the hepatocyte number preceding the decrease is expected because there was a considerable increase in cell density. Specifically, the number of hepatocytes increased from 1.58 x 102 cells/mm2 to 2.27 x 102 cells/mm2 because they were stimulated to divide by extracellular HS and HS proteoglycan. Although the hepatocytes decreased to 76% of the original number, that percentage was maintained at a significantly high level in comparison to the other two cultures. The activated HSC obviously secreted extracellular HS and HS proteoglycan and these two mitogens initially amplified hepatocyte proliferation (Uyama et al. 2002).

           

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In each type of culture, after random fields of hepatocytes were immunocytochemically evaluated, the BrdU labeling index was calculated. The BrdU L.I. revealed that the hepatocytes that were co-cultured had a higher rate of DNA synthesis than the hepatocytes that were mono-cultured. This effect is evident because the hepatocytes that were co-cultured were in the presence of HSC and the secreted growth factors stimulated DNA synthesis in the hepatocytes. In the mono-culture, the BrdU L.I. of hepatocytes was 2.41 at 24 h after plating and 11.3 at 72 h after plating. In the mixed co-culture, the BrdU L.I. of hepatocytes was significantly increased to 25.8 48 h after plating, but there was no significant increase at 72 h after plating.  The most dramatic increase of the BrdU L.I. was evident in the separated co-culture. In this type of culture, the BrdU L.I. was 47.5 and 48.5 at 24 h and 72 h after plating. These results indicate that the two co-cultures, with increased cell densities, had an effect on the DNA synthesis of hepatocytes. By having HSC and the mitogenic factors, extracellular HS and HS proteoglycan, present in the same culture, DNA synthesis in the hepatocytes is enhanced and as a result the hepatocytes proliferate (Uyama et al. 2002).

            Hepatic stellate cells, as well as hepatocytes, have a central and important role in the pathogenesis of liver fibrosis. The “activation” that HSC undergo, in which they transform to myofibroblastic cells, may also be facilitated by proteases that are produced by inflammatory cells. Mast cells (MC) accumulate in the sinusoids and the fibrotic septae during human fibrosis. The hepatic stellate cells that are activated recruit mast cells, therefore, mast cells support fibrogenesis by releasing fibrogenic mediators. Specifically, the major constituent of MC granules is the serine protease tryptase which is a mitogen for fibroblasts and increases collagen synthesis. Tryptase exerts its proliferative effects through proteinase-activated receptor (PAR)-2 and thrombin exerts its proliferative effects through proteinase-activated receptor (PAR)-1. Thrombin and MC tryptase are both increased in an injured liver. In one study, hepatic stellate

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cells were examined to reveal if they express proteinase-activated receptors-1 and -2 and if receptor agonists, thrombin and mast cell (MC) tryptase, influence stellate cell activation and proliferation (Gaca et al. 2002).

            Initially, hepatic stellate cells were isolated for reverse-transcription polymerase chain reaction (RT-PCR) and cell proliferation assays. The HSC were isolated from rats by collagenase perfusion and purified by density gradient separation and centrifugal elution. The HSC were maintained in a medium containing fetal calf serum (Gaca et al. 2002).

            In this study, cultured stellate cells were examined for expression of mRNA for proteinase-activated receptors-1 and -2 by RT-PCR. To perform the RT-PCR, rat skin fibroblasts and rat HSC were homogenized and total RNA was extracted. One μg of total
RNA was reverse-transcribed using the Moloney Murine Leukemia Virus reverse transcriptase and the resultant cDNA was diluted five fold for PCR. The polymerase chain reaction was performed for 35 cycles in the presence of MgCl2. The PCR products were amplified and were also analyzed by agarose gel electrophoresis and ethidium bromide staining (Gaca et al. 2002).

            In this study, cell proliferation assays were also performed to examine the effects of receptor agonists, thrombin and MC tryptase, on stellate cell proliferation. HSC were cultured, washed in serum-free medium, and incubated in fetal calf serum. The two PAR-1 and PAR-2 agonists were then added to the HSC for 24 h. 3H-thymidine was then added so that 3H-thymidine could be incorporated into HSC DNA (Gaca et al. 2002).

            The RT-PCR revealed that HSC express both proteinase-activated receptors-1 and -2 mRNA. PAR-1 mRNA was detectable in HSC at all times of culture and was even increased following transformation of these cells to myofibroblasts. PAR-2 was barely detectable before 7 days of culture, but was highly expressed in activated HSC after 14 days of culture. The verification that PAR-1 and PAR-2 were both expressed in activated HSC was encouraging and

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justified the attempt to perform cell proliferation assays. By experimentally proving that these two proteinase-activated receptors were present, the possibility that thrombin and MC tryptase influenced stellate cell activation was becoming a reality (Gaca et al. 2002).

            Cell proliferation assays also revealed that PAR-1 and PAR-2 agonists, thrombin and MC tryptase, induced proliferation of cultured HSC. The PAR-1 agonist, thrombin, increased 3H-thymidine incorporation into cellular DNA by 96%. The PAR-2 agonist, MC tryptase, also significantly increased 3H-thymidine incorporation into cellular DNA by 104%. These two results indicate that PAR-1 and PAR-2 agonists ultimately increase stellate cell proliferation (Gaca et al. 2002).

            In conclusion, hepatic stellate cell proliferation is regulated by the growth factors, extracellular heparan sulfate (HS) and HS proteoglycan, along with thrombin and MC tryptase. The mitogenic factors were revealed to regulate and stimulate hepatocyte and hepatic stellate cell proliferation in primary culture models and the BrdU labeling index. Extracellular HS and HS proteoglycan, which are secreted by hepatic stellate cells, amplify hepatocyte proliferation. The proteinase-activated receptor agonists, thrombin and MC tryptase, also regulate and increase stellate cell proliferation as demonstrated by RT-PCR and cell proliferation assays. By further studying and examining the crucial and significant role that hepatic stellate cells and hepatocytes play in fibrogenesis, advances will continually be made in elucidating their pathophysiology.

 

 

 

 

 

                                                                                                                                   

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References

Friedman, Scott L. “Liver Fibrosis-From Bench to Bedside.” Journal of Hepatology. 2003. 38: S38-S53.

Friedman, Scott L. “Molecular Regulation of Hepatic Fibrosis, and Integrated Cellular Response to Tissue Injury.” Journal of Biological Chemistry. 2000. 275(4): 2247-2250.

Gaca, Marianna D.A., et al. “Regulation of Hepatic Stellate Cell Proliferation and Collagen Synthesis by Proteinase-Activated Receptors.” Journal of Hepatology. 2002. 36: 362-369.

Marieb, Elaine N., and Jon Mallatt. Human Anatomy. 2nd ed. Menlo Park: Benjamin Cummings, 1996.

Nelson, David L., and Michael M. Cox. Lehninger Principles of Biochemistry. 3rd ed. New York: Worth Publishers, 2000.

Olaso, Elvira, and Scott L. Friedman. “Molecular Regulation of Hepatic Fibrogenesis.” Journal of Hepatology. 1998. 29: 836-847.

Uyama, Naoki, et al. “Regulation of Cultured Rat Hepatocyte Proliferation by Stellate Cells.” Journal of Hepatology. 2002. 36: 590-599.

 

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Copyright 2003 Melissa Potts and Koni Stone