Role of Albumin in Hepatic Stellate Cell Activation the Antifibrotic Effects of Albumin

Role of Albumin in Hepatic Stellate Cell Activation The Antifibrotic Effects of Albumin

Master’s Research Report 1 Author: B.W. Perridon. S2031582. Master’s student in Biomedical Sciences at University of Groningen. Internship: December 2013 – July 2014. Report: May 6, 2015. Department of Gastroenterology and Hepatology, University Medical Center Groningen. Supervisors: prof. dr. K.N. Faber and F.W. Haijer.

Abstract Introduction Liver fibrosis and cirrhosis are caused by a continuous wound healing response of the liver, characterized by scar formation and the deposition of excessive amounts of extracellular matrix components by hepatic myofibroblasts. Hepatic stellate cells (HSCs) are currently considered as the major source of the hepatic myofibroblasts. During fibrogenesis the role of the HSCs in the liver changes as they undergo a phenotypic transformation from their quiescent to an activated, myofibroblast-like phenotype. In pancreatic stellate cells, cells that are comparable to HSCs, it was suggested that albumin has a direct effect on (pancreatic) stellate cells activation. Whether albumin is produced by HSCs or whether they acquire it from extracellular sources is unknown, as well as its potential role in HSCs. Aims of the study were to investigate the presence and the source of albumin in HSCs and its effect on the activation of HSCs. Methods Primary HSCs, portal myofibroblasts, hepatocytes and Kupffer cells were isolated from male Wistar rats. The presence of albumin protein in HSCs cultured in different conditions was examined using Ponceau S staining and Western blotting. Immunofluorescence microscopy was used to study the intracellular localization of albumin. The production of albumin by HSCs and the expression of quiescent HSC markers (PPAR γ; LRAT) and HSC activation markers ( αSMA; collagen type 1a1) were studied using qPCR. Potential contamination of HSCs by hepatocytes was analyzed using hepatocyte markers (BAAT; BSEP). Uptake of albumin orthologs by HSCs was examined using Western blotting. Effects of albumin administration to culture-activated HSCs on the HSC activation markers were determined using qPCR. Results Western blotting and immunofluorescence microscopy revealed the presence and location of albumin in both quiescent and activated HSCs. Albumin mRNA levels decreased upon culture-activation of HSCs in a similar fashion as the hepatocyte markers BAAT and BSEP, suggesting that it was (virtually) absent in both HSC phenotypes. Primary rat HSCs only contained rat albumin when cultured in rat serum containing medium but contained bovine/calf albumin when cultured in fetal calf serum containing medium. Bovine/calf albumin dose-dependently reduced the expression of the HSC activation markers collagen type 1a1 and αSMA when added to the culture medium at concentrations above 22.5 g/L, which is important since the normal blood level of albumin ranges from 35 to 55 g/L and is thus markedly higher than the albumin concentration normally used in culture. Conclusion These data show that HSCs do not produce albumin but that they do actively take up albumin from extracellular sources, suppressing HSC activation in a concentration and time dependent manner. Based on the finding that albumin has antifibrotic properties, we propose a mechanism in which healthy hepatocytes attenuate liver fibrosis through albumin.

Table of contents Abstract ...... 1 Introduction ...... 3 Materials and methods ...... 6 Animals ...... 6 Cell isolation and culture ...... 6 Experimental design ...... 7 Protein isolation ...... 8 RNA isolation and cDNA synthesis ...... 8 Real-time quantitative PCR ...... 9 Western blot analysis ...... 9 Immunofluorescence microscopy ...... 10 Statistical analysis ...... 10 Results ...... 10 Albumin present in both quiescent and activated HSCs ...... 10 Albumin not expressed by HSCs ...... 12 Albumin taken up by HSCs ...... 16 HSC activation delayed by albumin ...... 16 HSC activation not only controlled by albumin ...... 18 Conclusions and discussion ...... 19 Bibliography ...... 23 Supplemental data ...... 27

Abbreviations: aHSC: activated hepatic stellate cell; αSMA: alpha-smooth muscle actin; BAAT: bile acid- CoA:amino acid N-acyltransferase; BSA: bovine serum albumin; BSEP: bile salt export pump; col-1: collagen type 1a1; ECM: extracellular matrix; FCS: fetal calf serum; HSC: hepatic stellate cell; LRAT: lecithin retinol acyltransferase; PMF: portal myofibroblast; PPAR γ: peroxisome proliferator activated receptor γ; PSC: pancreatic stellate cell; qHSC: quiescent hepatic stellate cell; RS: rat serum; TGF-β: transforming growth factor β.

Introduction The liver is a vital organ that plays a major role in a great variety of metabolic processes. It is involved in storage, synthesis and breakdown of small and complex molecules such as proteins, carbohydrates and lipids. The liver is also involved in clearance of potentially harmful compounds. The organ can deal with damage in a way no other organ can as it can completely regenerate from a small portion of healthy liver. The regenerated liver tissue (partly) reverses the loss of liver function caused by the damage. All populations of cells within the liver are able to regenerate and migrate to the regenerated tissue [Reviewed by [1] . In case of repetitive damage, in for example chronic liver diseases, the liver becomes fibrotic as it attempts to replace and repair damaged tissue. Apoptotic hepatocytes, Kupffer cells and inflammatory cells greatly contribute to this process. The hepatic tissue repair reaction is characterized by the formation of highly dynamic scar tissue and a shifted balance between the production and degradation of connective tissue. This results in the deposition of excessive amounts of extracellular matrix (ECM) components, mainly fibrillar collagen, by hepatic myofibroblasts [2, 3] . It has been speculated that hepatic myofibroblasts, normally absent in the liver, are able to promote liver regeneration of the damaged liver by producing growth factors for hepatic oval cells [4] and probably also for other cells involved in the regeneration. However, it is generally accepted that an accumulation of hepatic myofibroblasts causes fibrosis and can result in a progressive loss of liver function called cirrhosis. There is increasing evidence that hepatic fibrosis is reversible if the stimuli are successfully removed [5, 6] . Eventually liver fibrosis can progress to liver cirrhosis. In general perpetuation to cirrhosis takes months to years of liver disease. Cirrhosis is characterized by a loss of liver architecture that results from excessive fibrosis and the formation of pseudolobules, also called regeneration nodules, throughout the liver [Reviewed by [7] . The process of cirrhosis causes liver dysfunction and distorts the fundamental hepatic architecture making reversal more difficult.

Liver and its cell types The complex functions of the liver mentioned above are mainly performed by hepatocytes, also known as hepatic parenchymal cells. Hepatocytes are orchestrated in liver lobules, which are small functional units of the liver. Besides hepatocytes, liver lobules contain non-parenchymal cells like the sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells (HSCs) (Figure 1) . The sinusoidal endothelial cells form the sinusoids, blood vessels with a fenestrated endothelium which are separated from the hepatocytes by the space of Disse. Within the lumen of the sinusoids, the Kupffer cells reside. Kupffer cells are the resident macrophages within the liver and play a role in hemoglobin degradation, host defense, immune tolerance, liver regeneration and liver injury [Reviewed by [8] . There is evidence for bipotential stem cells in the liver, called hepatic oval cells or ovalocytes, which are thought to reside in the portal area and in the canals of Hering. During embryogenesis and during severe liver disease these cells are activated and differentiate into either hepatocytes or cholangiocytes [9] , the latter being the cell type that lines the bile ducts. To what extent these progenitor cells are relevant in liver regeneration and/or fibrosis is not known.

Figure 1: Schematic overview of a hepatic sinusoid In the liver, both arterial and portal venous blood flows from the portal blood vessels towards the central vein through liver-specific capillaries called sinusoids. These capillaries are formed by fenestrated sinusoidal endothelial cells and in the lumen of the sinusoids blood cells such as Kupffer cells, the residential macrophages of the liver, reside. The sinusoids are separated from the hepatocytes by the space of Disse, which allows the exchanges of oxygen, nutrients and waste products between the blood and the hepatocytes. Hepatic stellate cells are present in the space of Disse. On the other side of the hepatocytes, bile canaliculus collect various products such as bile salts produced and excreted by hepatocytes and drain bile into the hepatic bile duct towards the gallbladder and the intestine. The region that connects the bile canaliculus and the biliary tree is called ‘canals of Hering’. Portal myofibroblasts are believed to be located in the portal field, along the portal vein. The gray dashed line marks the portal field. Adapted from [10] .

In a healthy liver, HSCs reside in the space of Disse in a quiescent state. Quiescent HSCs (qHSCs) are able to store vitamin A (retinyl-esters) in lipid droplets [11-13] . When the liver is damaged, qHSCs become activated and highly proliferative as they undergo a phenotypic transformation. The activated HSCs (aHSCs) lose their vitamin A stores and migrate to the site of injury, downregulate their neural markers, upregulate mesenchymal markers [14] and start to synthesize large amounts of connective tissue proteins [2, 3] . In vivo , the transformation of HSCs into their myofibroblast-like phenotype is likely caused by paracrine stimulation by other, neighboring cell types. Hepatocyte damage for example leads to the recruitment of inflammatory blood cells and platelets as well as the infiltration and activation of Kupffer cells. Cytokines and reactive oxygen species secreted by these Kupffer cells are necessary for liver regeneration and do play a prominent role in the activation of HSCs as the influx of Kupffer cells coincides with the appearance of stellate cell activation markers. There is evidence indicating the contribution of activated Kupffer cells to the pathogenesis of different liver injuries [Reviewed by [8] . Other cell types including sinusoidal endothelium, hepatocytes, platelets and leukocytes are also thought to play a role in the activation process of HSCs [15] . After the transformation, the myofibroblast-like HSCs are thought to play an important role in liver fibrogenesis and are currently considered to be the major source of hepatic myofibroblasts in damaged liver [14, 16] . The origin of hepatic myofibroblasts has intensively been discussed and investigated. The population of hepatic myofibroblasts was shown to be heterogeneous [17-19] , indicating that HSCs are not the only source of hepatic myofibroblasts responsible for fibrosis development. Several other sources have been suggested. Studies provided evidence for a population of hepatic myofibroblasts derived from portal myofibroblasts (PMFs) [Reviewed by [20] . The connective tissue around portal tracts locates a heterogeneous, non-parenchymal cell population of PMFs. In culture, freshly isolated PMFs become activated as they undergo myofibroblastic differentiation and become motile and contractile [14] . These activated PMFs are increasingly associated with the production of ECM in fibrotic liver [19, 21] . However,

their role in liver homeostasis and their response to injury in vivo remain unclear [Reviewed by [20] . It is debated whether PMFs are a different cell type besides HSCs or not. Morphologically, PMFs and HSCs are similar, but vitamin A droplets are absent in PMFs [22] and their response to profibrogenic and mitogenic stimuli is different [21] . Several groups described differences in proteomics between these cell types [Reviewed by [23] . Striking differences in the ability to proliferate were found when PMFs and HSCs are cultured (unpublished data). Despite these differences, the existence of PMFs as a distinct cell type besides HSCs remains disputed. Recent evidence indicates that PMFs are only a minor contributor to the population of hepatic myofibroblasts as 82% to 96% of fibrogenic myofibroblasts were derived from HSCs in 7 models of fibrosis [24] . Other recent studies proposed that HSCs and PMFs occupy different niches which yield myofibroblasts with specialized functions dependent on the etiology of liver injury [Reviewed by [20] . The contribution of PMFs to the development of fibrosis is undefined and remains controversial [Reviewed by [20] . A small contribution to the population of hepatic myofibroblasts comes from bone marrow derived mesenchymal cells and hepatic bone marrow-derived mesenchymal stem cells [25-29] . The contribution of epithelial-to-mesenchymal transition, in which epithelial cells acquire features of mesenchymal cells and give rise to fully differentiated myofibroblasts, has been suggested [30-32] but more recent studies failed to detect any hepatic myofibroblasts originated from epithelial-to-mesenchymal transition [33, 34] . Another, unassessed theoretical source of hepatic myofibroblasts is the transition of endothelial-to- mesenchymal cells [35, 36] .

Thus far, a tremendous amount of research has demonstrated that HSCs are the most important cells in liver fibrosis owing to the considerable amount of ECM production [37]. Therefore HSCs became the major target for antifibrotic drugs [38]. Many drugs of which potent antifibrotic activities are shown in vitro do often show only minor effects in vivo as the drugs are not efficiently taken up by HSCs, do insufficiently accumulate around the target cells or may produce unwanted side effects that limits their therapeutic ratio [Reviewed by [39]. Cell-specific delivery can provide a solution to these problems and several kinds of targeted delivery system to target the HSC have been designed [Reviewed by [39]. The HSC-selective drug carrier mannose-6-phosphate (M6P)-modified human serum albumin (HSA) was designed [40] after an upregulation of the M6P/IGF-II receptors on HSCs in fibrotic rat livers was described [41-43]. The M6P- modified HSA is taken up by HSCs in fibrotic rat livers and therefore can be applied as a selective drug carrier to deliver intracellular acting antifibrotic drugs to HSCs [44]. Another way to tackle liver fibrosis without the use of antifibrotic drugs is to prevent the activation of HSCs by determining what compounds or conditions cause their activation. For example, despite the well-established concept that stellate cell activation is induced in vivo by the mentioned triggers, in vitro quiescent HSCs spontaneously activate without stimulation. In addition, activated myofibroblast-like liver cells are highly proliferative in cell culture without stimulation. Until now, there is no satisfactory explanation in literature available that explains this striking contradiction. Some studies suggest that culture plastic induces activation as special coated plates can retain HSCs in their quiescent phenotype [45-47]. Other studies even report deactivation of aHSCs when cultured in those coated plates [48, 49]. In our study we hypothesize, in addition, that the lack of a suppressive signal from hepatocytes ameliorates this activation process.

Albumin in pancreatic and hepatic stellate cells In a study on pancreatic stellate cells (PSCs), a protein band was observed on Western blots of whole cell lysate of quiescent PSCs at the position just below 70 kDa and the protein was identified as albumin [50] . Albumin, with a molecular weight of around 67 kDa, is the most abundant protein in human blood plasma (35 to 55 g/L) and is mainly produced by hepatocytes. In the blood, it binds and transports compounds and regulates pH and osmotic pressure. The study suggested the involvement of albumin in the formation of vitamin A droplets in PSCs [50] . As PSCs are morphologically comparable to HSCs [Reviewed by [51] it is possible that albumin is present in HSCs and is involved in processes comparable to those described

in PSCs. On Western blots of whole cell lysate of HSCs we reproducibly observed a clear band at the position just below 70 kDa (unpublished observation). It is not clear whether albumin is present in the HSCs or not and if so what role it has in HSCs. Therefore it is interesting to determine the potential presence and source of albumin in the HSCs. The production of albumin by HSCs was investigated before and albumin expression was observed in unpurified HSC isolates [52] . However, these HSC isolates were isolated using density gradient centrifugation resulting in a purity of around 70% [Reviewed by [52] , thus still containing contaminating cells and subcellular particles [53, 54] . After the HSC isolates were purified by fluorescence-activated cell sorting (FACS) based on the granularity of the cells as a measure of the presence of lipid droplets in the cells, no albumin expression was observed in HSCs by conventional PCR [52] . The study suggested that HSCs do not produce albumin themselves. However, after FACS sorting only a subpopulation of HSCs is obtained, which could lead to falsely negative results [52] . As mentioned above, albumin is produced by PSCs and the production of albumin by HSCs was suggested, mainly based on findings in PSCs as results for HSCs are barely shown [50] . Strong evidence whether albumin is produced by HSCs is absent and therefore we reconsider and study the idea that HSCs contain albumin either from endogenous or exogenous sources and may affect HSC activation.

The aims of this study were to investigate the presence of albumin in quiescent and activated HSCs at different time points during in vitro activation and to examine the production of albumin when present in the HSCs. Only little is known about the effects of albumin on the activation process of HSCs and myofibroblasts-like liver cells. In order to further clarify the important role of albumin in liver fibrosis we investigated whether albumin has antifibrotic effects on HSCs. The role of albumin in other liver cell types like PMFs, Kupffer cells and hepatocytes was also investigated.

Materials and methods Animals Specified pathogen-free male Wistar rats were purchased from Charles River Laboratories Inc. (Wilmington, MA, USA). Animals were kept under standard laboratory conditions and had free access to standard laboratory chow and water. Each experiment was performed in accordance with the guidelines of the local Committee for Care and Use of laboratory animals.

Cell isolation and culture Hepatic stellate cells Hepatic stellate cells were isolated from male Wistar rats (500–600 g) by pronase (Merck, Amsterdam, the Netherlands) and collagenase-P (Roche, Almere, the Netherlands) perfusion of the liver, followed by Nycodenz (Axis-Shield POC, Oslo, Norway) gradient centrifugation (13% w/v) as described previously [55] . Cells were then cultured in Iscove's Modified Dulbecco's Medium (IMDM) with Glutamax (Invitrogen, Breda, the Netherlands) supplemented with 20% heat-inactivated fetal calf serum (FCS; Invitrogen), 1 mM sodium pyruvate (Invitrogen), 1x MEM non-essential amino acids (Invitrogen), 50 μg/mL gentamycin (Invitrogen), 100 U/mL penicillin (Lonza, Vervier, Belgium), 10 μg/mL streptomycin (Lonza) and 250 ng/mL fungizone (Lonza) in a humidified incubator containing 5% CO 2 at 37 °C. This medium composition is used unless indicated otherwise. Other compositions of the medium used are a medium with 10% heat- inactivated FCS, a medium with 20% heat-inactivated rat serum instead of FCS and a medium without serum, all three with all the other supplements described above. To study the activation of HSCs, the isolated quiescent HSCs were cultured on plastic and were harvested at different time points for experiments by cell scraping. For experiments on activated HSCs, primary HSCs culture-activate in 7 days, grown to confluence and were passaged via trypsinization. The cells were used for experiments on activated HSCs.

Hepatocytes, portal myofibroblasts and Kupffer cells Hepatocytes were isolated from male Wistar rats (220–250 g) by a two-step collagenase perfusion technique as described previously [55, 56] . First the calcium-containing bridges between the cells are broken down by perfusion with Ca 2+ -free buffer. The second perfusion step is with a Ca 2+ -containing buffer with collagenase. Ca 2+ is necessary to activate the collagenase, which digests the collagen matrix of the liver. After isolation the hepatocytes were put in 1.5 ml cups and stored at −80 °C. These hepatocyte (t=0) samples were used in the experiments without being cultured. Portal myofibroblasts were isolated from the outgrowth of isolated intrahepatic bile duct and portal tree segments, which are residue products after hepatocyte isolation, as described earlier [57] . The cells were cultured in IMDM containing 20% FCS in the humidified incubator and were harvested at indicated time points. Kupffer cells were isolated from the non-parenchymal cell fraction obtained after hepatocyte isolation using a Percoll density cushion, as described previously [58] . The cells were allowed to adhere to the culture plates for 30 minutes in Hank's Balanced Salt Solution (HBSS) containing magnesium, calcium (Life Technologies) and 10% FCS. The unattached contaminating cells were removed by washing whereupon the attached Kupffer cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Life Technologies) containing 10% FCS in the humidified incubator [59] and were harvested at indicated time points.

Experimental design Experiments on quiescent HSCs Isolated primary HSCs were cultured in 6 wells plates in medium to which no extra compounds were added besides the ones described above. The number of cells put in the wells for RNA isolation and protein isolation for the 4 hours, 1 day and 3 days time points were 300,000 and 150,000, respectively. For the 7 days time point the number of cells were 100,000 and 75,000, respectively. The cells were put in the humidified incubator and the medium was refreshed every 3 to 4 days. The experiments were started after the attachment period of 4 hours. After these 4 hours and at the other indicated time points RNA and protein were isolated as described below. Samples for RNA isolation were stored at −80 °C and for protein isolation at −20 °C. All experimental conditions were performed in duplicate. The experiments were repeated multiple times with HSCs from different rats.

Experiments on activated HSCs The isolated primary quiescent HSCs were culture-activated on tissue culture plastic for at least 7 days. Experiments were started 24 hours after passaging the activated HSCs into 6 wells plates. Monolayers of cultured activated HSCs were exposed to the 20% FCS-IMDM medium, to the 20% rat serum-IMDM medium, to the serum-free IMDM medium or to the 10% FCS-IMDM medium enriched with the indicated concentrations of bovine serum albumin (Sigma; product A3059) or casein (Merck; product 102244). The 10% FCS-IMDM medium contains 10% (4.5 g/L) of the albumin found in the blood serum (35-55 g/L). A stock of 200% of the blood serum albumin concentration was prepared by dissolving bovine serum albumin to the 10% FCS-IMDM medium to an albumin end concentration of 90 g/L. From this stock a dilution series was made with concentrations of 200% (90 g/L), 100% (45 g/L), 50% (22.5 g/L) and 20% (9 g/L) of the serum concentration of albumin (SCA) by adding the enriched stock to the 10% FCS-IMDM medium. The casein enriched medium was prepared in a comparable way and had concentrations equivalent to those of albumin. The cells were put in the humidified incubator and the medium was refreshed every 3 to 4 days. At the indicated time points after the addition of the protein enriched medium, cells were harvested for the different assays. Prior to the RNA isolation the cells were rinsed three times with HBSS without calcium, magnesium and phenol red (Life Technologies) after which the Tri-reagent (Sigma-Aldrich) was added. Prior to protein isolation the cells were rinsed five times with HBSS just before

the later described lyses buffer was added. Each experimental condition was performed in duplicate wells, which were pooled during the isolations after scraping. Samples for RNA isolation were stored at −80 °C and for protein isolation at −20 °C. The experiments were performed only once or twice due to infections during cell culturing, using HSCs from different isolations.

Experiments on hepatocytes, portal myofibroblasts and Kupffer cells Freshly isolated hepatocytes were stored at −80 °C directly after isolation, without culturing and are therefore called t=0. Hepatocytes were taken from the freezer and defrost after which cells were used for RNA isolation and protein isolation to perform the different assays as described below. The experiments were performed three times using rat hepatocytes from different isolations. Isolated PMFs are cultured on tissue culture plastic in 20% FCS-IMDM medium. After 3 passages the cells were subcultured in 6 wells plates and after attachment the monolayers of PMFs were exposed to the 20% FCS-IMDM medium, to the 20% rat serum-IMDM medium or to the serum-free IMDM medium for 3 days. The cells are placed in the humidified incubator and after 3 days the cells were harvested and protein and RNA were isolated for the different assays described below. Each experimental condition was performed in duplicate wells, which were pooled during the isolations after scraping. These experiments on PMFs were performed only once. The isolated Kupffer cells were cultured for 4 hours and for 1 day in 6 wells plates in 10% FCS-RPMI medium. The Kupffer cells were harvested at the indicated time points and RNA was isolated from the cells for the different assays as described next. Each experimental condition was performed in sextuplicate wells which were pooled during the isolations after scraping. These experiments on Kupffer cells were performed only once. For all these cells, the RNA isolates were stored at −80 °C and the protein isolates at −20 °C.

Protein isolation Proteins were isolated using lysis buffer consisting of 25 mM HEPES (Sigma), 150 mM potassium acetate (Sigma), 2 mM EDTA (Sigma), 0.1% Nonidet P40 (Roche Biochemicals, Almere, The Netherlands), 10 mM sodium fluoride (Sigma), 50 mM PMSF (Sigma), 1 μg/mL Aprotinin (Roche Biochemicals), 1 μg/ml Pepstatin A (Roche Biochemicals), 1 μg/ml Leupeptin (Roche Biochemicals) and 1mM dithiothreitol (Roche Biochemicals), pH 7.5. The cells were scraped in the lysis buffer and the duplicate wells were pooled in 1.5 ml cups after scraping. The samples are put into liquid nitrogen and are then thawed by putting them in a 37 °C heating block for 10 minutes. This is repeated three to five times. The samples are centrifuged for 10 minutes at 12000xg at RT after which the supernatant with the proteins is transferred to new cups. The protein samples were stored at −20 °C until used for Western blot analysis.

RNA isolation and cDNA synthesis RNA was isolated using Tri-reagent (Sigma-Aldrich) according to the manufacturer's instructions. The cells were scraped in the Tri-reagent and the duplicate wells were pooled after scraping. Per 500 μl Tri- reagent 100 μl chloroform (Merck) was added to the sample. After vigorous mixing the samples were centrifuged at 12,000xg at 4 °C for 15 minutes. The aqueous phase was removed and put into new 1.5 ml cups. To these samples 250 μl of 2-isopropanol (Merck) was added and after mixing gently the samples are centrifuged 10 minutes at 12,000xg at 4 °C. The supernatant was removed and the RNA/DNA pellet was washed with ice-cold 75% ethanol and centrifuged for 5 minutes at 7,500xg at 4 °C. The supernatant was discarded and the pellet was air-dried for 5 to 15 minutes. The pellets were dissolved in 40 μl to 100 μl RNAse free water dependent on the size of the pellet. The RNA samples were stored at −80 °C. Prior to the production of cDNA by reverse transcription-Polymerase Chain Reaction (RT-PCR), the RNA concentration of the samples was quantified using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Breda, the Netherlands) for equal loading of RNA. Reverse transcription was performed on total

RNA using random nonamers (Sigma-Aldrich) in a final volume of 20 μl. Reverse transcription was performed in three steps: 10 minutes at 25 °C, 1 hour at 37 °C and 5 minutes at 95 °C on the GeneAmp® PCR System 9700 (Applied Biosystems, Nieuwekerk a/d IJssel, the Netherlands).

Real-time quantitative PCR Real-time quantitative Polymerase Chain Reaction (qPCR) detection was performed on the 7900HT Fast Real-Time PCR System with software version 2.3 (Applied Biosystems) initialized by 10 minutes at 95 °C, followed by 40 cycles (15 seconds at 95 °C for denaturation and 60 seconds at 60 °C for annealing and polymerization). Each sample was analyzed in duplicate wells. The mRNA levels of the housekeeping gene 18S were used as an internal control to which the other gene transcripts are corrected. The reaction mixture contained 900 nM sense and antisense primer and 200 nM labeled probe supplemented to qPCR mastermix plus-dTTP (Eurogentec, Maastricht, the Netherlands). The sequences of the primers (Invitrogen) and the probe (Eurogentec) used are listed in Table 1. Relative gene expressions were calculated using the comparative ( ∆∆ )Ct method, a quantitation approach in which the Ct values of the samples of interest were compared with a control, which is the first sample of the experiment, unless indicated otherwise. The Ct values of both the control and the other samples were normalized to an appropriate endogenous housekeeping gene 18S [60] .

Table 1: Sequences of rat PCR primers and probes used for real-time qPCR analysis Gene Sequence 18S Sense 5′-cgg cta cca cat cca agg a-3′ Antisense 5′-cca att aca ggg cct cga aa-3′ Probe 5′-cgc gca aat tac cca ctc ccg a-3′ Albumin Sense 5’-ggc gac ctg ttg gaa tgc-3’ Antisense 5’-gga gat agt ggc ctg gtt ctc a-3’ Probe 5’-ctt ggc aag gtc cgc cct gtc atc-3’ αSMA Sense 5′-gcc agt cgc cat cag gaa c-3′ Antisense 5′-cac acc aga gct gtg ctg tct t-3′ Probe 5′-ctt cac aca tag ctg gag cag ctt ctc ga-3′ BAAT Sense 5’-tgt aga gtt tct cct gag aca tcc taa-3’ Antisense 5’-gtc caa tct ctg ctc caa tgc-3’ Probe 5’-tgc caa ccc ctg ggc cca g-3’ BSEP Sense 5′-cca agc tgc caa gga tgc ta-3′ Antisense 5′-cct tct cca aca agg gtg tca-3′ Probe 5′-cat tat ggc cct gcc gca gca-3′ collagen type 1a1 Sense 5’-tgg tga acg tgg tgt aca agg t-3’ Antisense 5’-cag tat cac cct tgg cac cat-3’ Probe 5’-tcc tgc tgg tcc ccg agg aaa ca-3’ LRAT Sense 5’-act gtg gaa caa ctg cga aca c-3’ Antisense 5’-agg cct gtg tag ata gac act aat cc-3’ Probe 5’-ttg tga cct act gca gat acg gct c-3’ PPAR γ Sense 5’-cac aat gcc atc agg ttt gg-3’ Antisense 5’-gct ggt cga tat cac tgg aga tc-3’ Probe 5’-cca aca gct tct cct tct cgg cct g-3’

Western blot analysis Western blot analysis was performed as described previously [61] . The DC™ Protein Assay kit (Bio-Rad, Hercules, CA, USA) was used to determine protein concentration to ensure equal protein loading on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Western blot analysis by

SDS-PAGE was followed by protein transfer to Hybond ECL nitrocellulose membrane (Amersham Bioscience, Piscataway, NJ, USA) using a semidry blotting system. Ponceau S (Sigma-Aldrich, St. Louis, MO, USA) staining was used to confirm electrophoretic transfer of the proteins to the membrane. The membrane is blocked with a 1.0% casein PBS-0.1%Tween-20 solution or a 0.5% casein PBS- 0.1%Tween-20 solution as the protocol was adjusted after the first experiment. Specific proteins were detected using primary antibodies dissolved in PBS-0.1%Tween-20 with either a 1.0% or a 0.5% dissolved casein and were added to the membranes for at least 2 hours. The primary antibodies used were rabbit anti-albumin mouse (dilution 1:5000; ICN Biomedicals; product 645601), polyclonal rabbit anti- bovine serum albumin (dilution 1:2500 and 1:5000; Life Technologies; product A11133) and mouse anti- GAPDH (dilution 1:10000; Calbiochem, VWR, the Netherlands CB1001). The secondary antibodies goat anti-IgG rabbit-HRP (dilution 1:2000; Dako, Heverlee, Belgium; product P0448) and rabbit anti-IgG mouse-HRP (dilution 1:2000; Dako; product P0260) were diluted in PBS-0.1%Tween-20 and were added to the membranes for at least 1 hour. The horseradish peroxidases on the immunoblots reacted with SuperSignal® West Dura Extended Duration Substrate (Thermo Scientific) after which the protein bands were detected using a Chemidoc XRS system (Bio-Rad).

Immunofluorescence microscopy Immunofluorescence microscopy was performed as described previously [62] . Quiescent and activated HSCs were cultured on coverslips and fixed with 4% paraformaldehyde for 30 minutes at 4 °C. The cell membranes of the fixed cells were permeabilized by adding 1% Triton X-100 to the cells for 5 minutes. Polyclonal rabbit antibodies against bovine serum albumin (dilution series and dilution 1:200) alone or together with monoclonal mouse anti-αSMA antibodies (dilution 1:200; Sigma-Aldrich; product A2547) were use as primary antibodies, followed by secondary antibodies labeled with Alexa Fluor-488 goat anti- IgG rabbit (dilution 1:400; Invitrogen; product A11008) and/or Alexa Fluor-594 goat anti-IgG mouse (dilution 1:400; Invitrogen; product A11005), respectively. The coverslips were then fixed to microscope slides with 25 μl of Vectashield® mounting medium for fluorescence with dapi (Vector Laboratories) to mount the cells and to stain the nucleus, after which the coverslips were sealed to the microscope slide with nail polish. Images were captured with a Leica TCS SP2-AOBS confocal laser scanning microscope (Leica, Heidelberg, Germany).

Statistical analysis Statistical analyses of data were performed using Microsoft’s Office Excel 2003 software. Results are presented as the mean of multiple experiments ± standard deviation, unless indicated otherwise. Per graph the number of repetitions is stated as the experiments were not always performed 3 times independently. Statistical differences between groups were calculated using the unpaired two-tailed student’s t-test with equal variances when experiments were performed. A p-value of less than 0.05 was considered to be statistically significant.

Results Albumin present in both quiescent and activated HSCs To determine if HSCs contain albumin protein, we first analyzed freshly isolated rat qHSC, activating HSCs after 1, 3 and 7 days in culture and culture-activated HSCs by SDS-PAGE, Ponceau S staining (total protein stain) and Western blotting (specific for rat albumin). On the total protein stains of quiescent and activating HSCs we observed a protein band at the position just below 70 kDa and identified it as albumin using Western blot (Figure 2a). In whole cell lysates of aHSCs, no albumin was detected on Ponceau S staining nor on Western blots (Figure 2b). However, when total protein stains of two batches

of aHSCs from the same rat were compared, different protein bands on different heights were observed (Figure S1) . This contradictive observation caused doubts about the presence of albumin in aHSCs as on one Ponceau S staining (Figure S1b) a protein band was observed just below 70 kDa, possibly caused by albumin. These results are not included in the main report because of these doubts and are therefore put in the supplemental data to complete the report.

a Ponceau S: 70 kDa albumin, 67 kDa GAPDH, 37 kDa

Ladder HSC 4h 4h HSC 1d HSC 3d HSC 7d HSC 4h HSC 1d HSC 3d HSC 7d HSC

cultured in rat serum-enriched medium cultured in calf serum-enriched medium

Ponceau S: 70 kDa

albumin, 67 kDa

GAPDH

SF SF SF RS RS RS FCS FCS FCS FCS FCS aHSC aHSC aHSC aHSC aPMF aPMF aPMF Ladder Ladder

Figure 2: Albumin is present in qHSCs, but not in aHSCs on Western blot Ponceau S stainings and Western blots on albumin and GAPDH of qHSCs cultured in FCS- or RS-enriched medium (a) and aHSCs and PMFs cultured in FCS-, RS- or serum free medium and serum containing medium ( b). Whole cell lysates of rat qHSCs (a) cultured in FCS- or RS-enriched medium shows the protein band just below 70 kDa identified as albumin, which shows the presence of albumin in qHSCs (a). The specificity of the rabbit anti-mouse albumin antibodies is shown as bovine/calf albumin does not react with the antibodies (a, lane 6-9), where rat albumin does react when present ( a, lane 2-5). Western blot on whole cell lysates of rat aHSCs (b) cultured in serum free medium, calf serum- or rat serum-enriched medium shows albumin to be absent in aHSCs whereas other proteins or protein fragments do react with the rabbit anti-mouse albumin antibodies (b, lane 2-7). The specificity of the rabbit anti-mouse albumin antibodies is also shown (b, lane 8-9). The GAPDH loading control showed equal loading but as the different layers were not lined up properly, the height of the protein could not be determined with the markers of the ladder. The anti- GAPDH antibodies used here were used frequently in the lab and therefore we claim that the proteins observed are indeed GAPDH. RS: rat serum-enriched IMDM medium, FCS: fetal calf serum-enriched IMDM medium, SF: serum-free IMDM medium.

Next, we analyzed both HSC phenotypes by immunofluorescence microscopy in order to determine the intra- and/or extracellular distribution of albumin. In addition to the distribution, we controlled the presence of albumin in HSCs. Albumin was detected at different locations in both quiescent (2 days after isolation) and activated HSCs (8 days after isolation) (Figure 3) . In qHSCs the albumin seems to be localized in the cytoplasm (Figure 3a) , whereas in aHSCs almost no albumin was found in the cytoplasm but was mostly located in or nearby the nucleus and extracellular (Figure 3b) . Colocalization of albumin with autofluorescence vitamin A containing lipid droplets in the cytoplasm was observed. Staining of extracellular, membrane-bound albumin cannot be ruled out as the microscopic images are two dimensional and therefore it cannot be judged whether the labeled albumin is really inside the cells and organelles or not. The here presented data show that albumin is transiently detected in HSCs that undergo transdifferentiation to myofibroblasts in vitro , but that intracellular albumin levels in qHSCs and fully activated HSCs are very low.

a b

Figure 3: Albumin protein is present and detectable in both qHSCs and aHSCs Immunofluorescence microscopy on rat qHSCs, after isolation cultured for 2 days (a) and aHSCs #1, after isolation cultured for 8 days (b) and stained for albumin in green. Albumin was detected in both qHSCs as well as in aHSCs. Primary antibodies: rabbit anti- BSA antibodies 1:200 (a) and 1:50 (b) and secondary antibodies: Alexa Fluor ®488 goat anti-rabbit IgG (H+L) 1:400. Nucleus stained with dapi in blue. Magnification: 40x. Scale bar: 25 µm.

Albumin not expressed by HSCs HSC isolates are contaminated with albumin producing hepatocytes To determine the source of the intracellular albumin in HSCs, we first examined the possible production of albumin by freshly isolated rat HSCs and activating HSCs after 1, 3 and 7 days in culture by real-time qPCR. Messenger RNA expression of albumin was determined as a measure of albumin production by HSCs and was corrected against the expression of the housekeeping gene 18S. Almost no albumin production was found in HSC isolates (Figure 4a) compared to hepatocytes. The expression levels of albumin markedly reduced during activation, but the reduction was not significant.

It is known that HSC isolates obtained via density gradient centrifugation are contaminated with other liver cell types and subcellular particles as mentioned above. Hepatocytes are the main producers of albumin and therefore its expression is commonly used as a marker for these cells. To investigate if the HSC isolates are contaminated with hepatocytes, we determined the mRNA levels of the hepatocyte markers BAAT and BSEP in culture-activating HSC isolates. The mRNA expression levels of BAAT and BSEP decreased markedly in time during culture-activation (Figure 4b-c) and the reduction of BSEP was even significant (Figure 4c). Next, we determined the expression levels of HSC markers in these samples to track their activation progress. We observed increases in the expression of the activation markers αSMA (n=1) and collagen type 1a1 (n=1) (Figure 4d) and a decrease in the expression of the qHSC marker PPAR γ (n=1) (Figure S2) during HSC activation, which is in line with previous studies. These data show that HSC isolates are contaminated with hepatocytes and that the expression of BAAT and BSEP in HSC isolates decreased during culture-activation, along with comparable decreases in the albumin mRNA expression. In addition to the experiments in activating HSCs, we determined the expression levels of albumin, BAAT, BSEP and αSMA in fully activated HSCs by qPCR to investigate the hepatocyte contamination of these cells. Almost no albumin expression was detected in aHSCs (n=1) compared to the hepatocyte control (Figure 5a). The expressions of the other hepatocyte markers BAAT (n=1) and BSEP (n=1) were almost absent in aHSCs when compared to hepatocytes (Figure 5b-c). The expression of αSMA was measured to determine the activation state of the HSCs and αSMA expression (n=1) was found in the aHSCs and not in hepatocytes (Figure 5d). These data show almost no expression of hepatocyte markers in the aHSC samples, indicating that the hepatocyte contamination in culture-activated HSCs is minor to absent.

Albumin is not expressed by PMFs; not yet determined for Kupffer cells To investigate if other cell types of the liver cause the contamination of the HSC isolates, we determined the expression levels of albumin among other genes in PMFs and Kupffer cells. We also determined whether the cultured isolates of these cells contained hepatocytes contamination or not. The experiments on cultured PMFs showed no production of albumin, BAAT and BSEP at all by these cells compared to hepatocytes (Figure 5a-c) and did show the production of αSMA by PMFs (n=1) (Figure 5d). The experiments on Kupffer cells were performed with a low number of cells as the number of cells in the isolated Kupffer cell fraction was also low. The results from these experiments are not reliable as the expression levels of the housekeeping gene are lower than normal and are fluctuating a lot. Therefore, the results on Kupffer cells are included as supplemental data (Figure S3) . These data show that PMFs do not produce albumin and that hepatocyte contamination is minor to absent in the PMF samples. Possible albumin expression by Kupffer cells and possible hepatocyte contamination in Kupffer cell isolates are not determined yet.

a b

c d

Figure 4: Albumin expression in early activating qHSCs is due to hepatocyte contamination Quiescent HSCs were culture-activated for 4 hours, 1 day, 3 days and 7 days and mRNA expression levels of albumin ( a), BAAT ( b), BSEP ( c) and the activation markers αSMA and collagen type 1a1 (d) were analyzed and compared to hepatocytes ( a-c) and to the 4 hour time point ( d). Culture-activation of qHSCs reduced the expression of albumin ( a) and the two hepatocyte markers BAAT ( b) and BSEP ( c). This indicates that the qHSCs are contaminated with hepatocytes and that the contamination disappears during cultivation. The expression of the activation markers αSMA and collagen type 1a1 (d) increased during activation. Results are shown as mean ± SD of the denoted number of independent experiments, significant differences between HSC 4 hours and the other HSC time points are indicated with an asterisk (*) when p < 0.05. The differences between hepatocytes and HSCs in a-c were not significant.

a b

c d

Figure 5: Albumin is produced by hepatocytes and not by aHSCs and PMFs Fully activated HSCs, PMFs and hepatocytes were culture and mRNA expression levels of albumin ( a), BAAT ( b), BSEP ( c) and the activation maker αSMA (d) were analyzed. No expression of albumin ( a) and the two hepatocyte markers BAAT ( b) and BSEP ( c) was detected in aHSCs and PMFs compared to hepatocytes. The hepatocyte contamination found in early activating qHSCs shown in Figure 4 thus disappeared during cultivation. The expression levels of the activation markers αSMA ( d) are increased in aHSCs and PMFs compared to hepatocytes. Results are shown as mean ± SD of the denoted number of independent experiments. N.D.: not detected, the expression levels were too low to be measured.

Albumin taken up by HSCs To identify the source of the intracellular albumin in HSCs, secondly the possibility that albumin is taken up by the HSCs was examined as the albumin is most likely not produced by HSCs. The possibility that the albumin is taken up by HSCs was investigated by Western blot analysis. The antibodies used, rabbit anti-mouse albumin antibodies, do not bind to the bovine/calf albumin from the fetal calf serum (Figure 2a; Figure 2b, lane 8-9) and can therefore be used to examine the uptake of albumin. To determine if HSCs take up albumin, freshly isolated HSCs are cultured in medium containing rat serum or fetal calf serum. In all cultured qHSCs the earlier described albumin band was found on the membrane when stained with the Ponceau S, indicating the presence of albumin in all samples. Only the qHSCs cultured in rat serum containing medium showed the albumin band on Western blots (Figure 2a, lane 2- 5) , whereas no albumin was detected on Western blots when qHSCs were cultured in medium containing fetal calf serum (Figure 2a, lane 6-9) . The albumin bands observed on the Ponceau S stained membrane in these latter qHSCs are most likely caused by bovine/calf albumin. As a loading control GAPDH was used. Detection of bovine/calf albumin uptake by qHSCs was not performed as the antibodies against bovine serum albumin were not specific enough and did also bind to the rat albumin in the hepatocytes (Figure 6). Staining of extracellular, membrane bound albumin was not excluded. These data suggest that albumin is taken up by HSCs.

Ponceau S: 70 kDa

albumin, 67 kDa

Old Old Old BSA BSA BSA Fresh Fresh Fresh Hepa. Hepa. Hepa. Ladder Ladder medium medium medium medium medium

First antibody dilution: 1:5000 First antibody dilution: 1:2500

Figure 6: Detection of rat and bovine/calf albumin with rabbit anti-BSA antibodies Western blot on newly prepared medium (fresh medium), medium taken from cells (old medium), hepatocytes and bovine serum albumin to determine the specificity of the antibody and to determine differences between fresh and old medium. The rabbit anti-BSA antibodies do react with both rat albumin (in hepatocytes) and bovine serum albumin. Both concentrations of antibodies can be used to detect the bovine/calf and the rat albumin orthologs.

HSC activation delayed by albumin Next, fully activated rat HSCs were subcultured in bovine serum albumin enriched mediums with different concentrations of albumin for 1, 3 and 7 days in order to determine the effect of albumin on the expression of HSC activation markers by qPCR. The expression of collagen type 1a1 decreased as the albumin concentration increased within 1 day after stimulation with the enriched mediums and the decrease was significant when the albumin concentration was increased to half the blood serum concentration (50%; 22,5g/L) and higher (Figure 7a). After 3 days the decrease was significant when the albumin concentrations of the medium were equal to (100%; 45 g/L) and two times (200%; 90 g/L) the normal blood serum concentration of albumin (Figure 7a). Albumin administration did not affect the expression of αSMA within 1 day but after 3 days of stimulation a decreasing trend in the αSMA expression was observed with increasing albumin concentration in the medium (Figure 7b). These data show that increasing albumin concentration in the medium delays fibrosis as it decreased the expression of collagen type 1a1 and αSMA in aHSCs.

Figure 7: Albumin reduces the gene expression of the activation markers in aHSCs Fully activated HSCs were cultured in medium with different concentrations of albumin for 1 day, 3 days and 7 days and mRNA expression levels of the activation markers collagen type 1a1 (a) and αSMA (b) were analyzed. The albumin concentrations are denoted as a percentage of the blood serum concentration of albumin (SCA). Different percentage of the SCA were obtained by dissolving BSA to a 10% FCS-IMDM medium. As the albumin concentration increased to 50% SCA and higher concentrations the expression of collagen type 1a1 decreased significantly within 1 day compared to aHSCs on 10% FCS/SCA (a). Significance was found on day 3 when the 100% SCA sample and higher was compared to the 10% FCS sample. On the day 7 the effect of albumin was even bigger (a). Increasing concentrations of albumin do thus reduce the expression of collagen type 1a1. The same trend was found for αSMA, however the effect of albumin was less rapid and not significant (b). After 7 days the effect of albumin on the expression of αSMA is larger compared to earlier time points and is comparable to collagen type 1a1. However it is not possible to determine significance from a single (n=1) experiment. Results are shown as mean ± SD of the denoted number of independent experiments, significant differences from the 10% FCS/SCA of the same time point are indicated with an asterisk (*) when p < 0.05.

HSC activation not only controlled by albumin In addition to the effect of albumin on HSC activation, other compounds in the serum seemed to affect the activation of HSCs as we observed differences in the expression levels of collagen type 1a1 (Figure 8a) and αSMA (Figure 8b) between aHSCs cultured in medium with different concentrations of fetal calf serum but with equal concentrations of albumin. Over time, the effect of either the compounds in the serum increased or the effect of the albumin decreased as the differences in the mRNA expression levels of the fibrotic markers between the two conditions became bigger (Figure 8). No self-explanatory trend was observed and as the experiment is only performed once, significance cannot be measured. The experiment should be repeated to determine whether the results of this trial are reproducible or not. Culturing aHSCs in serum free IMDM medium resulted in altered expression patterns of the housekeeping gene 18S and all other genes tested compared to aHSCs cultured in the calf serum containing medium (Figure S4) . Whether this effect is caused by the absence of the albumin, the absence of the other compounds of the serum or is coincidence in the n=1 pilot is not known. The here presented data suggest that other serum compounds, besides albumin, influence the activation of HSCs but irrefutable evidence is not yet shown.

a b

Figure 8: Other serum compounds besides albumin are probably involved in HSC activation Fully activated HSCs were cultured in two different mediums both with 20% of the blood serum concentration of albumin for 1 day, 3 days and 7 days and mRNA expression levels of the activation markers collagen type 1a1 (a) and αSMA (b) were analyzed. The 20% SCA medium was obtained by dissolving BSA to a 10% FCS-IMDM medium and the 20% FCS medium is the medium to which 20% fetal calf serum was added. The expression levels of both collagen type 1a1 and αSMA fluctuate in time and differences between the two different 20% SCA mediums are found. For both graphs the expression of 10% FCS d1 of Figure 7 is used as reference and was set to 1.00. Results are shown as mean ± SD of the denoted number of independent experiments.

Conclusions and discussion In this study, we first investigated if albumin is present in HSCs. The presence of intracellular albumin in HSCs was suggested by Kim and colleagues [50] but was not shown previously. Here we show by Western blotting and immunofluorescence microscopy that albumin is present in both qHSCs and aHSCs. On Western blots the amount of albumin within the HSCs seems to fluctuate during activation, whether this fluctuation is reproducible or not has not been determined. The determination and identification of albumin protein in aHSCs was difficult due to contradictive observations in total protein stains of aHSCs from the same rat, possibly caused by the breakdown of (extracellular) protein and albumin by trypsin [63] . Therefore, trypsin was no longer used to harvest cells prior to protein isolation but was still used for passaging the cells. Immunofluorescence microscopy was performed to control albumins presence in HSCs and to determine the location of the albumin within these cells. We observed differences in the localization of albumin between the quiescent and activated phenotype of the HSCs. Albumin seemed to be present in the cytoplasm of qHSCs where it seemed to colocalize with the autofluorescence vitamin A containing lipid droplets, whereas almost no albumin was found in the cytoplasm of aHSCs in which it was mostly located in or nearby the nucleus instead. As the number and size of the vitamin A-containing lipid droplets decreased during activation so did the cytoplasmic immunofluorescence staining on albumin in HSCs. These findings are in line with findings in PSCs [50] . More research is required to determine whether albumin is indeed present in these lipid droplets or not. The possibility that albumin is not present in the cytoplasm but is bound extracellular to the cell membrane instead is not ruled out and needs to be investigated. How albumin ends up in the nucleus of aHSCs is not known as albumin is too big to pass the nuclear envelope via the nuclear pores without nuclear transport mechanisms. Generally considered, these nuclear pores enable proteins with a maximal molecular mass up to 40 kDa to diffuse freely into and out of the nucleus. The transport of larger proteins into the nucleus is a receptor mediated process that requires energy and that is dependent upon the presence of a nuclear location signal (NLS) in the protein [64-68] . As there is no NLS in albumin, nuclear translocation of albumin is unlikely. However, Wang and Brattain showed the nuclear translocation of proteins with sizes from 90 to 110 kDa without a NLS, contradicting the general, long-termed view on the maximal size for protein diffusion through the nuclear pore [69] . They suggest that many proteins do not require a NLS to diffuse through the nuclear pore. Other factors besides the protein size, like the association and formation of complexes with other proteins and physical compatibility between nuclear pore components and the protein, have been suggested to contribute to the cytoplasmic retention of the protein [69] . This might explain the very poor nuclear translocation of albumin frequently described [69] . Others have described the translocation of albumin from extranuclear sites, like the cell membrane and the cytosol, into the nucleus of cells in cortical slides [70] and of JB6 epithelial cells in response to oxidative stress [71] . Nuclear translocation of albumin was not observed in all cells of the cortical slides that took up albumin [70] , indicating that the intracellular location where the albumin ends up differs between cell types. Another possibility is the transport of albumin towards the nucleus, where it does not enter but accumulates nearby the nucleus instead. This translocation was observed in PSCs where small, tiny dots of fluorescent albumin were found in the perinuclear region of activated PSCs [50] . More research is required to determine if albumin can be translocated from the cytosol into the nucleus and how it is transported within the HSCs.

After albumin was shown to be present in HSCs, the origin of the albumin was determined next. Kim and colleagues concluded that the albumin present in PSCs was produced in these cells as expression of albumin was detected in PSC isolates [50] . They speculated that albumin is also produced by HSCs [50] . In our search on the source of the intracellular albumin of HSCs we therefore started by looking at the production of albumin by HSCs. Almost no expression of albumin was detected in qHSCs and for aHSCs

no expression of albumin was detected. The expression levels of albumin found are most likely due to contamination of the HSC isolates with hepatocytes as other hepatocyte markers showed comparable decreases in their expression during HSC activation. This is in line with earlier studies in which contamination of the HSC isolates with other cell types and subcellular particles was suggested [53, 54] as previously mentioned. The hepatocyte contamination gradually disappeared from the HSC isolates when cultured and was eventually undetectable in aHSCs. We ruled out portal myofibroblast as the albumin producing contaminating cells in the isolates as these cells did not produce albumin. Whether Kupffer cells express albumin and contaminate the HSC isolates still needs to be examined as problems with the cell yield and purity of the Kupffer cell isolations resulted in unreliable data with low expression levels of the housekeeping gene. To address these problems, the protocol for Kupffer cell isolation should be optimized and a more reliable housekeeping gene for the Kupffer cells is desired. Removal of the contaminating cells during cultivation of qHSCs explains the detected decrease in albumin expression in HSC isolates during culture-activation. We therefore conclude that the albumin expression in HSC isolates is due to contamination with other, albumin producing cells and that HSCs do not produce albumin themselves. As contamination of the HSC isolates was shown, the idea that the albumin in these isolates is produced solely by the contaminating cells and not by HSCs cannot be confirmed. A real pure fraction of freshly isolated qHSCs is needed to investigate and rule out the possibility that HSCs do produce albumin. The purity of the HSC isolates cannot be increase by culturing as seeding of quiescent HSCs results in gradual activation and development of activated phenotype. It is possible to increase the purity via cell sorting by FACS to obtain 96.6% ± 2.9% pure stellate cells [52] but the final yield of cells as well as their viability are two major concerns. Nevertheless these purified samples are desired to rule out the possible production of albumin by HSCs.

As HSCs do not produce albumin themselves, it is most likely that these cells take up albumin. The uptake of albumin was considered before for other cell types throughout the body and there is evidence for the cellular uptake of albumin by brain [70, 72, 73] , lung [74-76] and kidney [77-80] cells. The possibility that HSCs take up albumin was therefore investigated next. Western blot analysis revealed the presence of bovine/calf albumin or rat albumin in rat HSC samples when cultured in medium containing fetal calf serum or rat serum, respectively. We therefore conclude that HSCs do take up albumin from the medium. The possibility that the albumin was present on Western blots of HSCs due to the contamination was considered and was refuted by the detection of albumin in both qHSCs and aHSCs after culture purification on immunofluorescence microscopic images, indicating that these cells do indeed contain albumin. However, the possibility that a minor amount of the albumin is present due to contamination cannot be ruled out completely as no Western blots were performed to check for hepatocyte markers in these protein isolates of HSCs. Possible staining of extracellular albumin should be taken into account and needs to be determined. There is still a lot to learn about the handling of albumin by HSCs. Despite we showed that HSCs take up the albumin, the mechanism involved in the uptake of albumin is not known. Most of the studies on the uptake of albumin by other cell types throughout the body described that the cellular uptake of albumin is a selective and receptor mediated process of endocytosis [70, 73, 81] . Specific albumin binding proteins mediate in the endocytosis of albumin into caveolae in a process that is under tight hormonal and enzymatic regulation [78, 82, 83] . Several albumin-binding proteins have been identified, like gp18, gp30, SPARC and albondin (gp60) [84-87] . The proteins gp18 and gp30 appear to be the scavenger receptors of modified albumin, mediating the endocytosis and lysosomal degradation of modified albumin [88, 89] . These proteins are found in all organs examined [90] and are expressed by various cell types including fibroblasts, macrophages and endothelial cells [88, 89] . The proteins albondin and SPARC are functionally and immunologically related, containing common immunological epitopes [91, 92] . SPARC is probably not involved in the receptor mediated uptake of albumin as SPARC is not a cell surface protein

but is secreted by a variety of cells instead [86] . However, specific interactions of SPARC with endothelial cells matrix proteins have been described [93] . Albondin on the other hand is a cell surface protein, selectively expressed in tissues and is thought to play an important role in albumin binding to endothelium, facilitating the internalization and transcytosis of albumin through plasmalemmal vesicles without entering the lysosomal compartments [75, 76, 90, 92] . Beside these albumin binding proteins, the multiligand receptors megalin and cubilin [94-97] and the transforming growth factor-β (TGF-β) receptors [70] have been described to be involved in albumin endocytosis. The albumin endocytosis via the megalin-cubilin complex can be inhibited by a variety of other ligands and antibodies against megalin or cubilin in proximal tubular cells [95] . TGF-β1 can also reduce megalin-cubilin-mediated internalization of albumin without inducing a general downregulation of cell function [79] . TGF-β1 also affected intracellular trafficking, most likely through the classical type I and type II receptors and the Smad pathway [98] . In lung epithelial cells a previously described 73 kDa endothelial cell surface, albumin binding protein was identified as the 75 kDa TGF-β receptor type II [74] . Both TGF-β receptors are shown to play a crucial role in the binding and endocytosis of albumin, also initiating the activation of the Smad signaling pathway [74] . However, other mechanisms do also contribute to albumin endocytosis as a blockade of the TGF-β receptors did only partly block the albumin endocytosis [74] . It remains to be further studied which of the receptors is involved in the uptake of albumin by HSCs and what pathways are involved. As several albumin binding receptors are known, the possibility that albumin was only bound extracellular instead of being present within the HSCs should be considered as we did not exclude this completely by immunofluorescence microscopy.

The presence and the uptake of albumin in and by the HSC may indicate that albumin has intracellular functions in the cell. Traditionally albumin was not considered to be a signal molecule in its own right. Previous studies however showed that albumin exposure to cells does have intracellular and transcriptional effects and therefore this assumption has been challenged. Kim and colleagues showed that intracellular albumin might be an important stabilizing element for the correct handling of retinoid storage in stellate cells [50] . Changes in ion and transmitter homeostasis after albumin exposure have been described [73] , as the uptake of albumin was associated with potassium buffering capacity in astrocytes [70, 73] and with calcium buffering in endothelial cells [99] and JB6 epithelial cells via calmodulin [71] . Other biological properties of albumin, such as free radical scavenging and molecule transportation, have been reported [100] . The scavenging of reactive oxygen species by albumin is known to inhibit apoptosis in macrophages [101] and albumin is known to be involved in the regulation of cellular glutathione levels, also protecting the cell from oxidative damage [102] . The binding of albumin to an antioxidant response element in the nucleus, which controls the expression of antioxidant enzymes leading to partial compensation of the stress has been described [71, 103] . Albumin protected endothelial cells from apoptosis during serum starvation [104] , whereas albumin exposure to cultured renal podocytes increased apoptotic cell death by upregulating the proapoptotic nuclear factor kappa B (NF κB) pathway [80, 105] . Until now, the effect of albumin administration on HSCs was not known. Here, we showed that exposure of aHSCs to different concentrations of albumin in the culture medium reduced their activation state. The expression of collagen type 1a1 significantly decreased when the albumin concentration in the medium increased and the expression levels of αSMA also showed a decreasing trend. The decreased expression of these HSC activation markers indicates that albumin can delay the activation of HSCs, which was in line with observed morphological changes. Previous studies have shown that albumin affects the transcription of several genes in other cell types throughout the body. The effect of albumin on the gene expression of collagen type 1a1 mRNA among others was earlier described in the bone-forming osteoblastic cells [106], only access to abstract]. Albumin administration increased the transcription and release of some inflammatory cytokines in podocytes [80] and increased the synthesis of TGF-β in proximal tubular cells [107, 108] . A transcriptional

upregulation of the TGF-β receptor type II was found after albumin exposure to proximal tubular cells [77] . Albumin also induced the expression of collagen type 4a1, suggesting a functional effect of the upregulation of this TGF-β receptor [77] which indicates that albumin affects the TGF-β/Smad signal transduction pathway. Blocking the TGF-β receptors showed an effective blockade of the cellular responses to albumin exposure in brain cells [70] . TGF-β has been shown to locally stimulate collagen synthesis by interstitial fibroblasts and is important in the transdifferentiation to and proliferation of renal interstitial fibroblasts [109, 110] . Transfection of PSCs with expression plasmids for albumin showed increased resistance to the activating effects of the profibrogenic factor TGF-β [50] which also indicates that albumin interferes in the TGF-β/Smad signaling pathway. Whether albumin has similar effects on the TGF-β/Smad signal transduction pathway in HSCs and stimulates the activation of hepatic myofibroblasts and thus liver fibrosis through this pathway is not known. Albumin is also shown to be a downstream effector of the nuclear PPAR γ receptor in PSCs [111] , a receptor thought to cooperate in maintaining the quiescent phenotype of HSCs in normal liver. As the activation of PPAR γ modulates profibrogenic and proinflammatory actions in HSCs [112] , interference of albumin in this pathway might cause the observed antifibrotic effects of albumin in HSCs. Other studies demonstrate that albumin uptake in proximal tubular cells activates other signal transduction pathways such as the renin-angiotensin system [77, 113] , the STAT/Jak 2 [114] , the ERK1/2 and the MAPK pathways [115] . More studies are required to determine which of these signal transduction pathways links albumin to fibrosis and underlies the antifibrotic effects of albumin. Another possible explanation is that the effects of albumin on HSCs are not caused by one of these mechanisms but are instead caused by the increasing protein concentration in the medium in general, inducing a stress response in these cells to the hypertonic osmotic environment. To determine whether the effect is specific for albumin, it is necessary to repeat the experiment with another compound dissolved in the medium. The controls in our study turned out to be unsuitable and failed, as: (i) casein did not dissolve properly in the medium and when dissolved it was not possible to filter the casein enriched medium; (ii) sodium chloride diluted in the medium resulted in cell death at all conditions with equal osmotic pressures compared to the albumin concentrations; and lastly (iii) the idea to use denaturated albumin as a control (as no other proteins were available in the lab) turned out to be impossible as it is impossible to denaturate albumin at normal pH [116] . Other studies used dextran of a similar molecular weight to albumin [80] and Ficoll70 [92, 117] as oncotic controls. These compounds might be suitable and useful in the control study to determine whether the observed effects are specific for albumin.

Where albumin seems to delay activation, other compounds in the medium or the serum might stimulate the activation. This was suggested from the finding that the expression levels of the HSCs activation markers differed between cells cultured in mediums with equal albumin concentration but with different concentrations of the other serum compounds. However, there was no self-explanatory trend visible and significance could not be determined from the n=1 pilot study. This effect might also be not specific for albumin and might be caused by oncotic differences. Therefore the experiments with other compounds dissolved in the medium are important to support these theories.

The uptake of albumin by HSCs makes albumin a possible antifibrotic drug carrier. However as albumin is shown to be taken up by more cells than only HSCs this might also have unwanted side effects in other organs. The idea to use albumin as a drug carrier is not new as Beljaars and colleagues used modified albumin for this purpose, as was described in the introduction [40, 44]. Our findings indicate that these modifications are not necessary to accomplish the uptake of albumin as HSCs do also take up native albumin. As albumin has antifibrotic effect in its own right, the combination of albumin with antifibrotic drugs is worth to be considered.

In summary, we show that HSCs do contain albumin which they do not produce themselves but take up from the medium instead. We also show that albumin reduces the activation state of activated HSCs in a concentration and time dependent manner. As in culture conditions the HSCs are not surrounded by the albumin producing hepatocytes, this might explain why HSCs do activate without any stimulation in vitro . However, there is still a lot to learn about the internalization, the intracellular translocation and the intracellular effects of albumin and which pathways are involved in these processes. The findings of this study suggest that albumin might be a key player in the process of HSCs activation and that the lack of albumin in culture conditions causes a spontaneous activation of quiescent HSCs.

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Supplemental data To complete the work performed during the internship, some experiments with unreliable results are included in the supplemental data section. The data is unreliable because of low or fluctuating expression of the housekeeping gene and/or big differences between duplicate measurements. a b

Hepa Hepa Ladder Ladder Ladder Ladder aHSC #1 #1 aHSC #2 aHSC #3 aHSC #4 aHSC #1 aHSC #1 aHSC #1 aHSC #1 aHSC #1 aHSC #1 aHSC aHSC 7d #0 7d aHSC

Figure S1: Albumin protein is present on Ponceau S stains when aHSCs are harvested by cell scraping but not when harvested with trypsin Ponceau S stainings of the nitrocellulose membrane with proteins of whole cell lysates of rat aHSCs harvested with trypsin (a, lane 2-6) and by cell scraping (b, lane 2-7) clearly show different protein patterns. The presence of albumin protein in aHSCs from different ages/passages was tested in ( a) and the effect of an antifungal drug on HSCs was determined in ( b), where aHSCs were treated with different concentrations of the drug. The bands just below the 70 kDa band of the ladder (lane 1, top orange marker) in the aHSC samples are thought to be albumin. The albumin bands are absent in (a) but present in (b) which is caused by the different cell handlings (trypsin versus cell scraping) when harvesting the aHSCs. Lane 8 in (a) is the hepatocyte control.

a b

18s PPAR γ

(Ct values) (Ct values) HSC 4h 13,616 13,509 32,886 30,592 HSC 1d 13,037 13,167 32,683 31,549 HSC 3d 12,869 12,705 32,807 32,084 HSC 7d 12,161 12,170 32,845 32,605 -RT 29,075 29,193 34,596 34,298 water 33,925 33,675 N.D. N.D.

Figure S2: PPAR γ expression decreases when qHSC activate Quiescent HSCs were culture-activated for 4 hours, 1 day, 3 and 7 days and the mRNA expression levels of the quiescence marker PPAR γ were analyzed and compared to the 4 hour time point. Culture-activation of qHSCs reduced the expression of PPAR γ ( a). The raw data of both the 18S and the PPAR γ expression in the activating rat qHSCs is shown in ( b). Results are shown as mean ± SD.

a b

c d

e 18s Albumin BAAT LRAT αSMA (Ct values) (Ct values) Ct values) (Ct values) (Ct values) Kupffer cells 4h 28,882 28,915 36,688 36,896 N.D. N.D. N.D. N.D. N.D. N.D. Kupffer cells 1d 19,365 19,368 29,061 29,726 38,840 37,726 N.D. N.D. N.D. 39,588 Hepatocytes 13,846 13,744 24,038 24,969 29,743 29,752 38,768 39,262 N.D. N.D. -RT 33,826 33,911 N.D. N.D. N.D. N.D. N.D. N.D. N.D. 39,767 water 33,623 32,997 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Figure S3: Gene expression in Kupffer cells Kupffer cells were culture-activated for 4 hours and 1 day and the mRNA expression levels of albumin (a), BAAT ( b), LRAT (c) and αSMA (d) were analyzed and compared to hepatocytes. As the raw data shows, the expression of the housekeeping gene is that low (corresponding with high Ct values) that the experiment is not reliable. The raw data of 18S, albumin, BAAT, LRAT and αSMA expression in Kupffer cells is shown in ( e). Results are shown as mean ± SD, N.D.: not detected, the expression levels were too low to measure.

a1 a2

b1 b2

c1 c2

e 18s Albumin BAAT BSEP αSMA (Ct values) (Ct values) Ct values) (Ct values) (Ct values) aHSC 1d SF 13,610 13,829 36,249 36,896 39,308 39,071 37,959 N.D. 23,061 23,063 aHSC 2d SF 16,893 16,711 36,208 36,479 39,194 39,509 N.D. N.D. 26,307 26,339 aHSC 3d SF 16,561 16,021 36,017 36,552 39,364 N.D. N.D. N.D. 25,853 25,781 aHSC 4d SF 20,063 19,165 39,171 N.D. N.D. N.D. N.D. N.D. 27,744 27,799 aHSC 4d FCS 11,915 11,669 N.D. N.D. N.D. 38,908 38,675 N.D. 19,783 20,016 aPMF 3d FCS 13,721 13,922 35,977 35,413 N.D. N.D. N.D. N.D. 23,266 23,305 Hepatocytes 12,897 13,085 19,947 19,984 24,841 24,743 26,510 26,425 37,031 36,922 -RT 29,630 31,132 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. water 28,430 29,086 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Figure S4: Gene expression in aHSCs, PMFs and hepatocytes in serum free and/or serum containing culture conditions Fully activated HSCs (n=1), PMFs (n=1) and hepatocytes (n=1) were culture in medium with fetal calf serum (FCS/CS) or in serum free medium (SF). Messenger RNA expression levels of albumin ( a), BAAT ( b), BSEP ( c) and the activation maker αSMA (d) were analyzed. The expression levels of albumin ( a1), BAAT ( b1) and BSEP ( c1) in aHSCs cultured in FCS and SF conditions and aPMFs culture in FCS condition are much lower than their expression levels in hepatocytes. The aHSCs cultured in SF conditions show big differences between days and with the aHSCs cultured in FCS containing medium ( a2, b2, c2, d). Results are shown as mean ± SD of the denoted number of independent experiments. N.D.: not detected, the expression levels were too low to measure.