Articles in PresS. Am J Physiol Gastrointest Liver Physiol (September 2, 2010). doi:10.1152/ajpgi.00302.

2010

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5 ISOLATION OF PERIPORTAL, MID-LOBULAR AND
6 CENTRILOBULAR RAT LIVER SINUSOIDAL
7 ENDOTHELIAL CELLS ENABLES STUDY OF ZONATED
8 DRUG TOXICITY
9 Running title: Isolation of endothelial cells from different liver regions

10 Guanhua Xie, Lin Wang, Xiangdong Wang, Lei Wang, Laurie D. DeLeve

11 Division of Gastrointestinal and Liver Diseases and the Research Center for Liver Diseases,
12 University of Southern California Keck School of Medicine

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17 Keywords: liver circulation; drug-induced liver injury; acetaminophen; CD45;
18 endocytosis
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28 Contact information:
29 Laurie D. DeLeve, M.D., Ph.D.
30 USC Keck School of Medicine
31 Division of Gastrointestinal and Liver Diseases
32 2011 Zonal Avenue-HMR603
33 Los Angeles CA 90033
34 Tel 323-442-3248
35 Email: deleve@usc.edu
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Copyright © 2010 by the American Physiological Society.

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38 Abstract
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40 Many liver sinusoidal endothelial cell (LSEC) - dependent processes, including drug-

41 induced liver injury, ischemia-reperfusion injury, acute and chronic rejection, fibrosis,

42 and the HELLP syndrome, may have a lobular distribution. Studies of the mechanism of

43 this distribution would benefit from a reliable method to isolate LSEC populations from

44 different regions. We established and verified a simple method to isolate periportal, mid-

45 lobular and centrilobular LSEC. Three sub-populations of LSEC were isolated by

46 immunomagnetic separation based on CD45 expression. Results: Flow cytometry showed

47 that 78.2 ± 2.3% of LSEC were CD45 positive and LSEC could be divided into CD45

48 bright (28.6 ± 2.7% of total population), dim (49.6 ± 1.0%) and negative populations

49 (21.8 ± 2.3%). Immunohistochemistry confirmed that in vivo expression of CD45 in

50 LSEC had a lobular distribution with enhanced CD45 staining in periportal LSEC. Cell

51 diameter, fenestral diameter, number of fenestrae per sieve plate and per cell, porosity

52 and lectin uptake were significantly different in the sub-populations, consistent with the

53 literature. Endocytosis of low concentrations of the LSEC-specific substrate,

54 formaldehyde treated serum albumin, was restricted to CD45 bright and dim LSEC.

55 Acetaminophen was more toxic to the CD45 dim and negative populations than to the

56 CD45 bright population. Conclusions: CD45 is highly expressed in periportal LSEC, low

57 in mid-lobular LSEC and negative in centrilobular LSEC and this provides an easy

58 separation method to isolate LSEC from the 3 different hepatic regions. The LSEC sub-

59 populations obtained by this method are adequate for functional studies and drug toxicity

60 testing.

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63 Introduction

64 The liver sinusoidal endothelial cell (LSEC) has been implicated in many forms of, often

65 zonated, liver injury including drug-induced liver injury, ischemia-reperfusion injury,

66 acute and chronic rejection, fibrosis, and the HELLP (hemolytic anemia, elevated liver

67 enzymes, low platelet count) syndrome (1, 4, 5, 7, 8, 10, 11, 13, 16, 18, 20, 23, 26, 27,

68 30). A zonated injury that originates in the sinusoid could be due to differences in LSEC

69 across the lobule, but other possibilities include regional differences in metabolism by

70 neighboring hepatocytes and oxygen or substrate gradients across the lobule. A simple

71 method to isolate viable LSEC sub-populations would greatly facilitate in-depth studies

72 of LSEC-dependent, zonated injury, but would also allow studies of the physiology of

73 LSEC across the lobule. It is well established that there are differences between periportal

74 and centrilobular LSEC in morphology (24, 25, 29), antigen expression (22) and lectin

75 affinity (3). However to the best of our knowledge, the only functional difference that has

76 been demonstrated to-date is a lobular difference in endocytosis (2, 21).

77 CD45, the common leukocyte antigen, is expressed both in vivo and in vitro in normal rat

78 LSEC (15, 19). Furthermore, there is subpopulation heterogeneity in CD45 expression in

79 LSEC (19). The current study examines LSEC for expression pattern of CD45,

80 determines whether LSEC can be separated into periportal, mid-lobular and centrilobular

81 sub-populations based on CD45 expression level, examines differences in endocytosis in

82 the sub-populations, and investigates whether the sub-populations obtained are adequate

83 to study the zonated toxicity of acetaminophen.

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85 Materials and Methods

86 Reagents. Chemicals were obtained from the Sigma-Aldrich Corporation (St. Louis, MO)

87 unless stated otherwise. Antibodies: mouse anti-rat CD45 antibody (BD Pharmingen, San

88 Diego, CA, catalog no. 554875), fluorescein isothiocyanate (FITC)-conjugated mouse

89 anti-rat CD45 (BD Pharmingen, catalog no. 554877), PE-conjugated mouse anti-rat

90 CD45 (BD Pharmigen, catalog no. 554878), mouse anti-rat CD31 antibody (Abcam,

91 Cambridge, MA), rabbit anti-rat von Willebrand factor antibody (Santa Cruz

92 Biotechnology, Santa Cruz, CA), rabbit anti-mouse IgG FITC conjugate (BD

93 Pharmingen), donkey anti-mouse IgG, PE conjugate and donkey anti-rabbit IgG,

94 rhodamine conjugate (Santa Cruz Biotechnology). FITC labeled formaldehyde-treated

95 serum albumin (FITC-FSA) was a kind gift from Dr. Bård Smedsrød, University of

96 Tromsø, Norway.

97 Animals. Male Sprague-Dawley rats (body weight 220-250 g) were obtained from Bantin

98 and Kingman Laboratories (Fremont, CA). All protocols were reviewed and approved by

99 the Animal Care and Use Committee at the University of Southern California to ensure

100 ethical and humane treatment of the animals. This study followed the guidelines outlined

101 in the NIH “Guide for the Care and Use of Laboratory Animals” prepared by the National

102 Academy of Sciences and published by the National Institutes of Health (NIH publication

103 86-23 revised 1985).

104 Cell Isolation. LSEC were first isolated by collagenase perfusion, iodixanol density

105 gradient centrifugation, and centrifugal elutriation as previously described (12). Yields of

106 LSEC are on average 100 million SEC/10 gram liver with viability of >95% and purity

107 of > 98%, as determined by positive staining for fluorescent acetylated-low density
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108 lipoprotein and a negative peroxidase stain to reveal contaminating Kupffer cells. Total

109 LSEC were then further separated based on CD45 expression using immunomagnetic

110 separation using an autoMACS Pro® separator (Miltenyi Biotec, Auburn, CA) according

111 to the manufacturer’s instructions. After blocking with FcR blocking reagent, LSEC

112 were incubated with 0.2ml (1:10) of FITC-conjugated mouse anti-rat CD45 or PE-

113 conjugated mouse anti-rat CD45 for 45 minutes at 4°C with gentle shaking, washed and

114 then incubated with 0.2ml (1:10) anti-FITC MicroBeads (Miltenyi Biotec, Auburn, CA)

115 or anti-PE MicroBeads (Miltenyi Biotec) for another 45 minutes at 4°C with gentle

116 shaking. Cells were washed, resuspended in 0.5ml buffer and subjected to magnetic

117 separation. The autoMacs “Possel” program was used to separate CD45 bright cells from

118 CD45 dim and negative populations. CD45 dim and negative populations were further

119 separated using the autoMacs “Possel_s” program. Cells were cultured on rat-tail

120 collagen-coated plates (400,000 cells/cm2) in Dulbecco’s minimal essential medium

121 (DMEM) -low glucose with 10% fetal bovine serum (FBS). Cells usually attached to the

122 culture dish within 3 hours of plating.

123 Real-time PCR. Rat bone marrow mononuclear cells (BMMC) were obtained from rat

124 femur with depletion of red blood cells. Total RNA of LSEC and BMMC were prepared

125 using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s

126 instructions. Total RNA of RAEC (rat aortic endothelial cells) was purchased from Cell

127 Applications Inc. (San Diego, CA). cDNA was prepared using RT2 First Strand Kit from

128 SABiosciences (Frederick, MD). Real-time PCR was performed on the ABI Prism

129 7900HT sequence Detection System (Applied Biosystems, Carlsbad, CA) using Real-
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130 Time RT2qPCR Primer Assays (SABiosciences, Frederick, MD) with β-actin as a

131 reference control. All primers were purchased from SABiosciences.

132 Fluorescence staining. Normal rat liver was snap-frozen in liquid nitrogen and

133 embedded in OCT compound. Cryostat sections (10μm) were fixed with cold acetone for

134 10 min and blocked with 5% rabbit serum for 30 min. Sections were incubated with

135 CD45 antibody (1:50) for 2 hours followed by a rabbit anti-mouse IgG FITC conjugate

136 (1:100) for 45 min. Controls were stained with a mouse isotype control antibody, a

137 purified mouse IgG1, κ immunoglobulin (BD Pharmingen). Slides were mounted with

138 Prolong Gold Antifade reagent (Invitrogen, Carlsbad, CA). Images were taken using a

139 Zeiss LSM 510 confocal microscope (Carl Zeiss Micro Imaging, Thornwood, NY).

140 Immunocytochemistry. Cells were cultured overnight and coverslips were washed, fixed

141 in 4% paraformaldehyde (PF) and cold methanol, and incubated with mouse anti-rat

142 CD31 antibody (1:50), or rabbit anti-rat von Willebrand factor (vWF) antibody (1:50) for

143 2 hours at room temperature. Coverslips were washed in PBS and incubated with either

144 donkey anti-mouse IgG, PE conjugated (1:100), or donkey anti-rabbit IgG, rhodamine

145 conjugated (1:50), respectively, for 45 min at room temperature. Controls were incubated

146 with isotype control IgG at the same concentration as the primary antibody. Coverslips

147 were mounted on slides with Prolong Gold Antifade reagent (Invitrogen). Slides were

148 examined at 40x magnification using a Zeiss LSM 510 confocal microscope (Carl Zeiss

149 Micro Imaging). Each condition was examined in triplicate, and values were obtained by

150 counting the number of cells positive for CD31 or vWF in 15 randomly selected fields.
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151 Co-localization of CD45 with Wheat-germ agglutinin. CD45 staining was examined in

152 LSEC labeled with WGA by in vivo perfusion or after in vitro labeling. Preferential

153 uptake of WGA in periportal LSEC was confirmed by in vivo perfusion and confocal

154 imaging. The livers were perfused through the portal vein with 10 ml

155 tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectin from Triticum vulgaris

156 (wheat germ agglutinin, WGA, 10 µg/ml) at 2 ml/min. The livers were then cut into

157 pieces and fixed by 4% PF, 4% sucrose solution followed by immersion in 30% sucrose

158 overnight. The fixed livers were snap-frozen in liquid nitrogen, cut into 10 µm sections,

159 and mounted with Prolong Gold Antifade reagent (Invitrogen). Images were obtained at

160 20x magnification using a Zeiss LSM 510 confocal microscope (Carl Zeiss Micro

161 Imaging). In vivo labeling: one million LSEC, isolated from WGA-TRITC-perfused liver

162 as described above, were incubated with FITC mouse anti-rat CD45 (1:50) at 4°C for 30

163 minutes. In vitro labeling: One million LSEC isolated from non-WGA-perfused rat liver

164 were incubated with WGA-TRITC (10µg/ml) at 37°C for 10 minutes followed by FITC-

165 conjugated mouse anti-rat CD45 (1:50) at 4°C for 30 minutes. Flow cytometry: After

166 gating for viable cells, expression of CD45 and uptake of WGA was analyzed on

167 channels FL1 and FL2 using the FACSCalibur (Becton Dickinson, Franklin Lakes, NJ).

168 In some experiments, Forward Scatter (FSC) was also analyzed. 20,000 cells were

169 analyzed per sample.

170 Scanning Electron Microscopy (SEM). LSEC cultured on collagen-coated Thermanox

171 coverslips (Thermo Fisher Scientific, Rochester, NY) were fixed with 2% glutaraldehyde

172 (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M cacodylate buffer, PH 7.4 for 30

173 minutes. After washing, LSEC were treated with 1% tannic acid (Electron Microscopy
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174 Sciences) in 0.15M cacodylate buffer for 1 hour, post-fixed with 1% osmium tetroxide

175 (Ted Pella, Redding, CA) in 0.1M cacodylate buffer for 30 minutes, dehydrated with

176 graded alcohols, dried with hexamethyldisilazane (Ted Pella), sputter-coated with 10-nm

177 gold, and examined using a JSM-6390LV scanning electron microscope (JEOL, Tokyo,

178 Japan).

179 Quantitative Imaging. SEM images of 15 randomly selected LSEC per experiment were

180 analyzed. Fenestral diameter, number of fenestrae per sieve plate and per cell, and

181 porosity were determined using MetaMorph software (version 7.0; Molecular Devices,

182 Downingtown, PA). Total LSEC surface area and the open area of individual fenestrae

183 were quantified in square microns. These open areas were summed, divided by the total

184 areas of the LSEC surface examined, multiplied by 100 to give the porosity as percent of

185 open area, and averaged to give a single value per group. For cell diameter, one drop of

186 freshly isolated cells was added to a glass slide, 15 random pictures were taken at 20x

187 magnification by light microscopy and the diameter was measured using MetaMorph

188 software.

189 Endocytosis. In vivo uptake of FITC-FSA: Normal rat livers were perfused through the

190 portal vein with 10 ml FITC-FSA (2 µg/ml) at 2 ml/min. The livers were then cut into

191 pieces and fixed with 4% PF, 4% sucrose solution for 4 hours at room temperature

192 followed by immersion in 30% sucrose overnight at room temperature. The fixed livers

193 were snap frozen in liquid nitrogen, cut into 10 µm sections, and mounted with Prolong

194 Gold Antifade reagent (Invitrogen). Images were obtained using a Zeiss LSM 510

195 confocal microscope (Carl Zeiss Micro Imaging). In vitro uptake of FITC-FSA: LSEC

196 cultured on collagen coated coverslips overnight as indicated were washed and incubated
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197 with DMEM-low glucose with 1% FBS for 60 minutes. LSEC were then incubated with

198 FITC-FSA (0.5 μg/ml or 0.02 μg/ml) in DMEM-low glucose with 1% FBS for 10

199 minutes in a 37°C incubator. After washing, LSEC were incubated with DMEM-low

200 glucose with 1% FBS for another 60 minutes. LSEC were then washed in cold PBS and

201 fixed in 4% PF (4°C) for 30 minutes. Coverslips were washed with deionized water and

202 mounted with Prolong Gold Antifade reagent (Invitrogen). Images were obtained at 40x

203 magnification using a Zeiss LSM 510 confocal microscope (Carl Zeiss Micro Imaging).

204 Percentage of uptake of FITC-FSA by LSEC was examined in triplicate, and values were

205 obtained by counting the number of cells with positive labeling in 15 randomly selected

206 fields.

207 Drug Studies. Studies were performed in freshly cultured cells exposed overnight to

208 acetaminophen in DMEM with 10% FBS. Cells were allowed to adhere for at least 3

209 hours before the drug incubation. Studies for toxicity testing were performed in 96-well

210 plates and incubations were performed in a dark 37°C, CO2 incubator overnight. Cell

211 viability was assessed with the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

212 (MTT) assay as previously described (6). Viability is expressed as a percentage of

213 untreated control cells.

214 Statistics. Numerical data represent mean ± standard error of the mean (SE) from at least

215 three separate experiments. Values were compared by two-factor analysis of variance

216 (ANOVA) with replication using the Microsoft Excel Analysis ToolPak (Microsoft,

217 Redmond, WA). If ANOVA was significant, à posteriori analysis was done with Least

218 Significant Difference. Results with p < 0.05 were considered significant.

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220 Results

221 Real-time PCR of CD45 Expression by LSEC. Our previous studies have shown that

222 LSEC express CD45 by immunocytochemistry and immunohistochemistry (15). One

223 possibility is that surface expression of CD45 is due to endocytosis of cell fragments

224 containing CD45 and trogocytosis rather than due to endogenous expression of CD45 by

225 LSEC. Trogocytosis is a phenomenon in which the cell extracts surface molecules from

226 another cell and then expresses these antigens on their own cell surface (17); this process

227 was first described in leukocytes and was subsequently recognized in various cell types

228 including endothelial cells (14). To rule out that surface expression was simply due to

229 trogocytosis, gene expression of CD45 was examined in LSEC. CD45 mRNA was

230 examined in LSEC, RAEC and BMMC by reverse transcriptional PCR. There was almost

231 no expression of CD45 in RAEC, but marked expression was found in both LSEC and

232 BMMC (data not shown). CD45 levels were then compared in the 3 groups by real-time

233 PCR, which showed a nearly 18- and 25- fold increase of CD45 mRNA level in LSEC

234 and BMMC, respectively, compared to RAEC (Fig. 1).

235 Isolation of LSEC Sub-populations Based on CD45 Expression Level. By flow

236 cytometry 78.2 ± 2.3% of LSEC were CD45 positive using cells stained with isotype-

237 control antibody as control for gaiting (Fig. 2). LSEC could be divided into CD45 bright,

238 CD45 dim and CD45 negative populations (Fig. 2, Table 1). The CD45 bright

239 population has smaller forward scatter (FSC, a parameter related to the cell size) than

240 CD45 dim and CD45 negative populations (Fig. 2). Cells from the 3 sub-populations

241 were also isolated by autoMACS and cell diameter was measured on light microscopy

242 (Table 1), which confirmed the observation by flow cytometry that cell size differs
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243 between the three LSEC sub-populations. The increase in size of LSEC with decreased

244 expression of CD45 in the 3 sub-populations is consistent with sub-populations that are

245 periportal (CD45 bright), mid-lobular (CD45 dim) and centrilobular (CD45 negative) in

246 origin.

247 In vivo CD45 Expression. LSEC express CD45 in vivo (12) and CD45 staining co-

248 localizes with CD31 staining. On immunohistochemistry of control liver sections, CD45

249 showed a lobular distribution (Fig. 3) with greater staining in the periportal than in the

250 centrilobular regions.

251 CD45 Bright LSEC Co-localize with Wheat Germ Agglutinin Positive LSEC. Wheat

252 germ agglutinin (WGA) is a lectin that binds preferentially to mouse periportal LSEC (3)

253 and that localized to rat periportal LSEC by immunohistochemistry (Fig. 4A). LSEC that

254 took up WGA, either after in vivo perfusion or in vitro labeling, stained CD45 bright (Fig.

255 4B and 4C), confirming that CD45 bright cells are periportal LSEC.

256 CD31 and von Willebrand factor (vWF) Expression. The 3 LSEC sub-populations were

257 cultured overnight and stained for CD31 or von Willebrand factor (vWF). Fewer CD45

258 bright LSEC expressed CD31 than the CD45 dim and negative populations (Table 1).

259 vWF expression was similar in the 3 LSEC sub-populations (Table 1) and the percentage

260 LSEC positive for vWF was consistent with previous findings (9).

261 CD45 Bright, Dim and Negative LSEC Have Different Fenestral Patterns. Periportal

262 and centrilobular LSEC differ in size of fenestrae, number of fenestrae per sieve plate and

263 per cell, and porosity (24, 25, 28). The 3 LSEC sub-populations were cultured overnight

264 and examined by SEM for these four parameters (Fig. 5). Quantification showed that
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265 CD45 bright LSEC have larger fenestrae, fewer fenestrae per sieve plate and per cell, and

266 lower porosity than CD45 dim and negative populations (Table 2). CD45 dim LSEC have

267 significantly different porosity than CD negative LSEC (Table 2). This is consistent with

268 a separation into periportal, mid-lobular and centrilobular LSEC.

269 Endocytosis. Endocytosis is a key functional characteristic of LSEC. Preferential uptake

270 of FITC-FSA in periportal LSEC was confirmed by in vivo perfusion at low

271 concentration and confocal imaging (Fig. 6A). The 3 sub-populations of LSEC were

272 isolated, cultured overnight and then exposed to FITC-FSA 0.5 or 0.02 µg/ml for 10

273 minutes. Uptake of FITC-FSA, 0.5 µg/ml, was 100% in the 3 sub-populations (data not

274 shown). At a concentration of 0.02 µg/ml, there was no uptake of FITC-FSA in CD45

275 negative population while 86.9 ± 2.4% of CD45 bright and 84.0 ± 3.9% of CD45 dim

276 LSEC took up FITC-FSA (Fig. 6B).

277 Acetaminophen Toxicity. Acetaminophen is toxic to LSEC (11, 16, 26, 27) and causes

278 centrilobular hemorrhagic necrosis. The 3 sub-populations of LSEC were isolated and

279 exposed to acetaminophen, 1, 3 and 10 mM, overnight. Acetaminophen was significantly

280 more toxic to CD45 dim and negative LSEC than to CD45 bright LSEC (Fig. 7).

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283 Discussion

284 The current study demonstrates high CD45 gene expression in LSEC, establishes and

285 validates a simple method to separate LSEC into periportal, mid-lobular and centrilobular

286 sub-populations based on CD45 expression level and demonstrates that these sub-

287 populations can be used to study zonated LSEC-dependent processes. The 3 groups

288 selected by CD45 expression were identified based on differences in size of cell, size of

289 fenestrae, numbers of fenestrae per sieve plate and per cell, porosity, uptake of wheat

290 germ agglutinin, and endocytosis, which were previously reported to be different in

291 periportal and centrilobular LSEC (3, 24, 25, 29). The cell sizes and fenestral findings

292 differ quantitatively from previous reports, as the cells under study were isolated cells

293 that had been cultured overnight and previous descriptions examined LSEC in liver

294 sections.

295 CD45 is a transmembrane protein tyrosine phosphatase usually expressed on leukocytes.

296 The current study confirms the endogenous expression of CD45 in LSEC, which suggests

297 that LSEC may be of bone marrow origin. Immunohistochemistry also demonstrates that

298 CD45 expression in LSEC has a lobular gradient distribution. CD45 is highly expressed

299 in periportal LSEC, low in mid-lobular LSEC and negative in centrilobular LSEC. The

300 method described is novel in that it isolates cells from the 3 regions of the lobule,

301 obtained from a single liver and adequate for functional studies. The morphological

302 characteristics of the 3 sub-populations change as a continuum, which suggests that the

303 phenotypic differences might be due to either maturation of the LSEC as they stream

304 across the lobule or to the effect of oxygen tension on LSEC phenotype.
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305 The current study confirms that periportal and mid-lobular LSEC are CD45 positive.

306 Some of the more recently developed protocols to isolate LSEC begin with exclusion of

307 CD45+ cells to exclude Kupffer cells, but the current finding suggests that these

308 approaches will yield a sub-population of centrilobular LSEC.

309 In the current study, the CD45 dim and CD45 negative populations are similar in size,

310 CD31 and von Willebrand factor expression, and susceptibility to acetaminophen toxicity.

311 However, the CD45 dim population has lower porosity and higher endocytic activity than

312 CD45 negative populations, demonstrating that the CD45 dim and negative are distinct

313 populations.

314 It is well established that acetaminophen is toxic to LSEC in vitro (11, 16), although the

315 contribution of LSEC injury to in vivo liver injury still requires exploration. The

316 experiments with acetaminophen presented here are meant as proof of principle that the

317 cells obtained by CD45 immunomagnetic sorting are healthy enough to perform drug

318 toxicity studies. Acetaminophen-induced liver injury causes centrilobular hemorrhagic

319 necrosis. Consistent with this, the findings here demonstrate that acetaminophen is indeed

320 more toxic to LSEC in the mid-lobular and centrilobular region of the liver, consistent

321 with a role for LSEC in determining the centrilobular localization of acetaminophen-

322 induced liver injury. One of the important questions that can be answered by separating

323 LSEC into sub-populations is whether lobular differences in CYP2E1 and glutathione

324 turnover in LSEC determine the zonation of injury. In some mouse strains acetaminophen

325 is P450 activated by LSEC, whereas in other mouse strains the LSEC is simply a target of

326 acetaminophen metabolically activated by neighboring hepatocytes (11)). The current

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327 experiments demonstrate that acetaminophen is metabolically activated by rat LSEC

328 rather than requiring P450 activation by hepatocytes.

329 In summary, the current study describes and validates a simple method to isolate

330 periportal, mid-lobular, and periportal LSEC. The cells isolated by this method show

331 lobular differences in endocytosis and susceptibility to acetaminophen injury. This

332 method will permit future studies of zonated, LSEC-dependent processes.

333 Acknowledgements: The authors gratefully acknowledge Michelle MacVeigh-Aloni for

334 her invaluable assistance with immunostaining and confocal microscopy. This work was

335 supported by NIH grant DK66423 and DK46357, by the Histology and Microscopy sub-

336 core of the USC Research Center for Liver Diseases (P30DK048522), and the Non-

337 parenchymal Liver Cell sub-core of the Southern California Research Center for

338 Alcoholic Liver and Pancreatic Diseases and Cirrhosis (R24AA012885).

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348 Legends

349 Figure 1. Real-time PCR of CD45 expression in isolated LSEC. CD45 mRNA

350 expression in LSEC isolated from normal rat liver was quantified by real-time PCR. Rat

351 bone marrow mononuclear cells (BMMC) were used as a positive control and rat aortic

352 endothelial cells (RAEC) were used as a negative control. CD45 mRNA levels are

353 significantly higher in LSEC and BMMC than RAEC (n = 4; p< 0.0001 for comparison

354 of the 3 cell types by ANOVA; *p<0.001 for comparison of LSEC and BMMC vs RAEC

355 by Least Significant Difference).

356 Figure 2. Flow cytometry of CD45 expression in isolated LSEC. LSEC isolated

357 from normal rat liver were stained with isotype control antibody (left) or CD45-FITC

358 (right) and examined by flow cytometry. Scatter plots are gated on CD45 and are

359 representative of four independent experiments. The percentage of LSEC expressing

360 CD45 is shown in each plot. Three populations could be observed in the right plot: CD45

361 bright (oval in the upper right quadrant), CD45 dim (polygon in the upper right quadrant),

362 and CD45 negative (lower right quadrant).

363 Figure 3. Immunohistochemistry of control rat liver stained for CD45. Frozen rat

364 liver section was stained for CD45. Continuous staining was observed (arrow) along the

365 sinusoids around the portal triad (PT) with no stain observed along the sinusoids around

366 the central vein (CV). Scale bar: 50 μm.

367 Figure 4. Co-localization of wheat germ agglutinin (WGA) uptake with CD45

368 bright LSEC. (A) Livers were perfused with WGA-TRITC and examined under

369 confocal microscopy. Positive staining of LSEC, manifested as a continuous line along
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370 the sinusoids, was observed in the sinusoids around the portal triad (PT) with no staining

371 observed in the sinusoids around the central vein (CV). Scale bar: 50 μm. (B) LSEC

372 isolated from WGA-TRITC-perfused rat liver were incubated in vitro with CD45-FITC

373 and examined by flow cytometry. (C) LSEC isolated from non-WGA-perfused rat liver

374 were incubated in vitro with WGA-TRITC and CD45-FITC and examined by flow

375 cytometry. Plots are gated on CD45 and WGA, and the percentage of cells positive for

376 WGA only, for both CD45 and WGA, and for CD45 only are shown in the upper left,

377 upper right, and lower right quadrant, respectively. The examples shown are

378 representative of three independent experiments.

379 Figure 5. Scanning electron microscopy. LSEC were isolated and separated into CD45

380 bright, dim and negative sub-populations as described in Materials and Methods. Cells

381 were cultured overnight, processed, and examined by SEM at 5,000x magnification.

382 Note that the cells in the CD45 bright population (A) have fewer fenestrae than the cells

383 in the CD45 dim (B) and the CD45 negative (C) populations. Scale bar: 5 μm.

384 Figure 6. Uptake of FITC-FSA by LSEC. (A) Livers were perfused with FITC-FSA

385 and examined by confocal microscopy. The left panel was taken at 20x magnification

386 and the middle and right panels were taken at 40x magnification of the same field as the

387 left panel. Uptake of FITC-FSA (arrow) was observed in the sinusoids around the portal

388 triad (PT) with no uptake was observed in the sinusoids around the central vein (CV).

389 Scale bar: 50 μm. (B) CD45 bright, dim and negative populations were exposed to 0.02

390 µg/ml FITC-FSA as described in Materials and Methods. Uptake of FITC-FSA was

391 observed in both CD45 bright and dim populations, while no uptake was observed in the

392 CD45 negative population. DIC: differential interference contrast. Scale bar: 50 μm.
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393 Figure 7. Toxicity of acetaminophen to CD45 bright (periportal), CD45 dim (mid-

394 lobular) and CD45 negative (centrilobular) LSEC. CD45 bright (●), CD45 dim (○),

395 and CD45 negative (▼) LSEC were exposed to acetaminophen, 1, 3 and 10mM,

396 overnight as described in Materials and Methods. Acetaminophen is significantly more

397 toxic to CD45 dim and CD45 negative cells than to CD45 bright cells (n = 3; p< 0.0001

398 for comparison of the 3-sub-populations by ANOVA; p<0.001 for comparison of CD45

399 dim and negative vs CD45 bright at 3 and 10mM by Least Significant Difference).

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Table 1. Characterization of 3 LSEC sub-populations.

CD45 bright CD45 dim CD45 negative

(periportal) (mid-lobular) (centrilobular)

Percentage of total cells 28.6 ± 2.7% 49.6 ± 1.0% 21.8 ± 2.3%

Yield of cells per liver (106) 18.67 ± 2.54 35.42 ± 3.12 16.46 ± 2.06

Cell diameter (µm) 6.0 ± 0.1 6.8 ± 0.0* 7.1 ± 0.2*

Percentage of CD31 positive cells 66.5 ± 1.2% 90.5 ± 2.7%* 92.0 ± 2.1%*

Percentage of vWF positive cells 11.5 ± 0.5% 8.9 ± 1.8% 15.1 ± 0.7%

Three LSEC sub-populations were isolated as indicated. Flow cytometry of CD45

expression by unselected LSEC was used to determine the percentage of each population

and light microscopy was used to measure the size of the cells in the 3 sub-populations.

The percentage of CD31 and vWF positive cells was determined as described in the

Materials and Methods section. Values are means ± SE (n=3). p< 0.0001 for comparison

of cell diameter of the 3 sub-populations by ANOVA; p<0.0005 for comparison of

percentage of CD31 positive cells of the 3 sub-populations by ANOVA; * p<0.001 for

comparison of CD45 dim and negative vs CD45 bright by Least Significant Difference.
Table 2. Characterization of fenestration of 3 sub-populations of LSEC.

CD45 bright CD45 dim CD45 negative

(periportal) (mid-lobular) (centrilobular)

Diameter of fenestrae (nm) 155.4 ± 2.7 127.7 ± 2.4** 118.0 ± 2.1*

No. fenestrae per sieve plate$$ 8.5 ± 0.6 23.1 ± 2.0* 24.5 ± 1.8*

No. fenestrae per cell§ 306.8 ± 36.8 731.3 ± 89.5** 832.3 ± 56.9*

Porosity$$ 2.8 ± 0.3% 4.5 ± 0.3%* 5.7 ± 0.3%*,#

Three sub-populations of LSEC were isolated as indicated. Cells were cultured

overnight, processed and examined by SEM at 5,000x magnification. Data are mean ±

SE from analysis of 15 images from each experiment (n=3 for each group). Comparison

of the 3- sub-populations by ANOVA: $ p<0.005, $$ p<0.00001, §p<0.0001; * p<0.001,

**p<0.005 by Least Significant Difference compared to CD45 bright population, #

p<0.01 by Least Significant Difference compared to CD45 dim population.