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From the Departments of Internal Medicine,*
Surgery,
and
Pathology,
Kitasato Institute Medical Center
Hospital, Saitama; the Department of Internal
Medicine,
School of Medicine, Keio University,
Tokyo; and Lead Discovery Research Laboratories,¶
Sankyo Co., Ltd., Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two types of ET receptors have been identified and studied; endothelin A (ETAR) and B receptors (ETBR), both signal via GTP-binding proteins.10 Another type of endothelin receptor, ETCR, has been found but its function and distribution remains unknown. ETAR is found predominantly on vascular smooth muscle cells and the affinities for endothelins are in the order ET-1 > ET-2 > ET-3; the affinity for ET-1 is more than 100-fold that of ET-3.11 The primary response mediated by ETAR is vasoconstriction.11 ETBR stimulation leads to diverse responses depending, in part, on cell type. A study has proposed that ETBR-dependent relaxation and smooth muscle vasoconstriction are mediated by distinct ETBRs, termed ETB1 and ETB2, respectively.11 While ET receptors are detectable on all cell types in rat liver, they are far more numerous on hepatic stellate cells (HSCs) than on other hepatic cells such as sinusoidal endothelial cells (SECs), Kupffer cells, and hepatocytes.12,13 When ET-1 is perfused into the rat liver, its localization is consistent with binding to HSCs and SECs.14 Rat HSCs express both ETAR and ETBR, as established by mRNA analysis and competitive binding assays.13 Functional studies indicate that both receptors mediate biological effects.15-17 The ETBR receptor appears to be involved additionally in regulating growth of human myofibroblast-like stellate cells.16
While the data of ETR have largely been obtained using rat liver, there is relatively little information on human liver. No study so far has localized ETAR and ETBR in intact human liver tissue. Our present report is the first to investigate the expression and distribution of ETR subtypes in human normal liver and cirrhotic liver tissues at protein (immunohistochemistry, Western blot, immunoelectron microscopic study) and mRNA (in situ hybridization) levels.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As controls, wedge biopsy specimens from normal portions of the liver were obtained from 5 patients (4 males and 1 female; aged from 44 to 73 years with a mean of 57.3 years) who underwent surgical resection for metastatic liver carcinoma (4 colonic carcinomas and 1 gastric carcinoma). Cirrhotic liver specimens were obtained from gross cirrhotic portions surgically resected from 5 patients (5 males; aged from 62 to 75 years with a mean of 67.4 years) who had hepatocellular carcinoma combined with hepatitis C-related cirrhosis.
Immunoperoxidase Staining
Liver tissues (approximately 5 x 5 x 5 mm) were fixed in periodate-lysine-paraformaldehyde (PLP), rinsed in 0.01 mol/L phosphate buffer (pH 7.4)18 containing 15 to 30% sucrose, embedded in Tissue-Tek OCT-compound (Sakura Finetek Inc., Torrance, CA), and frozen at -80°C until use. Semithin sections 5 µm in thickness were cut using a cryostat, and incubated two days at 4°C with 1:50 dilution of anti-ETAR or anti-ETBR rabbit polyclonal antibodies (Immunobiological Lab., Fujioka, Japan).19 Then the sections were incubated with the EnVision system (DAKO, Glostrup, Denmark)20 at room temperature for 120 minutes. After repeated washes with phosphate-buffered saline (PBS), the sections were reacted with diaminobenzidine containing 0.01% H202, and counterstained with hematoxylin for light microscopic study.
Immunogold-Silver Staining Method for Light and Electron Microscopy
For light microscopy, the sections were immersed in three changes of 0.01% PBS (pH 7.4) for 15 minutes and then incubated for two days at 4°C in a moist chamber with anti-ETAR or anti-ETBR rabbit polyclonal antibodies diluted 1:50 with 0.01 mol/L PBS containing 1% bovine serum albumin (BSA). After treating with PBS for 15 minutes three times, the sections were incubated for 40 minutes with 10-nm colloidal gold-conjugated anti-rabbit IgG antibody (Cosmo Bio Co., Tokyo, Japan) diluted 1:100. The slides were developed with a developing solution (described below) for 50 minutes at 20°C in a dark room, washed in running tap water, briefly counterstained with 0.1% nuclear fast red in 5% aluminum sulfate aqueous solution, dehydrated, cleared and mounted in Biolet. The developing solution had two components. Solution A contained 45 ml of 20% gum arabic aqueous solution (Kanto Chemical Co., Tokyo, Japan) and 1 ml of 10% silver nitrate solution. The gum arabic solution was prepared by centrifugation a 20% solution at 18,000 rpm for 30 minutes at 0°C and separation of the supernatant for use. Solution B contained 200 mg of hydroquinone (Kanto Chemical Co., Tokyo, Japan) and 300 mg of citric acid monohydrate (Kanto Chemical Co., Tokyo, Japan) in 10 ml of distilled water. The working developing solution was prepared by mixing solution A and B in a dark room under illumination of a photographic safety lamp.21
For electron microscopy, the tissue specimens were treated with PBS for 15 minutes three times, fixed in 1.2% glutaraldehyde buffered with 0.01% phosphate buffer (pH 7.4) for 1 hour at 4°C, treated with graded series of ethanol solutions, and post-fixed with 1% osmium tetroxide in 0.01% phosphate buffer (pH 7.4). The liver tissues were embedded in Epon (Polyscience, Inc. Warrington, PA). Ultrathin sections cut with a diamond knife on a LKB ultramicrotome (Bromma, Sweden) were stained with uranyl acetate and observed under a transmission electron microscope (JEM-1200 EX, Tokyo, Japan) operated at an acceleration voltage of 80 kV.
Western Blotting
Western blotting was conducted using fresh control and cirrhotic liver tissues. Briefly, liver tissues were homogenized in 10 volumes of homogenization buffer (20 mmol/L Tris-HCl, pH 7.5, 5 mmol/L MgCl2, 0.1 mmol/L PMSF, 20 µmol/L pepstatin A, and 20 µmol/L leupeptin) using a polytron homogenizer at setting 7 for 90 seconds. The homogenates were centrifuged at 100,000 x g for 45 minutes. The membranes were washed three times, resuspended in 10 volumes of homogenization buffer, homogenized using a Teflon/glass homogenizer, and centrifuged. The membrane proteins thus obtained were used for immunoblotting. Proteins were separated on SDS/PAGE (4 to 20% gel) (Daiichi-Ikagaku, Tokyo, Japan) and transferred onto vinylidene difluoride membranes (Millipore, Bedford, MA). The blots were blocked with 5% (w/v) dried milk in PBS for 30 minutes, incubated with 5 µg/ml anti-ETAR or anti-ETBR antibody, washed in 0.1% Tween 20 in PBS, and incubated with 5000-fold diluted anti-rabbit goat IgG conjugated with horseradish peroxidase (Amersham-Pharmacia, Bioteck, Buckinghamshire, UK). Both primary and secondary antibodies were diluted with 0.1% Tween 20 in PBS, and incubation was at room temperature for 1 hour. The immunoreactive bands were visualized with the ECL Plus detection system (Amersham-Pharmacia).
In Situ Hybridization Technique
mRNA of ETR subtypes was detected in formalin-fixed, paraffin-embedded sections by in situ hybridization using peptide nucleic acid (PNA) probes22 and the catalyzed signal amplification (CSA) technique. Liver tissues were cut into 4 µm-thick sections and adhered to silanated, RNase-free glass slides (prepared by heating in an oven at 60°C for 30 minutes). The sections were dewaxed in xylene (twice for 15 minutes each), followed by a graded ethanol series, rehydrated in RNase-free distilled water, and incubated for 30 minutes in Target Retrieval Buffer (DAKO JAPAN, Kyoto, Japan) preheated and maintained at 95°C. The slides were allowed to cool at room temperature for 20 minutes. The sections were then digested with 20 µg/ml proteinase K (DAKO) at room temperature for 30 minutes. The slides were rinsed in distilled water and rapidly air-dried. After pretreatment, air-dried sections were covered with approximately 15 ml of hybridization solution containing 10% (w/v) dextran sulfate, 10 mmol/L NaCl, 30% (v/v) formamide, 0.1% (w/v) sodium pyrophosphate, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v) Ficoll, 5 mmol/L Na2-EDTA, 50 mmol/L Tris-HCl, pH 7.5, and 1 µg/ml PNA probe. ETBR antisense (FITC-ACC/TGT/CAA/CAC/TTA), ETAR sense (FITC-TTA/GTG/TTG/ACA/GGT), ETBR antisense (FITC-ACA/CAA/GGC/AGG/ACA), or ETBR sense (FITC-TGT/CCT/GCC/TTG/TGT) probe was used.23 The slides were evenly covered with the hybridization solution and incubated in a moist chamber at 43°C for 90 minutes. Following hybridization, the coverslips were removed, and the slides were transferred to pre-warmed TBS in a water bath at 49°C and washed for 30 minutes with gentle shaking (PNA hybridization kit; DAKO JAPAN, Kyoto, Japan). A non-isotopic, colorimetric signal amplification system (GenPoint Kit, DAKO JAPAN, Kyoto, Japan) was used to visualize specific hybridization signals. Briefly, tissue sections were incubated with a FITC-horseradish peroxidase reagent for 15 minutes, washed three times with TBST (150 mmol/L NaCl, 10 mmol/L Tris. pH 7.5, 1.1% v/v Tween 20), incubated with a solution containing H202 and biotinyl tyramide for 15 minutes, and washed three times with TBST.24 This step resulted in catalyzed signal amplification by additional deposition of biotin at the site of probe hybridization. The sections were then incubated in streptavidin-horseradish peroxidase for 15 minutes and washed three times in TBST. Colorimetric signals were localized after incubation in diaminobenzidine solution containing 0.01% H2O2, and counterstained with hematoxylin for light microscopic examination.
Semiquantitative Analysis
The immunogold labeling in the ultra-thin sections of peri-sinusoidal SECs and HSCs was quantitated using the Mac Measure program, version 1.61. SECs and HSCs around sinusoids were selected randomly, and the numbers of gold particles per unit length of membrane were counted. Statistical significance of the difference between control and cirrhotic samples was assessed by Student’s t-test, and P < 0.05 was regarded as indicating a significant difference. Data are expressed as means ± SEM.
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Results |
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In control liver tissue, immunohistochemical study demonstrated
that ETBR was moderately expressed on the hepatic sinusoidal lining
cells, particularly on the SECs and HSCs, whereas ETAR was only rarely
detected on the sinusoidal lining cells although it is found on portal
vessels (Figure 1)
. In cirrhotic liver
tissue, immunohistochemical study demonstrated overexpression of ETBR
on hepatic sinusoidal lining cells compared to control liver tissue,
while ETAR showed an increase but remained low in level (Figure 2)
. There was a patchy distribution of ET
receptors.
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. Western Blot
To confirm the immunohistochemical results, we investigated
receptor protein expression by Western blotting. Samples containing 15
µg of protein were subjected to SDS/PAGE (4 to 20% gels) and
analyzed by blotting. ETBR was found in abundance in cirrhotic
liver, but ETAR was not detected either in control or cirrhotic liver
(Figure 3)
.
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We examined the ultrastructural localization of ETRs on sinusoidal
lining cells by immunogold electron microscopy. In control liver
tissue, electron-dense gold particles showing the presence of ETAR were
found scantily on hepatic sinusoidal lining cells, while ETBR labeling
was evident on HSCs and SECs, mainly localized on the luminal side
(Figure 4)
. In cirrhotic liver tissue,
ETBR was significantly increased not only on HSCs but also on SECs
compared to control liver tissue, while ETAR labeling remained scanty
(Figure 5)
. In morphometric analysis of
immunogold particle labeling for ETAR and ETBR (Figure 6)
, ETAR labeling was extremely low on
HSCs (control; 0.2 ± 0.1/10 µm, cirrhosis; 0.1 ± 0.1, not
significant) and SECs (control; 0.1 ± 0.1/10 µm, cirrhosis;
0.1 ± 0.1, not significant) both in control and cirrhotic liver
tissues. On the other hand, ETBR labeling was considerably high in HSCs
and SECs in control liver, and was significantly increased on HSCs
(control; 2.2 ± 0.6, cirrhosis; 5.8 ± 1.6.
P < 0.01) and SECs (control; 4.8 ± 0.3,
cirrhosis; 6.8 ± 1.8, P < 0.05) in cirrhotic
liver. The increase in ETBR expression was
particularly marked in HSCs. Therefore, HSCs and SECs in normal human
liver tissue expressed predominantly ETBRs, and the expression of ETBRs
was strongly enhanced on HSCs and slightly enhanced on SECs in
cirrhotic liver.
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Next, we investigated the expression of endothelin receptors at
mRNA level by in situ hybridization using peptide nucleic
acid probe. In control liver tissue, ETAR mRNA was almost undetectable,
but ETBR mRNA was detected on hepatic sinusoidal lining cells (Figure 7)
. In cirrhotic liver tissue, ETBR mRNA
expression was enhanced on hepatic sinusoidal lining cells,
particularly on HSCs, compared with control liver tissue, while ETAR
mRNA remained almost undetectable (Figure 8)
. Similar results were obtained in all
control (n = 5) and cirrhotic
(n = 5) liver tissues.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelin receptors have been studied by various techniques such as endothelin binding assay and mRNA measurement. However, no study so far has localized endothelin subtypes directly in human liver. The commercial antibodies against ETAR and ETBR we used were recently developed in Japan; they were rabbit antibodies against the C-terminal of human ETA and ETB receptors. Some of the methods used in this paper (immunohistochemistry, immunoelectron microscopy) have been established using normal and cirrhotic rat liver25 using these antibodies and using the newly developed EnVision system, we were able to obtain rapid immunostaining of ETAR and ETBR in frozen liver tissue. We have verified these antibodies in rat liver tissues and clearly localized both ETAR and ETBR in rat liver by light and electron microscopy.25 The EnVision system uses a polymeric conjugate consisting of a large number of peroxidase and secondary antibody molecules bound directly to an activated dextran backbone. The polymeric conjugates hold up to 100 enzyme molecules and up to 20 antibody molecules per backbone,20 thus greatly increasing the sensitivity.
Immunogold-silver staining procedure not only allows ultrastructural localization of endothelin receptors but also easy quantitation of the immunogold-silver labeling in ultrathin sections of sinusoidal cells.21 In control liver, ultrastructural localization of ETAR was hardly detectable on HSCs and SECs, while ETBR was evident on HSCs as well SECs. In cirrhotic liver, ETBRs were strongly expressed on HSCs. A previous autoradiographic endothelin binding study showed rare or few grains on the portal vein but abundant grains in HSCs and SECs,14 which is consistent with our immunohistochemical results. ETBR was localized along the apical plasma membrane of endothelial cells.26 In the present study, ETBR was mainly localized at the luminal side of SECs and HSCs.
We used PNA probes in in situ hybridization. PNAs are pseudo-peptides with DNA-binding capability first reported as nucleotide analogs capable of binding, in a sequence-specific fashion, to DNA and RNA.27 In PNA, the sugar phosphate backbone found in DNA/RNA is replaced by a polyamide backbone, keeping the distance between the nucleotide bases the same as in DNA/RNA. Hybridization of PNA to DNA has been shown to obey the Watson-Crick rules, and the compounds have also been found to hybridize to their nucleic acid counterparts.28 The relatively hydrophobic PNA probes penetrate tissues more easily than the corresponding DNA/RNA oligonucleotide-based probes, thereby allowing development of fast protocols for in situ hybridization.22 While conventional non-radioactive ISH method is relatively insensitive, target amplification by PCR or signal amplification by CSA allows sensitive detection of few copies of nucleic acid sequences. Compared with PCR in situ amplification, CSA is simpler and faster and does not require thermal cycling or the specialized equipment.29 In a preliminary experiment, we failed to obtain any signal using RNA probes to detect ETBR mRNA (data not shown), but we obtained satisfactory signals with PNA probes using the CSA method. The results of our trial clearly demonstrate the advantages of PNA probes and the CSA method. In cirrhotic liver, ETBR mRNA was overexpressed on sinusoidal lining cells.
Only a few studies have characterized ETRs in human liver cultured cells using binding or PCR studies. The detailed binding kinetic studies of Pinzanni et al17 demonstrated that the progressive activation in HSCs in culture is associated with a progressive shift from a relative predominance of ETAR to relative predominance of ETBR, but the relative densities changed on serial subculture. A predominance of ETBR was observed when the cells had undergone complete transition to myofibroblast-like phenotype that resembles the in vivo condition of HSCs. Mallat et al16 reported 20% ETAR and 80% ETBR binding sites in HSCs with myofibroblastic phenotype. ET-1 binding to ETBRs causes a potent growth inhibition of human myoblastic HSCs. Their results support our in vivo studies that ETBR is expressed predominantly in HSCs and SECs. An immunohistochemical study showed increase of ETR in human cirrhotic liver associated with decrease in intralobular innervation, but detailed information on subtypes was not provided.30 A quantitative PCR in human liver tissue showed that both ETAR and ETBR mRNA levels were significantly increased in cirrhosis, correlating with increased portal pressure.31 Their result indicated a higher level of ETBR mRNA than ETAR mRNA, and the authors stated that their method only provided an estimate of the number of copies. Therefore, our results and previous findings generally agree that ETBR is the predominant endothelin receptor in sinusoidal lining cells, especially in HSC, although our in vivo localization studies demonstrated an extremely low level of ETAR. Using the same antibodies in light and electron microscopic studies, we have localized similar densities of ETAR and ETBR in HSCs and SECs of rat liver.25 There may be interspecies differences in ETR distribution. Results obtained from rat models may have to be interpreted with caution.
During liver injury, stellate cells undergo a process characterized by loss of retinoid droplets, enhanced collagen production, and expression of smooth muscle actin, which has been termed "activation."32 Following liver injury, activated HSCs are associated with increased proliferation and synthesis of the major components of liver fibrosis.33 Activated HSCs display a large number of ET binding sites that mediate at least two biological effects of ET-1; contraction15 and growth inhibition.16 ET-1 inhibits proliferation of activated HSCs and the growth inhibition is mediated by ETBR.16 ETBRs may be subdivided into two putative receptors, ETB1 and ETB2.11,34,35 ETB1 is considered to induce NO release from endothelial cells and to promote vasodilation.34 ETB2 may induce the contraction of vascular smooth muscles.34
In a simultaneously stained rat section, there is a homogeneous
distribution of ET receptors in rat liver (Figure 1, f and g)
. In
comparison, human liver tissue expresses ET receptors in a patchy
pattern, and the sites of positive staining are fewer than the number
of stellate cells (Figures 1 and 2)
. Again, these findings validate the
difference in ET distribution in human and rat livers. Our findings
suggest a highly heterogeneous population of cells in human liver with
only a minority being responsive to endothelin based on receptor
expression. We speculate that the baseline ET receptor levels are lower
in human liver than in rat liver, as confirmed by a low level of
protein (Western blot) and mRNA (in situ
hybridization) expression, and that the ET receptors are up-regulated
in selected cells during liver injury. This is shown by the
demonstration of ETB in areas of strong
inflammation and at the edge of nodules and fibrotic septa in cirrhotic
specimens.
Overexpression of ETBR expression in sinusoidal lining cells, especially on HSCs, in cirrhotic tissue may have several implications. It may amplify the growth inhibitory effect of ET-1 and play a role in negative control of liver fibrogenesis. On the other hand, Kaneda et al demonstrated that the parenchyma of ET-1 perfused liver in rat was distinguished histologically into two areas; one consisted of loosely woven plate of vacuolated hepatocytes with widened sinusoids, and the other with closely packed hepatic cell plates intervened by narrowed, collapsed sinusoids concomitant with constriction of preterminal portal venules.36 Increased ETBR may mediate cell contraction and increase hepatic sinusoidal microvascular tone, and contribute to portal hypertension.
The therapeutic potential of ET blockade for portal hypertension remains a controversial topic. In experimental liver injury in rat, blockage of ETAR has been shown to disturb hepatic microcirculation and worsen endotoxin-induced liver injury,37 while chronic blockade with a mixed antagonist Bosentan had no hemodynamic effects but increased the connective tissue content of cirrhotic liver.38 Thus, precise elucidation of ETR, especially ETBR-mediated paracrine/autocrine functions of ET is required to understand the role of ET and its antagonists or agonists. Morphological combined with functional studies are essential. Our fast protocols for demonstrating ETR protein and mRNA may be useful assets in these investigations.
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Acknowledgements |
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Footnotes |
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Accepted for publication June 28, 2001.
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