aquaporin-1 associated with hepatic arterial capillary proliferation on hepatic sinusoid in human...

BAS IC STUDIES

Aquaporin-1 associated with hepatic arterial capillaryproliferation on hepatic sinusoid in human cirrhotic liverHiroaki Yokomori1, Masaya Oda2, Kazunori Yoshimura3, Fumihiko Kaneko1 and Toshifumi Hibi4

1 Department of Internal Medicine, Kitasato Medical Center Hospital, Kitasato University, Saitama, Japan

2 Organized Center of Clinical Medicine, International University of Health and Welfare, Tokyo, Japan

3 Department of Rehabilitation, Nihon Institute of Medical Science, Saitama, Japan

4 Division of Gastroenterology and Hepatology, Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan

Keywords

Aquaporin-1 – hepatic arterial capillary –

liver cirrhosis – portal hypertension

Correspondence

Hiroaki Yokomori, MD, Department of

Internal Medicine, Kitasato Medical Center

Hospital, Kitasato University, Saitama 364-

8501, Japan

Tel: +81 485 93 1212

Fax: +81 485 93 1239

e-mail: [email protected]

Received 2 May 2011

Accepted 23 June 2011

DOI:10.1111/j.1478-3231.2011.02610.x

AbstractBackground: Aquaporins (AQPs) are key regulators not only of watertransport in the cytoplasm but also of angiogenesis. Although AQPs in thenormal hepatobiliary system have been studied in mammals, little is knownabout the localization and changes of AQPs in the hepatic microvascularsystem including sinusoids in cirrhotic liver, which might contribute toportal hypertension. Aims: We designed this study to examine the locali-zation of AQP1 in human cirrhotic liver. Methods: Surgical wedge biopsyspecimens were obtained from non-cirrhotic portions of human livers(normal control) and from cirrhotic livers (LC) (Child A-LC and ChildC-LC). Immunostaining, Western blotting, in situ hybridization (ISH) andlaser-captured microdissection (LCM) were conducted. Results: In controlliver tissue, AQP1 was localized mainly in the portal venules, hepatic arte-rioles and bile ducts in the portal tract, although AQP1 was detected onlyslightly in the sinusoids. In cirrhotic liver tissue, AQP1 expression was evi-dent, aberrantly observed on periportal sinusoidal endothelial cells corre-sponding to the capillarized sinusoids, on the proliferated arterialcapillaries opening into the sinusoid in the generating hepatic nodule andon proliferated bile ductules at the peripheral edge of nodules and fibroticsepta. In cirrhotic liver, overexpression of AQP1 at protein and mRNA lev-els was demonstrated, respectively, using Western blot and ISH. AQP-1 ofmRNA level in sinusoid was confirmed using LCM. Conclusions: Aberrantexpressions of AQP1 in periportal sinusoidal regions in human cirrhoticliver indicate the proliferation of arterial capillaries directly connected tothe sinusoids, contributing to microvascular resistance in cirrhosis.

Aquaporin-1 (AQP1) is a water channel protein that isexpressed widely in vascular endothelia, where itincreases cell membrane water permeability (1). At least13 AQPs (AQP0–AQP12) have been found in mammals,with many (AQP0, AQP1, AQP4, AQP5, AQP8, AQP9and AQP11) localized to the hepatobiliary system (2, 3).

Actually, AQP1 is widely expressed in continuousendothelial capillaries (1). The role of AQP1 in endo-thelial cell function remains unknown, but AQP1 hasbeen found recently to play a fundamental role as awater channel in cell migration, which is central todiverse biological phenomena including angiogenesis,wound healing, tumour spreading and organ regenera-tion (4). Hepatic microvascular subunits are severalpotential morphological sites for regulating blood flowthrough the sinusoids. These include the various seg-

ments of the afferent portal venules and hepatic arteri-oles, the sinusoids themselves, as well as central andhepatic venules (5). Little agreement exists as to whetherhepatic arterial blood pours directly into the sinusoids.Moreover, the roles of the hepatic arterial system in thecontrol of hepatic microcirculation remain unclear.However, increasing evidence indicates that hepaticarterioles demonstrate directly in the hepatic sinusoids(6–7), particularly by intravital microscopic observa-tions of hepatic microvascular hemodynamics (8),scanning electron microscopy (9–10) and enzymohisto-chemistry (11). In long-standing cirrhosis, collageniza-tion of the interstitial space and formation of abasement membrane at the margin of the microvascularchannels occur, with subsequent capillarization of thehepatic sinusoids (12). Rappaport et al. (13) showedthat the development of scars in cirrhotic liver is invari-ably accompanied by intense vascular proliferation, sug-All authors contributed equally to this work.

Liver International (2011)© 2011 John Wiley & Sons A/S1554

Liver International ISSN 1478-3223

gesting tissue remodelling and the fibrous repair path asthe way for the development of collateral flow in cirrho-sis. Their observations were confirmed by others, show-ing that, in cirrhotic tissues, the regenerative nodulesare surrounded by a dense arterial capillary plexus (14).However, mechanisms triggering the intense vascularproliferative response remain unknown. A previousreport described that impaired oxygen delivery to thehepatocytes might occur in cirrhosis as a result of intra-hepatic shunts or capillarization of the sinusoids, lead-ing to hepatocyte hypoxia (15).

Recently, results of a study that examined theexpression of hypoxia-induced VEGF during biliary-type liver fibrogenesis have implicated hypoxia as amajor factor in the induction of VEGF and in markedangiogenesis occurring at an early stage before theonset of cirrhotic lesions (16).

According to previously published data, in anexperimental cirrhotic liver examined in an immuno-histochemical and immunoelectron microscopic study,the expression of AQP-1 on liver sinusoidal endothe-lial cell (LSEC)s is especially located at zone 1 andperiportal microvascular endothelial cells. The AQP-1immunoreactivities were expressed aberrantly onLSECs in regenerative nodules of cirrhotic liver, whichmight be associated with capillarization of LSECs andremodelling in this region (17). At the molecular level,fibroblast growth factor (FGF) promotes the amoeboidphenotype to drive endothelial invasion through theextracellular matrix (ECM) in experimental cirrhoticliver (18). Little is known about AQP-1 localizationsand their changes in the hepatic microvascular system,including the sinusoids in human cirrhotic liver. Weattempted to clarify the localizations of AQP-1 in themicrovessels in normal and cirrhotic human livers.

Materials and methods

Materials

As controls, wedge biopsy specimens from normal por-tions of the liver were obtained from 10 patients (eightmen and two women; ages 54–73 years with a mean of65.7 years) who underwent surgical resection for meta-static liver carcinoma (eight colonic carcinomas andtwo gastric carcinomas). Cirrhotic liver specimens wereobtained from gross cirrhotic portions that had beensurgically resected from eight patients (eight men; aged62–75 years with a mean of 67.4 years) who had hepato-cellular carcinoma (HCC) concurrent with hepatitisC-related cirrhosis. The HCC underwent hepatectomy.Each patient was evaluated before the operation usingthe indocyanine green (ICG) clearance test, ICG <15%and Child–Pugh grading A (19). Five other cirrhoticpatients were diagnosed as HCC concurrent with hepa-titis C-related cirrhosis for liver transplantation. Thesepatients had HCC and Child–Pugh grading C cirrhosiswith three or fewer tumour nodules with maximal

diameter not exceeding 5 cm, and no apparent signs ofvascular invasion (20). Autopsies on five patients wereperformed within 3 h after death. Informed consentfor autopsies was received in each case from the familyof the deceased patient. Informed consent was pro-vided in every case from the patient’s family. Thisstudy was conducted according to Helsinki declaration.

Immunohistochemistry

Liver tissues (approximately 3 9 2 9 1 cm) were fixedin formalin and embedded in paraffin. Firstly, 4-lm sec-tions were cut from the paraffin blocks, deparaffinizedwith xylene and dehydrated using graded ethanol. Theywere incubated overnight at 4°C with 1:100 dilution ofanti-AQP1 rabbit polyclonal antibody (Alpha DiagnosticInternational Inc., San Antonio, TX, USA). Then the sec-tions were incubated (Envision; Dako Japan Inc., Tokyo,Japan) at room temperature for 30 min. After repeatedwashing with PBS, the sections were reacted with diam-inobenzidine containing 0.01% H2O2, and counter-stained with haematoxylin for light microscopic study.Moreover, we investigated CD34 dual staining forimmunohistochemical examination for capillary endo-thelial cell marker. Primary antibody with CD34 (Dako,Glostrup, Denmark) was used for 30 min at room tem-perature (RT) with subsequent incubation (Envision;Dako Japan Inc.). The bound antibody was visualizedusing peroxidase reaction in a 3,3′-diaminobenzoic tetra-hydrochloride (DAB) (brown) and H2O2 solution, or byalkaline phosphatase reaction for Fuchsin (Dako) (red).

Computer-assisted morphometric analysis of AQP-1labelled sinusoidal network

Sections labelled immunohistochemically with AQP-1were analysed morphometrically. Briefly, the immuno-stained sections were scanned using light microscopy atlow magnification (940), and AQP-1 immunostainingin sinusoidal lining cells was counted in a representativehigh magnification (9400; 0.152 mm2) field. Imageanalysis was done using the computer. Images were cap-tured and digitized using an internal frame grabberboard (Photoshop CS4/Photoshop CS4 Extended;Adobe Systems Inc., San Jose, CA, USA). This procedureconsisted of converting the captured image in pixels.The number of AQP-1-immunostained sinusoidal lin-ing cells per square millimetre was derived from imageanalysis (21). Twenty fields containing hepatic sinusoidswere measured for each control and cirrhotic liver tissuesample. Fisher’s PLSD was used for statistical analyses.

Western blotting

The tissue sample was homogenized in 10 volumes ofhomogenization buffer (20 mM Tris–HCl, pH 7.5,5 mM MgCl2, 0.1 mM PMSF, 20 lM pepstatin A and20 lM leupeptin) using a polytron homogenizer at set-

Liver International (2011)© 2011 John Wiley & Sons A/S 1555

Yokomori et al. AQP-1 overexpression in human cirrhosis

(a)(A) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

(m) (n) (o)

(p) (q) (r)

(B) (C)


AQP-1 overexpression in human cirrhosis Yokomori et al.

ting 7 for 90 s. The obtained membrane proteins wereused for immunoblotting. Proteins (20 lg/ml) wereseparated on SDS/PAGE and transferred onto polyviny-lidene difluoride membranes (Millipore, Billerica, MA,USA). The blots were blocked with 5% (w/v) dried milkin PBS for 30 min, and were then incubated with anti-AQP-1 (Alpha Diagnostic International Inc.) diluted1:5000 in 0.1% Tween 20 in PBS. The blots werewashed and incubated for 1 h at room temperaturewith horseradish peroxidase-conjugated goat anti-rabbitimmunoglobulin (Santa Cruz Biotechnology, Inc.,Santa Cruz, CA, USA). Protein bands were detectedusing an enhanced chemiluminescence detection system(ECL Plus; Amersham Biosciences, Uppsala, Sweden).After exposing the nitrocellulose sheets to Kodak XARfilm, glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) (Imgenex Corp., San Diego, CA, USA) was usedas a loading control for Western blot analysis.

In situ hybridization

Messenger RNA of AQP-1 was detected in formalin-fixed, paraffin-embedded sections using in situ hybrid-ization with peptide nucleic acid (PNA) probes andcatalysed signal amplification (CSA) technique (22).Then 4-lm liver sections were adhered to silanated,RNAse-free glass slides (prepared by heating in anoven at 55°C for 30 min). The sections were deparaffi-nized in xylene (twice for 15 min each), followed by agraded ethanol series. They were dehydrated inRNAse-free distilled water and were then incubated for30 min in Target Retrieval Buffer (Dako, Glostrup,Denmark) preheated and maintained at 95°C. Theslides were cooled at room temperature for 20 minand were then digested with 20 lg/ml proteinase K(Dako) at room temperature for 30 min. After rinsingin distilled water and rapid air-drying, the sectionswere covered with approximately 15 ll of hybridiza-tion solution containing 10% (w/v) dextran sulphate,10 mM NaCl, 30% (v/v) formamide, 0.1% (w/v)sodium pyrophosphate, 0.2% (w/v) polyvinylpyrroli-

done, 0.2% (w/v) Ficoll, 5 mM Na2EDTA, 50 mMTris–HCl, pH 7.5 and 10 lg/ml PNA probe. AQP-1(accession No: NM_198098.1) antisense (FITC-GCCA-GTGTAGTCAATAGC) and sense (FITC-GCTATTGA-CTACACTGGC) probes (prepared by OperonBiotechnology Co. Ltd., Tokyo, Japan) were used. Theslides were evenly covered with the hybridization solu-tion and incubated in a moist chamber at 50°C for90 min. Following hybridization, the coverslips wereremoved. The slides were transferred to pre-warmedTBS in a water bath at 49°C and washed for 30 minwith gentle shaking (PNA Hybridization Kit; DakoJapan, Tokyo, Japan). A non-isotopic, colorimetric sig-nal amplification system (GenPoint Kit; Dako Japan)was used to visualize specific hybridization signals. Sec-tions were incubated with a FITC-horseradish peroxi-dase reagent for 15 min and washed three times withTBST (150 mM NaCl, 10 mM Tris, pH 7.5, 1.1% vol/vol Tween 20). Then they were immersed in a solutioncontaining H2O2 and biotinyl tyramide for 15 min fol-lowed by three washes with TBST. This catalysed signalamplification step enhanced the deposition of biotin atthe site of probe hybridization. The sections were thenincubated in streptavidin-horseradish peroxidase for15 min and washed three times in TBST. Colour wasdeveloped by incubation in diaminobenzidine solutioncontaining 0.01% H2O2. The sections were counter-stained with haematoxylin for light microscopic exami-nation. As negative controls, we used untreated ratliver sections and sections obtained from a rat cirrhosismodel induced by injecting 200 mg/kg of thioaceta-mide (TAA) twice weekly for 24 weeks.

Laser capture microdissection (LCM)

Liver tissues were frozen immediately in optimal cut-ting temperature compound (Thermo Fisher ScientificInc., Suwanee, GA, USA) and stored at �80°C untiluse. After rapid staining with H&E, 6-lm sectionsfrom frozen sections were microdissected under a Pix-Cell II laser capture microscope with an infrared diode

Fig. 1. (A) Light microscopic distribution of aquaporin (AQP)1 in human control liver (a–f) and human cirrhotic liver (g–r) using immuno-peroxidase and alkaline phosphatase staining with haematoxylin counterstaining. The arrows indicate capillary endothelial cells. Thearrowheads mark the bile duct. Black arrowheads indicate the portal vein. White arrowheads show bile ductules. Black arrows indicate abile duct. White arrowheads indicate hepatic arteries. Left panel: low magnification (9100). Middle and right panels: high magnification(9400). C, central vein; P, portal vein. Lower panel: high magnification (9400). Upper panel: AQP-1 DAB reaction (a–c, g–i, m–o). Mid-dle panel: AQP-1 plus CD34 alkaline phosphatase (d–f, j–l, p–r). In control liver (a, 9100; b, 9400; c, 9400), reaction products showingAQP1 are localized mainly on endothelial cells of large vessels, the portal vein and bile ducts, and very sparsely on hepatic sinusoidal lin-ing cells. In Child A-LC (i, 9100; j, 9400, k, 9400), reaction products showing AQP1 are partially localized on hepatic sinusoidal endo-thelial cells and on abundantly proliferated bile ductules. They are partially localized on hepatic sinusoidal endothelial cells, onabundantly proliferated bile ductules and also partially on the proliferative arterial capillaries (*) directly connecting with the sinusoids inthe generating hepatic nodule. In Child C-LC (p, 9100; q, 9400; r, 9400), reaction products showing AQP-1 expressions are evident onthe proliferated arterial capillaries (*) in the peripheral region of fibrous septa as well as aberrantly on the sinusoid in the regenerativenodule surrounded by the broad fibrous septa. (B) Computer-assisted morphometric analysis of AQP-1 labelled sinusoidal network.(C) Western blot analysis of expression of aquaporin (AQP)-1 protein in human control and cirrhotic liver tissues. Samples containing20 lg protein were subjected to SDS/PAGE and were analysed using Western blotting. Lanes 1, 2 show a control liver. Lanes 3, 4 showa Child A-LC. Lanes 5, 6 show a Child C-LC. Positions of molecular mass markers (kDa) are shown. The AQP1 protein expression ismarkedly more intense in Child C-LC.



laser (Arcturus Engineering Inc., Mountain View, CA,USA), as described in an earlier report (23). Briefly, aspecific polymer film mounted on optically transparentcaps (CapSure TF-100; Arcturus Engineering, Inc.) wasplaced on the section. Sinusoidal components werecaptured by focal melting of membranes using thelaser beam under visual control. The caps with cap-tured epithelial cells were used as a lid for 500-lLmicrocentrifuge tubes containing RNA extractionbuffer (Arcturus Engineering, Inc.).

RNA extraction and amplification

Total RNA from captured epithelial cells was extractedusing a Pico Pure RNA isolation kit (Arcturus Engi-neering, Inc.) according to the manufacturer’s recom-mendations. After deoxyribonuclease treatment(Invitrogen Corp., Carlsbad, CA, USA), RNA waseluted and stored at �80°C until use. All total RNAsamples were first tested for quality (Agilent Bioanalyz-er 2100B with RNA Pico Lab Chip Kit; Agilent Tech-nologies Inc., Palo Alto, CA, USA) and weresubsequently amplified (RiboAmp OA RNA Amplifica-tion Kit; Arcturus Engineering, Inc.). The amplifiedRNA quality was evaluated again (RNA Pico Lab ChipKit; Agilent Technologies Inc., Santa Clara, CA, USA).

Reverse-transcription polymerase chain reaction analysis

Approximately 2 lg of total RNA from sinusoidal cellswas reverse-transcribed using the StrataScript first-strand synthesis system (Stratagene, La Jolla, CA,USA). Complementary DNA (cDNA) was amplifiedwith 18S primers and SYBR Green polymerase chainreaction (PCR) master mix (Applied Biosystems, Fos-ter City, CA, USA) using PCR with an iCycler real-time PCR detection system (Bio-Rad Laboratories Inc.,Hercules, CA, USA) for 40 cycles. Relative RNA levelswere calculated using software (iCycler; Applied Bio-systems) and a standard equation AQP-1 (RNA acces-sion no: NM_198098.1) (prepared by Takara Bio Inc.,Otsu, Shiga, Japan): forward primer 5′-GCACCGT-CAAGGCTGAGAAC-3′; reverse primer 5′-TGGTGAA-GACGCCAGTGGA-3′. The relative amount of mRNAwas calculated Using GAPDH (RNA accession no:NM_002046.3) (forward primer; 5′-GGTGATCACA-CACAACTTCAGCAA-3′; reverse primer 5′-TTCACG-CGGTCTGTGAGGTC-3′) as a control.

Immunogold–silver staining method for electronmicroscopy

Liver tissues were fixed in periodate–lysine–parafor-maldehyde (PLP). Semi-thin 5-lm sections were (i)immersed for 15 min in three changes of 0.01% phos-phate buffered saline (PBS, pH 7.4), (ii) incubatedwith anti-AQP-1 rabbit polyclonal antibody diluted1:500 in 0.01 M phosphate buffered saline containing

1% bovine serum albumin overnight at 4°C in a mois-ture chamber, (iii) treated for 15 min in PBS threetimes, (iv) incubated in 1.4 nm colloidal gold-conju-gated anti-rabbit IgG antibody (Nanoprobes Inc.,Yaphank, NY, USA) diluted 1:50 for 40 min and (v)physically developed using a silver enhancement kit(Nanoprobe Silver Enhancement Kit; Nanoprobes Inc.,Yaphank, NY, USA) (24). For transmission electronmicroscopy, the materials were treated for 15 min PBSthree times and fixed in 1.2% glutaraldehyde bufferedwith 0.01% phosphate buffer (pH 7.4) for 1 h at 4°C,followed by a graded series of ethanol solutions andpost-fixation with 1% osmium tetroxide in 0.01%phosphate buffer (pH 7.4). After embedding in Epon,ultrathin sections were cut using a diamond knife onan LKB ultra microtome. They were stained with ura-nyl acetate and observed under a transmission electronmicroscope (JEM-1200 EX; JEOL, Tokyo, Japan) with80-kV acceleration voltage.

Semi-quantitative analysis

Densitometrical analyses of Western blot and RT-PCRwere performed using software (Scion Image ver. Beta4.0.2; Scion Corp., Frederick, MD, USA). One-wayanalysis of variance with a Bonferroni–Dunn post-hocanalysis was used to determine statistical differencesbetween individual groups (SuperANOVA; AbacusConcepts Inc., Berkeley, CA, USA). Differences wereinferred as significant when the probability of chanceexplaining the results was <5% (P < 0.05).

The immunogold labelling of peri-sinusoidal SECsand capillary endothelial cell (EC) in the ultrathin sec-tions was quantified using software (Mac MeasureProgram; NIH Image ver. 1.62, National Institute ofMental Health, Bethesda, MD, USA). We investigateda single specimen and counted two separate platesfrom each of six patients of control, Child-A LC andChild-C LC samples. The SECs or capillary endothelialcells around sinusoids (94000) were selected ran-domly, and the gold particles per unit length of mem-brane were counted (n = 12). Statistical significance ofthe difference between control and cirrhotic (LC-Aand LC-C) samples was assessed using Student’s t-test;P < 0.05 was regarded as indicating a significant dif-ference. Data are expressed as mean ± SEM.

Results

Immunohistochemical findings

In control liver tissue, AQP-1 was mainly localized inthe portal terminal venules and bile ducts, whereasAQP-1 was detected only slightly in the sinusoids(Fig. 1-A a,b,c). In human control liver samples, AQP-1 plus CD34 dual staining is expressed only in capillaryendothelial cells of portal areas and the periportal areas(Fig. 1-A d,e,f). In Child A-LC, reaction products



showing AQP1 are partially localized on hepatic sinu-soidal endothelial cells and on abundantly proliferatedbile ductules. They are partially localized on hepaticsinusoidal endothelial cells, on abundantly proliferatedbile ductules and also partially on the proliferative arte-rial capillaries directly connecting with the sinusoids inthe generating hepatic nodule (Fig. 1-A g,h,i). TheAQP-1 and dual CD34 expression are proliferative cap-illary vessels that are partially connected to the sinusoid(Fig. 1-A j,k,l). In Child C-LC (Fig. 1-A m,n,o), reac-tion products showing AQP-1 expression are readilyevident on the proliferated arterial capillaries in theperipheral region of fibrous septa as well as aberrantlyon the sinusoid in the regenerative nodule surroundedby the broad fibrous septa, indicating the capillariza-tion of the hepatic sinusoid. Furthermore, AQP-1 anddual CD34 expressions are prominent on proliferativearterial capillaries onto the sinusoid (Fig. 1-A p,q,r).

Using morphometric measurements, AQP-1 immuno-staining was 1.3578 ± 0.472 pixels/mm3 in control,4.4273 ± 0.895 pixels/mm3 in Child-A cirrhosis and11.685 ± 1.976 pixels/mm3 in Child-C cirrhosis. It was

(a)(A)

(c)

(b) (d)

(e) (g)

(f) (h)

(i) (k)

(j)

(b) (c)

(d)

(a)(B)

(l)

Fig. 2. (A) In situ hybridization for detection of aquaporin (AQP)1mRNA in control normal and Child A and C cirrhotic liver tissues.Peptide nucleic acid probes (a, c, d, f, g, i: AQP1 anti-sense, b, e,h: AQP1 sense) were used with FITC labelling. Sections werecounterstained with haematoxylin. Arrows indicate capillary endo-thelial cells. Arrowheads show the bile duct. Images with theanti-sense probe are also shown (a, d, g: 9100; c, f, i: 9400).a, c: Normal control liver tissue, immunoperoxidase-positive sub-stances are located on the portal vein and bile duct, but areobserved only slightly on hepatic sinusoidal endothelial cells.d, f: Child A-cirrhotic liver tissue. Immunoperoxidase positive sub-stances are partially localized on hepatic sinusoidal endothelialcells and on abundantly proliferated bile ductules. They are par-tially localized on hepatic sinusoidal endothelial cells, and also onthe proliferative arterial capillaries directly connecting with thesinusoids in the generating hepatic nodule. g–i: Child C-cirrhoticliver tissue. Immunoperoxidase-positive substances showing thepresence of AQP1 are observed in numerous sinusoidal endothe-lial cells, in proliferated arterial capillaries opening into the sinu-soid and in proliferated bile ductules in the generating hepaticnodule. (B) Reverse transcriptase-polymerase chain reaction (RT-PCR) for AQP-1 in the sinusoidal component separated by micro-dissection. a: Liver tissue sample of Child C-cirrhotic liver tissuebefore removal of the target sinusoidal cells using LCM. b: Child-C cirrhotic liver tissue sample after removal of sinusoidal cellsusing LCM. c: LCM-captured sinusoidal cells attached to transferfilm. d: The AQP-1 mRNA level is significantly higher in Child-CLC samples than in either control or Child-A LC samples fromliver parenchyma. Gel photograph of PCR for GAPDH and AQP-1showing PCR products of the expected sizes: lanes 1,2 (controlsinusoidal components), lanes 3,4 (sinusoidal component of ChildA-LC, cirrhosis sinusoidal components), lanes 5,6 (sinusoidal com-ponents Child C-LC, cirrhosis sinusoidal components). (a) AQP-1and (b) GAPDH.



(a) (b)

(c) (d)

(e) (f)

(g)



significantly higher in Child C than in either Child A(P < 0.001) or control (P < 0.001). Furthermore, itwas significantly higher in control than in Child-Acirrhosis (P < 0.001) (Fig. 1-B).

Western blotting

To confirm the immunohistochemical results, we inves-tigated the protein expression of AQP-1 in normaland cirrhotic liver tissues using Western blotting. TheAQP-1 protein was expressed abundantly in Child C-LCand only moderately in control liver tissues (Fig. 1-C).

In situ hybridization

Next, we investigated the expression of AQP-1 atmRNA level using in situ hybridization with peptidenucleic acid probe. In control liver tissue, AQP-1mRNA was located at the portal vein and vascularECs, but it was scarcely detected on hepatic SECs(Fig. 2-A a–d). Similar results were obtained for allcontrol samples (n = 5). In Child A-cirrhotic liver tis-sue, AQP-1 mRNA expression was enhanced in prolif-erated arterial capillaries opening into the sinusoid inthe generating hepatic nodule, and proliferated bileductules at the edge of nodules and capillary artery infibrotic septa (Fig. 2-A e–h). Similar results wereobtained for all cirrhotic (n = 5) liver tissues. In fact,AQP1 are partially localized on hepatic sinusoidalendothelial cells and on abundantly proliferated bileductules. They are partially localized on hepatic sinu-soidal endothelial cells, on abundantly proliferated bileductules and also partially on the proliferative arterialcapillaries. In child-C cirrhotic liver tissue, reactionproducts showing AQP-1 expression are prominent onthe proliferated arterial capillaries in the peripheralregion of fibrous septa as well as aberrantly on thesinusoid in the regenerative nodule surrounded by thebroad fibrous septa (Fig. 2-A i–l).

Moreover, in this study, we analysed the pathophys-iology of cirrhosis by investigating the expression levelsof messenger RNAs (mRNA) using quantitativereverse-transcription polymerase chain reaction (RT-PCR). Laser capture microdissection (LCM) enabled

us to collect tissue samples from distinct areas of theliver, specifically the liver parenchyma. RT-PCR forAQP-1 was conducted in the sinusoidal componentseparated by LCM.

Laser capture microdissection-polymerase chain reaction

In expression of AQP-1 mRNAs in the liver sinusoid,one area of liver sinusoid was captured from each sec-tion using LCM (Fig. 2-B a–c). The quantities of sinu-soids captured from the specimens of normal controlmRNAs were not detected in most of the liver sinusoi-dal sample (Fig. 2-B d), although AQP-1 mRNAs werepresent in surgical (LC-A) and transplant LC liver(LC-C) samples. The sinusoidal component of ChildC-cirrhotic samples indicates greater expression ofAQP-1 than of either control or Child A-cirrhoticsinusoidal (Fig. 2-B e).

Immunogold–silver enhancement

To clarify the ultrastructural localization of AQP-1and how the expression is altered in the cirrhotic liver,we conducted an immunoelectron microscopic study.In control liver tissue, electron dense gold particlesshowing the presence of AQP-1 were mainly vascularendothelial cells (Fig. 3a), but were mainly localizedon the hepatic SECs (Fig. 3b). In Child A-LC, electrondense gold particles showing the presence of AQP-1were increased slightly on sinusoidal endothelial cells(Fig. 3c) compared with control liver. Moreover, theywere found not only on the proliferated capillary ECsbut also on the SECs (Fig. 3d). In Child C-LC, elec-tron dense gold particles showing the presence ofAQP-1 on the proliferated capillary ECs were increasedcompared with control and Child-A liver (Fig. 3e,f).In morphometric analysis of immunogold-labelledAQP-1 around hepatic sinusoids (Fig. 3d), the AQP-1labelling was low in LSECs of control liver tissue (1.2± 0.1/2 lm). In Child A-LC, AQP-1 labelling washigher in ECs (8.2 ± 0.5/2 lm, P < 0.05). In ChildC-LC, AQP-1 labelling was significantly higher in ECs(18.2 ± 1.7/2 lm, P < 0.01) compared with those ofnormal control and Child A-LC (Fig. 3g).

Fig. 3. Ultrastructural localization of aquaporin (AQP)1 using immunogold electron microscopy in human control liver (a, b), Child A-LC(c, d), and Child C-LC (e, f), and morphometric analysis of immunogold labelling for AQP1 (g). Electron micrographs were stained withuranyl acetate. Arrowheads show gold and silver particles; e, sinusoidal endothelial cell; H, hepatic stellate cell; S, hepatic sinusoid; c,capillary arteriole. Bar denotes 2 lm. (a, b) Control liver: Electron dense immunoreactive products indicate localizations of AQP1 areendothelial cells of portal venule (PVn) (a), but they are only slightly evident on sinusoidal endothelial cells (b). (c, d) Child A-LC. Electrondense gold particles showing that the presence of AQP-1 was slightly increased on sinusoidal endothelial cells compared with controlliver tissues and that it was found not only on the capillary endothelial cells in sinusoid (d). (e, f) Child C-LC. Electron dense gold particlesshowing the presence of AQP-1 on the proliferated capillary endothelial cells were more numerous than in control or Child-A liver (e)samples. Moreover, proliferative capillary endothelial cell is found in conjunction with the sinusoidal endothelial cell in sinusoid (f). (g)Morphometric analysis: Immunogold particles around hepatic sinusoids from 12 electron micrographs were counted. In Child C-cirrhotichuman liver, AQP1 expression is considerably greater in proliferated capillary arteriole samples than in control or Child A-cirrhotic liversamples.



Discussion

This study demonstrated overexpression of AQP1 inhuman cirrhotic liver. Compared with this finding,studies using experimental models of cirrhosis havereported contrasting results related to aquaporin-2expression in the kidney (25). A human study revealedthat urinary aquaporin-2 excretion is lower in patientswith cirrhosis than in healthy subjects (26). A progres-sive decrease in urinary aquaporin-2 excretion wasobserved as the severity of cirrhosis increased fromcompensated cirrhosis to cirrhosis with ascites andhepatorenal syndrome. Urinary AQP-1 excretion isreportedly extremely low in patients with hepatorenalsyndrome (26). However, no difference exists in uri-nary excretion of AQP-1 between healthy subjects andpatients with cirrhosis with or without ascites.

In a previous study, a prominent increase of pseudo-sinusoidal-appearing microvessels with strong AQP-1expression was observed in HCCs. In addition, in cho-langiocarcinomas, a minor increase of AQP-1-express-ing microvessels was apparent in comparison with thesurrounding tumour-free parenchyma (27). Moreover,it is important (a) to correlate HCC with the type andgrade of change and the occurrence of arterialization,and (b) to evaluate whether they might be relevant tothe differential diagnosis between high-grade dysplasiaand early HCC.

Moreover, Fig. 1C appears to show AQP1 in lanes 5and 6 to be at a lower molecular weight than thatdetected in lanes 1–4. The AQPs are well described aspost-translationally modified, a factor that is impor-tant in their processing, translocation and eventualfunction. The presence of an N-glycosylated isoform ofAQP1 at 35–45 kDa has been reported before. In thatreport, Ticozzi-Valerio et al. note the underexpressionof AQP1 protein and its glycosylated isoforms inhomogenate and subcellular fraction obtained fromRCC tissue compared with the adjacent normal cortex.They might express another isoform of AQP-1 withlower molecular weights (28). Therefore, in our study,the difference of molecular weight of AQP1 from aChild A- and Child C-LC might reflect these differentdisease states.

This report is the first to document AQP-1 expres-sion in proliferated arterial capillaries opening into thesinusoids and at peripheral edges of nodule and fibro-tic septa. Two contradictory concepts of blood supplyto regenerative nodules in experimental cirrhosis havebeen reported: one emphasizes the importance of por-tal blood supply (29); the other holds that the hepaticarterial blood supply (13, 30) contributes to functionalcirculatory changes. The differences in vascularchanges between the portal vein and hepatic artery incirrhosis are explainable by the anatomical and physio-logical differences between these vessels.

The liver receives its blood supply from both thehepatic artery and the portal vein. These vessels lead to

numerous small branches, associated closely with thebiliary tree. Each intrahepatic bile duct is accompaniedby a branch of the hepatic artery, and is surroundedby a well-developed vascular network: The peribiliaryvascular plexus (31). The hepatic artery and portal veinalso undergo marked proliferation, presumably to sup-port the increased nutritional and functional demandsof the proliferated bile ducts (32). The molecular mech-anisms of these vascular changes remain to be deter-mined. The intimate relation between the intrahepaticbile ducts and hepatic arteries might also be criticallyimportant for development of hepatobiliary diseases(33). Anatomical modifications of the hepatic vascularstructures, particularly in experimentally induced bili-ary cirrhosis, have also been documented. Most inves-tigators have observed neovascularization of arterialbranches. Rearrangement of portal venous branches hasalso been noted under identical conditions, although noagreement exists as to the characteristics of thesechanges (13, 34, 35). Using light microscopy and scan-ning electron microscopic vascular corrosion cast tech-nique to study the architecture of the peribiliary plexusin rats with common bile duct ligation, Gaudio et al.(35) observed significant microvasculature proliferationextending from the peribiliary plexus of the bile tracts.Vascular proliferation coincided with the extension ofportal tract connective tissue. No evidence of vascularproliferation or other morphological modifications wasfound at the level of sinusoids around the portal tracts.Gouw et al. (36) examined the vessels around the int-rahepatic large bile ducts (peribiliary vascular plexus)in humans using histological, immunohistochemicaland scanning electron microscopic observations. Theyobserved extra-portal extension of bile ductules in jux-taposition with capillaries, and showed that proliferatedbile ductules maintained their vasculature during theregenerative processes by increased expression ofVEGF, a potent inducer of angiogenesis. The AQP-1water channels, present in apical and basolateral plasmamembrane domains of bile duct epithelial cells, medi-ate water movement in these cells (37). In a humanstudy, AQP-1 was expressed intensely on smaller prolif-erated bile ducts in cholestatic liver. Moreover, AQP-1expression has been demonstrated in injured bile ductepithelial cells undergoing degeneration attributable tonegative feedback of the decreased bile flow (38).

AQP-1 expression was found in the epithelia of allsmall bile ducts, in erythrocytes, and noted in endo-thelial cells of capillaries, central veins, minor branchesof the portal vein, and rarely in small branches of thehepatic artery in whole liver. AQP-1 protein levels byWestern blot were abundant in Child C-LC and wereonly moderate in the control liver. We investigatedsinusoidal components by LCM and examined 40cycles PCR in LCM tissue. The sinusoidal componentof Child C-cirrhotic samples indicates greater expres-sion of AQP-1 than of either control or Child A-cir-rhotic sinusoidal components.



The AQP1 expression is associated preferentiallywith microvessels of multiple myeloma, and the high-est degree of expression occurs in active multiple mye-loma, consistent with enhanced angiogenesis (39). Therecent finding that AQP1 is necessary for growth fac-tor-induced angiogenesis introduces a new perspectiveto previously described interaction of this protein withthe angiogenic hormone oestrogen, posing a challeng-ing question related to the contribution of AQP1 tothe pro-angiogenic effects of oestrogen. Oestrogen actsin endothelial cells partially by upregulating the expres-sion of VEGF (40). Moreover, AQP1 is involved inhypoxia-inducible angiogenesis in retinal vascular endo-thelial cells through a mechanism that is independentof the VEGF signalling pathway (41). Recently, overex-pression of AQP-1 promotes FGF-induced dynamicmembrane blebbing of LSECs (18).

Actually, CD34 is a 110-kDa, heavily glycosylatedtransmembrane protein that is present in hematopoi-etic progenitor cells and endothelial cells (42). A trendof increased CD34 positive area and intensity is moreapparent in cirrhotic livers than in normal livers (43).As a result of its preferential expression on the surfaceof regenerating or migrating endothelial cells, CD34has been used as a marker of proliferating endothelialcells in sprouting during angiogenesis (44). It has beendemonstrated recently that bone-marrow-derivedCD34 positive endothelial progenitor cells migratethrough blood circulation. They are incorporated intothe angiogenesis site (45). This information is usefulfor characterizing useful BMCs for patients withcirrhosis. Gordon et al. recently showed that theCD34-positive cell can improve liver function in liverdisease (46). The molecular mechanisms for prolifer-ated bile ductules and arterial capillary endothelial cellsremain unclear. In this study, AQP1 was localizedmainly on the proliferated arterial capillaries in LC,suggesting that AQP1 might induce angiogeneticresponses, thereby enhancing the flow of arterial bloodinto the sinusoids, leading to increased sinusoidalmicrovascular resistance, and contributing to exagger-ating portal hypertension in cirrhosis.

Acknowledgements

No conflicts of interest were declared.

Authors’ contributions

HY conceptualized and designed the study, analysedand interpreted the data and wrote the manuscript.HY and MO collected the clinical materials. HY andKY conducted analyses using immunohistochemistry,electron microscopy and Western blots. HY, KY, MOand TH assisted in the analysis and interpretation ofdata. HY supervised the research and edited the manu-script. All authors critically reviewed the manuscript,

and read and approved the final version of the manu-script.

References

1. Nielsen S, Smith BL, Christensen EI, et al. Distributionof the aquaporin CHIPS in secretary and resorptive epi-thelia and capillary endothelia. Proc Natl Acad Sci USA1993; 90: 7275–9.

2. Matsuzaki T, Tajika Y, Ablimit A, et al. Aquaporins in thedigestive system. Med Electron Microsc 2004; 37: 71–80.

3. Portincasa P, Palasciano G, Svelto M, et al. Aquaporins inthe hepatobiliary tract. Which, where and what they do inhealth and disease. Eur J Clin Invest 2008; 38: 1–10.

4. Saadoun S, Papadopoulos MC, Chikuma MH, et al.Impairment of angiogenesis and cell migration by tar-geted aquaporin-1 gene disruption. Nature 2005; 434:786–92.

5. McCuskey RS, Reilly FD. Hepatic microvasculature:dynamic structure and its regulation. Semin Liver Dis1993; 13: 1–12.

6. Rappaport AM, Black RG, Lucas CC, et al. Normal andpathologic microcirculation of the living mammalianliver. Rev Int Hepatol 1966; 16: 813–28.

7. Oda M, Yokomori H, Han JY. Regulatory mechanismsof hepatic microcirculatory hemodynamics: hepatic arte-rial system. Clin Hemorheol Microcic 2006; 34: 11–26.

8. McCuskey RS. A dynamic and static study of hepaticarterioles and hepatic sphincters. Am J Anat 1966; 119:455–77.

9. Kardon RH, Kessel RG. Three-dimensional organizationof the hepatic microcirculation in the rodent as observedby scanning electron microscopy of corrosion casts. Gas-troenterology 1980; 79: 72–81.

10. Yamamoto K, Sherman I, Phillips MJ, et al. Three-dimensional observations of the hepatic arterial termi-nations in rat, hamster and human liver by scanningelectron microscopy of microvascular casts. Hepatology1985; 5: 452–6.

11. Kazemoto S, Oda M, Kaneko H ,et al. Enzymohisto-chemical demonstration of arterial capillaries in rat liver.Microcirculation Annual 1992; 8: 109–10. In: Tsuchiya,M., Asano, M., Sato, N, eds. Microcirculation Annual1997, Tokyo: Nihon-Igakukan, 1992.

12. Orrego H, Blendis LM, Crossley IR, et al. Correlation ofintrahepatic pressure with collagen in the Disse spaceand hepatomegaly in humans and in the rat. Gastroenter-ology 1981; 80: 546–56.

13. Rappaport AM, MacPhee PJ, Fisher MM, et al. The scar-ring of the liver acini (cirrhosis): tridimensional and mi-crocirculatory consideration. Virchows Arch (PatholAnat) 1983; 402: 107–37.

14. Yamamoto T, Kobayashi T, Phillips M. Perinodular arte-riolar plexus in liver cirrhosis: scanning electron micros-copy of microvascular casts. Liver 1984; 4: 50–4.

15. Morgan D, McLean A. Therapeutic implications ofimpaired hepatic oxygen diffusion in chronic liver dis-ease. Hepatology 1991; 14: 1280–2.

16. Rosmorduc O, Wendum D, Corpechot C, et al.Hepatocellular hypoxia-induced vascular endothelialgrowth factor expression and angiogenesis in experimen-tal biliary cirrhosis. Am J Pathol 1999; 155: 1065–73.



17. Yokomori H, Oda M, Yoshimura K, et al. Aberrantexpressions of aquaporin-1 in association with capillar-ized sinusoidal endothelial cells in cirrhotic rat liver.Med Mol Morphol 2010; 43: 6–12.

18. Huebert RC, Vasdev MM, Shergill U, et al. Aquaporin-1facilitates angiogenic invasion in the pathological neovas-culature that accompanies cirrhosis. Hepatology 2010; 52:238–48.

19. Lam CM, Fan ST, Lo CM, et al. Major hepatectomy forhepatocellular carcinoma in patients with an unsatisfac-tory indocyanine green clearance test. Br J Surg 1999; 86:1012–17.

20. Mazzaferro V, Regalia E, Doci R, et al. Liver transplanta-tion for the treatment of small hepatocellular carcinomasin patients with cirrhosis. N Engl J Med 1996; 334: 693–9.

21. Hamilton PW, Allen DC. Morphometry in histopathol-ogy. J Pathol 1995; 175: 369–79.

22. Yokomori H, Oda M, Yasogawa Y, et al. Enhanced expres-sions of endothelin B receptor at protein and gene levels inhuman cirrhotic liver. Am J Pathol 2001; 159: 1353–62.

23. Emmert-Buck MR, Bonner RF, Smith PD, et al. Lasercapture microdissection. Science 1996; 274: 998–1001.

24. Humbel BM, Sibon OC, Stierhof Y-D, et al. Ultra-smallgold particles and silver enhancement as a detection sys-tem in immunolabeling and in situ hybridization. J His-tochem Cytochem 1995; 43: 735–7.

25. Asahina Y, Izumi N, Enomoto N, et al. Increased geneexpression of water channel in cirrhotic rat kidneys. Hep-atology 1995; 21: 169–73.

26. Esteva-Font C, Baccaro ME, Fernandez-Llama P, et al.Aquaporin-1 and aquaporin-2 urinary excretion in cir-rhosis: relationship with ascites and hepatorenal syn-drome. Hepatology 2006; 44: 1555–63.

27. Mazal PR, Susani M, Wrba F, Haitel A. Diagnostic sig-nificance of aquaporin-1 in liver tumors. Hum Pathol2005; 36: 1226–31.

28. Ticozzi-Valerio D, Raimondo F, Pitto M, et al. Differen-tial expression of AQP1 in microdomain-enrichedmembranes of renal cell carcinoma. Proteomics Clin Appl2007; 1: 588–97.

29. Alpert L, Mitra SK. Anatomical and functional circula-tory changes in experimental cirrhosis. Am J Pathol 1966;48: 579–95.

30. Mcindoe AH. Vascular lesions of portal cirrhosis. ArchPathol Lab Med 1928; 5: 23–42.

31. LaRusso NF. Morphology, physiology and biochemistryof biliary epithelia. Toxicol Pathol 1996; 24: 84–9.

32. Masyuk TV, Erik L, Ritman EL, et al. Hepatic artery andportal vein remodeling in rat liver. Vascular response toselective cholangiocyte proliferation. Am J Pathol 2003;162: 1175–82.

33. Kono N, Nakanuma Y. Ultrastructural and immunohis-tochemical studies of the intrahepatic peribiliary capillaryplexus in normal livers and extrahepatic biliary obstruc-tion in human beings. Hepatology 1992; 15: 411–8.

34. Terada T, Ishida F, Nakanuma Y. Vascular plexusaround intrahepatic bile ducts in normal livers and por-tal hypertension. J Hepatol 1989; 8: 139–49.

35. Gaudio E, Onori P, Pannarale L, et al. Hepatic microcir-culation and peribiliary plexus in experimental biliarycirrhosis: a morphological study. Gastroenterology 1996;111: 1118–24.

36. Gouw A, van den Heuvel M, Boot M, et al. Dynamics ofthe vascular profile of the finer branches of the biliarytree in normal and diseased human livers. J Hepatol2006; 45: 393–400.

37. Roberts SK, Yano M, Ueno Y, et al. Cholangiocytesexpress the aquaporin CHIP and transport water via achannel-mediated mechanism. Proc Natl Acad Sci USA1994; 91: 13009–13.

38. Tamai K, Fukushima K, Ueno Y, et al. Differentialexpressions of aquaporin proteins in human cholestaticliver diseases. Hepatol Res 2006; 34: 99–103.

39. Vacca A, Frigeri A, Ribatti D, et al. Microvessel overex-pression of aquaporin 1 parallels bone marrow angiogen-esis in patients with active multiple myeloma. Br JHaematol 2001; 113: 415–21.

40. Clapp C, de la Escalera GM. Aquaporin-1: a novel pro-moter of tumor angiogenesis. Trends Endocrinol Metab2006; 17: 1–2.

41. Kaneko K, Yagui K, Tanaka A, et al. Aquaporin 1 isrequired for hypoxia-inducible angiogenesis in humanretinal vascular endothelial cells. Microvasc Res 2008; 75:297–301.

42. Fina L, Molgaard HV, Robertson D, et al. Expression ofthe CD34 gene in vascular endothelial cells. Blood 1990;75: 2417–26.

43. Di Carlo I, Fraggetta F, Lombardo R, et al. CD 34expression in chronic and neoplastic liver diseases. Pan-minerva Med 2002; 44: 365–67.

44. Schlingemann RO, Rietveld FJ, de Waal RM, et al. Leu-kocyte antigen CD34 is expressed using a subset of cul-tured endothelial cells and on endothelial abluminalmicroprocesses in the tumor stroma. Lab Invest 1990; 62:690–6.

45. Asahara T, Murohara T, Sullivan A, et al. Isolation ofputative progenitor endothelial cells for angiogenesis. Sci-ence 1997; 275: 964–7.

46. Levicar N, Pai M, Habib NA, et al. Long-term clinicalresults of autologous infusion of mobilized adult bonemarrow derived CD34+ cells in patients with chronicliver disease. Cell Prolif 2008; 41: 115–25.



aquaporin-1 associated with hepatic arterial capillary proliferation on hepatic sinusoid in human...

Documents