Aquaporin-1 Promotes Angiogenesis, Fibrosis, and Portal Hypertension Through Mechanisms Dependent on Osmotically Sensitive MicroRNAs
2011; Elsevier BV; Volume: 179; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.06.045
ISSN1525-2191
AutoresRobert C. Huebert, Kumaravelu Jagavelu, Helen Hendrickson, Meher M. Vasdev, Juan Pablo Arab, Patrick L. Splinter, Christy E. Trussoni, Nicholas F. LaRusso, Vijay H. Shah,
Tópico(s)Apelin-related biomedical research
ResumoChanges in hepatic vasculature accompany fibrogenesis, and targeting angiogenic molecules often attenuates fibrosis in animals. Aquaporin-1 (AQP1) is a water channel, overexpressed in cirrhosis, that promotes angiogenesis by enhancing endothelial invasion. The effect of AQP1 on fibrogenesis in vivo and the mechanisms driving AQP1 expression during cirrhosis remain unclear. The purpose of this study was to test the effect of AQP1 deletion in cirrhosis and explore mechanisms regulating AQP1. After bile duct ligation, wild-type mice overexpress AQP1 that colocalizes with vascular markers and sites of robust angiogenesis. AQP1 knockout mice demonstrated reduced angiogenesis compared with wild-type mice, as evidenced by immunostaining and endothelial invasion/proliferation in vitro. Fibrosis and portal hypertension were attenuated based on immunostaining, portal pressure, and spleen/body weight ratio. AQP1 protein, but not mRNA, was induced by hyperosmolality in vitro, suggesting post-transcriptional regulation. Endothelial cells from normal or cirrhotic mice were screened for microRNA (miR) expression using an array and a quantitative PCR. miR-666 and miR-708 targeted AQP1 mRNA and were decreased in cirrhosis and in cells exposed to hyperosmolality, suggesting that these miRs mediate osmolar changes via AQP1. Binding of the miRs to the untranslated region of AQP1 was assessed using luciferase assays. In conclusion, AQP1 promotes angiogenesis, fibrosis, and portal hypertension after bile duct ligation and is regulated by osmotically sensitive miRs. Changes in hepatic vasculature accompany fibrogenesis, and targeting angiogenic molecules often attenuates fibrosis in animals. Aquaporin-1 (AQP1) is a water channel, overexpressed in cirrhosis, that promotes angiogenesis by enhancing endothelial invasion. The effect of AQP1 on fibrogenesis in vivo and the mechanisms driving AQP1 expression during cirrhosis remain unclear. The purpose of this study was to test the effect of AQP1 deletion in cirrhosis and explore mechanisms regulating AQP1. After bile duct ligation, wild-type mice overexpress AQP1 that colocalizes with vascular markers and sites of robust angiogenesis. AQP1 knockout mice demonstrated reduced angiogenesis compared with wild-type mice, as evidenced by immunostaining and endothelial invasion/proliferation in vitro. Fibrosis and portal hypertension were attenuated based on immunostaining, portal pressure, and spleen/body weight ratio. AQP1 protein, but not mRNA, was induced by hyperosmolality in vitro, suggesting post-transcriptional regulation. Endothelial cells from normal or cirrhotic mice were screened for microRNA (miR) expression using an array and a quantitative PCR. miR-666 and miR-708 targeted AQP1 mRNA and were decreased in cirrhosis and in cells exposed to hyperosmolality, suggesting that these miRs mediate osmolar changes via AQP1. Binding of the miRs to the untranslated region of AQP1 was assessed using luciferase assays. In conclusion, AQP1 promotes angiogenesis, fibrosis, and portal hypertension after bile duct ligation and is regulated by osmotically sensitive miRs. Liver cirrhosis is the final common end point in a variety of toxic, metabolic, infectious, and autoimmune forms of chronic liver disease. Progression toward end-stage liver disease is characterized by an exaggerated wound healing response to long-term injury, culminating in regenerative nodules of hepatocytes surrounded by a dense scar of extracellular matrix.1Schuppan D. Afdhal N.H. Liver cirrhosis.Lancet. 2008; 371: 838-851Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar In concert with this progressive fibrogenesis, pathological changes in the hepatic angioarchitecture also occur and are thought to promote fibrosis, portal hypertension, and their clinical sequelae.2Paternostro C. David E. Novo E. Parola M. Hypoxia, angiogenesis and liver fibrogenesis in the progression of chronic liver diseases.World J Gastroenterol. 2010; 16: 281-288Crossref PubMed Scopus (96) Google Scholar, 3Iwakiri Y. Grisham M. Shah V. Vascular biology and pathobiology of the liver: report of a single-topic symposium.Hepatology. 2008; 47: 1754-1763Crossref PubMed Scopus (40) Google Scholar, 4Huebert R.C. Shah V. Hepatic Sinusoidal Endothelial Cells.in: Dufour J.F. Clavien P.A. Springer-Verlag, Berlin, Heidelberg2009: 79-91Google Scholar Despite intensive investigations and significant insights into the basic mechanisms driving these processes, no effective anti-fibrotic therapies are yet available for use in patients with chronic liver diseases. Thus, further mechanistic insights into liver fibrogenesis and coinciding events, such as pathological angiogenesis, are needed to identify potential anti-fibrotic targets and translate those into advances in clinical care. Aquaporins (AQPs) are a class of integral membrane channel proteins that facilitate the rapid, transmembrane flux of water that occurs passively and bidirectionally in response to local osmotic gradients. These proteins have well-characterized roles in epithelial secretion, absorption, and cell volume regulation.5Huebert R.C. Splinter P.L. Garcia F. Marinelli R.A. LaRusso N.F. Expression and localization of aquaporin water channels in rat hepatocytes: evidence for a role in canalicular bile secretion.J Biol Chem. 2002; 277: 22710-22717Crossref PubMed Scopus (138) Google Scholar, 6Ma T. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels.J Biol Chem. 1998; 273: 4296-4299Crossref PubMed Scopus (504) Google Scholar, 7Liu X. Bandyopadhyay B.C. Nakamoto T. Singh B. Liedtke W. Melvin J.E. Ambudkar I. A role for AQP5 in activation of TRPV4 by hypotonicity: concerted involvement of AQP5 and TRPV4 in regulation of cell volume recovery.J Biol Chem. 2006; 281: 15485-15495Crossref PubMed Scopus (190) Google Scholar, 8Li Y.H. Eto K. Horikawa S. Uchida S. Sasaki S. Li X.J. Noda Y. Aquaporin-2 regulates cell volume recovery via tropomyosin.Int J Biochem Cell Biol. 2009; 41: 2466-2476Crossref PubMed Scopus (16) Google Scholar More recently, they have also been implicated in localized protrusions of plasma membranes, cell motility, and angiogenesis.9Saadoun S. Papadopoulos M.C. Hara-Chikuma M. Verkman A.S. Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption.Nature. 2005; 434: 786-792Crossref PubMed Scopus (646) Google Scholar, 10La Porta C. AQP1 is not only a water channel: it contributes to cell migration through Lin7/beta-catenin.Cell Adh Migr. 2010; 4: 204-206Crossref PubMed Scopus (24) Google Scholar Researchers11Huebert R.C. Vasdev M.M. Shergill U. Das A. Huang B.Q. Charlton M.R. LaRusso N.F. Shah V.H. Aquaporin-1 facilitates angiogenic invasion in the pathologic neovasculature that accompanies cirrhosis.Hepatology. 2010; 52: 238-248Crossref PubMed Scopus (48) Google Scholar, 12Yokomori H. Oda M. Yoshimura K. Watanabe S. Hibi T. Aberrant expressions of aquaporin-1 in association with capillarized sinusoidal endothelial cells in cirrhotic rat liver.Med Mol Morphol. 2010; 43: 6-12Crossref PubMed Scopus (6) Google Scholar, 13Xian Z.H. Cong W.M. Wang Y.H. Wang B. Wu M.C. Expression and localization of aquaporin-1 in human cirrhotic liver.Pathol Res Pract. 2009; 205: 774-780Crossref PubMed Scopus (5) Google Scholar have demonstrated robust overexpression of AQP1 in both human and rodent chronic liver disease. The increased expression during cirrhosis is localized to the pathological neovasculature and promotes dynamic membrane protrusions that facilitate invasion through the dense extracellular microenvironment associated with that disease. However, direct in vivo evidence of a contribution of AQP1 to liver fibrogenesis is lacking. The molecular mechanisms driving the increased expression of AQP1 during cirrhosis are unknown; however, as our results will suggest, the mechanism may involve epigenetic responses to osmotic stress within the endothelium. microRNAs (miRs) are small noncoding nucleic acids 21 to 23 nucleotides long that have emerged as important post-transcriptional regulators of protein expression that affect a variety of developmental and pathobiological processes. miRs are initially transcribed by RNA polymerase II as monocistronic or polycistronic primary miRs and are further processed within and outside the nucleus into functionally active mature miRs that act by binding to target messenger RNAs and regulating stability or translational efficiency. The mechanisms regulating miR expression remain largely unclear, but there is precedent for the concept of osmotically sensitive miRs in human, zebra fish, and plant responses to osmotic stress.14Lee H.J. Palkovits M. Young 3rd, W.S. miR-7b, a microRNA up-regulated in the hypothalamus after chronic hyperosmolar stimulation, inhibits Fos translation.Proc Natl Acad Sci U S A. 2006; 103: 15669-15674Crossref PubMed Scopus (79) Google Scholar, 15Uney J.B. Lightman S.L. MicroRNAs and osmotic regulation.Proc Natl Acad Sci U S A. 2006; 103: 15278-15279Crossref PubMed Scopus (7) Google Scholar, 16Huang W. Liu H. Wang T. Zhang T. Kuang J. Luo Y. Chung S.S. Yuan L. Yang J.Y. Tonicity-responsive microRNAs contribute to the maximal induction of osmoregulatory transcription factor OREBP in response to high-NaCl hypertonicity.Nucleic Acids Res. 2011; 39: 475-485Crossref PubMed Scopus (42) Google Scholar, 17Flynt A.S. Thatcher E.J. Burkewitz K. Li N. Liu Y. Patton J.G. miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos.J Cell Biol. 2009; 185: 115-127Crossref PubMed Scopus (100) Google Scholar, 18Zhao B. Ge L. Liang R. Li W. Ruan K. Lin H. Jin Y. Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor.BMC Mol Biol. 2009; 10: 29Crossref PubMed Scopus (307) Google Scholar Furthermore, conceptual precedent for osmoregulation of AQP1 exists in recent articles19Lanaspa M.A. Andres-Hernando A. Li N. Rivard C.J. Cicerchi C. Roncal-Jimenez C. Schrier R.W. Berl T. The expression of aquaporin-1 in the medulla of the kidney is dependent on the transcription factor associated with hypertonicity, TonEBP.J Biol Chem. 2010; 285: 31694-31703Crossref PubMed Scopus (45) Google Scholar showing increased AQP1 expression in response to osmolality in renal epithelial cells. Therefore, the aims of the present study were to test the effect of genetic deletion of AQP1 on liver angiogenesis, fibrosis, and portal hypertension in an established murine model of cirrhosis and to examine the mechanisms by which AQP1 is overexpressed in cirrhotic endothelia. The experimental results demonstrate a prominent role for endothelial cell AQP1 in liver fibrogenesis after bile duct ligation (BDL) and propose a novel mechanism driving AQP1 expression involving osmotically regulated miRs. Mice with global genetic knockout of AQP1 in a CD1 background were a gift from the laboratory of Dr. Alan Verkman (University of California San Francisco, San Francisco, CA).6Ma T. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels.J Biol Chem. 1998; 273: 4296-4299Crossref PubMed Scopus (504) Google Scholar Fibrosis was induced at the age of 8 to 10 weeks by common BDL using a well-established protocol with appropriate Institutional Animal Care and Use Committee approval.20Semela D. Das A. Langer D.A. Kang N. Leof E. Shah V.H. Platelet-derived growth factor signaling through ephrin-B2 regulates hepatic vascular structure and function.Gastroenterology. 2008; 135: 671-679Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar Experiments were performed 4 weeks after BDL. Animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals by the National Academy of Sciences. Freshly isolated mouse hepatic sinusoidal endothelial cells (mHSEC) were obtained using an immunomagnetic bead isolation protocol and characterized using 3,3'-dioctadecylindocarbocyanine labeled low-density lipoprotein and staining for von Willebrand's factor (vWF), as previously described.11Huebert R.C. Vasdev M.M. Shergill U. Das A. Huang B.Q. Charlton M.R. LaRusso N.F. Shah V.H. Aquaporin-1 facilitates angiogenic invasion in the pathologic neovasculature that accompanies cirrhosis.Hepatology. 2010; 52: 238-248Crossref PubMed Scopus (48) Google Scholar, 21LeCouter J. Moritz D.R. Li B. Phillips G.L. Liang X.H. Gerber H.P. Hillan K.J. Ferrara N. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1.Science. 2003; 299: 890-893Crossref PubMed Scopus (562) Google Scholar, 22Follenzi A. Benten D. Novikoff P. Faulkner L. Raut S. Gupta S. Transplanted endothelial cells repopulate the liver endothelium and correct the phenotype of hemophilia A mice.J Clin Invest. 2008; 118: 935-945PubMed Google Scholar The cells were cultured on collagen-coated plastic tissue culture dishes in endothelial cell media (ScienCell, Carlsbad, CA) containing 5% fetal bovine serum, 1% penicillin-streptomycin, and 1% ECGS supplement (ScienCell). Primary human hepatic sinusoidal endothelial cells (HHSECs; ScienCell), or transformed sinusoidal endothelial cells (TSECs), an SV40-immortalized mouse cell line derived from sinusoidal endothelial cells,23Huebert R.C. Jagavelu K. Liebl A.F. Huang B.Q. Splinter P.L. LaRusso N.F. Urrutia R.A. Shah V.H. Immortalized liver endothelial cells: a cell culture model for studies of motility and angiogenesis.Lab Invest. 2010; 90: 1770-1781Crossref PubMed Scopus (47) Google Scholar were grown on uncoated plastic dishes in the same media. In some experiments, cells were incubated in an experimentally modified hypoxia chamber or in the presence of altered osmolality of the culture media. Osmolality was altered using variable concentrations of sodium chloride in the culture media and verified using an osmometer. Western blot analyses were performed as previously described.5Huebert R.C. Splinter P.L. Garcia F. Marinelli R.A. LaRusso N.F. Expression and localization of aquaporin water channels in rat hepatocytes: evidence for a role in canalicular bile secretion.J Biol Chem. 2002; 277: 22710-22717Crossref PubMed Scopus (138) Google Scholar Briefly, mouse liver, mouse sinusoidal endothelial cells, or TSECs were homogenized in lysis buffer and cleared by centrifugation. Each sample, 50 to 100 μg, was denatured, electrophoresed, transferred, blocked with milk, and incubated with antibodies to AQP1 (1:1000; α Diagnostic International, San Antonio, TX), β-actin (1:10,000; Sigma, St. Louis, MO), or total extracellular signal–regulated kinase (1:1000; BD Biosciences, Franklin Lakes, NJ) overnight at 4°C. Horseradish peroxidase–conjugated secondary antibodies (GE Healthcare, Piscataway, NJ) were used at 1:5000. Protein was detected using chemiluminescence (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and autoradiography. Immunofluorescence (IF) was performed as previously described.5Huebert R.C. Splinter P.L. Garcia F. Marinelli R.A. LaRusso N.F. Expression and localization of aquaporin water channels in rat hepatocytes: evidence for a role in canalicular bile secretion.J Biol Chem. 2002; 277: 22710-22717Crossref PubMed Scopus (138) Google Scholar Liver was harvested, sliced, embedded, and flash frozen on dry ice. Sections were cut to 4 to 8 μm. Sections were fixed, quenched, blocked, and incubated with antibodies against AQP1 (1:500; α Diagnostics International), CD31 (1:250), vWF (1:250), vascular endothelial growth factor receptor 2 (VEGFR2; 1:250), and endothelial nitric oxide synthase (eNOS; 1:250). Fluorescently tagged secondary antibodies were used at 1:500. In some experiments, nuclear counterstaining was performed with TOTO-3 (Invitrogen, Carlsbad, CA). Slides were mounted with Vectashield (Vector, Burlingame, CA) and imaged by scanning laser confocal microscopy (Carl Zeiss MicroImaging, Berlin, Germany). Similar procedures were used to stain cultured cells using four-well chamber slides. Whole liver was harvested, sliced, formalin fixed, and embedded in paraffin. Sections were cut to 4 to 8 μm and antigen unmasked with hot citrate buffer. After quenching of endogenous peroxidase, the sections were blocked and incubated with antibody against AQP1 (1:500; α Diagnostics International) overnight at 4°C. The remaining steps were performed using an immunoperoxidase detection kit (Vector Laboratories) and counterstaining with hematoxylin. Cell proliferation rates of mHSEC, HHSECs, and TSECs were measured in 96-well plates using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI), which is a colorimetric method for determining the number of viable cells. The AQueous One Solution contains a tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; [MTS] and an electron coupling reagent (phenazine methosulfate; PES). The MTS tetrazolium compound is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. Assays were performed by adding the AQueous One Solution directly to culture wells, incubating for 1 to 4 hours, and measuring optical density at 490 nm with a plate reader at baseline and 48 hours to calculate the proliferation rate. Portal pressure was directly measured using a digital blood pressure analyzer (Digi-Med, Louisville, KY) with a computer interface. Once the analyzer was calibrated, a 16-gauge catheter attached to the pressure transducer was inserted into the portal vein and sutured in place. The pressure was continuously monitored, and the average portal pressure was recorded. On sacrifice of the animal, the spleen was removed and weighed and the spleen/body weight ratio was calculated. Whole blood was obtained from each experimental animal via a heart puncture technique and transferred into 3.5-mL serum separating tubes. Specimens were processed and analyzed for serum transaminase and bilirubin levels by the Mayo Clinic Special Studies Laboratory (Rochester, MN), a clinically validated reference laboratory. Total RNA (including miRs) was isolated from endothelial cells derived from BDL or sham operated on mice using the MirVana miRNA Isolation Kit (Ambion, Inc., Austin, TX), according to the manufacturer's instructions. The samples were delivered to the Mayo Clinic Advanced Genomic Technology Center Microarray Shared Resource, where they were further analyzed. Briefly, the samples were hybridized to the GeneChip miRNA Array (Affymetrix, Santa Clara, CA), which interrogates miRs from >70 species, including the entire known murine transcriptome of miRs. Data were extracted, manually curated, and analyzed using QC Toolbox software (Affymetrix). Further rational processing of array data included assessment of relative expression levels in BDL versus sham endothelial cells. Down-regulated targets were analyzed using TargetScan (Whitehead Institute for Biomedical Research, Cambridge, MA) and Microcosm software (European Bioinformatics Institute, Cambridgeshire, UK) to identify down-regulated targets that had potential binding sites within the untranslated regions of AQP1 mRNA. TargetScan predicts biological targets of miRs by searching for the presence of conserved eight- and seven-base pair sites that match the seed region of each miR. Predictions are ranked based on the predicted efficacy of targeting, as calculated using the context scores of the sites. Microcosm uses an algorithm to identify potential binding sites for a given miR using dynamic alignment to identify highly complementary sequences. The algorithm uses a weighted scoring system and prioritizes complementarity at the 5′ end of the miR. Total RNA (including miRs) was isolated as previously outlined. RNA was reverse transcribed using the miScript RT-PCR system (Qiagen), and cyber green–based real-time RT-PCR was performed using miScript Primer Assays (Qiagen) or AQP1-specific primers, according to the manufacturer's instructions. Hydroxyproline content was quantified from whole liver tissue using a colorimetric assay, as described elsewhere.24Yang L. Chan C.C. Kwon O.S. Liu S. McGhee J. Stimpson S.A. Chen L.Z. Harrington W.W. Symonds W.T. Rockey D.C. Regulation of peroxisome proliferator-activated receptor-gamma in liver fibrosis.Am J Physiol Gastrointest Liver Physiol. 2006; 291: G902-G911Crossref PubMed Scopus (108) Google Scholar Briefly, frozen tissue was weighed and processed using hydrochloric acid hydrolysis and chloramine-T–dimethylaminobenzaldehyde incubation, and absorbance at 561 nm was recorded and compared with a standard prepared from commercial hydroxyproline (Sigma). Complementary oligonucleotides were designed to amplify a 300-bp portion of the untranslated region of mouse AQP1. The primers were synthesized to contain SpeI and HindIII restriction enzyme digestion sites and used to amplify the region of interest using RT-PCR. The amplified fragment was digested with SpeI and HindIII and ligated into the multiple cloning site of the pMIR-REPORT Luciferase vector (Ambion, Inc.). Chinese hamster ovary cells were transfected with the reporter construct and, in some experiments, cotransfected with miR precursors for miR-666, miR-708, or a control miR. This was followed by assessment of luciferase activity 24 hours after transfection. Luciferase activity was normalized to the expression of a control TK Renilla construct. RNA from TSECs overexpressing either AQP1 or a LacZ control gene was isolated using the QiaShredder and RNeasy kits (Qiagen), according to the manufacturer's instructions. RNA was used for reverse transcription using the RT2 kit (SA Biosciences, Frederick, MD). Cyber green–based real-time quantitative RT-PCR was performed with the Endothelial Cell Biology Array (SA Biosciences), according to the manufacturer's instructions. Array data were processed using the PCR Array Data Analysis Web Portal (SA Biosciences). Data are presented as the mean ± SEM. Bar graphs, blots, and micrographs represent typical experiments reproduced at least three times. Data analysis was performed using Graph Stat Prizm software (GraphPad Software, Inc., La Jolla, CA). Data were analyzed for normal gaussian distribution using the Kolmogorov-Smirnov normality test. For paired and normally distributed data, statistical analyses were performed using two-tailed Student's t-tests. For normally distributed multiple comparisons, statistical analyses were performed using one-way analysis of variance with a Tukey post-test. For all analyses, P < 0.05 was considered statistically significant. To directly test the role of AQP1 in chronic liver disease, we used mice from a CD1 background with global genetic deletion of AQP1 or age-matched, wild-type controls and induced cirrhosis and portal hypertension in these mice. We confirmed deletion of AQP1 in total liver lysates from knockout animals by using Western blot analysis (Figure 1A). We induced fibrosis in wild-type mice using BDL, a model of cholestatic liver injury, and confirmed that AQP1 protein levels were significantly increased after BDL using Western blot analysis (Figure 1B) and IF (Figure 1, C and D). Immunohistochemistry (IHC) for AQP1 in these animals demonstrated a similar increase in AQP1 protein levels and showed specific staining of vascular structures, including sinusoids, the portal vein, and the hepatic artery (Figure 1, E and F). IF costaining showed colocalization of AQP1 with several vascular markers (Figure 1, G–J; see also Supplemental Figure S1 at http://ajp.amjpathol.org), including CD31 (Figure 1G), vWF (Figure 1H), VEGFR2 (Figure 1I), and eNOS (Figure 1J). To test the effects of AQP1 deletion on the pathological angiogenesis that accompanies fibrosis, we measured endothelial cell invasion and proliferation, both required for angiogenesis. Liver endothelial cells were isolated from wild-type or AQP1 knockout mice after BDL or sham surgery and subjected to invasion and proliferation assays in vitro. Endothelial cell purity was validated using uptake of diI-labeled acetylated low-density lipoprotein and staining for vWF (see Supplemental Figure S2 at http://ajp.amjpathol.org). Cells isolated from wild-type animals showed markedly increased invasion after BDL, an effect that was largely absent in the knockout animals (Figure 2, A–E). Proliferation assays demonstrated a similar effect (Figure 2F). We also measured proliferation in two sinusoidal endothelial cell lines (TSECs and HHSECs) transduced with retroviral AQP1 or control gene and again found small, but statistically significant, increases in proliferation after overexpression of AQP1 (see Supplemental Figure S3A at http://ajp.amjpathol.org). We used IF staining for vWF in wild-type and knockout mice subjected to either BDL or sham surgery and quantified the fluorescence signals. We found significantly increased neovascularization after BDL in the wild-type animals, an effect that was partially abrogated in AQP1 knockout animals (Figure 2G). A similar effect was noted on staining for CD31 (see Supplemental Figure S3, B–F, at http://ajp.amjpathol.org). To assess fibrogenesis, we immunostained for a standard panel of morphological markers of extracellular matrix deposition, stellate cell activation, and fibrosis, as well as complementary histochemical and biochemical approaches. After BDL, IF staining showed a prominent increase in the extracellular matrix components, collagen (Figure 3, A–E) and fibronectin (Figure 3F), that were partially inhibited in the AQP1 knockout animals. A well-accepted histochemical stain for fibrosis, Sirius red, showed similar effects (Figure 3G). Smooth muscle actin (a marker of stellate cell activation), H&E staining, and a biochemical correlate of cross-linked collagen, hydroxyproline content, further corroborated these findings (see Supplemental Figure S4 at http://ajp.amjpathol.org). Collectively, these results strongly support a role for endothelial cell AQP1 in fibrogenesis and its associated angiogenesis. We next assessed the level of portal hypertension in wild-type and AQP1 knockout mice after BDL using two complementary approaches. After direct cannulation of the portal vein and connection to a pressure transducer, we directly measured portal pressure. The BDL-induced increase in portal pressure seen in wild-type mice was reduced in the absence of AQP1 (Figure 4A). As a complementary approach, we also measured spleen/body weight ratio, an indirect assessment of portal hypertension. AQP1 knockout mice demonstrated splenomegaly at baseline (data not shown), possibly due to lack of AQP1 in red blood cells and impaired hematopoiesis.25Zeidel M.L. Ambudkar S.V. Smith B.L. Agre P. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.Biochemistry. 1992; 31: 7436-7440Crossref PubMed Scopus (518) Google Scholar Nevertheless, after normalizing BDL values to the corresponding sham group, we again saw a significantly smaller increase in spleen size in AQP1 knockout animals compared with wild-type controls (Figure 4B). To broadly assess liver inflammation, which is closely associated with, and frequently parallels, angiogenesis, we also measured transaminase (aspartate aminotransferase and alanine aminotransferase) levels in the serum of these animals. There was a substantially smaller increase in both transaminases in AQP1 knockout animals after BDL (45% and 58% reduction for aspartate aminotransferase and alanine aminotransferase, respectively), albeit not statistically significant (Figure 4, C and D). This is consistent with the concept that the angiogenic neovasculature may be a source of inflammatory cytokines, driving chronic inflammation and progression of fibrosis. Total bilirubin levels were increased after BDL but unchanged in knockouts compared with wild-type controls (Figure 4E). Given the robust overexpression of AQP1 in human cirrhosis11Huebert R.C. Vasdev M.M. Shergill U. Das A. Huang B.Q. Charlton M.R. LaRusso N.F. Shah V.H. Aquaporin-1 facilitates angiogenic invasion in the pathologic neovasculature that accompanies cirrhosis.Hepatology. 2010; 52: 238-248Crossref PubMed Scopus (48) Google Scholar, 12Yokomori H. Oda M. Yoshimura K. Watanabe S. Hibi T. Aberrant expressions of aquaporin-1 in association with capillarized sinusoidal endothelial cells in cirrhotic rat liver.Med Mol Morphol. 2010; 43: 6-12Crossref PubMed Scopus (6) Google Scholar, 13Xian Z.H. Cong W.M. Wang Y.H. Wang B. Wu M.C. Expression and localization of aquaporin-1 in human cirrhotic liver.Pathol Res Pract. 2009; 205: 774-780Crossref PubMed Scopus (5) Google Scholar and in animal models and the functional consequences on liver angiogenesis, fibrosis, and portal pressure, we next sought to elucidate a molecular mechanism whereby AQP1 protein expression is increased during cirrhosis. We consistently saw that, although ECs isolated from cirrhotic mice expressed high levels of AQP1, this expression was rapidly lost in culture and that cells in long-term culture, such as the TSEC cell line, do not express AQP1 under normal culture conditions (see Supplemental Figure S5 at http://ajp.amjpathol.org). We anticipated that this could be the result of changes in the endothelial microenvironment in the context of cirrhosis. We explored two previously described AQP1 regulatory mechanisms, hypoxia and altered osmolality. Indeed, both have been linked to AQP expression19Lanaspa M.A. Andres-Hernando A. Li N. Rivard C.J. Cicerchi C. Roncal-Jimenez C. Schrier R.W. Berl T. The expression of aquaporin-1 in the medulla of the kidney is dependent on the transcription factor associated with hypertonicity, TonEBP.J Biol Chem. 2010; 285: 31694-31703Crossref PubMed Scopu
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