Hepatocyte and stellate cell deletion of liver fatty acid binding protein reveals distinct roles in fibrogenic injury

THE JOURNAL • RESEARCH • www.fasebj.org Hepatocyte and stellate cell deletion of liver fatty acid binding protein reveals distinct roles in fibrogenic injury Elizabeth P. Newberry,* Yan Xie,* Carlos Lodeiro,* Roberto Solis,* William Moritz,* Susan Kennedy,* Lauren Barron,† Emily Onufer,† Gianfranco Alpini,‡,§ Tianhao Zhou,‡,§ William S. Blaner,{ Anping Chen,k and Nicholas O. Davidson*,1 *Gastroenterology Division, Department of Medicine, and †Pediatric Surgery Division, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri, USA; ‡Department of Medical Physiology and §Department of Internal Medicine, Texas A&M University, Temple, Texas, USA; {Department of Medicine, Columbia University, New York, New York, USA; and kDepartment of Pathology, Saint Louis University School of Medicine, St. Louis, Missouri, USA Liver fatty acid binding protein (L-Fabp) modulates lipid trafficking in enterocytes, hepatocytes, and hepatic stellate cells (HSCs). We examined hepatocyte vs. HSC L-Fabp deletion in hepatic metabolic adaptation and fibrotic injury. Floxed L-Fabp mice were bred to different transgenic Cre mice or injected with adeno-associated virus type 8 (AAV8) Cre and fed diets to promote steatosis and fibrosis or were subjected to either bile duct ligation or CCl4 injury. Albumin-Cre–mediated L-Fabp deletion revealed recombination in hepatocytes and HSCs; these findings were confirmed with 2 other floxed alleles. Glial fibrillary acid protein–Cre and platelet-derived growth factor receptor b-Cre–mediated L-Fabp deletion demonstrated recombination only in HSCs. Mice with albumin promoter–driven Cre recombinase (Alb-Cre)–mediated or AAV8-mediated L-Fabp deletion were protected against food withdrawal–induced steatosis. Mice with Alb-Cre–mediated L-Fabp deletion were protected against high saturated fat–induced steatosis and fibrosis, phenocopying germline L-Fabp2/2 mice. Mice with HSC-specific LFabp deletion exhibited retinyl ester depletion yet demonstrated no alterations in fibrosis. On the other hand, fibrogenic resolution after CCl4 administration was impaired in mice with Alb-Cre–mediated L-Fabp deletion. These findings suggest cell type–specific roles for L-Fabp in mitigating hepatic steatosis and in modulating fibrogenic injury and reversal.—Newberry, E. P., Xie, Y., Lodeiro, C., Solis, R., Moritz, W., Kennedy, S., Barron, L., Onufer, E., Alpini, G., Zhou, T., Blaner, W. S., Chen, A., Davidson, N. O. Hepatocyte and stellate cell deletion of liver fatty acid binding protein reveal distinct roles in fibrogenic injury. FASEB J. 33, 4610–4625 (2019). www.fasebj.org ABSTRACT: KEY WORDS: fibrogenesis • steatohepatitis • mouse models The global epidemic of obesity is associated with a variety of comorbidities, including nonalcoholic fatty liver disease, which encompasses a spectrum of pathology ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis, and whose progression ABBREVIATIONS: a-SMA, a-smooth muscle actin; AAV8, adeno-associated virus type 8; Alb-Cre, albumin promoter–driven Cre recombinase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BA, bile acid; BDL, bile duct ligation; FA, fatty acid; FFA, free fatty acid; Gfap-Cre, glial fibrillary acid protein promoter–driven Cre recombinase; GFP, green fluorescent protein; HSC, hepatic stellate cell; LD, lipid droplet; L-Fabp, liver fatty acid binding protein; MCD, methionine-choline–deficient; NASH, nonalcoholic steatohepatitis; Pdgfrb-Cre, platelet-derived growth factor receptor b promoter–driven Cre recombinase; Plin5, perilipin 5; TFF, transfat fructose; Tg, triglyceride; VLDL, very low density lipoprotein 1 Correspondence: Gastroenterology Division, Department of Medicine, Washington University, 425 South Euclid Ave., Campus Box 8124 St. Louis, MO 63110, USA. E-mail: nod@wustl.edu doi: 10.1096/fj.201801976R 4610 • cell-specific Cre-deletion among patients is both variable and unpredictable (1, 2). Among the most significant histologic characteristics predicting outcomes of patients with advanced forms of nonalcoholic fatty liver disease/NASH is the presence of hepatic fibrosis, where it is believed that steatohepatitis triggers a variety of pathways that both initiate and sustain fibrogenic injury [reviewed in Friedman et al. (2) and Greuter et al. (3)]. These inflammatory and fibrogenic pathways reflect interactions among a number of liver cell types, including hepatocytes, macrophages/Kupffer cells, and lymphocyte subsets, whose signals converge on hepatic stellate cells (HSCs), which are the major fibrogenic effector cell type (4). In their resting, quiescent state, HSCs contain abundant neutral lipids (including retinol and retinyl esters) localized within lipid droplets (LDs) (4). However, upon HSC activation there is loss of LDs, along with the induction of fibrogenesis, enhanced proliferation, increased expression of a-smooth muscle actin (a-SMA), 0892-6638/19/0033-4610 © FASEB and overproduction of extracellular matrix proteins, including aI(I) collagen. Whether the loss of lipid species and depletion of HSC LDs is required for, coincident with, or merely an incidental epiphenomenon in HSC activation is unresolved, with prior studies offering conflicting interpretations. Studies in mice lacking liver X receptors (LXRab2/2 or LXRb2/2) showed altered HSC LD formation, increased retinoid turnover, and LD depletion with HSC activation and exaggerated fibrosis after injury (5, 6). On the other hand, HSCs from mice lacking lecithin-retinol acyltransferase (Lrat2/2) showed virtually complete depletion of both retinoid stores and LDs but demonstrated no change in hepatic fibrosis after either bile duct ligation (BDL) or carbon tetrachloride (CCl4) injury (7). Other work has suggested a functional link between HSC LD mobilization and activation, coupled to reduced expression of one of the major LD proteins, perilipin 5 (Plin5) (8). Plin5 is a LD-associated protein that facilitates the storage of fatty acid (FA) within LDs as triglyceride and the oxidation of FA via physical interactions with mitochondria (9). Studies in passaged HSCs demonstrated that exogenous Plin5 significantly increased intracellular lipid content, restored LDs, and attenuated intracellular oxidative stress and HSC activation, supporting the concept that preserving lipid content by modulating turnover of HSC LDs may promote quiescence (10, 11). Those conclusions were supported by studies in one line of Plin52/2 mice that showed altered hepatic lipid content with increased hepatic inflammation (12), although the findings regarding increased inflammation were not replicated in other reports of Plin52/2 mice (13, 14). Liver fatty acid binding protein (L-Fabp) (Fabp1), a member of a family of cytosolic, multiligand lipid-binding proteins, has been shown to facilitate the intracellular, and in some cases nuclear, trafficking and utilization of fatty acids and other lipids. We showed previously that L-Fabp is abundantly expressed in hepatocytes and quiescent HSCs and that activation of HSCs is coupled to decreased L-Fabp expression, coincident with LD depletion (15). Studies in cultured HSCs from germline L-Fabp2/2 mice demonstrated progressive LD depletion, along with features of activation (including increased collagen 1 mRNA abundance), yet those mice were protected from high fat diet–induced hepatic steatosis and fibrosis (15). Those observations suggest that germline L-Fabp deletion is associated with reduced steatosis that in turn mitigates fibrogenic injury in vivo, despite findings in cultured HSCs demonstrating that germline L-Fabp deletion promotes an activated phenotype in vitro. Here we report the impact of cell-specific, Cre-mediated deletion of L-Fabp from hepatocytes vs. HSCs in several models of fibrogenic injury. Our findings support a protective role of hepatocyte-specific L-Fabp deletion in models of hepatic steatosis and lipid-mediated injury. Mice with HSC-specific L-Fabp deletion exhibit retinyl ester depletion yet no significant alterations in fibrogenic phenotype. On the other hand, fibrogenic resolution after CCl4 administration was impaired in mice with hepatocyte L-Fabp deletion, suggesting distinct, cell-specific roles for L-Fabp in fibrogenic injury and in fibrosis reversal and repair. CELL-SPECIFIC L-Fabp DELETION IN FIBROGENIC INJURY MATERIALS AND METHODS Reagents Adeno-associated virus type 8 (AAV8)-Tbg-cre (Penn Vector Core, Philadelphia, PA, USA) was injected (1–5 3 1011 viral genome copies/mouse, intravenously injected) to drive hepatocyte-specific expression of cre. Mice were studied 2–8 wk after AAV8 Cre injection. L-Fabp and Fabp2 (I-Fabp) antibodies (Western blot) were previously characterized (16) or were obtained from Abcam (Cambridge, MA, USA) (ab7847, IHC). Other antibodies include Mttp (612022; BD Biosciences, San Jose, CA, USA), a-SMA (A2547; MilliporeSigma, St. Louis, MO, USA), albumin (ab83465; Abcam), and Gapdh (SC-25778; Santa Cruz Biotechnology, Dallas, TX, USA). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were determined using kits (A526, A561) from Teco Diagnostic (Anaheim, CA, USA). Triglyceride, cholesterol, free FA (FFA), and glucose levels in liver and serum were determined using kits from Wako (Fuji Film Wako Diagnostics, Mountain View, CA, USA), with lipid extractions performed as previously described (17). Serum b-hydroxybutyrate was determined using a b-hydroxybutyrate (Ketone Body) Colorimetric Assay Kit (700190; Cayman Chemical, Ann Arbor, MI, USA). Bile acid (BA) levels were measured using a Total Bile Acids Colorimetric Assay Kit (GWB-BQK090; GenWay, San Diego, CA, USA). Thiobarbituric acid reactive substances levels were measured using an assay kit (0801192; ZeptoMetrix, Buffalo, NY, USA). Generation of L-Fabp f/f mice Homologous 59 (6.8 kb) and 39 (1 kb) arms were amplified by PCR using a mouse BAC as template. A Lox P site was introduced into the 59 arm by PCR ;1.2 kb upstream of L-Fabp Exon 1. Fragments were cloned into the targeting vector containing a Frtflanked Neo cassette and the 39 LoxP site (18). These elements were inserted into an intronic region ;400 nt after Exon 1. Integrity of the Lox P sites and Exon 1 was confirmed by sequencing and by incubation of plasmid DNA with recombinant Cre recombinase in vitro. The targeting construct was transfected into SCC10 129x1Sv/J ES cells (Washington University Mouse Stem cell core). Recombinant clones were selected with G418 and screened by PCR for correct targeting. One ES clone was expanded and injected into blastocysts (Washington University Mouse cell core), resulting in 4 high-percentage chimeras. Chimeras were bred to C57BL/6J mice (000664; The Jackson Laboratory, Bar Harbor, ME, USA) and subsequently to Flp1 Tg (009086; The Jackson Laboratory) mice to remove the Frtflanked Neo cassette in the targeted allele. Mice were backcrossed 8 generations to C57BL/6J, and genetic background was verified using SNP genome scanning (143 SNP panel; The Jackson Laboratory). Outcrossed C57BL/6/129 SvJ mice (mixed background) were used for initial studies (including food restriction, cocoa butter diet) with outcrossed or littermate controls; other studies (gallstone susceptibility, BDL) used C57BL/6J congenic mice, with in-house backcrossed L-Fabp f/f mice as controls. Oligonucleotides The following primers were used to detect recombination of L-Fabp gene: P1, 59-ACACAGACCTTCCACGCTATC-39; P2, 59CAAACACTCTCTAAACTGTGAG-39. To investigate whether Alb cre–mediated deletion of floxed alleles occurs in other conditional lines, we examined Mttp expression and locus recombination in Mttp f/f Alb cre mice (19). Mttp is required for the synthesis and secretion of lipoprotein particles from the intestine 4611 and liver (20). The following primers spanning the deleted region were used to detect recombination: M1, 59-GAACCAGGATTGCTTTAAG-39; M2, 59-TCAGAGAAGACTTACAAG-39. The following primers within the deleted region were used as control: C1, 59-AGACCAATCGCTCTAAAGGCA-39; C2, 59AAGACCCCATTTGCTCAGGT-39. Other primer pairs are as described in our previous publications (15, 21). Cre deletor and reporter mice Platelet-derived growth factor receptor b promoter–driven Cre recombinase (Pdgfrb-Cre) mice were generated by Ralf Adams (22) and generously provided by Dean Shepherd (University of California–San Fransisco, San Fransisco, CA, USA). Albumin Cre (23) (003574), Gfap Cre (24) (004600), and green fluorescent protein (GFP)/b-Gal reporter (25) (008606) transgenic mice were purchased from The Jackson Laboratory (Table 1). Animal studies Mouse were housed in individually ventilated cages in a full barrier facility (12-h light/dark cycle) with corncob bedding and fed standard, low-fat rodent chow (PicoLab 20, 5053; LabDiet, St. Louis, MO, USA) with free access to food and water unless otherwise noted. All interventions were performed during the light cycle. All animal protocols were approved by the Washington University Animal Care and Use Committee and conformed with guidelines outlined by the National Institutes of Health (Bethesda, MD, USA). For high-fat diet studies, female mice were fed a 20% cocoa butter diet (CSMD761; MP Biomedicals, Solon, OH, USA) for 12 wk and were weighed weekly. To promote fibrogenesis, male mice were fed a diet containing 22% hydrogenated vegetable oil (TD.06303; Envigo, Huntingdon, United Kingdom) and given fructose-containing water (45% glucose, 55% fructose, 42 g/L) for 10 or 26 wk. To induce steatohepatitis, male mice (C57BL/6J congenic) were fed a methionine-choline– deficient (MCD) diet (960439; MP Biomedicals) for 3 wk. For food withdrawal studies, food was removed 48 h prior to tissue collection. For all other studies, mice were held without food for 4 h. Carbon tetrachloride (CCl4, 289116; MilliporeSigma) was diluted 1:4 in olive oil and injected biweekly for 4 wk (final dose of 1 ml CCl4/g body weight, intraperitoneally injected). Mice were killed 48 h or 4 wk after the final CCl4 injection. To induce gallstone formation, mice were fed a lithogenic diet containing 1.2% cholesterol and 0.5% cholic acid (02960393; MP Biomedicals) for 7 d. vicryl suture, and the skin was closed with wound clips. Mice were given Buprenorphine-SR (1 mg/kg, subcutaneously injected; Zoopharm, Windsor, CO, USA) at the time of surgery for postoperative analgesia. HSC isolation (differential centrifugation) Livers were perfused in situ using collagenase (C5138, 50 mg/ml; MilliporeSigma) in HBSS at 37°C and then manually disrupted. Cells were filtered through a 100 mm nylon cell strainer and centrifuged (50 g, 5 min) to obtain hepatocytes (pellet) and HSCs (supe). HSCs were collected (2000 rpm), pooled (2–4 mice/pool), and purified using a discontinuous OptiPrep gradient as previously described (D1556; MilliporeSigma) (15). For HSC isolation for RNA or protein studies, HSCs were doubly purified using a second OptiPrep gradient. Cells were collected, washed, and counted. For stellate cell immunohistochemistry, cells were transferred to slides using cytospin columns (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. HSC isolation (laser capture) Liver tissue was frozen in optimal cutting temperature compound, and tissue sections were immunostained with an antibody to desmin (85033, 1:100 d; Abcam) to identify hepatic stellate cells. Desmin-positive HSCs were dissected using laser capture microdissection (LMD7000; Leica Microsystems, Buffalo Grove, IL, USA), and RNA was isolated using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific) as previously described (26). Cholangiocyte isolation Freshly isolated mouse cholangiocytes (pooled samples of total and large cholangiocytes) were prepared from wild-type C57BL/ 6N mice using a rat IgG2a monoclonal antibody against an unknown antigen expressed by all cholangiocytes (a gift from Dr. R. Faris, Brown University, Providence, RI, USA) as previously described (27) by centrifugal elutriation using immunoaffinity isolation and verified using CK-19 immunohistochemical staining. Lipidomic analyses BDL Mice were anesthetized with isoflurane (4% induction, 1.5–3% maintenance). In each mouse, the abdomen was shaved and sterilized, the liver was lifted, and the bile duct was ligated with 2 ligatures using 5/0 suture. The peritoneum was closed with Liver tissue was homogenized in PBS and extracted using a modified Bligh-Dyer method in the presence of internal standards. Extracted FFA was further derivatized with amino methyl phenyl pyridium to increase sensitivity. Measurement of lipids [triglyceride (Tg), diacylglycerol, ceramide, and FFA] was TABLE 1. Mouse lines used, including expected target cell population, original description, and designation Strain Figure designation Modification Targeted cell population Reference L-Fabp f/f L-Fabp f/f Alb Cre Tg L-Fabp f/f Gfap Cre Tg L-Fabp f/f Pdgfrb Cre Tg L-Fabp f/f AAV8 Cre GFP/b-gal reporter Mttp f/f Mttp f/f Alb Cre Tg f/f Alb Cre Gfap Cre Pd Cre AAV8 Cre GFP/b-gal Mttp f/f Mttp f/f Alb cre Floxed allele Floxed allele, transgene Floxed allele, transgene Floxed allele, transgene Floxed allele, adeno-associated virus Transgene Floxed allele Floxed allele, transgene None Hepatocytes, HSC HSC, cholangiocytes HSC Hepatocytes Cre-expressing cells None Hepatocytes, HSC Current 23 24 22 Current 25 19 Current 4612 Vol. 33 March 2019 The FASEB Journal x www.fasebj.org NEWBERRY ET AL. performed with a 10A HPLC system (Shimadzu, Kyoto, Japan) and a SIL-20AC HT auto-sampler (Shimadzu) coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific) operated in selected reaction monitoring mode under ESI (+). Data processing was conducted with Xcalibur (Thermo Fisher Scientific). For analysis of BA species, liver tissue was homogenized in water and BA precipitated in the presence of deuterated internal standards. The extracts were separated by column-switching HPLC on a Security Guard Gemini C18 (4 3 3 mm; Phenomenex, Torrance, CA, USA) and ACE Excel Super C18 column (3 mm, 50 3 4.6 mm; Advanced Chromotograpy Technologies, LTD, Aberdeen, Scotland). BAs and their internal standards were detected by a tandem mass spectrometer (Sciex 4000QTrap; Applied Biosystems, Foster City, CA, USA) equipped with an electrospray ion source in negative ion mode and multiple-reaction monitoring detection. For stellate cell retinoid analyses, pelleted cells were homogenized, and retinyl acetate added as an internal standard. Retinoids were extracted into hexane and suspended for injection onto 4.6 3 250 mm Ultrasphere C18 HPLC columns (Fullerton; Beckman Coulter, Brea, CA, USA) preceded by a C18 guard column (MilliporeSigma) using 70% acetonitrile–15% methanol–15% methylene chloride as the running solvent flowing at 1.8 ml/min. Retinol and retinyl esters (retinyl palmitate, oleate, linoleate, and stearate) were detected at 325 nm and identified using authentic standards. Concentrations of retinol and retinyl esters were quantitated by comparing integrated peak areas against purified standards, corrected for recovery of internal standard. The data were plotted as nmol retinoid per million cells. Microscopy Sirius red staining and F4/80 immunostaining (ab16911; Abcam) were quantitated in 8–10 fields per mouse, across 2–3 distinct pieces of liver tissue using Nuance (21) quantitation software. A specific spectral library was created and used for the analysis of each experimental cohort. Stained area was expressed as a percentage of total area and averaged. Statistical analysis Data are presented as mean 6 SE unless otherwise noted. Oneway ANOVA followed by Tukey’s multiple comparisons test was performed using Prism v.7 for Mac OS X (GraphPad, La Jolla, CA, USA). For experiments involving only 2 genotypes, unpaired, 2-tailed Student’s t tests were performed (GraphPad Prism). RESULTS Cell-specific L-Fabp deletion in hepatocytes vs. HSCs We generated a floxed L-Fabp allele (Fig. 1A) and verified Cre-mediated recombination in whole liver after albumin promoter–driven Cre recombinase (Alb-Cre)– mediated L-Fabp deletion (Fig. 1B), with reduction of L-Fabp mRNA and protein abundance (Fig. 1C, D). Those features were recapitulated (as expected) after hepatocyte-specific Cre delivery using AAV8-Tbg Cre (Fig. 1C, D). In contrast, we observed no reduction of L-Fabp protein or mRNA in whole liver extracts after HSCspecific [glial fibrillary acid protein promoter–driven Cre recombinase (Gfap-Cre), (Pdgfrb-Cre)] L-Fabp deletion (Fig. 1C, D), likely due to the dominance of hepatocyte L-Fabp expression. Hepatic Tg content was CELL-SPECIFIC L-Fabp DELETION IN FIBROGENIC INJURY decreased in mice with Alb-Cre–mediated L-Fabp deletion (Fig. 1E, F), with targeted lipidomic profiling showing reduced abundance of a range of Tg FA species (Fig. 1G). Overall there were no changes in total hepatic lipid content, hepatic fibrosis, or Tg species distribution in mice with Gfap-Cre–mediated L-Fabp deletion compared with flox controls (Fig. 1E–G), and there was no difference in FA species in any of the genotypes (data not shown). We found an increase in hepatic ceramides with C24:1 and C16:0 FA species from mice with AlbCre–mediated L-Fabp deletion with no changes in mice with Gfap-Cre–mediated L-Fabp deletion vs. flox controls (Fig. 1H). We next examined L-Fabp recombination specifically in HSCs after crosses of the floxed allele with different Cre drivers. Because prior work suggested that Gfap-Cre is active in mouse cholangiocytes [reviewed by Greenhalgh et al. (28)] as well as HSCs, we compared expression of L-Fabp in whole liver and purified cholangiocytes isolated from wild-type mice. Our findings revealed ,1% relative L-Fabp mRNA abundance in cholangiocytes compared with whole liver and virtually no L-Fabp expression in large cholangiocytes (Fig. 2A–C). Nevertheless, we undertook selected studies of the phenotypes reported below using an additional HSC-specific knockout approach, Pdgfrb-Cre–mediated L-Fabp deletion, to complement the findings with GfapCre–mediated L-Fabp deletion. We found that both Gfap-Cre–mediated and PdgfrbCre–mediated L-Fabp deletion produced recombination in HSCs (Fig. 2A), with loss of L-Fabp protein and mRNA in isolated HSCs, purified by either differential centrifugation or laser capture microdissection (Fig. 2B). To our surprise, we observed partial L-Fabp recombination in HSCs with Alb-Cre–mediated L-Fabp deletion (Fig. 2A), accompanied by loss of both L-Fabp mRNA and protein in HSCs (Fig. 2B), but no recombination or change in L-Fabp mRNA in HSC from AAV8-cre–injected mice. Turning to HSC lipid content, we found that retinyl ester content was significantly reduced in HSCs from mice with GfapCre-mediated or Pdgfrb-Cre–mediated L-Fabp deletion and in mice with Alb-Cre–mediated deletion, compared with flox mice and AAV8 Tbg-Cre–treated mice (Fig. 2D, left panel), with no change in retinol content by genotype (Fig. 2D, right panel). We observed reduced abundance of 16:0 as well as 18:0, 18:1, and 18:2 FA species in retinyl esters in all genotypes after HSC deletion of L-Fabp (Alb-Cre, Gfap-Cre, Pdgfrb-Cre) (Fig. 2E). These findings demonstrate that Alb-Cre–mediated L-Fabp deletion attenuates complex neutral lipid accumulation in hepatocytes and stellate cells, whereas HSC-specific deletion of L-Fabp (Gfap-Cre, PdgfrbCre) predominantly attenuates stellate cell retinyl ester content. Alb-Cre–driven recombination in HSCs We considered several possibilities for the observation that L-Fabp expression is reduced in both HSCs and hepatocytes from Alb-Cre Tg mice, with a corresponding decrease in retinyl ester content. First, it is 4613 Figure 1. Hepatocyte-specific deletion of L-Fabp. A) Schematic diagram of conditional L-Fabp targeting strategy with and without (top) Cre-mediated recombination. Lox P sequences (triangles) were introduced on either side of Exon1, resulting in deletion of Exon 1 and the initiator methionine in the presence of Cre. P1 and P2 denote the location of primers used to detect recombination in genomic DNA (gDNA). B) PCR amplification (P1/P2 primers) of gDNA isolated from livers of L-Fabp f/f and Alb cre mice showing deletion of the region between Lox P elements in presence of Cre recombinase. C, D) L-Fabp mRNA (C ) and protein (D) expression in livers of L-Fabp f/f mice, in L-Fabp f/f mice expressing liver (Alb) or stellate cell specific (Gfap, Pd) Cre transgenes, and in mice injected with hepatocyte specific AAV8-Tbg Cre (AAV8-Cre). Expression of Gapdh and Fabp2 (I-Fabp) is shown as controls. Immunohistochemistry (D, right panel) shows the absence of L-Fabp protein in hepatocytes L-Fabp f/ f Alb cre mice. E) Hepatic Tg, total cholesterol (Chol), and FFA in livers of 12 wk, chow-fed female mice on a mixed C57BL/6J 129/ SvJ background. F) Sirius red–stained fibrotic area in livers of 12 wk, chow-fed female mice as percent of total area. G) Relative abundance of major Tg species (.80% of all species) in livers of chow-fed female L-Fabp f/f, Alb Cre, and Gfap Cre mice (n = 4/ genotype). H ) Relative abundance of ceramide species in livers of female chow-fed mice (n = 4/genotype). possible that hepatocyte deletion triggers HSC activation, a phenotype shown previously to coincide with reduced L-Fabp expression and loss of retinoidcontaining LDs (15). However, we found no evidence of increased HSC activation at baseline as assessed by fibrogenic gene expression or Sirius red staining (Fig. 2F, G). Moreover, hepatocyte-specific Cre delivery using AAV8-Tbg did not decrease L-Fabp mRNA or HSC retinyls (Fig. 2B, D). 4614 Vol. 33 March 2019 Another explanation is that the Alb-cre transgene is active in HSCs because albumin mRNA is detectable in both freshly isolated and cultured HSCs (29). We pursued this possibility by turning to 2 additional conditional alleles generated using Alb-Cre–mediated genetic deletion. First, we bred floxed GFP/b-gal reporter mice to Alb-Cre Tg mice and observed GFP expression in .50% HSCs from the offspring, with no expression in the absence of Alb-Cre (Fig. 2H). In addition, we examined HSC The FASEB Journal x www.fasebj.org NEWBERRY ET AL. Figure 2. HSC-specific deletion of L-Fabp. A) PCR amplification (P1/P2 primers) of genomic DNA from stellate cells from L-Fabp f/f mice, Alb Cre, Gfap Cre, and Pd Cre mice and from L-Fabp f/f mice injected with AAV8 Tbg cre. Note the partial deletion of the Lox P–flanked region in HSCs from mice expressing Alb Cre but not AAV8 Tbg Cre. Based on differences in PCR product size and staining intensity, recombination efficiency is estimated to be ;50%. B) L-Fabp mRNA (left, middle) and protein (right) expression in HSCs isolated by differential centrifugation (left, right) or laser capture microdissection (middle) from L-Fabp f/f and Alb Cre, Gfap Cre, and Pd Cre mice. For Western blot (right), expression of Gapdh is shown as a loading control. C ) Relative expression of L-Fabp mRNA in pools of isolated C57BL/6N cholangiocytes (total or large) expressed relative to L-Fabp mRNA levels in whole liver (C57BL/6J mice). D) Total retinyl ester (left) and retinol levels in freshly isolated HSC (continued on next page) CELL-SPECIFIC L-Fabp DELETION IN FIBROGENIC INJURY 4615 expression in a line of conditional microsomal triglyceride transfer protein (Mttp) knockout mice after Alb-Cre– mediated genetic deletion (21). HSCs from wild-type mice exhibited Mttp protein, whereas HSCs from Mttp-Alb Cre mice showed Mttp recombination and complete loss of Mttp protein (Fig. 2I). Taken together, these findings, along with other reports showing Cre expression in HSCs (29), suggest that Alb-Cre–mediated genetic deletion not only targets hepatocytes but may also directly modify gene expression in HSCs. Metabolic adaptations after cell-specific L-Fabp deletion Our prior observations in germline L-Fabp2/2 mice revealed distinct metabolic phenotypes, including attenuated hepatic steatosis upon having food withheld (16) or with chronic high saturated fat feeding (17, 30–32) and demonstrated increased cholesterol gallstone susceptibility after lithogenic diet feeding (33). Hepatic triglyceride content was reduced in mice starved for 48 h with both Alb-Cre and AAV8-Tbg Cre–mediated L-Fabp deletion (Fig. 3A, B), with no differences noted in mice with GfapCre–mediated L-Fabp deletion. In line with this result, serum ketone levels were reduced in mice with Alb-Cre– mediated L-Fabp deletion (Fig. 3C, left panel), with no changes in serum FFA levels in any genotype (Fig. 3C, right). We also observed a trend toward reduced weight gain and protection against high-fat diet–induced hepatic steatosis in mice with Alb-Cre–mediated L-Fabp deletion (Fig. 3D, E), along with reduced serum glucose (Fig. 3F). Turning to a different model of altered hepatic lipid metabolism, we observed increased lithogenic diet–induced cholesterol gallstone formation in mice with Alb-Cre– mediated L-Fabp deletion (Fig. 3G, H). These findings, using 3 different dietary models of altered hepatic lipid metabolism, demonstrate that mice with Alb-Cre–mediated L-Fabp deletion phenocopy mice with germline L-Fabp deletion and suggest that hepatocyte (vs. intestinal or stellate cell) L-Fabp is likely the dominant mediator of these adaptations. Protection against diet-induced hepatic steatosis and fibrogenic injury in models of cell-specific L-Fabp deletion Our earlier work indicated that freshly isolated HSCs from germline L-Fabp2/2 mice were depleted of LDs and exhibited an activated phenotype when studied in culture (15). However, neither germline L-Fabp2/2 mice (17, 32, 34, 35) nor mice with conditional L-Fabp deletion mediated by Alb-Cre or Gfap-Cre exhibit features of spontaneous hepatic fibrosis (Fig. 2F–G). The findings regarding retinyl ester depletion in those conditional L-Fabp knockout lines suggested, however, that these mice might be prone to exaggerated fibrosis after dietinduced steatotic injury. Accordingly, we turned to 2 different dietary models of steatosis shown to promote hepatic fibrogenesis: high transfat/fructose (TFF) feeding and MCD diet feeding. We fed the high-TFF diet to groups of mice with Alb-Cre– and Gfap-Cre– mediated L-Fabp deletion and floxed controls. After 10 wk, we observed reduced hepatic Tg content (Fig. 4A) in mice with Alb-Cre–mediated L-Fabp deletion and reduced fibrosis as inferred from Sirius staining compared with floxed mice and with mice with Gfap-Cre– mediated L-Fabp deletion (Fig. 4B, C). There were no differences by genotype in the expression of several fibrogenic mRNAs (Fig. 4D) and no differences in transaminases, which were variably elevated (Fig. 4E). Mice studied at 26 wk revealed no differences by genotype in hepatic Tg content (Fig. 4F), although the reduced fibrosis observed at 10 wk in mice with AlbCre–mediated L-Fabp deletion persisted at this later time point (Fig. 4G, H). There were again no differences by genotype in the expression of several fibrogenic mRNAs (Fig. 4I), but the expression of hepatic TNF-a and IL-1b mRNA was reduced in mice with Alb-Cre– mediated L-Fabp deletion (Fig. 4J), with no differences in transaminase levels by genotype (Fig. 4K). These findings suggest that Alb-Cre–mediated (but not GfapCre–mediated) L-Fabp deletion is at least partially protective against high TFF diet–induced fibrogenic injury, implying that stellate cell L-Fabp deletion alone does not modify the steatotic-fibrogenic phenotype associated with prolonged high TFF feeding. We then examined a second model of diet-induced steatosis and fibrogenic injury, namely MCD diet feeding, using mice with Pdgfrb-Cre–mediated L-Fabp deletion as a model of HSC-specific deletion. Mice of all genotypes exhibited similar responses in terms of weight loss after MCD diet feeding (Fig. 5A), but mice with Alb-Cre– mediated L-Fabp deletion exhibited attenuated steatosis (Fig. 5B). There were no differences by genotype in transaminase levels or in hepatic oxidative stress as inferred by thiobarbituric acid reactive substances content (Fig. 5C, D). There were also no differences in fibrogenic mRNAs by genotype (Fig. 5E), although mice with Alb-Cre–mediated pools from L-Fabp f/f, Alb Cre, Gfap Cre, Pd Cre and AAV8-Tbg Cre injected L-Fabp f/f mice (n = 2–6 pools per genotype). E ) Relative abundance of individual retinyl ester species. *P , 0.05, **P , 0.01, ***P , 0.001. F ) Baseline fibrogenic gene expression in liver of chow-fed female mice (;12 wk, n = 4–7/genotype). G) Sirius red–stained area in liver tissue from chow-fed female mice (n = 3/genotype). H ) To examine whether Albumin Cre promotes recombinase expression in HSC, Alb cre Tg mice were crossed with mice expressing a floxed GFP/b-gal Cre reporter transgene (top). GFP/b-gal protein is expressed after deletion of intervening LoxP flanked stop sequences by Cre. Expression of GFP was detected by immunostaining in HSCs isolated from Alb Cre Tg mice (right) but not in HSCs expressing only the reporter. I ) Alb-Cre induces partial recombination of Mttp gene. Schematic diagram (left) shows the location of primers spanning the deleted region (M1/M2, to detect recombination) and within the deleted region (C1/C2, control). Partial recombination of Mttp locus is detected in HSCs isolated from Mttp f/f Alb Cre mice, with reduced amplification of the control PCR product (middle panel) and no expression of Mttp protein in HSCs (right). Gapdh is shown as a loading control. 4616 Vol. 33 March 2019 The FASEB Journal x www.fasebj.org NEWBERRY ET AL. Figure 3. Metabolic adaptation in L-Fabp f/f Alb Cre Tg mice. A) Hepatic Tg content (left) and liver size (expressed as liver/body ratio, right) in male mice after 48 h without food (n = 4–8). B) Hepatic Tg in AAV8-Tbg-Cre injected L-Fabp f/f and uninjected female control mice after 48 h without food (n = 4–5 female mice per group). C ) Serum b-hydroxybutyrate (left) and FFA levels after 48 h without food (n = 4–8 male mice/genotype). D) Weight gain in female mice fed a 20% cocoa butter diet for 12 wk (n = 7 f/f, n = 10 Alb Cre). Initial and final body weights were not significantly different by genotype. E ) Hepatic Tg (left) and liver/ body ratio (right) in mice fed a cocoa butter diet. F ) Serum glucose in cocoa butter–fed mice (n = 6–7/genotype). G) Gallstones in situ (left) and examined under polarized light microscopy in male L-Fabp f/f and Alb-Cre mice (C57BL/6J background) fed a lithogenic diet for 7 d. Representative images are shown. H ) Quantitation of gallstone incidence (left) and score (right) (n = 8–9 mice/genotype). *P , 0.05 vs. f/f control, **P , 0.01, ***P , 0.001. L-Fabp deletion exhibited reduced abundance of F4/80positive cells (Fig. 5F, H) and reduced fibrosis (Fig. 5G, H), whereas mice with Pdgfrb-Cre–mediated L-Fabp deletion exhibited a trend toward reduced fibrosis (Fig. 5G, H). These findings again suggest that Alb-Cre–mediated L-Fabp deletion is protective against hepatic steatosis and fibrogenic injury with MCD diet feeding. Pdgfrb-Cre– mediated stellate cell L-Fabp deletion does not modify the steatotic phenotype with this model but revealed a trend (P = 0.21) toward reduced fibrosis. Cell-specific L-Fabp deletion does not modify fibrogenic phenotype after BDL Our observations suggest that hepatic fibrogenesis might be slightly attenuated with MCD diet feeding in mice CELL-SPECIFIC L-Fabp DELETION IN FIBROGENIC INJURY with stellate cell L-Fabp deletion. Accordingly, we reasoned that the loss of retinyl esters from HSCs in mice with Alb-Cre–mediated, Gfap-Cre–mediated, and PdgfrbCre–mediated L-Fabp deletion might predispose those genotypes to exaggerated fibrosis in experimental settings independent of hepatic steatosis. In the first approach to test this possibility, mice of all genotypes were subjected to BDL. We observed an increase in bile infarct numbers in mice with Alb-Cre–mediated L-Fabp deletion (Fig. 6A) compared with all other genotypes. There were no differences by genotype in serum ALT or BA levels (Fig. 6B, C), and serum bilirubin determinations confirmed a comparable cholestatic phenotype in all genotypes (Fig. 6D), with no differences in fibrosis by genotype (Fig. 6E). Mice with Alb-Cre–mediated L-Fabp deletion exhibited higher total hepatic BA content (Fig. 6F) with increased tauromuricholic acid but otherwise comparable 4617 Figure 4. Reduced steatosis and fibrosis in L-Fabp f/f Alb cre Tg male mice fed a TFF diet for 10 (A–E ) or 26 (F–K ) weeks. A) Hepatic Tg in L-Fabp f/f, Alb Cre, and Gfap Cre mice fed a TFF diet for 10 wk. B) Quantitation of Sirius red–stained area, expressed as a percentage of total area in both TFF-fed (left) and chow-fed mice. C ) Representative images of Sirius red–stained sections showing periportal fibrosis. Original magnification, 3400. D) Relative expression of fibrogenic genes in livers of mice fed a TFF diet for 10 wk (n = 6 mice/genotype). E ) Serum ALT and AST levels after 10 wk TFF feeding. F ) Hepatic Tg in male LFabp f/f, Alb Cre, and Gfap Cre mice fed a TFF diet for 26 wk. G) Quantitation of Sirius red–stained area expressed as a percentage of total area. H ) Representative images of Sirius red staining. I ) Relative expression of fibrogenic genes in livers of mice fed a TFF diet for 26 wk (n = 8 mice per genotype). J ) Relative expression of Tnf-a and IL-1b in livers of TFF-fed mice. K ) Serum ALT and AST after 26 wk TFF feeding. *P , 0.05 between groups. distribution of BA species (Fig. 6G). These data suggest that Alb-Cre–mediated L-Fabp deletion in the setting of BDL increases bile infarct area and hepatic BA content 4618 Vol. 33 March 2019 but not the fibrosis accompanying BDL, whereas stellate cell L-Fabp deletion does not modify any of those phenotypes. The FASEB Journal x www.fasebj.org NEWBERRY ET AL. Figure 5. Reduced steatosis and inflammation in male L-Fabp f/f Alb Cre Tg mice fed an MCD diet. A) Weight loss by genotype during MCD diet feeding. Differences are not significant by genotype. B) Hepatic Tg in male L-Fabp f/f, Alb Cre, and Pd Cre mice fed an MCD diet. C ) Serum ALT (left) and AST in MCD diet–fed mice. D) Quantitation of thiobarbituric acid reactive substances (Tbars) in livers of MCD diet–fed mice. E ) Expression of fibrogenic and inflammatory genes in livers of MCD diet–fed mice (n = 5 mice/genotype). F ) Quantitation of F4/80-stained area in the livers of MCD diet–fed mice expressed as percentage of total area. G) Quantitation of Sirius red–stained area expressed as a percentage of total area. H ) Representative images of Sirius red and F4/80 staining. *P , 0.05, **P , 0.01. Cell-specific deletion of L-Fabp modifies fibrotic injury and impairs reversal of fibrosis after CCl4 administration We created a second model of fibrogenic injury by treating mice with CCl4 for 4 wk. Serum transaminases were variably elevated in all 3 genotypes (Fig. 7A), and there was increased Sirius red staining (Fig. 7B) and increased hepatic hydroxyproline content (Fig. 7C), suggesting CELL-SPECIFIC L-Fabp DELETION IN FIBROGENIC INJURY comparable fibrogenic injury by genotype despite statistically increased collagen1a1 mRNA abundance and a trend toward increased a-SMA mRNA and protein expression in mice with Gfap-Cre–mediated L-Fabp deletion (Fig. 7D, E). Although these findings indicated no differences in CCl4–mediated fibrogenesis by genotype, we considered the possibility that the reversal of established fibrosis might be impaired in genotypes with decreased stellate 4619 Figure 6. Altered BA metabolism in L-Fabp f/f Alb Cre mice after BDL. A) Quantitation of hepatic BA infarct area 2 wk after BDL expressed as percentage total area. Representative hematoxylin and eosin–stained images (original magnification, 340) showing infarct regions. B–D) Serum ALT (B), serum BA (C ), and serum bilirubin (D) levels 2 wk after BDL. For comparison, bilirubin levels in serum of control (no BDL) mice are shown (D). E ) Quantitation of Sirius red–stained fibrotic area in liver 2 wk after BDL, with representative images (original magnification, 3400). F ) BA content in liver tissue 2 wk after BDL. G) Relative abundance of hepatic BA species in L-Fabp f/f and f/f Alb Cre mice by mass spectrometry. Pie charts (right) show that changes in the relative abundance of the major 2 species (TMCA, TCA) likely accounts for increased BA content in Alb Cre mice. *P , 0.05. cell L-Fabp expression. To address this question, we treated mice of all genotypes (including Pdgfrb Cre and AAV8 Tbg Cre–mediated L-Fabp deletion) with CCl4 for 4 wk as previously described and studied them after 4 weeks off CCl4 to allow the injury to resolve. We again observed variably elevated transaminase levels, but this time we found that mice with Alb-Cre–mediated L-Fabp deletion exhibited sustained ALT and AST elevations compared with all other genotypes (Fig. 8A). In addition, livers from mice with Alb-Cre–mediated L-Fabp deletion exhibited greater Sirius staining (Fig. 8B, C) and increased hydroxyproline content (Fig. 8D). There was a variable increase in residual fibrosis in mice with Gfap-Cre–mediated L-Fabp deletion but no residual accumulation of hydroxyproline and no changes with other genotypes (Fig. 8C) when compared 4620 Vol. 33 March 2019 with floxed controls. There were also no alterations in hepatic mRNA abundance for fibrogenic markers or for candidate genes involved in remodeling or inflammation by genotype (Fig. 8E, F). Taken together, the findings suggest that reversal of established fibrosis after CCl4 is impaired in mice with Alb-Cre–mediated L-Fabp deletion, whereas mice with either stellate cell–specific (Gfap-Cre, Pdgfrb-Cre) or hepatocyte-specific (AAV8 Tbg Cre) L-Fabp deletion do not exhibit impaired fibrosis reversal. DISCUSSION The current studies were undertaken with a view to understanding the cell-specific role of L-Fabp as a modifier The FASEB Journal x www.fasebj.org NEWBERRY ET AL. Figure 7. CCl4–induced injury is increased in male mice with hepatocyte or HSC deletion of L-Fabp. A) Serum ALT (left) and AST levels in L-Fabp f/f, Alb Cre, and Gfap Cre mice after 4 wk of CCl4 treatment (biweekly intraperitoneal injection, 1 ml/g body weight). B) Sirius red–stained fibrotic area (% of total) in liver after CCl4 injury. Representative images are shown (original magnification, 3200). C ) Hydroxyproline content of liver tissue. D) Relative expression of Col1a1 (left) and a-SMA mRNA in liver of CCl4–treated animals. E ) Quantitation of Western blot analyses of a-SMA protein in livers of f/f, Alb cre, and Gfap cre mice (left). Abundance of a-SMA protein was normalized to expression of albumin and expressed relative to levels in L-Fabp f/f analyzed on same gel. *P , 0.05. of traits associated with steatotic and fibrogenic injury in mice. Our prior work in germline L-Fabp2/2 mice revealed several metabolic traits, including protection against steatosis with prolonged food withdrawal (16) and after sustained high saturated fat feeding (17, 30–32) along with increased susceptibility to lithogenic diet–induced gallstone formation (33). These traits were apparent in mice after Alb-Cre–mediated L-Fabp deletion, suggesting a dominant role for hepatocyte L-Fabp in modulating those metabolic adaptations; these findings are consistent with studies using primary hepatocytes isolated from L-Fabp2/2 mice, which indicated altered fatty acid uptake and LD formation and CELL-SPECIFIC L-Fabp DELETION IN FIBROGENIC INJURY turnover (21). We offer a note of caution in this interpretation, however, because the metabolic adaptations to dietary fat and cholesterol supplementation in germline L-Fabp2/2 mice are highly influenced by the background genetic strain (C57BL/6J vs. 6N) (34–36). Our findings confirmed this divergence in mice that were incompletely backcrossed into the C57BL/6J background (32). Because of the intense interest in understanding the mediators and pathways involved in hepatic fibrogenesis and the central role of HSCs as fibrogenic effectors (2, 4), we explored the consequences of stellate cell–specific deletion of L-Fabp. Prior work in germline 4621 Figure 8. Reversal of CCl4–induced fibrosis is delayed in male L-Fabp f/f Alb Cre mice. A) Serum ALT (left) and AST levels 4 wk after final CCl4 injection. B) Sirius red–stained liver tissue showing residual fibrosis after 4 wk (original magnification, 3400). C ) Quantitation of Sirius red–stained fibrotic area. D) Hydroxyproline content of liver tissue. E ) Expression of fibrogenic genes in liver 4 wk after final CCl4 injections (n = 5/genotype). F ) Expression of genes related to inflammation and ECM degradation (n = 5–6 per genotype). *P , 0.05, **P , 0.01, ***P , 0.001. L-Fabp2/2 mice indicated that cultured HSCs exhibited LD depletion and a progressive activation phenotype (15, 37). However, notwithstanding the findings in cultured HSCs indicating an activated phenotype, those germline L-Fabp2/2 mice were protected against steatosis and fibrosis after prolonged feeding of a fibrogenic high-fat/fructose diet (15), and to our knowledge there is no indication that any of the reported lines of germline L-Fabp2/2 mice in any genetic background exhibit spontaneous fibrosis. As alluded to above, mice with germline deletion of lecithin-retinol acyltransferase (Lrat2/2) also exhibited no baseline fibrosis and no exaggerated fibrogenic response to injury (7). However, germline Lrat2/2 mice exhibited reduced carcinogeninduced hepatic tumorigenesis and defective liver regeneration after partial hepatectomy, suggesting that HSC retinoids may be key signaling molecules regulating cell proliferation and differentiation in the context of injury (7, 38). Those findings raise the possibility that other growth-related phenotypes may arise in the context of HSC-specific L-Fabp deletion, which will be pursued in future studies. Our previous observations in germline L-Fabp2/2 mice in the C57BL/6J background revealed no defect in liver regeneration after partial hepatectomy (39). A related objective of our study concerns the unexpected observation that Alb-Cre–mediated L-Fabp deletion led to genetic recombination not only in hepatocytes (as expected) but also in HSCs, as determined in cells isolated by either differential ultracentrifugation or laser capture microdissection. The line of AlbCre mice used for these studies (003574) has been 4622 Vol. 33 March 2019 extensively documented as producing robust, liverspecific (and presumed hepatocyte-specific) Cre expression coincident with early postnatal development (23). Our findings in several different flox lines suggest that at least partial recombination may occur in HSCs with this transgene. Other researchers have similarly shown evidence of cre expression and genomic modification in both cultured and freshly isolated HSCs from Alb cre mice (40). Moreover, there is precedent from other studies to suggest that cells with mesenchymal characteristics and morphology, and with the capacity to express albumin, have been identified from human livers (41) and from rat pancreatic and hepatic stellate cells (42). These findings serve as a cautionary note to investigators trying to dissect possibly distinct roles of cell-specific deletion in mouse liver. Thus, from the perspective of understanding the roles of hepatocyte vs. stellate cell loss of gene function, future studies might consider the use of AAV8 Tbg (or other hepatocytespecific promoters) Cre vs. a stellate cell Cre driver (Pdgfrb or Gfap) (28, 43). Our findings demonstrate an attenuated fibrogenic phenotype with Alb-Cre–mediated L-Fabp deletion in the setting of steatotic injury models (high transfat/ fructose and MCD diets), which most likely reflects the effects of decreased steatosis and lipotoxic injury. We observed a trend toward reduced fibrosis with MCD diet feeding in mice with Pdgfrb-Cre–mediated L-Fabp deletion, but there was substantial variability that precluded firm conclusions. However, the absence of an effect on fibrosis in any of the other models tested (steatotic and nonsteatotic) leads us to conclude that The FASEB Journal x www.fasebj.org NEWBERRY ET AL. any impact of stellate cell–specific L-Fabp deletion in modulating fibrogenic injury is unlikely to be biologically relevant. We observed no differences in fibrosis by genotype in response to BDL, although we found increased numbers of bile infarcts and increased total hepatic BA content with a shift in species in the livers of mice with Alb-Cre–mediated L-Fabp deletion. The mechanism underlying these changes may reflect changes in intrahepatic BA transport because L-Fabp is known to bind conjugated BAs and to participate in the regulation of biliary lipid secretion (33, 36). Recent work in liver-specific b-catenin knockout mice demonstrated a similar phenotype of increased bile infarcts after BDL, but in that situation total hepatic BA content was reduced rather than increased and inflammation and fibrosis were attenuated (44). Thus, the biologic implications of increased number of bile infarcts after BDL in mice with Alb-Cre–mediated L-Fabp deletion and the impact in other models of cholestatic injury remain to be addressed. We also examined the role of hepatocyte vs. stellate cell L-Fabp in the process of fibrosis reversal. There is increasing interest in identifying therapeutic targets for fibrosis, with the emergence of clinical data to support fibrosis reversal in the setting of cholestatic and inflammatory liver diseases [reviewed by Greuter et al. (3)] and the early promise of therapeutic approaches in NASH (45). Our findings demonstrate greater residual fibrosis after cessation of CCl4 treatment in the livers of mice with Alb-Cre–mediated L-Fabp deletion along with residual accumulation of hydroxyproline as a surrogate for hepatic collagen abundance. The mechanisms and mediators underlying this observation are not immediately apparent, but we can invoke several possibilities from other findings in this report as well as prior results. We observed that hepatic C24:1 and C16:0 ceramide content was significantly increased in mice with Alb-Cre– mediated L-Fabp deletion (Fig. 1H), which is of interest because other researchers have demonstrated that hepatocyte-derived exosomes, enriched in C16: 0 ceramides, are a potent activator of macrophages (46). It will be of interest to examine the profile of serum and hepatocyte-derived exosomes from mice with Alb-Cre–mediated L-Fabp deletion to pursue this question more directly. Other work, as well as our own, has shown altered hepatic endocannabinoid production from germline L-Fabp2/2 mice (31, 47), which is relevant because endocannabinoids are potent inflammatory modulators with an emerging role as targets for liver fibrosis (48, 49). The possibility that Alb-Cre–mediated L-Fabp deletion alters the profile of endocannabinoid production from hepatocytes and/or stellate cells will require formal investigation. Another possibility is suggested from our previous work using antisense L-Fabp oligonucleotides to reduce hepatic L-Fabp expression in the liver of mice with impaired secretion of VLDL (21). Those studies demonstrated attenuation of hepatic lipid content with antisense L-Fabp oligonucleotide treatment but revealed exaggerated inflammation and increased CELL-SPECIFIC L-Fabp DELETION IN FIBROGENIC INJURY hepatic fibrosis, suggesting that the production, trafficking, or release of lipotoxic inflammatory mediators might be enhanced with acute reductions in L-Fabp abundance. We also know very little about the characteristics and fate of the liver myofibroblasts in any of the stellate cell L-Fabp knockout lines during regression of fibrosis. It will be of interest to know if those activated HSCs undergo apoptosis or if they are capable of undergoing reversion to a quiescent state as described in other models (50, 51). Those and other questions concerning the origins of HSCs and the requirement for LDs and retinoid stores during development and in response to injury will likely require lineage tracing studies as outlined by others (51). ACKNOWLEDGMENTS This work was supported by the U.S. National Institutes of Health (NIH) National Institute of Diabetes and Digestive and Kidney Diseases (DK 56260, and DK 52574, to N.O.D), and NIH National Heart, Lung, and Blood Institute (HL 38180, to N.O.D.); Veterans Affairs (VA) Research Career Scientist Award and VA Merit Award 5I01BX000574 from the United States Department of Veteran’s Affairs (to G.A.); and Biomedical Laboratory Research and Development Service Grants DK 068437 and DK 101251 (to W.S.B.). Portions of this material are the result of work supported with resources and the use of facilities at the Central Texas Veterans Health Care System, Temple, Texas. The content is the responsibility of the authors alone and does not necessarily reflect the views or policies of the Department of Veterans Affairs or the United States Government. The authors declare no conflicts of interest. AUTHOR CONTRIBUTIONS E. P. Newberry, Y. Xie, G. Alpini, W. S. Blaner, A. Chen, and N. O. Davidson developed the study concept and designed the study; E. P. Newberry, Y. Xie, C. Lodeiro, R. Solis, W. Moritz, S. Kennedy, L. Barron, E. Onufer, G. Alpini, T. Zhou, W. S. Blaner, A. Chen, and N. O. Davidson acquired, analyzed, and interpreted the data; E. P. Newberry and N. O. Davidson drafted the manuscript; E. P. Newberry, Y. Xie, G. Alpini, W. S. Blaner, A. Chen, and N. O. Davidson critically reviewed the manuscript; G. Alpini, W. S. Blaner, and N. O. Davidson obtained funding; N. O. Davidson supervised the study; and all authors had access to the study data, and have reviewed and approved the final manuscript. REFERENCES 1. Zhang, X. J., She, Z. G., and Li, H. 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USA 109, 9448–9453 Received for publication September 17, 2018. Accepted for publication November 26, 2018. 4625