Figures
Abstract
Chronic liver injury leads to fibrosis, cirrhosis, and loss of liver function. Liver cirrhosis is the 12th leading cause of death in the United States, and it is the primary risk factor for developing liver cancer. Fibrosis and cirrhosis result from activation of hepatic stellate cells (HSCs), which are the primary collagen producing cell type in the liver. Here, we show that platelet-derived growth factor receptor α (PDGFRα) is expressed by human HSCs, and PDGFRα expression is elevated in human liver disease. Using a green fluorescent protein (GFP) reporter mouse strain, we evaluated the role of PDGFRα in liver disease in mice and found that mouse HSCs express PDGFRα and expression is upregulated during carbon tetrachloride (CCl4) induced liver injury and fibrosis injection. This fibrotic response is reduced in Pdgfrα heterozygous mice, consistent with the hypothesis that liver fibrosis requires upregulation and activation of PDGFRα. These results indicate that Pdgfrα expression is important in the fibrotic response to liver injury in humans and mice, and suggest that blocking PDGFRα–specific signaling pathways in HSCs may provide therapeutic benefit for patients with chronic liver disease.
Citation: Hayes BJ, Riehle KJ, Shimizu-Albergine M, Bauer RL, Hudkins KL, Johansson F, et al. (2014) Activation of Platelet-Derived Growth Factor Receptor Alpha Contributes to Liver Fibrosis. PLoS ONE 9(3): e92925. https://doi.org/10.1371/journal.pone.0092925
Editor: Johannes Haybaeck, Medical University Graz, Austria
Received: December 17, 2013; Accepted: February 27, 2014; Published: March 25, 2014
Copyright: © 2014 Hayes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants R01CA127228 (to J.S. Campbell), the Herbert S. Coe Foundation, American College of Surgeons Louis C. Argenta Fellowship, the American Surgical Association Foundation Fellowship (to K.J. Riehle), and the Howard Hughes Medical Institute program in Molecular Medicine and T32HL007312 (to B.J. Hayes). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Chronic liver injury is a major cause of morbidity and mortality in the US and worldwide, due to complications of liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) [1]. To date, there are no effective treatments for patients with liver fibrosis, so a better understanding of pathways that regulate fibrosis has great clinical potential [2]. Many inflammatory cytokines and growth factors are released during liver injury, including platelet derived growth factors (PDGFs), which are potent mitogens for hepatic stellate cells (HSCs) [2], [3]. The PDGF family of ligands and receptors plays a central role in repair after injury, and are key regulators of the formation of connective tissue [4], [5]. Elevated platelet-derived growth factor receptor (PDGFR) expression is detected in human heart disease, pulmonary fibrosis, and kidney fibrosis [6]–[8], and blocking PDGFR signaling decreases collagen deposition after myocardial infarct, in pulmonary fibrosis, and in kidney fibrosis [9]–[11]. Thus, targeting the PDGF pathway may modulate liver fibrosis.
There are five known functional ligand dimers in the PDGF family, -AA, -AB, -BB, -CC, and –DD, which bind cell surface receptor tyrosine kinases comprised of PDGFRα and PDGFRβ subunits [12]. PDGFs stimulate the migration and proliferation of mesenchymal cells during development [13]. Loss of PDGFRs leads to significant abnormalities in mice [14], [15]. PDGFRβ is critical to vascular and hematopoietic development, and cell specific deletion or activation of PDGFRβ results in failure or increased pericyte and vascular smooth muscle cell coverage of blood vessels in mice [14], [16], [17]. PDGFRα is required for migration and survival of neural crest cells and for skeletal development, and cell specific deletion of PDGFRα decreases β-cell proliferation in the pancreas and ventricular septation of the heart [15], [18], [19]. Constitutive activation of PDGFRα causes fibrosis that is particularly noticeable in intestine, skin, muscle and heart, but activation has to be conditionally induced in late prenatal or adult animals, as constitutive PDGFRα activation causes lethality [5]. Deleting one allele of Pdgfrα in mice does not affect development, unlike the observed phenotype in homozygous knockout mice [15], [20]. PDGF signal transduction pathways play a prominent role in fibrosis [21]. It has been suggested that PDGFRα signaling is more likely to induce fibrosis than PDGFRβ [22], however this notion has not been conclusively demonstrated in the liver. In summary, PDGF signaling is tightly regulated by abundance and degree of signal transduction, and perturbing either results in developmental defects and organ dysfunction.
In the present study we analyzed PDGFR in human liver disease, human liver cell lines, and a mouse model of liver injury and fibrosis. We found increased PDGFRα in human liver specimens with fibrosis and cirrhosis. PDGFRα is primarily expressed in HSCs, and Pdgfrα expression increased in injured mouse livers. We investigated the role of PDGFRα in liver fibrosis using mice with only one allele of Pdgfrα, and found that reducing Pdgfrα copy number inhibits liver fibrosis in mice. Together our data suggest that PDGFRα inhibitors could be an effective means to reduce liver fibrosis in patients.
Materials and Methods
Animals
Mice were housed in a specific pathogen-free environment overseen by the Department of Comparative Medicine at the University of Washington with IACUC approval under protocol #4295-01. Mice that express nuclear localized green fluorescent protein (GFP) driven by the endogenous Pdgfrα promoter, PdgfrαnGFP, were purchased from the Jackson Laboratory (007669) [20]. Either wild type (WT) littermates that retain both Pdgfrα alleles, or control mice, i.e. male C57BL/6 mice purchased from the Jackson Laboratory (000664), were used as experimental controls. To induce fibrosis, mice were injected (i.p.) with 10 μl/g body weight CCl4 (Sigma-Aldrich) diluted in olive oil 10% (v/v), either one time (acute injury) or twice weekly for four or six weeks (chronic injury). Olive oil-injected animals served as controls for CCl4-injected mice. Animals were sacrificed using CO2 inhalation. The Institutional Animal Care and Use Committee of the University of Washington, which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care International, approved all experiments.
Human Liver Samples
Human liver and HCC specimens were obtained from the University of Washington Medical Center after IRB-approval. IHC was performed on liver specimens from patients with cirrhosis who underwent liver transplantation surgery at the University of Washington Medical Center from 1989 to 2002, HSD #23602 (MMY) [23]. Immunoblot analysis was performed on resected liver specimens collected after receiving informed consent IRB #31281 (RSY). All samples were de-identified of any patient information. Specimens were either fixed in formalin or frozen at −80°C until use.
Immunohistochemistry (IHC) and Histological Staining
Formalin-fixed liver tissue was processed and embedded in paraffin using standard protocols, and IHC was performed as previously described [24], using the primary antibodies listed in Table S1. A board-certified clinical liver pathologist (MMY) reviewed all human samples and determined the presence of cirrhosis and/or tumor and assessed for PDGFRα and PDGFRβ immunoreactivity. To quantify fibrosis, formalin-fixed liver tissue was stained with picrosirius red. For morphometric analysis, picrosirius red area was imaged under polarized light [25]. Images were analyzed using NIH image J software to convert pixels to binary values and determine the relative number of positive and negative pixels.
Immunoblotting
Tissues were homogenized in a 1% Triton-x 100 lysis buffer and processed as described [26]. Membranes were incubated with primary antibodies overnight at 4°C, and then with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. Primary antibodies used in this study are listed in Table S1.
Immunofluorescence (IF) and ex-vivo Imaging
Livers were fixed in 4% paraformaldehyde overnight, and tissues were frozen in optimum cutting temperature compound for cryosectioning. IF was performed using standard techniques, with liver sections incubated overnight with the primary antibodies listed in Table S1. Immune complexes were detected with goat Alexa 633 conjugated anti-rat IgG (A-21094, Life Technologies) and goat Alexa 546 conjugated anti-rabbit IgG (A-11010, Life Technologies) antibodies. Sections were mounted with SlowFade Gold (S36936, Life Technologies) and imaged with a Leica SL confocal microscope (Leica Microsystems, Keck Center UW). For ex-vivo imaging, freshly harvested livers were analyzed as previously described [27]. Images were captured using a Zeiss 510 Meta confocal microscope. In PdgfrαWT/nGFP mice, GFP fluorescence was used to report PDGFRα positive cells [20].
Cell Culture and Proliferation Assay
Liver cell lines (Table S2) were grown in a 37°C incubator with 95% humidity and 5% CO2 in DMEM (Life Technologies) with 10% FBS. Confluent cells were split and allowed to attach to plates as described [28]. Cells were serum-starved for 24 hours then stimulated with 10 ng/ml PDGF-AA, -AB,-BB, or –CC (R&D systems) for 24 hours. [3H]Thymidine (1 μCi/ml final concentration) was added to the media for the final 3 hours of stimulation. Unincorporated [3H] thymidine was removed from the cells, and trichloroacetic acid was used to precipitate protein-bound DNA. DNA was solubilized in NaOH, quantified using a scintillation counter, and measured in triplicate.
RNA Expression Analysis
RNA was extracted from cells or liver tissue using Trizol (15596-018, Life Technologies) as described by the manufacturer. Reactions contained cDNA synthesized from 0.5 μg RNA using MMLV (28025-013, Life Technologies), and Taqman Universal Mastermix II (4440040, Life Technologies). Cycling conditions were 95°C for 10 min, and 49 cycles of 95°C for 15 sec, 60°C for 60 sec with a final extension at 72°C for 1 min. Data are represented as delta delta Ct values after normalization to Gapdh mRNA levels. Primers used in this experiment are listed in Table S3.
Results
Expression of PDGFRα in Human Cirrhosis and HCC
Previous studies have demonstrated that over expression of PDGF ligands induces fibrosis in mice [29]–[31], and elevated expression of PDGFRβ in chronic liver disease has been reported [32]–[34]. As PDGF ligands can activate both PDGFRα and PDGFRβ, we sought to investigate the role of PDGFRα in chronic liver injury. 80–90% of human HCC arise in the setting of a cirrhotic liver, in which HSCs have been activated [35], so we first performed IHC analysis to determine whether PDGFRα and PDGFRβ levels are elevated in human cirrhosis and HCC. Fibrotic and cirrhotic livers had focal perisinusoidal immunoreactivity for PDGFRα, which was stronger in steatotic and cirrhotic livers, while normal adult liver had relatively little PDGFRα immunoreactivity (Figure 1 A–D). PDGFRβ immunoreactivity was also increased in the fibrotic and cirrhotic areas compared to un-injured liver (data not shown). Table S4 summarizes PDGFRα and PDGFRβ immunoreactivity in diseased human liver specimens, 77% of which demonstrated increased PDGFRα immunoreactivity and 56% of which demonstrated increased PDGFRβ immunoreactivity. Using a separate set of specimens, we compared PDGFR protein levels in grossly dissected HCC tumors to those of adjacent non-tumor livers from the same patients by immunoblot analysis. PDGFRα protein was frequently detected in the non-tumor tissue (Figure S1, Table S5). One specimen, patient 5, had detectable PDGFRα protein by immunoblot in the tumor (Table S5). IHC analysis of this specimen demonstrated PDGFRα immunoreactive cells within the tumor (Figure 1 E, F), but these cells did not have the histological appearance of hepatocytes, suggesting that non-parenchymal cells (NPCs) had invaded the parenchymal tumor and account for the PDGFRα immunoreactivity observed in this tumor by immunoblot analysis. Taken together, our data suggest that PDGFRα is expressed primarily in fibrotic and cirrhotic livers, predominantly in NPCs.
A) Uninjured (non-diseased) human liver demonstrate focal PDGFRα immunoreactivity (arrow) in NPCs but not hepatocytes. Resected tumor specimens show PDGFRα positive cells within fibrotic areas (arrow) (B), and cirrhotic areas (C), with sinusoidal PDGFRα immunoreactivity (arrows) within fibrotic septa (D). E and F) A resected tumor specimen shows stromal PDGFRα immunoreactivity (arrows). All scale bars are 50 μm.
Expression of PDGFRα and PDGFRβ in Human Liver and Stellate Cell Lines
We next analyzed mRNA transcripts from human liver cell lines, and found that both PDGFRα and PDGFRβ mRNA are expressed in non-diseased human liver (Figure 2), but that the human HSC line LX-1 has a significantly higher relative expression of PDGFRα (Figure 2A). LX-2 cells, a LX-1 subclone, express PDGFRβ, albeit to variable levels, and LX-1 cells express little to no PDGFRβ (Figure 2B). Transcription of both PDGFRs is reduced in human hepatoma cell lines compared to whole liver, suggesting that PDGFRs are predominantly expressed in NPCs. We next stimulated various cell lines with PDGF ligands, and found that PDGF -AA, -AB, -BB, and –CC lead to robust proliferation in stellate cells, but these ligands had little effect on the hepatocyte or hepatoma cell lines tested (Table S6).
A) Relative PDGFRα mRNA expression is greater in LX1 HSCs compared to LX2 and hepatocyte cell lines. B) PDGFRβ mRNA expression in LX2 HSC is variable, but similar to whole liver and hepatocyte cell lines. PDGFR expression was normalized to 18S ribosomal RNA and reported as fold increase by the ΔΔCt method, normalized to adult human liver. Error bars indicate standard error of the mean, n = 3 separate cultures.
Increased Expression of Pdgfrα and Pdgfrβ in Mice after CCl4 induced Hepatocyte Injury
To investigate the role of PDGFRα in liver injury and fibrosis, we used the well-established model of CCl4 injection, in which HSCs are activated in response to necroinflammatory injury to hepatocytes [36]. CCl4 injury to rats has been shown to induce Pdgfrs mRNA in the liver [37]. To determine whether Pdgfr expression is induced after liver injury in mice, WT mice were injected with a single dose of CCl4. We found that Pdgfrα expression increased during 72 hours after injury (Figure 3A). CCl4 injection also induced expression of Pdgfrβ, although to a differing extent and with a different time course than Pdgfrα (Figure 3B). Thus, acute CCl4 exposure induces Pdgfr expression in the liver.
Expression of Pdgfrα (A) and Pdgfrβ (B) increases after a single injection of CCl4 in C57BL/6 mice. Values are represented as means with SEM; n = 3 mice per group; and data was analyzed by Kruskal-Wallis non-parametric ANOVA * = p<0.05.
Cells Expressing Pdgfrα and Pdgfrβ Respond to CCl4 Injury
To determine the liver cell type that expresses PDGFRα in response to liver injury, we used transgenic PdgfrαWT/nGFP mice, in which the endogenous Pdgfrα promoter initiates transcription of nuclear-restricted GFP reporter in place of the Pdgfrα gene [20]. These mice have one copy of Pdgfrα replaced by GFP and are thus heterozygous for Pdgfrα. While a single injection of CCl4 induces necrosis and injury that is repaired within seven days, repeated injection of CCl4 induces liver fibrosis [36]. Vehicle-injected PdgfrαWT/nGFP mice have histologically normal liver (Figure 4A). A single CCl4 injection increases the density of small cells with a high nuclear to cytoplasmic ratio, suggestive of inflammatory cells, around central veins at 72 hours, while chronic injection increases the density of cells between portal veins (Figure 4B, C). As shown in Figure 4D, vehicle-injected PdgfrαWT/nGFP mice have an even distribution of PDGFRα-positive cells throughout the liver lobule. 72 hours after a single injection of CCl4, however, PDGFRα-positive cells have a higher density around central veins at areas of hepatocyte injury (Figure 4E). We found that after six weeks of twice weekly CCl4 injections, PDGFRα positive cells are detected around and between portal veins, where fibrotic bands form (Figure 4F). As PDGFRβ is expressed in quiescent and activated HSCs [38], we next determined whether both PDGFRs are expressed in the same cell type. PDGFRα positive cells in chronic CCl4 injected PdgfrαWT/nGFPmouse livers (Figure 5A, D) co-localize with PDGFRβ immunoreactive cells (Figure 5B, E), indicating that activated HSCs express both receptors (Figure 5C, F) after chronic CCl4 injection.
Oil injection A) does not lead to necrosis around central veins (CV), while areas of necrosis are visible (arrow) 72 hrs after CCl4 injection (B) as determined by H&E. C) NPCs are visible in areas between portal veins (PV, arrow). D) PDGFRα positive cells (green nuclei) are evenly distributed throughout the liver after oil injection. E) PDGFRα positive cells localize around the CV 72 hr after CCl4 injury. F) PDGFRα positive cells align with fibrotic bands that develop between portal triads after chronic CCl4 injury. Scale bars are 100 μm.
PdgfrαWT/nGFP mice were injected with CCl4 twice weekly for six weeks. PDGFRα-expressing cells are identified by nuclear-localized GFP (green). PDGFRβ-expressing cells are identified by IF (PDGFRβ; magenta). A) PDGFRα-positive cells are aligned between portal veins (PV). B) PDGFRβ is expressed in the same periportal area as PDGFRα-positive cells, as shown in the merged image (C). A–C) Scale bars are 100 μm. D–F) Higher magnification shows that PDGFRα and PDGFRβ co-localize in the same cell, based upon co-localization of the GFP and PDGFRβ signal. Scale bars are 10 μm.
Our data from human tissue using IHC (Figure 1) and from mice using a GFP reporter (Figure 5) indicated that HSCs are the predominant liver cell type that expresses PDGFRα. In order to confirm these findings, we stained for specific liver cell epitopes using IF in combination with nuclear-GFP expression in PdgfrαWT/nGFPmice [20]. Images of livers from PdgfrαWT/nGFPmice indicate that PDGFRα and PDGFRβ co-localize in the same cells (Figure 5, Figure S2A), and that these cells also express desmin (Figure S2B) and cellular retinol binding protein 1 (CRBP-1, Figure S2C), proteins expressed in HSCs. Furthermore, GFP is not detected in Kupffer cells that express F4/80 expression (Figure S2D), or endothelial cells as identified by CD31 expression (Figure S2E). Hepatocytes, identified morphologically by fluorescence as described [39], were also negative for GFP (Figure S2F). Taken together, these data suggest that HSCs are the primary liver cell type which express PDGFRα and PDGFRβ.
Reducing PDGFRα Expression Reduces Fibrosis in Mice
After confirming that Pdgfrα expression increases with CCl4-induced liver injury, and that PDGFRα-positive HSCs are activated by CCl4 exposure, we utilized PdgfrαWT/nGFPmice to evaluate the functional significance of Pdgfrα expression during liver fibrosis. PdgfrαWT/nGFPmice, which are heterozygous for Pdgfrα expression, are phenotypically normal [20] and have normal liver architecture (Figure 4A). Uninjured and CCl4-injected PdgfrαWT/nGFP and C57BL/6 mice were analyzed for transcriptional changes in genes associated with chronic liver injury. At baseline and after 4 weeks of twice weekly injections of CCl4, Pdgfrα expression is decreased in PdgfrαWT/nGFP mice compared to C57BL/6 mice (Figure 6A). Pdgfrβ expression was equivalently expressed in both genotypes of uninjured and chronic CCl4 injected mice (Figure 6B). Expression of smooth muscle α-actin (Acta2), an epitope that is upregulated when HSCs are activated [40], [41], increased when C57BL/6 mice were injected with CCl4 for 4 weeks, but PdgfrαWT/nGFP mice had greatly reduced Acta2 expression after chronic liver injury (Figure 6C). Fibrillar collagen 1a1 (Col1a1) expression was equivalent in uninjured mice of the two genotypes, but significantly reduced in chronically injured PdgfrαWT/nGFP mice (Figure 6D). Conversely, collagen 4 (Col4, Figure 6E) and tissue inhibitor of metalloproteinase 1 (Timp1, Figure 6F) expression was increased after chronic CCl4 exposure, but was not significantly reduced in PdgfrαWT/nGFP mice compared to wild type mice.
Livers from PdgfrαWT/nGFP and C57BL/6 mice that were either uninjured (controls) or treated for 4 weeks with CCl4 were used to prepare total liver RNA. A) Compared to C57BL/6 mice, expression of Pdgfrα is decreased in PdgfrαWT/nGFP mice in uninjured mice and after chronic CCl4. B) Expression of Pdgfrβ does not differ between C57BL/6 and PdgfrαWT/nGFP mice. C) Expression of Acta2 is increased in uninjured PdgfrαWT/nGFP mice compared to C57BL/6 and decreased after chronic CCl4 between PdgfrαWT/nGFP and C57BL/6 mice. D) Expression of Col1a1 is similar in uninjured PdgfrαWT/nGFP mice compared to C57BL/6 and decreased after chronic CCl4 between PdgfrαWT/nGFP and C57BL/6 mice. E) Expression of Col4 is similar in PdgfrαWT/nGFP mice compared to C57BL/6 in both uninjured and chronic CCl4 injected mice. F) Expression of Timp1 is increased to a similar level in both genotypes. Samples were processed as described in figure 2. Values are represented as means with SEM, and were analyzed by Mann-whitney non-parametric U test * = p<0.05, n = 3–6 mice per time point.
Reduced mRNA expression of Col1a1 in PdgfrαWT/nGFP mice after chronic CCl4 injection was accompanied by a reduction in liver fibrosis, as assessed by picrosirius red staining, a histochemical assay for tissue fibrosis. C57BL/6 mice injected with vehicle for 4 weeks had little to no fibrosis (Figure 7A, D), but developed periportal fibrosis after 4 weeks of twice weekly CCl4 injections (Figure 7B, D). Significantly less collagen was deposited in chronically injured PdgfrαWT/nGFP mice (Figure 7C, D). These results demonstrate that liver fibrosis in response to chronic CCl4 injection is dependent on normal expression of PDGFRα, and are consistent with the hypothesis that liver fibrosis is regulated in part by PDGFRα ligands.
Mice were injected with olive oil or CCl4 twice weekly for 4 weeks and collagen was detected in liver tissue by picrosirius red staining. A) Liver from a mouse injected with oil shows little collagen deposition. B) C57BL/6 mice develop fibrosis (arrow) after 4 weeks of CCl4 injections. C) PdgfrαWT/nGFP mice develop less fibrosis (arrow) than C57BL/6 mice after 4 weeks of CCl4 injections. D) Quantification of picrosirius red positive area. Values are represented as means with SEM, and were analyzed by Mann-Whitney non-parametric U test * = p<0.05, n = 3–6 mice per time point. Scale bars are 100 μm.
Discussion
PDGFRs stimulate proliferation, migration, and survival of mesenchymal cells, and increased activation of PDGFRs leads to organ fibrosis [21], [42]. Elevated expression of PDGFRs is associated with liver fibrosis and cirrhosis, so we sought to determine whether PDGFRα regulates liver fibrogenesis using mice that have one allele of Pdgfrα (PdgfrαWT/nGFP). We found that mice with decreased Pdgfrα expression have less liver fibrosis after chronic CCl4 injury. In addition, and consistent with the notion that PDGFRα regulates the liver’s response to injury, patients with liver disease have elevated expression of PDGFRα and PDGFRβ. In conjunction, GFP localization in PdgfrαWT/nGFP mice indicates that PDGFRα-positive HSCs migrate to sites of injury following CCl4 injection. These data all suggest that PDGFRα is involved in the activation of HSCs after hepatocyte injury.
PDGFRs are thought to play a central role in activating HSCs and promoting liver fibrosis and cirrhosis [33], [34], [43]; whether PDGFRα and PDGFRβ play independent roles in fibrogenesis is not known. We and others observe that Pdgfrβ expression increases in WT mice after acute liver injury by CCl4, implicating PDGFRβ in HSC activation. Thus it is surprising that mice which systemically express a hyperactive PDGFRβ allele do not develop more liver fibrosis than WT mice after 4 weeks of CCl4 injections [44]. Our data indicate that hepatocyte injury induces Pdgfrα expression above uninjured liver in both mice and humans, corroborating previously published studies [34], [37]. Our results demonstrate that expression of Pdgfrα and Pdgfrβ are both increased after chronic CCl4 liver injury, while reducing Pdgfrα copy number reduces Pdgfrα expression but not Pdgfrβ expression in PdgfrαWT/nGFP mice. Reduced Pdgfrα expression in PdgfrαWT/nGFP mice corrrelates with significantly reduced Col1a1 and Acta2 expression, as well as reduced picrosirius red staining, even though Pdgfrβ expression remains elevated. PDGFRα and PDGFRβ appear to affect HSCs differentially, despite being co-localized in the same liver cell type. Further studies will be necessary to dissect the receptor-specific contributions of PDGF signaling pathways in HSCs and in liver fibrosis.
Small perturbations in the PDGF signaling pathway, whether due to changes in expression of ligand or receptor, appear to have a large impact on specific diseases. Support for this notion is found in genetic evidence from rodents, which suggests that small changes in PDGFR activity in vivo are capable of significantly affecting a cell’s function. For example in development, chimerism studies show that both Pdgfrβ+/− and Pdgfrα+/− embryonic stem cells are deficient in contributing cells to the embryo [15], [45], and adult mice have a decreased number of progenitor cells in mice heterozygous Pdgfrs [46], [47]. Heterozygous Pdgfrα mice have been bred to mice with mutations in PDGF ligands [48] or mutations in immediate early genes directly downstream of PDGFRα [49], resulting in additive effects. However, deletion of one allele of Pdgfrα and the resultant heterozygosity does not affect development [15], [20]. These studies suggest that a single copy of Pdgfrα is usually sufficient for development, although under certain circumstances two alleles of Pdgfrα are required. In the current study, we found that PdgfrαWT/nGFP mice have reduced fibrosis and reduced expression of the profibrotic genes Acta2 and Col1a1 after chronic CCl4 injury. Our data indicate that in chronic liver injury, PDGFRα plays a critical role in the development of fibrosis, but that other pathways also contribute to fibrogenesis. Expression of Col4 and Timp1 were reduced in PdgfrαWT/nGFP mice but not to a significant extent, suggesting that expression of these genes could be more reliant on PDGFRα independent pathways, or heterogeneity in populations of HSCs.
We also sought to better define the role of PDGFRα in liver fibrosis by utilizing both human specimens and mouse models. Using a variety of experimental approaches, increased PDGFRα was seen in cirrhotic human livers and in mice with chemically-induced liver fibrosis. Although no preclinical rodent model fully recapitulates human liver fibrosis, there appears to be comparable molecular pathophysiology between humans and mice. We chose to utilize a knock-in mouse model expressing nuclear-GFP driven by the Pdgfrα promoter in order to discriminate between cells located in close proximity to each other, specifically different NPC populations in liver sinusoids [20]. We did not observe nuclear-GFP expression in hepatocytes, Kupffer cells, or LSECs, thus we conclude that the majority of PDGFRα is expressed in HSCs in the mouse liver, consistent with our observation that human liver specimens express PDGFRα primarily in NPCs. Our IHC data are further supported by data from the Human Protein Atlas (http://www.proteinatlas.org/ENSG00000134853/cancer), which demonstrates that NPCs are positive for PDGFRα by IHC in both normal liver and HCC [50].
We and others posit that selectively targeting PDGFRα in liver fibrosis and cirrhosis could reduce the proliferation, migration, and survival of the activated HSCs cells that contribute to collagen deposition. Therapeutic blockade of PDGFRα signaling may have a broad impact in the treatment of liver fibrosis, as four of the five PDGF ligand dimers, PDGF-AA, -AB, -BB, and –CC, bind and activate PDGFRα [12]. Targeting PDGFRβ, on the other hand, would completely inhibit only signal transduction induced by PDGF-DD, and could disrupt necessary functions of PDGFRβ in the liver. Targeting both PDGFRs with multi-kinase inhibitors, such as imatinib or sorafenib, leads to severe off target effects [51], [52]. The breadth of multi-kinase inhibitor activity thus likely leads to inhibition of beneficial signal transduction, either via PDGFRβ or other kinases. In summary, our data suggest that PDGFRα has a specific role in liver fibrosis in mice and in humans, and suggest that further mechanistic evaluation of PDGFRα function in the liver has the potential to uncover new anti-fibrotic therapies.
Supporting Information
Figure S1.
Expression of PDGFRs in human liver tumor and non-tumor tissue. An immunoblot of PDGFRα detects PDGFRα in non-tumor tissue while an immunoblot for PDGFRβ shows protein in both tumor and surrounding tissue. Albumin was used as a loading control.
https://doi.org/10.1371/journal.pone.0092925.s001
(TIF)
Figure S2.
HSCs express PDGFRα. PDGFRα-driven nuclear GFP is expressed in liver cells that are immunoreactive for common HSC markers: A) PDGFRβ (red), B) Desmin (red), and C) cellular retinol binding protein 1 (CRBP-1) (red). PDGFRα and CRBP-1 are not expressed in cells that stain for D) the Kupffer cell marker F4/80 (blue) or E) the endothelial cell marker CD31(blue). F) PDGFRα positive cells (green) are distinct from hepatocytes (yellow). Scale bars are 10 μm.
https://doi.org/10.1371/journal.pone.0092925.s002
(TIF)
Table S2.
Human and mouse hepatocyte and stellate cell lines used in this study. *references as PMID number.
https://doi.org/10.1371/journal.pone.0092925.s004
(DOCX)
Table S3.
Primers used for real time analysis.
https://doi.org/10.1371/journal.pone.0092925.s005
(DOCX)
Table S4.
Summary of PDGFRα and PDGFRβ immunoreactivity in human liver specimens. Resected liver specimens with HCCs were formalin-fixed, paraffin embedded, and evaluated for the presence of cirrhosis and HCC. IHC for PDGFRα and PDGFRβ was performed as described in Materials and Methods. Relative staining intensity is indicated as weak (+), moderate (++), strong (+++), or absent (0).
https://doi.org/10.1371/journal.pone.0092925.s006
(DOCX)
Table S5.
Immunoblot detection of PDGFR expression in macroscopically dissected human tumors and surrounding liver. HCCs (Tumor) and surrounding liver (Non-Tumor) were macrodissected from patients, frozen, and processed for immunoblot analysis as described in Materials and Methods. Intensity is indicated as present (+) or absent (0).
https://doi.org/10.1371/journal.pone.0092925.s007
(DOCX)
Table S6.
PDGF stimulates proliferation1 in stellate cell lines, but not primary hepatocytes or hepatoma cell lines. 1Cell proliferation was measured by DNA synthesis using tritiated thymidine incorporation [28]. The data is the average of three different experiments that were each done in triplicates. Fold change represents the increase when compared to unstimulated cells for each cell line. 2“Positive control” indicates that DNA synthesis was stimulated in each cell line or primary culture with a growth factor previously reported to simulate proliferation. Growth factors used for each cell and the concentrations are as follows: mouse hepatocytes, EGF (20 ng/mL); AML12 cells, EGF (20 ng/mL); NMH cells, HB-EGF (20 ng/mL); rat stellate cells (2G), 1% fetal calf serum; human stellate cells (LX-2), 1% FCS; SK-Hep (human hepatoma cells of endothelial origin), 10% fetal calf sera.
https://doi.org/10.1371/journal.pone.0092925.s008
(DOCX)
Acknowledgments
The authors would like to thank Dr. Dan Bowen-Pope for critically reviewing this manuscript, Melissa Johnson, Heidi Kenerson and Jagadambika Gunaje for experimental assistance, and Dr. Nelson Fausto for valuable input in the early stages of this project. The authors also thank Dr. Scott L. Friedman for generously sharing the LX1 and LX2 cells.
Author Contributions
Conceived and designed the experiments: BJH KJR JSC. Performed the experiments: BJH MS RLB KLH FJ JSC. Analyzed the data: BJH MMY KJR JSC. Contributed reagents/materials/analysis tools: WMM MMY RSY. Wrote the paper: BJH KJR JSC WMM.
References
- 1. Starr SP, Raines D (2011) Cirrhosis: diagnosis, management, and prevention. Am Fam Physician 84: 1353–1359.
- 2. Bataller R, Brenner DA (2005) Liver fibrosis. J Clin Invest 115: 209–218.
- 3. Hernandez-Gea V, Friedman SL (2011) Pathogenesis of liver fibrosis. Annu Rev Pathol 6: 425–456.
- 4. Pierce GF, Tarpley JE, Tseng J, Bready J, Chang D, et al. (1995) Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds. J Clin Invest 96: 1336–1350.
- 5. Olson LE, Soriano P (2009) Increased PDGFRalpha activation disrupts connective tissue development and drives systemic fibrosis. Dev Cell 16: 303–313.
- 6. Chong JJ, Reinecke H, Iwata M, Torok-Storb B, Stempien-Otero A, et al. (2013) Progenitor cells identified by PDGFR-alpha expression in the developing and diseased human heart. Stem Cells Dev 22: 1932–1943.
- 7. Antoniades HN, Bravo MA, Avila RE, Galanopoulos T, Neville-Golden J, et al. (1990) Platelet-derived growth factor in idiopathic pulmonary fibrosis. J Clin Invest 86: 1055–1064.
- 8. Floege J, Eitner F, Alpers CE (2008) A new look at platelet-derived growth factor in renal disease. J Am Soc Nephrol 19: 12–23.
- 9. Zymek P, Bujak M, Chatila K, Cieslak A, Thakker G, et al. (2006) The role of platelet-derived growth factor signaling in healing myocardial infarcts. J Am Coll Cardiol 48: 2315–2323.
- 10. Abdollahi A, Li M, Ping G, Plathow C, Domhan S, et al. (2005) Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis. J Exp Med 201: 925–935.
- 11. Chen YT, Chang FC, Wu CF, Chou YH, Hsu HL, et al. (2011) Platelet-derived growth factor receptor signaling activates pericyte-myofibroblast transition in obstructive and post-ischemic kidney fibrosis. Kidney Int 80: 1170–1181.
- 12. Andrae J, Gallini R, Betsholtz C (2008) Role of platelet-derived growth factors in physiology and medicine. Genes Dev 22: 1276–1312.
- 13. Hoch RV, Soriano P (2003) Roles of PDGF in animal development. Development 130: 4769–4784.
- 14. Soriano P (1994) Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev 8: 1888–1896.
- 15. Soriano P (1997) The PDGF alpha receptor is required for neural crest cell development and for normal patterning of the somites. Development 124: 2691–2700.
- 16. Mellgren AM, Smith CL, Olsen GS, Eskiocak B, Zhou B, et al. (2008) Platelet-derived growth factor receptor beta signaling is required for efficient epicardial cell migration and development of two distinct coronary vascular smooth muscle cell populations. Circ Res 103: 1393–1401.
- 17. Olson LE, Soriano P (2011) PDGFRβ signaling regulates mural cell plasticity and inhibits fat development. Dev Cell 20: 815–826.
- 18. Chen H, Gu X, Liu Y, Wang J, Wirt SE, et al. (2011) PDGF signalling controls age-dependent proliferation in pancreatic β-cells. Nature 478: 349–355.
- 19. Tallquist MD, Soriano P (2003) Cell autonomous requirement for PDGFRalpha in populations of cranial and cardiac neural crest cells. Development 130: 507–518.
- 20. Hamilton TG, Klinghoffer RA, Corrin PD, Soriano P (2003) Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol Cell Biol 23: 4013–4025.
- 21. Bonner JC (2004) Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev 15: 255–273.
- 22. Iwayama T, Olson LE (2013) Involvement of PDGF in fibrosis and scleroderma: recent insights from animal models and potential therapeutic opportunities. Curr Rheumatol Rep 15: 304.
- 23. Yeh MM, Larson AM, Campbell JS, Fausto N, Rulyak SJ, et al. (2007) The expression of transforming growth factor-alpha in cirrhosis, dysplastic nodules, and hepatocellular carcinoma: an immunohistochemical study of 70 cases. Am J Surg Pathol 31: 681–689.
- 24. Campbell JS, Johnson MM, Bauer RL, Hudkins KL, Gilbertson DG, et al. (2007) Targeting stromal cells for the treatment of platelet-derived growth factor C-induced hepatocellular carcinogenesis. Differentiation 75: 843–852.
- 25. Junqueira LC, Bignolas G, Brentani RR (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11: 447–455.
- 26. Campbell JS, Riehle KJ, Brooling JT, Bauer RL, Mitchell C, et al. (2006) Proinflammatory cytokine production in liver regeneration is Myd88-dependent, but independent of Cd14, Tlr2, and Tlr4. J Immunol 176: 2522–2528.
- 27.
Wright JH, Johnson MM, Shimizu-Albergine M, Bauer RL, Hayes BJ, et al.. (2013) Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: Evidence for stromal induction of hepatocellular carcinoma. Int J Cancer.
- 28. Argast GM, Campbell JS, Brooling JT, Fausto N (2004) Epidermal growth factor receptor transactivation mediates tumor necrosis factor-induced hepatocyte replication. J Biol Chem 279: 34530–34536.
- 29. Thieringer F, Maass T, Czochra P, Klopcic B, Conrad I, et al. (2008) Spontaneous hepatic fibrosis in transgenic mice overexpressing PDGF-A. Gene 423: 23–28.
- 30. Czochra P, Klopcic B, Meyer E, Herkel J, Garcia-Lazaro JF, et al. (2006) Liver fibrosis induced by hepatic overexpression of PDGF-B in transgenic mice. J Hepatol 45: 419–428.
- 31. Campbell JS, Hughes SD, Gilbertson DG, Palmer TE, Holdren MS, et al. (2005) Platelet-derived growth factor C induces liver fibrosis, steatosis, and hepatocellular carcinoma. Proc Natl Acad Sci U S A 102: 3389–3394.
- 32. Ikura Y, Morimoto H, Ogami M, Jomura H, Ikeoka N, et al. (1997) Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol 32: 496–501.
- 33. Wong L, Yamasaki G, Johnson RJ, Friedman SL (1994) Induction of beta-platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture. J Clin Invest 94: 1563–1569.
- 34. Pinzani M, Milani S, Herbst H, DeFranco R, Grappone C, et al. (1996) Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis. Am J Pathol 148: 785–800.
- 35. Tiribelli C, Melato M, Crocè LS, Giarelli L, Okuda K, et al. (1989) Prevalence of hepatocellular carcinoma and relation to cirrhosis: comparison of two different cities of the world–Trieste, Italy, and Chiba, Japan. Hepatology 10: 998–1002.
- 36. Weber LW, Boll M, Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 33: 105–136.
- 37. Borkham-Kamphorst E, Kovalenko E, van Roeyen CR, Gassler N, Bomble M, et al. (2008) Platelet-derived growth factor isoform expression in carbon tetrachloride-induced chronic liver injury. Lab Invest 88: 1090–1100.
- 38. Breitkopf K, Roeyen C, Sawitza I, Wickert L, Floege J, et al. (2005) Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC). Cytokine 31: 349–357.
- 39.
Johnson S, Rabinovitch P (2012) Ex vivo imaging of excised tissue using vital dyes and confocal microscopy. Curr Protoc Cytom Chapter 9: Unit 9.39.
- 40. Rockey DC, Boyles JK, Gabbiani G, Friedman SL (1992) Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J Submicrosc Cytol Pathol 24: 193–203.
- 41. Yamaoka K, Nouchi T, Marumo F, Sato C (1993) Alpha-smooth-muscle actin expression in normal and fibrotic human livers. Dig Dis Sci 38: 1473–1479.
- 42. Heldin CH, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79: 1283–1316.
- 43. Friedman SL, Arthur MJ (1989) Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest 84: 1780–1785.
- 44. Krampert M, Heldin CH, Heuchel RL (2008) A gain-of-function mutation in the PDGFR-beta alters the kinetics of injury response in liver and skin. Lab Invest 88: 1204–1214.
- 45. Crosby JR, Seifert RA, Soriano P, Bowen-Pope DF (1998) Chimaeric analysis reveals role of Pdgf receptors in all muscle lineages. Nat Genet 18: 385–388.
- 46. McKinnon RD, Waldron S, Kiel ME (2005) PDGF alpha-receptor signal strength controls an RTK rheostat that integrates phosphoinositol 3′-kinase and phospholipase Cgamma pathways during oligodendrocyte maturation. J Neurosci 25: 3499–3508.
- 47. Bell RD, Winkler EA, Sagare AP, Singh I, LaRue B, et al. (2010) Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging. Neuron 68: 409–427.
- 48.
Andrae J, Ehrencrona H, Gallini R, Lal M, Ding H, et al.. (2013) Analysis of mice lacking the heparin-binding splice isoform of PDGF-A. Mol Cell Biol.
- 49. Schmahl J, Raymond C, Soriano P (2007) PDGF signaling specificity is mediated through multiple immediate early genes. Nat Genet 39: 52–60.
- 50. Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, et al. (2010) Towards a knowledge-based Human Protein Atlas. Nat Biotechnol 28: 1248–1250.
- 51. Teo YL, Ho HK, Chan A (2013) Risk of tyrosine kinase inhibitors-induced hepatotoxicity in cancer patients: A meta-analysis. Cancer Treat Rev 39: 199–206.
- 52. Hasinoff BB, Patel D (2010) The lack of target specificity of small molecule anticancer kinase inhibitors is correlated with their ability to damage myocytes in vitro. Toxicol Appl Pharmacol 249: 132–139.