Activation of Platelet-Derived Growth Factor Receptor Alpha Contributes to Liver Fibrosis

<div><p>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 <i>Pdgfrα</i> heterozygous mice, consistent with the hypothesis that liver fibrosis requires upregulation and activation of PDGFRα. These results indicate that <i>Pdgfrα</i> 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.</p></div

Chronically injured <i>Pdgfrα^WT/nGFP</i> mice have less collagen deposition than C57BL/6 mice.

<p>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) <i>Pdgfrα^WT/nGFP</i> 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.</p

PDGFRα positive cells form fibrotic bands after chronic CCl₄ injection in <i>Pdgfrα^WT/nGFP</i> mice.

<p>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.</p

PDGFRα-positive cells co-localize with PDGFRβ-positive cells in chronic CCl₄ injured liver.

<p><i>Pdgfrα^WT/nGFP</i> 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.</p

Compared to C57BL/6 mice, chronically CCl₄ injured <i>Pdgfrα^WT/nGFP</i> mice have reduced transcription of fibrotic genes.

<p>Livers from <i>Pdgfrα^WT/nGFP</i> 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 <i>Pdgfrα</i> is decreased in <i>Pdgfrα^WT/nGFP</i> mice in uninjured mice and after chronic CCl₄. B) Expression of <i>Pdgfrβ</i> does not differ between C57BL/6 and <i>Pdgfrα^WT/nGFP</i> mice. C) Expression of <i>Acta2</i> is increased in uninjured <i>Pdgfrα^WT/nGFP</i> mice compared to C57BL/6 and decreased after chronic CCl₄ between <i>Pdgfrα^WT/nGFP</i> and C57BL/6 mice. D) Expression of <i>Col1a1</i> is similar in uninjured <i>Pdgfrα^WT/nGFP</i> mice compared to C57BL/6 and decreased after chronic CCl₄ between <i>Pdgfrα^WT/nGFP</i> and C57BL/6 mice. E) Expression of <i>Col4</i> is similar in <i>Pdgfrα^WT/nGFP</i> mice compared to C57BL/6 in both uninjured and chronic CCl₄ injected mice. F) Expression of <i>Timp1</i> is increased to a similar level in both genotypes. Samples were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092925#pone-0092925-g002" target="_blank">figure 2</a>. 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.</p

PDGF receptors are expressed in hepatic stellate cell lines.

<p>A) Relative <i>PDGFRα</i> mRNA expression is greater in LX1 HSCs compared to LX2 and hepatocyte cell lines. B) <i>PDGFRβ</i> mRNA expression in LX2 HSC is variable, but similar to whole liver and hepatocyte cell lines. <i>PDGFR</i> 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.</p