Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells
Sheng Cao, … , Debabrata Mukhopadhyay, Vijay H. Shah
Sheng Cao, … , Debabrata Mukhopadhyay, Vijay H. Shah
Published June 23, 2010
Citation Information: J Clin Invest. 2010;120(7):2379-2394. https://doi.org/10.1172/JCI41203.
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Research Article Hepatology

Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-β signaling in hepatic stellate cells

Abstract

PDGF-dependent hepatic stellate cell (HSC) recruitment is an essential step in liver fibrosis and the sinusoidal vascular changes that accompany this process. However, the mechanisms that regulate PDGF signaling remain incompletely defined. Here, we found that in two rat models of liver fibrosis, the axonal guidance molecule neuropilin-1 (NRP-1) was upregulated in activated HSCs, which exhibit the highly motile myofibroblast phenotype. Additionally, NRP-1 colocalized with PDGF-receptor β (PDGFRβ) in HSCs both in the injury models and in human and rat HSC cell lines. In human HSCs, siRNA-mediated knockdown of NRP-1 attenuated PDGF-induced chemotaxis, while NRP-1 overexpression increased cell motility and TGF-β–dependent collagen production. Similarly, mouse HSCs genetically modified to lack NRP-1 displayed reduced motility in response to PDGF treatment. Immunoprecipitation and biochemical binding studies revealed that NRP-1 increased PDGF binding affinity for PDGFRβ-expressing cells and promoted downstream signaling. An NRP-1 neutralizing Ab ameliorated recruitment of HSCs, blocked liver fibrosis in a rat model of liver injury, and also attenuated VEGF responses in cultured liver endothelial cells. In addition, NRP-1 overexpression was observed in human specimens of liver cirrhosis caused by both hepatitis C and steatohepatitis. These studies reveal a role for NRP-1 as a modulator of multiple growth factor targets that regulate liver fibrosis and the vascular changes that accompany it and may have broad implications for liver cirrhosis and myofibroblast biology in a variety of other organ systems and disease conditions.

Authors

Sheng Cao, Usman Yaqoob, Amitava Das, Uday Shergill, Kumaravelu Jagavelu, Robert C. Huebert, Chittaranjan Routray, Soha Abdelmoneim, Meher Vasdev, Edward Leof, Michael Charlton, Ryan J. Watts, Debabrata Mukhopadhyay, Vijay H. Shah

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Figure 2

NRP-1 is enriched within low-buoyant-density membrane microdomains and colocalizes with PDGFRβ.

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NRP-1 is enriched within low-buoyant-density membrane microdomains and c...
(A) LX2 cells transduced with PDGFRβ retrovirus were cotransduced with Ad-NRP-1–RFP. Cells were immunostained with NRP-1 (red) and PDGFRβ (green) Abs and photographed under a confocal microscope. The top row shows ×20 magnification and the bottom row shows ×63 magnification of the cropped cell, with further magnification of the cropped area (representative photomicrographs or Western blot are from 3 independent experiments). (B) HSC lysates were prepared for sucrose density gradient fractionation studies, and equal volumes of each fraction were analyzed by SDS-PAGE (top). White line indicates splicing of a noncontiguous sample from the same membrane. NRP-1 was also found to be enriched within low-buoyant-density fractions as assessed by levels of NRP-1 per microgram protein within each fraction when SDS-PAGE fraction signal was normalized for protein concentration (bottom). Separation and purity of membrane fractions were determined by immunoblotting for caveolin-1, a marker for low-buoyant-density membranes (n = 3). TPM, total plasma membrane. (C) Cyclodextrin, a compound that disrupts low-buoyant-density vesicle formation, inhibits PDGF-dependent hHSC chemotaxis. Cyclodextrin inhibition of PDGF-induced HSC chemokinesis was studied using time-lapse video microscopy followed by analysis using MetaMorph Imaging software. The right 2 bars show a reversible effect of cyclodextrin, thus reducing the likelihood of cell toxicity relating to the compound (n = 3, *P < 0.05).