Cao S, Yaqoob U, Das A, Shergill U, Jagavelu K, Huebert RC, et al. Neuropilin-1 promotes cirrhosis of the rodent and human liver by enhancing PDGF/TGF-beta signaling in hepatic stellate cells. J Clin Invest 2010;120:2379-2394. (Reprinted with permission.)
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 beta (PDGFRbeta) 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-beta-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 PDGFRbeta-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.
Chronic liver disease afflicts millions of patients and is among the 10 leading causes of death in the United States.1 The great majority of chronic liver disease is caused by hepatitis B, hepatitis C, nonalcoholic fatty liver disease, and alcoholic liver disease. In most cases, these diseases progress slowly over several decades in characteristic stages, with hepatic fibrosis setting the stage for the development of cirrhosis and, in some cases, hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) have emerged as the main profibrogenic cell type in the liver, and the transformation from quiescent, vitamin A storing to activated HSCs with a myofibroblastic phenotype is believed to be a key event in the progression to fibrosis and cirrhosis. Numerous studies have characterized signaling pathways that contribute to HSC activation such as transforming growth factor β (TGFβ), platelet-derived growth factor (PDGF), angiotensin II, lipopolysaccharide/Toll-like receptor 4, hedgehog signaling, cannabinoids, leptin, and adiponectin, among many others.2 Evidence from transgenic and knockout mice as well as pharmacological studies have revealed PDGF and TGFβ as probably the two most important contributors to HSC activation and liver fibrosis.2 In HSCs, the binding of PDGF to the PDGF cell surface receptor stimulates several profibrogenic signaling cascades, including the phosphoinositide 3-kinase (PI3K)–AKT-p70S6 kinase, the mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK) pathway, and the Ras/MEK/extracellular signal-regulated kinase (ERK) pathway to stimulate HSC proliferation and motility.3, 4 TGFβ binds the TGFβ receptor complex to promote HSC activation both through Smad transcription factors as well as Smad-independent pathways such as Ras-MEK-ERK and TGFβ-activated kinase 1/ MAPK kinase–p38/JNK.5
The study of Cao et al.6 introduces neuropilin-1 (NRP-1) as a new element in profibrogenic signaling pathways in HSCs and suggests that NRP-1 serves as an important amplifier of the two major profibrogenic signaling pathways, PDGF and TGFβ. Neuropilins were first discovered as receptors for class 3 semaphorins, polypeptides with key roles in the nervous system such as axonal guidance.7 Subsequently, it was found that neuropilins are also involved in other signaling pathways such as vascular endothelial growth factor (VEGF) signaling. Recent evidence, including the results presented by Cao et al., also imply a role for NRP-1 in the cellular response to PDGF and TGFβ.8, 9 NRP-1 has a very short intracellular domain that lacks a defined signaling role. It is therefore widely believed that NRP-1 mediates functional responses as a result of complex formation with other receptors (e.g., plexins and VEGF receptors).7 NRP-1 functions are best studied in the nervous system and the vasculature, and knockout mice demonstrate decreased neural vascularization and hypoplasia of segments of the arch arteries and dorsal aorta and die during embryogenesis.10
Because HSCs express many neural markers such as neural cell adhesion molecule and glial fibrillary acidic protein,2 the expression of NRP-1 in activated HSCs as demonstrated by Cao et al. is not entirely surprising. Cao et al. not only show that NRP-1 increases in HSCs isolated from CCl4-treated and bile duct–ligated livers but also demonstrate an up-regulation of NRP-1 in cirrhotic livers from patients with hepatitis C virus and nonalcoholic steatohepatitis. The clinically most relevant result of the study by Cao et al. is the reduction of liver fibrosis as assessed by hydoxyproline levels and multiple fibrogenesis markers such as Col1a1, α smooth muscle actin, and Tgfβ messenger RNA by an inhibitory NRP-1 antibody. Further mechanistic studies revealed that small interfering RNA knockdown of NRP-1 inhibited PDGF-induced chemotaxis independently of VEGF receptor and Sema3a, whereas overexpression of NRP-1 increased HSC motility. Moreover, in vitro binding studies demonstrated that NRP-1 increases PDGF binding affinity for PDGFR-expressing cells. HSCs from NRP-1–deleted mice exhibited decreased migration in response to PDGF, whereas overexpression of NRP-1 promoted selective activation of Rac1 in the presence of PDGF without affecting Akt and ERK activity. Interestingly, Rac activity was diminished in c-Abl–deficient mouse embryonic fibroblasts overexpressing NRP-1, suggesting that NRP-1 directs the PDGFR signals to Rac1 through its ability to bind and activate c-Abl (Fig. 1). Furthermore, Cao et al. investigate the role of NRP-1 in the regulation of collagen deposition induced by the PDGF and TGF-β pathways. Surprisingly, both cytokines induce collagen deposition after overexpression of NRP-1. Collagen deposition is inhibited in NRP-1– and c-Abl–deficient mouse embryonic fibroblasts after treatment with PDGF or TGF-β, suggesting the presence of an NRP-1/c-Abl pathway in enhancing collagen deposition. In a recently published parallel study, the same group demonstrated that NRP-1 mediates R-SMAD signaling via TGFβ11 and suggested that NRP-1 amplifies TGFβ-induced myofibroblast activation by increasing the profibrogenic Smad2/3 pathway and suppressing the antifibrogenic Smad 1/5 pathway. In summary, these studies convincingly establish NRP-1 as an amplifier for profibrogenic signaling pathways such as PDGF and TGF-β, leading to increased HSC activation and fibrosis in the liver.
A few questions have yet to be answered, however. Culture-activated HSCs and HSC cell lines employed for mechanistic experiments in this study may differ significantly from in vivo–activated HSCs.12, 13 In this regard, additional in vivo studies may be helpful to further delineate whether NRP-1 promotes HSC activation and liver fibrosis acting through its role as a VEGF and semaphorin coreceptor. Notably, the two antibodies employed in the present studies differ in their epitope binding, with NRP-1a blocking semaphoring binding and NRP-1b blocking VEGF binding. Because NRP-1b antibody reduced CCl4-induced liver fibrosis, one needs to consider whether the VEGF blocking abilities of this antibody played a role in the improved fibrosis observed in vivo. Importantly, angiogenesis has been suggested to contribute to hepatic fibrosis.14 Although Cao et al. investigated the role of NRP-1 in regulating the ability of HSCs to promote the formation of vascular tubes, they did not investigate angiogenesis in vivo. Moreover, the current study did not employ genetic methods to inhibit NRP-1 expression in vivo. The floxed NRP-1 mice employed for in vitro experiments in this study should ideally be used to delete NRP-1 in HSCs during liver fibrosis. Finally, it is intriguing that NRP-1 was strongly up-regulated in HSCs from CCl4-treated livers but only very moderately in HSCs isolated form bile duct–ligated livers. Thus, it would be important to study the transcriptional regulation of NRP-1 in HSCs as well as the functional contribution of NRP-1 in additional models such as bile duct ligation or genetic models of liver fibrosis. With this additional information, future studies can possibly attempt to target NRP-1 in patients and to “hit three birds with one stone”: namely PDGF, TGFβ, and most likely also VEGF signaling. Antibodies to human NRP-1 are currently studied in phase l trials and might be available for antifibrotic therapies in the near future. In view of several studies showing antitumor effects of NRP-1 inhibition,15, 16 it would also be interesting to investigate whether NRP-1 is expressed in HCCs or the hepatic tumor microenvironment, and whether it promotes growth or angiogenesis of HCC.