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.

Angiogenesis defines the growth of new blood vessels from preexisting vascular endothelial networks and corresponds to the wound healing process that is typified by the process of liver fibrosis. Liver fibrosis is also associated with increased endotoxin within the gut lumen and its associated portal circulation. However, the interrelationship of gut endotoxin and its receptor, toll‐like receptor 4 (TLR4), with liver fibrosis and associated angiogenesis remains incompletely defined. Here, using complementary genetic, molecular, and pharmacological approaches, we provide evidence that the pattern recognition receptor that recognizes endotoxin, TLR4, which is expressed on liver endothelial cells (LECs), regulates angiogenic responses both in vitro and in vivo. Mechanistic studies have revealed a key role for a cognate TLR4 effector protein, myeloid differentiation protein 88 (MyD88), in this process, which culminates in extracellular protease production that regulates the invasive capacity of LECs, a key step in angiogenesis. Furthermore, TLR4‐dependent angiogenesis in vivo corresponds to fibrosis in complementary liver models of fibrosis. Conclusion: These studies provide evidence that the TLR4 pathway in LECs regulates angiogenesis through its MyD88 effector protein by regulating extracellular protease production and that this process is linked to the development of liver fibrosis. (HEPATOLOGY 2010;)

Background Paracrine signaling between hepatic stellate cells (HSC) and liver endothelial cells (LEC) modulates fibrogenesis, angiogenesis, and portal hypertension. However, mechanisms regulating these processes are not fully defined. Sorafenib is a receptor tyrosine kinase inhibitor that blocks growth factor signaling in tumor cells but also displays important and not yet fully characterized effects on liver nonparenchymal cells including HSC and LEC. The aim of this study was to test the hypothesis that sorafenib influences paracrine signaling between HSC and LEC and thereby regulates matrix and vascular changes associated with chronic liver injury. Results Complementary magnetic resonance elastography, micro-CT, and histochemical analyses indicate that sorafenib attenuates the changes in both matrix and vascular compartments that occur in response to bile-duct ligation induced liver injury in rats. Cell biology studies demonstrate that sorafenib markedly reduces cell to cell apposition and junctional complexes, thus reducing the proximity typically observed between these sinusoidal barrier cells. At the molecular level, sorafenib down-regulates angiopoietin-1 and fibronectin, both released by HSC in a manner dependent on the transcription factor KLF6, suggesting that this pathway underlies both matrix and vascular changes associated with chronic liver disease. Conclusion Collectively, our results demonstrate that sorafenib inhibits both matrix restructuring and vascular remodeling that accompany chronic liver diseases and characterize cell and molecular mechanisms underlying this effect. These data may help to refine future therapies for advanced gastrointestinal and liver diseases characterized by abundant fibrosis and neovascularization.

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMPdependent kinase (PKG) via NO/cGMP paracrine signaling. Although PKG is implicated in integrin-mediated cell adhesion to extracellular matrix, whether or how PKG signaling regulates the assembly of focal adhesion complexes (FA) and migration of HSC is not known. With the help of complementary molecular and cell biological approaches, we demonstrate here that activation of PKG signaling in HSC inhibits vascular tubulogenesis, migration/chemotaxis, and assembly of mature FA plaques, as assessed by vascular tubulogenesis assays and immunofluorescence localization of FA markers such as vinculin and vasodilator-stimulated phosphoprotein (VASP). To determine whether PKG inhibits FA assembly by phosphorylation of VASP at Ser-157, Ser-239, and Thr-278, we mutated these putative phosphorylation sites to alanine (VASP3A, phosphoresistant mutant) or aspartic acid (VASP3D, phosphomimetic), respectively. Data generated from these two mutants suggest that the effect of PKG on FA is independent of these three phosphorylation sites. In contrast, activation of PKG inhibits the activity of small GTPase Rac1 and its association with the effector protein IQGAP1. Moreover, PKG activation inhibits the formation of a trimeric protein complex containing Rac1, IQGAP1, and VASP. Finally, we found that expression of a constitutively active Rac1 mutant abolishes the inhibitory effects of PKG on FA formation. In summary, our data suggest that activation of PKG signaling in pericytes inhibits FA formation by inhibiting Rac1. nitric oxide; vasodilator-stimulated phosphoprotein; cGMP-dependent kinase; hepatic stellate cells NITRIC OXIDE (NO) generated by endothelial cells plays a major role in the control of vessel tone and vascular architecture (2, 10). In perivascular cells (pericytes), NO exerts its biological effects mainly by activating the cGMP-dependent protein kinase (PKG) via NO/cGMP/PKG paracrine signaling. Similar to pericytes in other vascular beds, liver-specific pericytes hepatic stellate cells (HSC) are also regulated by the NO/cGMP/PKG paracrine signaling. For instance, NO/cGMP/PKG signaling inhibits the contractility, migration, and survival of HSC in vitro (27,29,38). Indeed, impaired NO production or attenuated response of HSC to NO stimulation in animal models of liver cirrhosis suggest that a defective NO/cGMP/PKG signaling cascade is implicated in the pathophysiology of liver fibrosis and ensuing portal hypertension (3,12,15,17).Focal adhesions (FA) are dynamic protein complexes that connect extracellular matrix to cells and convey these external stimuli into intracellular signals, which is fundamental for cell adhesion, migration, and survival (5, 14, 31, 45). Although it is known that PKG signaling in smooth muscle cells mediates FA disassembly induced by counter-adhesive extracellular matrix proteins such as thrombospondin and tenascin (34), and that PKG inhibits integ...