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.

Increasing evidence suggests that hepatic fibrosis and pathological angiogenesis are interdependent processes that occur in parallel. Endothelial cell invasion is requisite for angiogenesis, and thus studies of the mechanisms governing liver endothelial cell (LEC) invasion during cirrhosis are of great importance. Emerging research implicates amoeboid-type motility and membrane blebbing as features that may facilitate invasion through matrix-rich microenvironments. Aquaporins (AQPs) are integral membrane water channels, recognized for their importance in epithelial secretion and absorption. However, recent studies also suggest links between water transport and cell motility or invasion. Therefore, the purpose of this study was to test the hypothesis that AQP-1 is involved in amoeboid motility and angiogenic invasion during cirrhosis. AQP-1 expression and localization was examined in normal and cirrhotic liver tissues derived from human and mouse. AQP-1 levels were modulated in LEC using retroviral overexpression or small interfering RNA (siRNA) knockdown and functional effects on invasion, membrane blebbing dynamics, and osmotic water permeability were assayed. Results demonstrate that AQP-1 is upregulated in the small, angiogenic, neovasculature within the fibrotic septa of cirrhotic liver. AQP-1 overexpression promotes fibroblast growth factor (FGF)-induced dynamic membrane blebbing in LEC, which is sufficient to augment invasion through extracellular matrix. Additionally, AQP-1 localizes to plasma membrane blebs, where it increases osmotic water permeability and locally facilitates the rapid, trans-membrane flux of water. Conclusion: AQP-1 enhances osmotic water permeability and FGF-induced dynamic membrane blebbing in LEC and thereby drives invasion and pathological angiogenesis during cirrhosis. (HEPATOLOGY 2010;52:238-248)

Changes in hepatic vasculature accompany fibrogenesis, and targeting angiogenic molecules often attenuates fibrosis in animals. Aquaporin-1 (AQP1) is a water channel, overexpressed in cirrhosis, that promotes angiogenesis by enhancing endothelial invasion. The effect of AQP1 on fibrogenesis in vivo and the mechanisms driving AQP1 expression during cirrhosis remain unclear. The purpose of this study was to test the effect of AQP1 deletion in cirrhosis and explore mechanisms regulating AQP1. After bile duct ligation, wild-type mice overexpress AQP1 that colocalizes with vascular markers and sites of robust angiogenesis. AQP1 knockout mice demonstrated reduced angiogenesis compared with wild-type mice, as evidenced by immunostaining and endothelial invasion/proliferation in vitro. Fibrosis and portal hypertension were attenuated based on immunostaining, portal pressure, and spleen/body weight ratio. AQP1 protein, but not mRNA, was induced by hyperosmolality in vitro, suggesting post-transcriptional regulation. Endothelial cells from normal or cirrhotic mice were screened for microRNA (miR) expression using an array and a quantitative PCR. miR-666 and miR-708 targeted AQP1 mRNA and were decreased in cirrhosis and in cells exposed to hyperosmolality, suggesting that these miRs mediate osmolar changes via AQP1. Binding of the miRs to the untranslated region of AQP1 was assessed using luciferase assays. In conclusion, AQP1 promotes angiogenesis, fibrosis, and portal hypertension after bile duct ligation and is regulated by osmotically sensitive miRs.