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.

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These studies elucidate novel HSC-signaling pathways that regulate microvascular structure and function in liver.

Hepatic stellate cells (HSCs) contribute to portal hypertension through multiple mechanisms that include collagen deposition, vasoconstriction, and regulation of sinusoidal structure. Under normal physiologic conditions, endothelial nitric oxide (NO) synthase-derived NO exerts paracrine effects on HSCs; however, in cirrhosis, NO generation is impaired in association with concomitant HSC activation and changes in sinusoidal structure, events that contribute significantly to the development of portal hypertension. These concepts, in combination with recent evidence that induction of HSC-selective apoptosis may represent a useful target for treatment of chronic liver disease, led us to examine if NO may further limit HSC function through apoptosis. Indeed, both NO donors and endothelial NO synthase overexpression promoted HSC apoptotic pathways. HSC death conferred by NO occurred through mitochondrial membrane depolarization and through a caspase-independent pathway. Furthermore, NO-induced apoptosis of HSC did not occur through the canonical pathways of soluble guanylate cyclase or protein nitration, but rather through the generation of superoxide and hydroxyl radical intermediates. Lastly, HSC isolated from rats after bile duct ligation were more susceptible to NO-induced apoptosis. These data indicate that NO promotes HSC apoptosis through a signaling mechanism that involves mitochondria, is mediated by reactive oxygen species, and occurs independent of caspase activation. Conclusion: We postulate that NO-dependent apoptosis of HSCs may maintain sinusoidal homeostasis, and may represent an additional beneficial effect of NO donors for therapy of portal hypertension.

The farnesoid X receptor (FXR) signaling pathway regulates bile acid and cholesterol homeostasis. Here, we demonstrate, using a variety of gain-and loss-of-function approaches, a role of FXR in the process of cell motility, which involves the small heterodimeric partner (SHP)-dependent up-regulation of matrix metalloproteinase-9. We use this observation to reveal a transcriptional regulatory mechanism involving the SP/KLF transcription factors, SP2 and KLF6. Small interference RNA-based silencing studies in combination with promoter, gel shift, and chromatin immunoprecipitation assays indicate that SP2 and KLF6 bind to the matrix metalloproteinase-9 promoter and together function to maintain this gene in a silenced state. However, upon activation of FXR, SHP interacts with SP2 and KLF6, disrupting the SP2/KLF6 repressor complex. Thus, together, these studies identify a mechanism for antagonizing Sp/KLF protein repression function via SHP, with this process regulating endothelial cell motility. Farnesoid X Receptor (FXR)3 is a member of the nuclear receptor superfamily of transcription factors. In response to ligand binding, FXR regulates expression of genes involved in bile acid, cholesterol, and triglyceride metabolism (1-9). FXR heterodimerizes with the 9-cis-retinoic acid receptor ␣, which allows binding to a specific DNA sequence composed of two inverted hexamer repeats separated by one nucleotide (IR-1), thereby regulating target gene transcription (10 -12). An alternate mechanism of regulation occurs through FXR-dependent up-regulation of the atypical nuclear receptor, small heterodimeric partner (SHP). Although SHP lacks a DNA binding domain, it regulates transcription by several putative mechanisms that are not fully understood (13-15).Recent work has delineated a requisite role for Sp/KLF transcription factors for diverse biological functions (16 -19). The Sp/KLF family contains 24 identified members, including SP1-8 and KLF1-16, which bind with varying affinity to GCrich DNA sequences of target gene promoters (20). Interestingly, these proteins may function as transcriptional activators or repressors, although the mechanisms by which specificity of effect is achieved are not well defined.Because bile acids such as chenodeoxycholic acid (CDCA) are the natural ligands for FXR (21-23), prior investigations have largely been pursued in cells with active bile acid signaling, such as hepatocytes, cholangiocytes, and enterocytes (4,24,25). However, more recent work has expanded the scope of this nuclear receptor system into a diversity of cell types and functions, including vascular wall cells (26 -28), thereby suggesting potentially important and heretofore unrecognized actions that may be achieved by the FXR pathway. In this study, we delineate a signaling pathway by which FXR promotes endothelial cell motility through transcriptional activation of matrix metalloproteinase-9 (MMP-9). This pathway requires SHP inhibition of an SP2/KLF6 repressor complex. Thus, whereas SP2 and KLF6 repress MMP-9 promoter acti...