Complementary vascular and matrix regulatory pathways underlie the beneficial mechanism of action of sorafenib in liver fibrosis.

UNLABELLED: Paracrine signaling between hepatic stellate cells (HSCs) and liver endothelial cells (LECs) 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 HSCs and LECs. The aim of this study was to test the hypothesis that sorafenib influences paracrine signaling between HSCs and LECs and thereby regulates matrix and vascular changes associated with chronic liver injury. Complementary magnetic resonance elastography, micro-computed tomography, 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-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 HSCs in a manner dependent on the transcription factor Kruppel-like factor 6 , suggesting that this pathway underlies both matrix and vascular changes associated with chronic liver disease. CONCLUSION: Collectively, the results of this study 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.

Protein kinase G signaling disrupts Rac1-dependent focal adhesion assembly in liver specific pericytes.

Nitric oxide (NO) regulates the function of perivascular cells (pericytes), including hepatic stellate cells (HSC), mainly by activating cGMP and cGMP-dependent 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.

Endothelial cell toll-like receptor 4 regulates fibrosis-associated angiogenesis in the liver.

UNLABELLED: 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.

Sternal osteomyelitis secondary to Aspergillus fumigatus after cardiothoracic surgery.

Sternal Osteomyelitis from Aspergillus fumigatus in immunocompetent patients is extremely rare with limited number of cases reported so far. Here we discuss the case of a 65-year-old female with osteomyelitis of the sternum caused by Aspergillus fumigatus after undergoing coronary artery bypass graft surgery. Patient was treated with surgical debridement and prolonged antifungal therapy; however, the course was complicated due to poor adherence to antifungal therapy.

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

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.