Citations

Abstract The tumor microenvironment, including stromal myofibroblasts and associated matrix proteins, regulates cancer cell invasion and proliferation. Here, we report that neuropilin-1 (NRP-1) orchestrates communications between myofibroblasts and soluble fibronectin that promote α5β1 integrin–dependent fibronectin fibril assembly, matrix stiffness, and tumor growth. Tumor growth and fibronectin fibril assembly were reduced by genetic depletion or antibody neutralization of NRP-1 from stromal myofibroblasts in vivo. Mechanistically, the increase in fibronectin fibril assembly required glycosylation of serine 612 of the extracellular domain of NRP-1, an intact intracellular NRP-1 SEA domain, and intracellular associations between NRP-1, the scaffold protein GIPC, and the nonreceptor tyrosine kinase c-Abl that augmented α5β1 fibronectin fibril assembly activity. Analysis of human cancer specimens established an association between tumoral NRP-1 levels and clinical outcome. Our findings indicate that NRP-1 activates the tumor microenvironment, thereby promoting tumor growth. These results not only identify new molecular mechanisms of fibronectin fibril assembly but also have important implications for therapeutic targeting of the myofibroblast in the tumor microenvironment. Cancer Res; 72(16); 4047–59. ©2012 AACR.

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.

Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 μg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase ( P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 μM), an inhibitor of MEK, which is a kinase upstream from Erk ( P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity ( P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation ( P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.

Angiogenesis occurs through a convergence of diverse signaling mechanisms with prominent pathways that include autocrine effects of endothelial nitric oxide (NO) synthase (eNOS)-derived NO and vascular endothelial growth factor (VEGF). However, the redundant and distinct roles of NO and VEGF in angiogenesis remain incompletely defined. Here, we use the partial hepatectomy model in mice genetically deficient in eNOS to ascertain the influence of eNOS-derived NO on the angiogenesis that accompanies liver regeneration. While sinusoidal endothelial cell (SEC) eNOS promotes angiogenesis in vitro, surprisingly the absence of eNOS did not influence the angiogenesis that occurs after partial hepatectomy in vivo. While this observation could not be attributed to induction of alternate NOS isoforms, it was associated with induction of VEGF signaling as evidenced by enhanced levels of VEGF ligand in regenerating livers from mice genetically deficient in eNOS. However, surprisingly, mice that were genetically heterozygous for deficiency in the VEGF receptor, fetal liver kinase-1, also maintained unimpaired capacity for liver regeneration. In summary, inhibition of VEGF- and NO-dependent angiogenesis does not impair liver regeneration, indicating signaling redundancies that allow liver regeneration to continue in the absence of this canonical vascular pathway.

Objective— Farnesoid X Receptor (FXR) mediates important signaling functions of bile acids in diverse cell types including those residing in the vascular wall. Indeed, recent work has identified FXR as a potential regulator of vascular structure and function in part through transcriptional activation of MMP-9. However, the signal transduction pathways linking bile acids to changes in actin cytoskeleton that are responsible for bile acid-induced vascular cell migration remain unexplored. Methods and Results— The FXR agonist and prototypical bile acid, chenodeoxycholic acid (CDCA), significantly increased endothelial cell (EC) motility, as analyzed by time lapse video microscopy, and tube formation, an in vitro correlate for angiogenesis. Increased cell motility was associated with prominent increases in focal adhesion (FA) plaques and was inhibited by FXR or MMP-9 siRNA, indicating a FXR-MMP-9-dependency of this signaling pathway. Mechanistically, incubation of cells with CDCA was associated with phosphorylation of a key FA protein, Focal Adhesion Kinase (FAK) at Y397 but not at Y576/577, or Y925. Studies using a site-specific phosphorylation mutant (phosphodeficient) of FAK revealed that FAK phosphorylation at tyrosine residue −397 was required for CDCA induced activation of the downstream FA assembly protein, paxillin. Lastly, siRNA-based silencing of FAK as well as phosphodeficient FAK mutant inhibited CDCA induced upregulation of MMP-9, cell motility, and vascular tube formation. Conclusion— Thus, this study demonstrates a pivotal role for FAK in the process of FXR-induced and MMP-9-dependent EC motility and vascular tube formation.

Endothelial cell-based angiogenesis requires activation of survival signals that generate resistance to external apoptotic stimuli, such as tumor necrosis factor-alpha (TNF-α), during pathobiologic settings. Mechanisms by which this is achieved are not fully defined. Here, we use a model in which the multifunctional cytokine nitric oxide counterbalances TNF-α-induced apoptosis, to define a role for membrane trafficking in the process of endothelial cell survival signaling. By perturbing dynamin GTPase function, we identify a key role of dynamin for ensuing downstream endothelial cell survival signals and vascular tube formation. Furthermore, nitric oxide is directly demonstrated to promote dynamin function through specific cysteine residue nitrosylation, which promotes endocytosis and endothelial cell survival signaling. Thus, these studies identify a novel role for dynamin as a survival factor in endothelial cells, through a mechanism by which dynamin S-nitrosylation regulates the counterbalances of TNF-α-induced apoptosis and nitric oxide-dependent survival signals, with implications highly relevant to angiogenesis.

Nitric oxide (NO) is a highly diffusible and short-lived physiological messenger. Despite its diffusible nature, NO modifies thiol groups of specific cysteine residues in target proteins and alters protein function via S-nitrosylation. Although intracellular S-nitrosylation is a specific posttranslational modification, the defined localization of an NO source (nitric oxide synthase, NOS) with protein S-nitrosylation has never been directly demonstrated. Endothelial NOS (eNOS) is localized mainly on the Golgi apparatus and in plasma membrane caveolae. Here, we show by using eNOS targeted to either the Golgi or the nucleus that S-nitrosylation is concentrated at the primary site of eNOS localization. Furthermore, localization of eNOS on the Golgi enhances overall Golgi protein S-nitrosylation, the specific S-nitrosylation of N -ethylmaleimide-sensitive factor and reduces the speed of protein transport from the endoplasmic reticulum to the plasma membrane in a reversible manner. These data indicate that local NOS action generates organelle-specific protein S-nitrosylation reactions that can regulate intracellular transport processes.

ABSTRACT Vascular endothelial growth factor receptor‐2 (VEGFR‐2, also known as KDR) is a receptor tyrosine kinase (RTK) regulating mitogenic, chemotactic, permeability, and survival signals in vascular endothelial cells (EC) in response to its ligand, vascular permeability factor/VEGF (VPF/VEGF), arguably the most important angiogenic cytokine. However, the compartmentalization of KDR in EC and the mechanisms regulating this process have not been well defined. Here, we demonstrate that KDR is present on the plasma membrane, on endosomes, and in the perinuclear region of EC and colocalizes with early endosomal antigen (EEA1), caveolin‐1, and dynamin‐2, a signal transducing GTPase involved in receptor endocytosis. Furthermore, we also observed that dynamin‐2 coimmunoprecipitates with KDR and is required for EC signaling/survival. Interestingly, EC overexpressing a mutant form of dynamin deficient in GTP binding (K44A) caused a selective inhibition in KDR protein level and endosomal vesicle formation and induced cell cycle arrest by inducing p21. Taken together, our findings suggest that dynamin‐2 regulates KDR expression and function and hence plays an important role in VPF/VEGF mediated angiogenesis.

Heat shock protein 90 (HSP90), an essential component of several signal transduction systems, participates in the activation of endothelial nitric oxide synthase (eNOS) in cells. The objective of the current study was to determine if HSP90 and eNOS were functionally interdependent and colocalized in the cerebral circulation. The authors used isometric force recording, cyclic 3′5′-guanosine monophosphate (cGMP) radioimmunoassay (RIA), and immunogold electron microscopy (EM) to study canine basilar artery. They found that geldanamycin (0.1 to 10 μg/mL), a selective HSP90 inhibitor, caused concentration-dependent contractions in arterial rings (n = 6 dogs). Contractions to geldanamycin were unaffected by a cyclooxygenase inhibitor, indomethacin (10 μmol/L; P < 0.05, n = 6). Functional evidence for interaction between HSP90 and nitric oxide (NO)-mediated signaling included observations that the contractile effect of geldanamycin was the following: (1) endothelium-dependent, (2) abolished by N g -nitro-l-arginine methylester (L-NAME; 0.3 mmol/L), and (3) nonadditive with the contractile effect of this NOS inhibitor ( P < 0.01, n = 6 for each). Furthermore, RIA showed significant reduction in cGMP levels in arteries treated with geldanamycin (3 μg/mL; P < 0.02, n = 8), whereas immunogold EM demonstrated areas of colocalization of HSP90 and eNOS selectively in the cytoplasm of endothelial cells. The current findings suggest that in cerebral arteries, endothelial HSP90 plays an important role in modulation of basal NO-mediated signaling. This interaction may be particularly important in stress-induced up-regulation of HSP90 with subsequent alteration of vasomotor function.