M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo

BACKGROUND: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. METHODOLOGY AND RESULTS: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. CONCLUSION: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo

Perturbation of Mouse Retinal Vascular Morphogenesis by Anthrax Lethal Toxin

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis

Hepatitis C Virus Sensitizes Host Cells to TRAIL-Induced Apoptosis by Up-Regulating DR4 and DR5 via a MEK1-Dependent Pathway

BACKGROUND: Hepatitis C virus (HCV) is the leading cause of liver fibrosis, cirrhosis and hepatocellular carcinoma. It is believed that continuous liver cell apoptosis contributes to HCV pathogenesis. Recent studies have shown that HCV infection can sensitize host cells to TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis, but the mechanism by which HCV regulates the TRAIL pathway remains unclear. METHODS AND RESULTS: Using a sub-genomic replicon and full length virus, JFH-1, we demonstrate that HCV can sensitize host cells to TRAIL-induced apoptosis by up-regulating two TRAIL receptors, death receptor 4 (DR4) and death receptor 5 (DR5). Furthermore, the HCV replicon enhanced transcription of DR5 via Sp1, and the HCV-mediated up-regulation of DR4 and DR5 required MEK1 activity. HCV infection also stimulated the activity of MEK1, and the inhibition of MEK1 activity or the knockdown of MEK1 increased the replication of HCV. CONCLUSIONS: Our studies demonstrate that HCV replication sensitizes host cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 via a MEK1 dependent pathway. These findings may help to further understand the pathogenesis of HCV infection and provide a therapeutic target

Adiponectin Upregulates Prolyl-4-Hydroxylase α1 Expression in Interleukin 6-Stimulated Human Aortic Smooth Muscle Cells by Regulating ERK 1/2 and Sp1

Adiponectin is an anti-atherogenic adipokine that inhibits the development of plaque by mechanisms that are not completely understood. Extracellular matrix (ECM) may have a role in the pathogenesis of atherosclerosis. We explored the effect and mechanisms of adiponectin on the synthesis of prolyl-4-hydroxylase (P4H) in interleukin 6 (IL-6)-stimulated human aortic smooth muscle cells (HASMCs). P4Hα1 mRNA level was quantified by RT-PCR, the protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and P4Hα1 were quantified by western blot analysis, and activation of specific protein 1 (Sp1) was determined by electrophoretic mobility shift assay and subcellular localization of Sp1 by immunofluorescence analysis. Adiponectin significantly increased P4Hα1 mRNA and protein levels in IL-6-stimulated HASMCs in a dose- and time-dependent manner. As well, ERK1/2 and Sp1 played a crucial role in the effect of adiponectin upregulating P4Hα1 expression in IL-6-stimulated HASMCs. Adiponectin abrogated the effects of IL-6 on collagen III level, which may indicate that P4Hα1 is essential for folding the procollagen polypeptide chains into stabilized collagen. Adiponectin attenuates IL-6–inhibited P4Hα1 synthesis and stabilizes collagen formation in HASMCs through a Sp1-ERK1/2-P4Hα1-dependent pathway

Inhibition of metastasis, angiogenesis, and tumor growth by Chinese herbal cocktail Tien-Hsien Liquid

<p>Abstract</p> <p>Background</p> <p>Advanced cancer is a multifactorial disease that demands treatments targeting multiple cellular pathways. Chinese herbal cocktail which contains various phytochemicals may target multiple dys-regulated pathways in cancer cells and thus may provide an alternative/complementary way to treat cancers. Previously we reported that the Chinese herbal cocktail Tien-Hsien Liguid (THL) can specifically induce apoptosis in various cancer cells and have immuno-modulating activity. In this study, we further evaluated the anti-metastatic, anti-angiogenic and anti-tumor activities of THL with a series of <it>in vitro </it>and <it>in vivo </it>experiments.</p> <p>Methods</p> <p>The migration and invasion of cancer cells and endothelial cells was determined by Boyden chamber transwell assays. The effect of THL on pulmonary metastasis was done by injecting CT-26 colon cancer cells intravenously to syngenic mice. The <it>in vitro </it>and <it>in vivo </it>microvessel formation was determined by the tube formation assay and the Matrigel plug assay, respectively. The <it>in vivo </it>anti-tumor effect of THL was determined by a human MDA-MB-231 breast cancer xenograft model. The expression of metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA) was measured by gelatin zymography. The expression of HIF-1α and the phosphorylation of ERK1/2 were determined by Western blot.</p> <p>Results</p> <p>THL inhibited the migration and invasion ability of various cancer cells <it>in vitro</it>, decreased the secretion of MMP-2, MMP-9, and uPA and the activity of ERK1/2 in cancer cells, and suppressed pulmonary metastasis of CT-26 cancer cells in syngenic mice. Moreover, THL inhibited the migration, invasion, and tube formation of endothelial cells <it>in vitro</it>, decreased the secretion of MMP-2 and uPA in endothelial cells, and suppressed neovascularization in Matrigel plugs in mice. Besides its inhibitory effect on endothelial cells, THL inhibited hypoxia-induced HIF-1α and vascular endothelial growth factor-A expression in cancer cells. Finally, our results show that THL inhibited the growth of human MDA-MB-231 breast cancer xenografts in <it>NOD-SCID </it>mice. This suppression of tumor growth was associated with decreased microvessel formation and increased apoptosis caused by THL.</p> <p>Conclusion</p> <p>Our data demonstrate that THL had broad-spectra anti-cancer activities and merits further evaluation for its use in cancer therapy.</p

Chronic hyperglycemia induces trans-differentiation of human pancreatic stellate cells and enhances the malignant molecular communication with human pancreatic cancer cells

BACKGROUND: Diabetes mellitus is linked to pancreatic cancer. We hypothesized a role for pancreatic stellate cells (PSC) in the hyperglycemia induced deterioration of pancreatic cancer and therefore studied two human cell lines (RLT-PSC, T3M4) in hyperglycemic environment. METHODOLOGY/PRINCIPAL FINDINGS: The effect of chronic hyperglycemia (CHG) on PSCs was studied using mRNA expression array with real-time PCR validation and bioinformatic pathway analysis, and confirmatory protein studies. The stress fiber formation (IC: αSMA) indicated that PSCs tend to transdifferentiate to a myofibroblast-like state after exposure to CHG. The phosphorylation of p38 and ERK1/2 was increased with a consecutive upregulation of CDC25, SP1, cFOS and p21, and with downregulation of PPARγ after PSCs were exposed to chronic hyperglycemia. CXCL12 levels increased significantly in PSC supernatant after CHG exposure independently from TGF-β1 treatment (3.09-fold with a 2.73-fold without TGF-β1, p<0.05). The upregualtion of the SP1 transcription factor in PSCs after CHG exposure may be implicated in the increased CXCL12 and IGFBP2 production. In cancer cells, hyperglycemia induced an increased expression of CXCR4, a CXCL12 receptor that was also induced by PSC's conditioned medium. The receptor-ligand interaction increased the phosphorylation of ERK1/2 and p38 resulting in activation of MAP kinase pathway, one of the most powerful stimuli for cell proliferation. Certainly, conditioned medium of PSC increased pancreatic cancer cell proliferation and this effect could be partially inhibited by a CXCR4 inhibitor. As the PSC conditioned medium (normal glucose concentration) increased the ERK1/2 and p38 phosphorylation, we concluded that PSCs produce other factor(s) that influence(s) pancreatic cancer behaviour. CONCLUSIONS: Hyperglycemia induces increased CXCL12 production by the PSCs, and its receptor, CXCR4 on cancer cells. The ligand-receptor interaction activates MAP kinase signaling that causes increased cancer cell proliferation and migration

Italo Calvino and the organizational imagination: Reading social organization through urban metaphors

© 2014 Taylor & Francis. This article explores the way in which uses or abuses of urban metaphors can inform differing polities and ethics of human organization. From its earliest inception, the city has taken on a metaphorical significance for human communities; being, at one and the same time, a discursive textual product of culture and, reciprocally, a provider of artefacts and architecture that produces culture and meaning. The city can be interpreted as a trope that operates bidirectionally in cultural terms. It is a sign that can be worked to serve the principles of both metonymy and synecdoche. In metonymical or reductive form, the city has the propensity to become weighty and deadening. The work of Michael Porter on competitive strategy is invoked to illustrate this effect. In the guise of synecdoche, on the other hand, the city offers imaginative potential. Drawing inspiration from the literary works of Italo Calvino (in particular, his novel Invisible Cities), the article attempts to reveal the fecundity of the city for interpreting technologically mediated organizational life. Calvino's emphasis on the principle of ‘lightness’ provides a link to the social theoretical writing of Boltanski and Chiapello on the ‘projective city’. A synthesis of these two stylistically different literatures yields a novel way of critically approaching and understanding the reticular form and emerging ethics of contemporary human organization

A neuropathologic diagnosis of Alzheimer's disease in an older adult with HIV-associated neurocognitive disorder.

We discuss the challenges associated with diagnosing neurodegenerative disorders in older adults living with HIV, illustrated through a case report where neurologic co-diagnosis of Alzheimer's disease (AD) and HIV-associated Neurocognitive Disorder (HAND) are considered. The patient was followed and evaluated for over 4 years and underwent post-mortem neuropathologic evaluation. Further work is needed to identify diagnostic tests that can adequately distinguish HAND from early stage neurodegenerative disorders among older adults living with HIV and cognitive changes