Adiponectin inhibits vascular endothelial growth factor-induced migration of human coronary artery endothelial cells

AIMS: Vascular endothelial growth factor (VEGF)-induced endothelial cell migration and angiogenesis are associated with the vascular complications of diabetes mellitus, and adiponectin is an abundant plasma adipokine that exhibits salutary effects on endothelial function. We investigated whether adiponectin suppresses VEGF-induced migration and related signal transduction responses in human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: Using a modified Boyden chamber technique and a monolayer 'wound-healing' assay, both the recombinant adiponectin globular domain and full-length adiponectin protein potently suppressed the migration of HCAEC induced by VEGF. Adiponectin did not increase endothelial cell apoptosis, as measured by terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labelling assay. Adiponectin also suppressed VEGF-induced reactive oxygen species generation, activation of Akt, the mitogen-activated protein kinase ERK and the RhoGTPase RhoA, and induction of the formation of actin stress fibres and focal cellular adhesions. VEGF-stimulated cell migration was inhibited by activation of adenylyl cyclase with forskolin, and adiponectin treatment increased cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) enzymatic activity. Pharmacological inhibition of either adenylyl cyclase or PKA significantly abrogated the effect of adiponectin globular domain to suppress VEGF-induced cell migration. CONCLUSION: Adiponectin suppresses VEGF-stimulated HCAEC migration via cAMP/PKA-dependent signalling, an important effect with implications for a regulatory role of adiponectin in vascular processes associated with diabetes and atherosclerosis.     

Inhibition of angiogenesis by vitamin D-binding protein: characterization of anti-endothelial activity of DBP-maf

Angiogenesis is a complex process involving coordinated steps of endothelial cell activation, proliferation, migration, tube formation and capillary sprouting with participation of intracellular signaling pathways. Regulation of angiogenesis carries tremendous potential for cancer therapy. Our earlier studies showed that vitamin D-binding protein-macrophage activating factor (DBP-maf) acts as a potent anti-angiogenic factor and inhibits tumor growth in vivo. The goal of this investigation was to understand the effect of DBP-maf on human endothelial cell (HEC) and the mechanism of angiogenesis inhibition. DBP-maf inhibited human endothelial cell (HEC) proliferation by inhibiting DNA synthesis (IC(50) = 7.8 +/- 0.15 microg/ml). DBP-maf significantly induced S- and G0/G1-phase arrest in HEC in 72 h. DBP-maf potently blocked VEGF-induced migration, tube-formation of HEC in a dose dependent manner. In addition, DBP-maf inhibited growth factor-induced microvessel sprouting in rat aortic ring assay. Moreover, DBP-maf inhibited VEGF signaling by decreasing VEGF-mediated phosphorylation of VEGFR-2 and ERK1/2, a downstream target of VEGF signaling cascade. However, Akt activation was not affected. These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting. DBP-maf exerts its effect by inhibiting VEGR-2 and ERK1/2 signaling cascades. Understanding the cellular and molecular mechanisms of anti-endothelial activity of DBP-maf will allow us to develop it as an angiogenesis targeting novel drug for tumor therapy.     

Protective Actions of Globular and Full-Length Adiponectin on Human Endothelial Cells: Novel Insights into Adiponectin-Induced Angiogenesis

Background/Aims: Adiponectin levels are decreased in diabetes and atherosclerosis. Coexisting hyperglycaemia and systemic inflammation predisposes to dysregulated angiogenesis and vascular disease. We investigated the effect of globular adiponectin (gAd) and full-length adiponectin (fAd) on angiogenesis and pro-angiogenic molecules, i.e. matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF), in human microvascular endothelial cells (HMEC-1). Methods: Angiogenesis was assessed by studying capillary tube formation in HMEC-1 on growth factor-reduced Matrigel. Endothelial cell migration assay was performed in a modified Boyden chamber. Results: Endothelial cell proliferation, in vitro migration and angiogenesis were significantly increased by gAd (mediated by AdipoR1, AMPK-Akt pathways), and gAd significantly increased MMP-2, MMP-9 and VEGF expression levels. The effect of gAd on VEGF appears to be mediated by AdipoR1, whilst the effect of gAd on MMP-2 and MMP-9 appears to be mediated by AdipoR1 and AdipoR2. Only endothelial cell proliferation was significantly increased by fAd in human microvascular endothelial cells and appears to be mediated by AdipoR2. No significant effects on MMP-2, MMP-9 and VEGF were observed. Importantly, gAd decreased glucose and C-reactive protein-induced angiogenesis with a concomitant reduction in MMP-2, MMP-9 and VEGF in HMEC-1 cells. Conclusion: We report novel insights into the mechanisms of adiponectin on angiogenesis.     

Identification of ARIA regulating endothelial apoptosis and angiogenesis by modulating proteasomal degradation of cIAP-1 and cIAP-2

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. By using a retrovirus-mediated signal sequence trap method, we identified a previously undescribed gene, termed ARIA (apoptosis regulator through modulating IAP expression), which regulates endothelial apoptosis and angiogenesis. ARIA was expressed in blood vessels during mouse embryogenesis, as well as in endothelial cells both in vitro and in vivo. ARIA is a unique protein with no homology to previously reported conserved domain structures. Knockdown of ARIA in HUVECs by using small interfering RNA significantly reduced endothelial apoptosis without affecting either cell migration or proliferation. ARIA knockdown significantly increased inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression, although their mRNA expression was not changed. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the antiapoptotic effect of ARIA knockdown. Using yeast 2-hybrid screening, we identified the interaction of ARIA with 20S proteasome subunit α-7. Thereafter, we found that cIAP-1 and cIAP-2 were degraded by proteasomes in endothelial cells under normal condition. Overexpression of ARIA significantly reduced cIAP-1 expression, and this reduction was abolished by proteasomal inhibition in BAECs. Also, knockdown of ARIA demonstrated an effect similar to proteasomal inhibition with respect to not only expression but also subcellular localization of cIAP-1 and cIAP-2. In vivo angiogenesis studied by Matrigel-plug assay, mouse ischemic retinopathy model, and tumor xenograft model was significantly enhanced by ARIA knockdown. Together, our data indicate that ARIA is a unique factor regulating endothelial apoptosis, as well as angiogenesis, presumably through modulating proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells.     

Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis

Obesity is rapidly becoming a pandemic and is associated with increased carcinogenesis. Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related carcinogenesis. Recently, we reported the oncogenic role of leptin including its potential to increase tumor invasiveness and migration of hepatocellular carcinoma (HCC) cells. In the present study we investigated whether adiponectin could antagonize the oncogenic actions of leptin in HCC. We employed HCC cell lines HepG2 and Huh7, the nude mice-xenograft model of HCC, and immunohistochemistry data from tissue-microarray to demonstrate the antagonistic role of adiponectin on the oncogenic actions of leptin. Adiponectin treatment inhibited leptin-induced cell proliferation of HCC cells. Using scratch-migration and electric cell-substrate impedance-sensing-based migration assays, we found that adiponectin inhibited leptin-induced migration of HCC cells. Adiponectin treatment effectively blocked leptin-induced invasion of HCC cells in Matrigel invasion assays. Although leptin inhibited apoptosis in HCC cells, we found that adiponectin treatment induced apoptosis even in the presence of leptin. Analysis of the underlying molecular mechanisms revealed that adiponectin treatment reduced leptin-induced Stat3 and Akt phosphorylation. Adiponectin also increased suppressor of cytokine signaling (SOCS3), a physiologic negative regulator of leptin signal transduction. Importantly, adiponectin significantly reduced leptin-induced tumor burden in nude mice. In HCC samples, leptin expression significantly correlated with HCC proliferation as evaluated by Ki-67, whereas adiponectin expression correlated significantly with increased disease-free survival and inversely with tumor size and local recurrence. CONCLUSION: Collectively, these data demonstrate that adiponectin has the molecular potential to inhibit the oncogenic actions of leptin by blocking downstream effector molecules.     

Adiponectin inhibits endothelial synthesis of interleukin-8

Adiponectin is an antiatherogenic adipokine that inhibits inflammation by mechanisms that are not completely understood. We explored the effect of adiponectin on endothelial synthesis of interleukin-8 (IL-8), a pro-inflammatory chemokine that plays a role in atherogenesis. Adiponectin decreased the secretion of IL-8 from human aortic endothelial cells (HAEC) stimulated with tumor necrosis factor-alpha (TNF-alpha). Adiponectin also inhibited IL-8 mRNA expression induced by TNF-alpha. Phosphorylation of IkappaB-alpha was decreased by adiponectin, but phosphorylation of ERK, SAPK/JNK, and p38MAPK were unaffected. Adiponectin increased intra-cellular cAMP levels in HAEC in a dose-dependent manner; PKA activity was also increased. The inhibitory effect of adiponectin on TNF-alpha-induced IL-8 synthesis was inhibited by pretreatment with Rp-cAMP, a PKA inhibitor. These observations suggest that adiponectin inhibits IL-8 synthesis through inhibition of a PKA dependent NF-kappaB signaling pathway. We also showed that adiponectin enhances Akt phosphorylation. The inhibitory effect of adiponectin on TNF-alpha-induced IL-8 synthesis was abrogated in part by pretreatment with the PI3 kinase inhibitor LY294002 or by Akt siRNA transfection, suggesting that Akt activation might inhibit IL-8 synthesis induced by TNF-alpha. We conclude that inhibition of NF-kappaB and activation of Akt phosphorylation may mediate adiponectin inhibition of atherosclerosis.     

Usnic acid inhibits breast tumor angiogenesis and growth by suppressing VEGFR2-mediated AKT and ERK1/2 signaling pathways

Tumor growth depends on angiogenesis and inducing angiogenesis is one of the most important hallmarks in the cancer development. Treatment with small molecules that inhibit angiogenesis has been an effective strategy for anti-cancer therapy. Some anti-angiogenic factors are derived from traditional Chinese herbs. Usnic acid (UA), an active compound mainly found in lichens, has shown some biological and physiological activities. However, the role and mechanism of UA in tumor angiogenesis are still unknown. The aim of this study was to assess the effects of UA on tumor angiogenesis. In this study, we demonstrated that UA strongly inhibited in vivo angiogenesis in a chick embryo chorioallantoic membrane assay and vascular endothelial growth factor-induced mouse corneal angiogenesis model. In a mouse xenograft tumor model, UA suppressed Bcap-37 breast tumor growth and angiogenesis without affecting mice body weight. In an in vitro assay, UA not only significantly inhibited endothelial cell proliferation, migration and tube formation, but also induced morphological changes and apoptosis in endothelial cells. In addition, UA inhibited Bcap-37 tumor cell proliferation. Moreover, western blot analysis of cell signaling molecules indicated that UA blocked vascular endothelial growth factor receptor (VEGFR) 2 mediated Extracellular signal-regulated protein kinases 1 and 2(ERK1/2) and AKT/P70S6K signaling pathways in endothelial cells. These results provided the first evidence of the biological function and molecular mechanism of UA in tumor angiogenesis.     

Down-modulation of TNFSF15 in ovarian cancer by VEGF and MCP-1 is a pre-requisite for tumor neovascularization

Persistent inflammation and neovascularization are critical to cancer development. In addition to upregulation of positive control mechanisms such as overexpression of angiogenic and inflammatory factors in the cancer microenvironment, loss of otherwise normally functioning negative control mechanisms is likely to be an important attribute. Insights into the down-modulation of such negative control mechanisms remain largely unclear, however. We show here that tumor necrosis factor superfamily-15 (TNFSF15), an endogenous inhibitor of neovascularization, is a critical component of the negative control mechanism that operates in normal ovary but is missing in ovarian cancer. We show in clinical settings that TNFSF15 is present prominently in the vasculature of normal ovary but diminishes in ovarian cancer as the disease progresses. Vascular endothelial growth factor (VEGF) produced by cancer cells and monocyte chemotactic protein-1 (MCP-1) produced mainly by tumor-infiltrating macrophages and regulatory T cells effectively inhibits TNFSF15 production by endothelial cells in vitro. Using a mouse syngeneic tumor model, we demonstrate that silencing TNFSF15 by topical shRNA treatments prior to and following mouse ovarian cancer ID8 cell inoculation greatly facilitates angiogenesis and tumor growth, whereas systemic application of recombinant TNFSF15 inhibits angiogenesis and tumor growth. Our findings indicate that downregulation of TNFSF15 by cancer cells and tumor infiltrating macrophages and lymphocytes is a pre-requisite for tumor neovascularization.     

Gossypin, a pentahydroxy glucosyl flavone, inhibits the transforming growth factor beta-activated kinase-1-mediated NF-kappaB activation pathway, leading to potentiation of apoptosis, suppression of invasion, and abrogation of osteoclastogenesis

Gossypin, a flavone originally isolated from Hibiscus vitifolius, has been shown to suppress angiogenesis, inflammation, and carcinogenesis. The mechanisms of these activities, however, are unknown. Because nuclear factor-kappaB (NF-kappaB) is associated with inflammation, carcinogenesis, hyperproliferation, invasion, and angiogenesis, we hypothesized that gossypin mediates its effects through modulation of NF-kappaB activation. In the present study, we demonstrate that gossypin (and not gossypetin, an aglycone analog) inhibited NF-kappaB activation induced by inflammatory stimuli and carcinogens. Constitutive NF-kappaB activation in tumor cells was also inhibited by this flavone. Inhibition of I kappa B alpha kinase by gossypin led to the suppression of I kappa B alpha phosphorylation and degradation, p65 nuclear translocation, and NF-kappaB-regulated gene expression. This, in turn, led to the down-regulation of gene products involved in cell survival (IAP2, XIAP, Bcl-2, Bcl-xL, survivin, and antiFas-associated death domain-like interleukin-1 beta-converting enzyme-inhibitory protein), proliferation (c-myc, cyclin D1, and cyclooxygenase-2), angiogenesis (vascular endothelial growth factor), and invasion (matrix metalloprotease-9). Suppression of these gene products by gossypin enhanced apoptosis induced by tumor necrosis factor and chemotherapeutic agents, suppressed tumor necrosis factor-induced cellular invasion, abrogated receptor activator of NF-kappaB ligand-induced osteoclastogenesis, and vascular endothelial growth factor-induced migration of human umbilical vein endothelial cells. Overall, our results demonstrate that gossypin inhibits the NF-kappaB activation pathway, which may explain its role in the suppression of inflammation, carcinogenesis, and angiogenesis.