DHEA-S inhibits human neutrophil and human airway smooth muscle migration

Airway diseases such as asthma, emphysema, and chronic bronchitis are, in part, characterized by reversible airflow obstruction and inflammation. In severe disease, marked decreases in lung function are associated with airway smooth muscle proliferation and airway neutrophilia. Inhaled glucocorticoids attenuate increased airflow obstruction and airway inflammation that occur, in part, due to increased smooth muscle migration and proliferation, as well as the airway neutrophilia. Glucocorticoids, however, have adverse side effects and, in some patients, are ineffective despite high doses. Recent research has explored the effects of non-traditional steroids on attenuation of inflammation associated with airway diseases. These non-traditional steroids have improved side effect profiles in comparison to glucocorticoid therapy. Our studies assessed effects of dehydroepiandrosterone-3-sulfate (DHEA-S) on migration of both human peripheral blood neutrophils (PMN) and human airway smooth muscle cells (HASM). DHEA-S dose-dependently inhibited chemotaxis of PMN and HASM while having no effect on the phosphorylation levels of Akt, ERK1/2, p38 MAPK or PKC, canonical positive regulators of cell migration. These studies demonstrate direct effects of DHEA-S on cell migration, thereby suggesting that DHEA-S may attenuate airway inflammation and cell migration.     

Oleic acid and adipokines synergize in inducing proliferation and inflammatory signalling in human vascular smooth muscle cells

In the context of obesity, perivascular fat produces various adipokines and releases free fatty acids, which may induce inflammation and proliferation in the vascular wall. In this study we investigated how adipokines, oleic acid (OA) and the combined treatment regulate human vascular smooth muscle cell (hVSMC) proliferation and migration and the underlying signalling pathways. Adipocyte‐conditioned media (CM) generated from human adipocytes induces a prominent proliferation and migration of hVSMC. Autocrine action of adiponectin totally abolishes CM‐induced proliferation. Furthermore, OA but not palmitic acid induces proliferation of hVSMC. CM itself does not contain fatty acids, but CM in combination with OA markedly enhances proliferation of hVSMC in a synergistic way. Both the nuclear factor (NF)‐κB and the mammalian target of rapamycin (mTOR) pathway were synergistically activated under these conditions and found to be essential for hVSMC proliferation. Expression of iNOS and production of nitric oxide was only enhanced by combined treatment inducing a marked release of VEGF. Combination of OA and VEGF induces an additive increase of hVSMC proliferation. We could show that the combination of CM and OA led to a synergistic proliferation of hVSMC. Expression of iNOS and production of nitric oxide were only enhanced under these conditions and were paralleled by a marked release of VEGF. These results suggest that the combined elevated release of fatty acids and adipokines by adipose tissue in obesity might be critically related to hVSMC dysfunction, vascular inflammation and the development of atherosclerosis.     

Fas cross-linking induces apoptosis in human airway smooth muscle cells

Hypertrophy and hyperplasia lead to excess accumulation of smooth muscle in the airways of human asthmatic subjects. However, little is known about mechanisms that might counterbalance these processes, thereby limiting the quantity of smooth muscle in airways. Ligation of Fas on the surface of vascular smooth muscle cells and nonmuscle airway cells can lead to apoptotic cell death. We therefore tested the hypotheses that 1) human airway smooth muscle (HASM) expresses Fas, 2) Fas cross-linking induces apoptosis in these cells, and 3) tumor necrosis factor (TNF)-alpha potentiates Fas-mediated airway myocyte killing. Immunohistochemistry using CH-11 anti-Fas monoclonal IgM antibody revealed Fas expression in normal human bronchial smooth muscle in vivo. Flow cytometry using DX2 anti-Fas monoclonal IgG antibody revealed that passage 4 cultured HASM cells express surface Fas. Surface Fas decreased partially during prolonged serum deprivation of cultured HASM cells and was upregulated by TNF-alpha stimulation. Fas cross-linking with CH-11 antibody induced apoptosis in cultured HASM cells, and this effect was reduced by long-term serum deprivation and synergistically potentiated by concomitant TNF-alpha exposure. TNF-alpha did not induce substantial apoptosis in the absence of Fas cross-linking. These data represent the first demonstration that Fas is expressed on HASM and suggest a mechanism by which Fas-mediated apoptosis could act to oppose excess smooth muscle accumulation during airway remodeling in asthma.     

Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alpha-dependent expression of thrombospondin-1

The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis.     

Rapamycin Inhibits Proliferation of Hemangioma Endothelial Cells by Reducing HIF-1-Dependent Expression of VEGF

Hemangiomas are tumors formed by hyper-proliferation of vascular endothelial cells. This is caused by elevated vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we show that elevated VEGF levels produced by hemangioma endothelial cells are reduced by the mTOR inhibitor rapamycin. mTOR activates p70S6K, which controls translation of mRNA to generate proteins such as hypoxia inducible factor-1 (HIF-1). VEGF is a known HIF-1 target gene, and our data show that VEGF levels in hemangioma endothelial cells are reduced by HIF-1α siRNA. Over-expression of HIF-1α increases VEGF levels and endothelial cell proliferation. Furthermore, both rapamycin and HIF-1α siRNA reduce proliferation of hemangioma endothelial cells. These data suggest that mTOR and HIF-1 contribute to hemangioma endothelial cell proliferation by stimulating an autocrine loop of VEGF signaling. Furthermore, mTOR and HIF-1 may be therapeutic targets for the treatment of hemangiomas.     

Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

Background  

Expression of the urokinase plasminogen activator receptor (UPAR) has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC), airway smooth muscle (HASM) and peripheral cells).     

Conditioned media from adipocytes promote proliferation,migration, and invasion in melanoma and colorectal cancer cells

Epidemiological evidence suggests that obesity can significantly increase the risk of various cancers, although the mechanisms underlying this link are completely unknown. Here, we analyzed the effect of adipocytes on melanoma and colon cancer cells proliferation, migration, and invasion. The potential effects of conditioned media (CM) obtained from differentiated mouse 3T3-L1 cells and human adipose tissue-derived mesenchymal stem cells (hAMSC) on the proliferation, migration, and invasion of B16BL6 melanoma and colon 26-L5 cancer cells were investigated. The 3T3-L1 and hAMSC CM increased cell proliferation, migration, and invasion in both the cell lines. In addition, adipocytes CM increased matrix metalloproteinase 9 (MMP-9) and MMP-2 activity in both B16BL6 and colon 26-L5 cells. These effects were found to be associated with an increased expression of various oncogenic proteins in B16BL6 and colon 26-L5 cells. Also, adipocyte CM induced Akt and mTOR activation in both tumor cell lines, and the pharmacological inhibition of Akt and mTOR blocked the CM induced Akt as well as mTOR activation and CM-stimulated melanoma and colon cancer cell proliferation, migration, and invasion. These data suggest that adipocyte promotes melanoma and colon cancer progression through modulating the expression of diverse proteins associated with cancer growth and metastasis as well as modulation of the Akt/mTOR signaling.     

Functional expression of the GABAB receptor in human airway smooth muscle

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via both ionotropic (GABA(A)/GABA(C)) and metabotropic (GABA(B)) receptors (R). In addition to their location on neurons, GABA and functional GABA(B) receptors have been detected in nonneuronal cells in peripheral tissue. Although the GABA(B)R has been shown to function as a prejunctional inhibitory receptor on parasympathetic nerves in the lung, the expression and functional coupling of GABA(B) receptors to G(i) in airway smooth muscle itself have never been described. We detected the mRNA encoding multiple-splice variants of the GABA(B)R1 and GABA(B)R2 in total RNA isolated from native human and guinea pig airway smooth muscle and from RNA isolated from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the GABA(B)R1 and GABA(B)R2 proteins in human native and cultured airway smooth muscle. The GABA(B)R1 protein was immunohistochemically localized to airway smooth muscle in guinea pig tracheal rings. Baclofen, a GABA(B)R agonist, elicited a concentration-dependent stimulation of [(35)S]GTPgammaS binding in HASM homogenates that was abrogated by the GABA(B)R antagonist CGP-35348. Baclofen also inhibited adenylyl cyclase activity and induced ERK phosphorylation in HASM. Another GABA(B)R agonist, SKF-97541, mimicked while pertussis toxin blocked baclofen's effect on ERK phosphorylation, implicating G(i) protein coupling. Functional GABA(B) receptors are expressed in HASM. GABA may modulate an uncharacterized signaling cascade via GABA(B) receptors coupled to the G(i) protein in airway smooth muscle.     

Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. HIF-1 alpha overexpression may be beneficial in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic heart disease. To address this issue, human peripheral blood mononuclear cells (PBMNCs) were induced to differentiate into endothelial progenitor cells (EPCs), and then were transfected with either an HIF-1 alpha-expressing or a control vector and cultured under normoxia or hypoxia. Hypoxia-induced HIF-1 alpha mRNA and protein expression was increased after HIF-1 alpha transfection. This was accompanied by VEGF mRNA induction and increased VEGF secretion. Hypoxia-stimulated VEGF mRNA induction was significantly abrogated by HIF-1 alpha-specific siRNA. Functional studies showed that HIF-1 alpha overexpression further promoted hypoxia-induced EPC differentiation, proliferation and migration. The expressions of endothelial cell markers CD31, VEGFR2 (Flk-1) and eNOS as well as VEGF and NO secretions were also increased. Furthermore, in an in vivo model of hindlimb ischemia, HIF-1 alpha-transfected EPCs homed to the site of ischemia. A higher revascularization potential was also demonstrated by increased capillary density at the injury site. Our results revealed that endothelial progenitor cells ex vivo modification by hypoxia inducible factor-1 alpha gene transfection is feasible and may offer significant advantages in terms of EPC expansion and treatment efficacy.     

Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.