Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression

BACKGROUND: The vasoconstricting peptide endothelin-1 (ET-1) has been associated with atherosclerotic cardiovascular disease, vascular smooth muscle cell (VSMC) growth stimulation, and intimal thickening. ET-1 binds 2 receptor subtypes, endothelin A and B, and the ETA receptor mediates vasoconstriction and VSMC growth. This study aims to quantitatively assess arterial remodeling variables and compare them with changes in ET-1, ETA, and ETB expression in the internal mammary artery (IMA). METHODS AND RESULTS: Specimens from 55 coronary artery disease (CAD) patients (45 men, 10 women; mean age 65 years) and 14 control IMA specimens (from 7 men and 7 women; mean age 45 years) were collected. IMA cross sections were assessed by histochemical and immunohistochemical staining methods to quantify the levels of medionecrosis, fibrosis, VSMC growth, ET-1, ETA, ETB, and macrophage infiltration. The percentage area of medionecrosis in the patients was almost double that in the controls (31.85+/-14.52% versus 17.10+/-9.96%, P=0.0006). Total and type 1 collagen was significantly increased compared with controls (65.8+/-18.3% versus 33.7+/-13.7%, P=0.07, and 14.2+/-10.0% versus 4.8+/-2.8%, P=0.01, respectively). Despite ACE and/or statin therapy, ET-1 expression and cell cycling were significantly elevated in the patient IMAs relative to the controls (46.27+/-18.46 versus 8.56+/-8.42, P=0.0001, and 37.29+/-12.88 versus 11.06+/-8.18, P=0.0001, respectively). ETA and ETB staining was elevated in the patient vessels (46.88+/-11.52% versus 18.58+/-7.65%, P=0.0001, and 42.98+/-7.08% versus 34.73+/-5.20%, P=0.0067, respectively). A mild presence of macrophages was noted in all sections. CONCLUSIONS: Elevated distribution of collagen indicative of fibrosis coupled with increased cell cycling and high levels of ET-1 and ETA expression in the absence of chronic inflammation suggests altered IMA VSMC regulation is fundamental to the remodeling process.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

The α5 subunit regulates the expression and function of α4*-containing neuronal nicotinic acetylcholine receptors in the ventral-tegmental area

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.

Differential expression of VEGF ligands and receptors in prostate cancer

BACKGROUND Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. METHODS The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. RESULTS Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. CONCLUSIONS These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer.

Epigenetic regulation of chemokine/chemokine receptor expression

Epigenetic regulation of gene expression is an important event for normal cellular homeostasis. Gene expression may be "switched" on or "turned" off via epigenetic means through adjustments in DNA architecture. These structural alterations result from changes to the DNA methylation status in addition to histone posttranslational modifications such as acetylation and methylation. Drugs which can alter the status of these epigenetic markers are currently undergoing clinical trials in a wide variety of diseases, including cancer.We illustrate the treatment of cell lines with histone deacetylase (HDi) and DNA methyltransferase inhibitors and the subsequent RNA isolation and reverse transcriptase polymerase chain reaction for several members of the CXC (ELR(+)) chemokine family. In addition we describe a chromatin immunoprecipitation assay to determine the association between chromatin transcription markers and DNA following pretreatment of cell cultures with an HDi, Trichostatin A (TSA). This assay allows us to determine whether treatment with TSA dynamically remodels the promoter region of our selected genes, as judged by the differences in the PCR product between our treated and untreated samples.

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results Serum TXB2 levels were correlated with VEGF (p<0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p<0.0001) and microvessel density (CD-31; p<0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p<0.0001) and female patients (p<0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p<0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.

Estrogen receptor β activation impairs mitochondrial oxidative metabolism and affects malignant mesothelioma cell growth in vitro and in vivo

Estrogen receptor (ER)-β has been shown to possess a tumor suppressive effect, and is a potential target for cancer therapy. Using gene-expression meta-analysis of human malignant pleural mesothelioma, we identified an ESR2 (ERβ coding gene) signature. High ESR2 expression was strongly associated with low succinate dehydrogenase B (SDHB) (which encodes a mitochondrial respiratory chain complex II subunit) expression. We demonstrate that SDHB loss induced ESR2 expression, and that activated ERβ, by over-expression or by selective agonist stimulation, negatively affected oxidative phosphorylation compromising mitochondrial complex II and IV activity. This resulted in reduced mitochondrial ATP production, increased glycolysis dependence and impaired cell proliferation. The observed in vitro effects were phenocopied in vivo using a selective ERβ agonist in a mesothelioma mouse model. On the whole, our data highlight an unforeseen interaction between ERβ-mediated tumor suppression and energy metabolism that may be exploited to improve on the therapy for clinical management of malignant mesothelioma.

Acquired differential regulation of caspase-8 in cisplatin-resistant non-small-cell lung cancer

Failure to efficiently induce apoptosis contributes to cisplatin resistance in non-small-cell lung cancer (NSCLC). Although BCL-2-associated X protein (BAX) and BCL-2 antagonist killer (BAK) are critical regulators of the mitochondrial apoptosis pathway, their requirement has not been robustly established in relation to cisplatin. Here, we show that cisplatin can efficiently bypass mitochondrial apoptosis block caused by loss of BAX and BAK, via activation of the extrinsic death receptor pathway in some model cell lines. Apoptosis resistance following cisplatin can only be observed when both extrinsic and intrinsic pathways are blocked, consistent with redundancy between mitochondrial and death receptor pathways in cisplatin-induced apoptosis. In H460 NSCLC cells, caspase-8 cleavage was shown to be induced by cisplatin and is dependent on death receptor 4, death receptor 5, Fas-associated protein with death domain, acid sphingomyelinase and ceramide synthesis. In contrast, cisplatin-resistant cells fail to activate caspase-8 via this pathway despite conserving sensitivity to death ligand-driven activation. Accordingly, caspase-8 activation block acquired during cisplatin resistance, can be bypassed by death receptor agonism.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

Effect of exercise training on skeletal muscle cytokine expression in the elderly

Aging is associated with increased circulating pro-inflammatory and lower anti-inflammatory cytokines. Exercise training, in addition to improving muscle function, reduces these circulating pro-inflammatory cytokines. Yet, few studies have evaluated changes in the expression of cytokines within skeletal muscle after exercise training. The aim of the current study was to examine the expression of cytokines both at rest and following a bout of isokinetic exercise performed before and after 12 weeks of resistance exercise training in young (n = 8, 20.3 ± 0.8 yr) and elderly men (n = 8, 66.9 ± 1.6 yr). Protein expression of various cytokines was determined in muscle homogenates. The expression of MCP-1, IL-8 and IL-6 (which are traditionally classified as ‘pro-inflammatory’) increased substantially after acute exercise. By contrast, the expression of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 increased only slightly (or not at all) after acute exercise. These responses were not significantly different between young and elderly men, either before or after 12 weeks of exercise training. However, compared with the young men, the expression of pro-inflammatory cytokines 2 h post exercise tended to be greater in the elderly men prior to training. Training attenuated this difference. These data suggest that the inflammatory response to unaccustomed exercise increases with age. Furthermore, regular exercise training may help to normalize this inflammatory response, which could have important implications for muscle regeneration and adaptation in the elderly.

Integrin αvβ3 upregulates integrin-linked kinase expression in human ovarian cancer cells via enhancement of ILK gene transcription

We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.

Mediating orientation and self-expression in the world of consumption : Australian and German lifestyle journalists' professional views

Despite having experienced rapid popularity over the past two decades, lifestyle journalism is still somewhat neglected by academic researchers. So far mostly explored as either part of wider lifestyle programming, particularly on television, or in terms of individual sub-fields, such as travel, fashion or food journalism, lifestyle journalism is in need of scholarly analysis particularly in the area of production, based on the increasing importance which the field has in influencing audiences’ ways of life. This study explores the professional views of 89 Australian and German lifestyle journalists through in-depth interviews in order to explore the ways in which they engage in processes of influencing audiences’ self-expression, identities and consumption behaviors. The article argues that through its work, lifestyle journalism is a significant shaper of identities in today’s consumer societies.

Silencing of ghrelin receptor expression inhibits endometrial cancer cell growth in vitro and in vivo

Ghrelin is a 28-amino acid peptide hormone produced predominantly in the stomach but also in a range of normal cell types and tumors, where it has endocrine, paracrine, and autocrine roles. Previously, we have demonstrated that ghrelin has proliferative and antiapoptotic effects in endometrial cancer cell lines, suggesting a potential role in promoting tumor growth. In the present study, we investigated the effect of ghrelin receptor, GHSR, and gene silencing in vitro and in vivo and characterized ghrelin and GHSR1a protein expression in human endometrial tumors. GHSR gene silencing was achieved in the Ishikawa and KLE endometrial cancer cell lines, using a lentiviral short-hairpin RNA targeting GHSR. The effects of GHSR1a knockdown were further analyzed in vivo using the Ishikawa cell line in a NOD/SCID xenograft model. Cell proliferation was reduced in cultured GHSR1a knockdown Ishikawa and KLE cells compared with scrambled controls in the absence of exogenously applied ghrelin and in response to exogenous ghrelin (1,000 nM). The tumor volumes were reduced significantly in GHSR1a knockdown Ishikawa mouse xenograft tumors compared with scrambled control tumours. Using immunohistochemistry, we demonstrated that ghrelin and GHSR1a are expressed in benign and cancerous glands in human endometrial tissue specimens, although there was no correlation between the intensity of staining and cancer grade. These data indicate that downregulation of GHSR expression significantly inhibits endometrial cancer cell line and mouse xenograft tumour growth. This is the first preclinical evidence that downregulation of GHSR may be therapeutic in endometrial cancer.

Expression, regulation and putative nutrient-sensing function of taste GPCRs in the heart

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and are enriched in myocytes, which we corroborated using in situ hybridization. Tas1r1 gene-targeted mice (Tas1r1Cre/Rosa26tdRFP) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart.