BRCA1 transcriptionally regulates genes associated with the basal breast cancer phenotype

Background Ten to twenty per cent of breast tumours exhibit a basallike genetic profile and these tumours carry a poor prognosis. Breast tumours which contain germline mutations for BRCA1 commonly exhibit a molecular profile similar to basal breast tumours. BRCA1 is a tumour suppressor gene which is mutated in up to 5–10% of breast cancer cases and is involved in multiple cellular processes including DNA damage control, cell cycle checkpoint control, apoptosis, ubiquitination and transcriptional regulation.

Methods Microarray-based profiling was carried out using the HCC1937EV and HCC1937BR breast cancer cell lines. Basal gene and protein expression levels were analysed by qRT-PCR and western blotting. ChIP analyses were performed and demonstrated that BRCA1 regulates basal gene expression through a transcriptional mechanism involving c-myc.

Results We have previously carried out microarray-based expression profiling to examine differences in gene expression when BRCA1 is reconstituted in BRCA1 mutated HCC1937 breast cancer cells. We observed that p-cadherin and the cytokeratin 5 and cytokeratin 17 genes, which are strongly correlated with the basal phenotype, are differentially expressed when BRCA1 is reconstituted. In addition, qRT-PCR and ChIP analysis of BRCA1 reconstituted cells show that BRCA1 represses the expression of these basal genes by a transcriptional mechanism. Furthermore, abrogation of endogenous BRCA1 protein in the T47D cell line using siRNA results in reexpression of these basal genes, suggesting that BRCA1 expression levels may be important in basal gene expression. We have also demonstrated that BRCA1 is physically associated with the promoter regions of basal genes through an association with c-myc. Consequently, we have confirmed that siRNA inhibition of c-myc in T47D cells results in re-expression of these genes.

Conclusions Our results suggest that BRCA1 is involved in the transcriptional regulation of genes associated with the basal phenotype and that BRCA1 controls basal gene expression through a transcriptional mechanism involving c-myc. Further work is now concentrating on defining the relationship between BRCA1 and basal gene expression and how this may affect clinical responses to breast cancer chemotherapy.

Dysregulated intracellular signaling impairs CTGF-stimulated responses in human mesangial cells exposed to high extracellular glucose

High ambient glucose activates intracellular signaling pathways to induce the expression of extracellular matrix and cytokines such as connective tissue growth factor (CTGF). Cell responses to CTGF in already glucose-stressed cells may act to transform the mesangial cell phenotype leading to the development of glomerulosclerosis. We analyzed cell signaling downstream of CTGF in high glucose-stressed mesangial cells to model signaling in the diabetic milieu. The addition of CTGF to primary human mesangial cells activates cell migration which is associated with a PKC-zeta-GSK3beta signaling axis. In high ambient glucose basal PKC-zeta and GSK3beta phosphorylation levels are selectively increased and CTGF-stimulated PKC-zeta and GSK3beta phosphorylation was impaired. These effects were not induced by osmotic changes. CTGF-driven profibrotic cell signaling as determined by p42/44 MAPK and Akt phosphorylation was unaffected by high glucose. Nonresponsiveness of the PKC-zeta-GSK3beta signaling axis suppressed effective remodeling of the microtubule network necessary to support cell migration. However, interestingly the cells remain plastic: modulation of glucose-induced PKC-beta activity in human mesangial cells reversed some of the pathological effects of glucose damage in these cells. We show that inhibition of PKC-beta with LY379196 and PKC-beta siRNA reduced basal PKC-zeta and GSK3beta phosphorylation in human mesangial cells exposed to high glucose. CTGF stimulation under these conditions again resulted in PKC-zeta phosphorylation and human mesangial cell migration. Regulation of PKC-zeta by PKC-beta in this instance may establish PKC-zeta as a target for constraining the progression of mesangial cell dysfunction in the pathogenesis of diabetic nephropathy.

Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells

We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.

Role of basal extracellular Ca2+ entry during 5-HT-induced vasoconstriction of canine pulmonary arteries.

1. Measurements of artery contraction, cytosolic [Ca(2+)], and Ca(2+) permeability were made to examine contractile and cytosolic [Ca(2+)] responses of canine pulmonary arteries and isolated cells to 5-hydroxytryptamine (5-HT), and to determine the roles of intracellular Ca(2+) release and extracellular Ca(2+) entry in 5-HT responses. 2. The EC(50) for 5-HT-mediated contractions and cytosolic [Ca(2+)] increases was approximately 10(-7) M and responses were inhibited by ketanserin, a 5-HT(2A)-receptor antagonist. 3. 5-HT induced cytosolic [Ca(2+)] increases were blocked by 20 microM Xestospongin-C and by 2-APB (IC(50)=32 microM inhibitors of InsP(3) receptor activation. 4. 5-HT-mediated contractions were reliant on release of InsP(3) but not ryanodine-sensitive Ca(2+) stores. 5. 5-HT-mediated contractions and cytosolic [Ca(2+)] increases were partially inhibited by 10 microM nisoldipine, a voltage-dependent Ca(2+) channel blocker. 6. Extracellular Ca(2+) removal reduced 5-HT-mediated contractions further than nisoldipine and ablated cytosolic [Ca(2+)] increases and [Ca(2+)] oscillations. Similar to Ca(2+) removal, Ni(2+) reduced cytosolic [Ca(2+)] and [Ca(2+)] oscillations. 7. Mn(2+) quench of fura-2 and voltage-clamp experiments showed that 5-HT failed to activate any significant voltage-independent Ca(2+) entry pathways, including store-operated and receptor-activated nonselective cation channels. Ni(2+) but not nisoldipine or Gd(3+) blocked basal Mn(2+) entry. 8. Voltage-clamp experiments showed that simultaneous depletion of both InsP(3) and ryanodine-sensitive intracellular Ca(2+) stores activates a current with linear voltage dependence and a reversal potential consistent with it being a nonselective cation channel. 5-HT did not activate this current. 9. Basal Ca(2+) entry, rather than CCE, is important to maintain 5-HT-induced cytosolic [Ca(2+)] responses and contraction in canine pulmonary artery.

Vasculogenic and osteogenesis-enhancing potential of human umbilical cord blood endothelial colony-forming cells

Umbilical cord blood-derived endothelial colony-forming cells (UCB-ECFC) show utility in neovascularization, but their contribution to osteogenesis has not been defined. Cocultures of UCB-ECFC with human fetal-mesenchymal stem cells (hfMSC) resulted in earlier induction of alkaline phosphatase (ALP) (Day 7 vs. 10) and increased mineralization (1.9×; p <.001) compared to hfMSC monocultures. This effect was mediated through soluble factors in ECFC-conditioned media, leading to 1.8-2.2× higher ALP levels and a 1.4-1.5× increase in calcium deposition (p <.01) in a dose-dependent manner. Transcriptomic and protein array studies demonstrated high basal levels of osteogenic (BMPs and TGF-ßs) and angiogenic (VEGF and angiopoietins) regulators. Comparison of defined UCB and adult peripheral blood ECFC showed higher osteogenic and angiogenic gene expression in UCB-ECFC. Subcutaneous implantation of UCB-ECFC with hfMSC in immunodeficient mice resulted in the formation of chimeric human vessels, with a 2.2-fold increase in host neovascularization compared to hfMSC-only implants (p = .001). We conclude that this study shows that UCB-ECFC have potential in therapeutic angiogenesis and osteogenic applications in conjunction with MSC. We speculate that UCB-ECFC play an important role in skeletal and vascular development during perinatal development but less so in later life when expression of key osteogenesis and angiogenesis genes in ECFC is lower.

Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells

The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-?B. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF.

XBP1 mRNA splicing triggers an autophagic response in endothelial cells through BECLIN-1 transcriptional activation

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3ß (LC3-ßII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1a (IRE1a)-dependent manner. Knockdown of XBP1 or IRE1a by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3ß expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt -537 to -755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1.

Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3

Galectin-9 expression in endothelial cells can be induced in response to inflammation. However, the mechanism of its expression remains unclear. In this study, we found that interferon-? (IFN-?) induced galectin-9 expression in human endothelial cells in a time-dependent manner, which coincided with the activation of histone deacetylase (HDAC). When endothelial cells were treated with the HDAC3 inhibitor, apicidin, or shRNA-HDAC3 knockdown, IFN-?-induced galectin-9 expression was abolished. Overexpression of HDAC3 induced the interaction between phosphoinositol 3-kinase (PI3K) and IFN response factor 3 (IRF3), leading to IRF3 phosphorylation, nuclear translocation, and galectin-9 expression. HDAC3 functioned as a scaffold protein for PI3K/IRF3 interaction. In addition to galectin-9 expression, IFN-? also induced galectin-9 location onto plasma membrane, which was HDAC3-independent. Importantly, HDAC3 was essential for the constitutive transcription of PI3K and IRF3, which might be responsible for the basal level of galectin-9 expression. The phosphorylation of IRF3 was essential for galectin-9 expression. This study provides new evidence that HDAC3 regulates galectin-9 expression in endothelial cells via interaction with PI3K-IRF3 signal pathway.

Tumour necrosis factor-alpha (TNF-alpha) increases nuclear factor kappaB (NFkappaB) activity in and interleukin-8 (IL-8) release from bovine mammary epithelial cells

Epithelia play important immunological roles at a variety of mucosal sites. We examined NFkappaB activity in control and TNF-alpha treated bovine mammary epithelial monolayers (BME-UV cells). A region of the bovine IL-8 (bIL-8) promoter was sequenced and a putative kappaB consensus sequence was identified bioinformatically. We used this sequence to analyse nuclear extracts for IL-8 specific NFkappaB activity. As a surrogate marker of NFkappaB activation, we investigated IL-8 release in two models. Firstly in BME-UV monolayers, IL-8 release in the presence of pro- and anti-inflammatory agents was determined by enzyme-linked immunosorbent assay (ELISA). Secondly, we measured IL-8 secretion from a novel model of intact mucosal sheets of bovine teat sinus. IL-8 release into bathing solutions was assessed following treatment with pro- and anti-inflammatory agents. TNF-alpha enhanced NFkappaB activity in bovine mammary epithelial monolayers. p65 NFkappaB homodimer was identified in both control and TNF-alpha treated cells. Novel sequencing of the bovine IL-8 promoter identified a putative kappaB consensus sequence, which specifically bound TNF-alpha inducible p50/p65 heterodimer. TNF-alpha induced primarily serosal IL-8 release in the cell culture model. Pre-treatment with anti-TNF or dexamethasone inhibited TNF-alpha induced IL-8 release. High dose interleukin-1beta (IL-1beta) induced IL-8 release, however significantly less potently than TNF-alpha. Bovine mammary mucosal tissue released high basal levels of IL-8 which were unaffected by TNF-alpha or IL-1beta but inhibited by both dexamethasone and anti-TNF. These data support a role for TNF-alpha in activation of NFkappaB and release of IL-8 from bovine mammary epithelial cells.

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells

PURPOSE: The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). METHODS: Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. RESULTS: Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. CONCLUSION: These results illustrate the internalization and intracellular trafficking by RVECs of insulin and LDL through highly efficient RME, and they provide evidence for at least two possible fates for the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well-known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a vascular endothelial growth factor receptor (VEGFR) and PI3K/Akt dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Over-expression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 Protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3 and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

Manganese Superoxide Dismutase Is a Promising Target for Enhancing Chemosensitivity of Basal-Like Breast Carcinoma

AIMS: Although earlier reports highlighted a tumor suppressor role for manganese superoxide dismutase (MnSOD), recent evidence indicates increased expression in a variety of human cancers including aggressive breast carcinoma. In the present article, we hypothesized that MnSOD expression is significantly amplified in the aggressive breast carcinoma basal subtype, and targeting MnSOD could be an attractive strategy for enhancing chemosensitivity of this highly aggressive breast cancer subtype.

RESULTS: Using MDA-MB-231 and BT549 as a model of basal breast cancer cell lines, we show that knockdown of MnSOD decreased the colony-forming ability and sensitized the cells to drug-induced cell death, while drug resistance was associated with increased MnSOD expression. In an attempt to develop a clinically relevant approach to down-regulate MnSOD expression in patients with basal breast carcinoma, we employed activation of the peroxisome proliferator-activated receptor gamma (PPARγ) to repress MnSOD expression; PPARγ activation significantly reduced MnSOD expression, increased chemosensitivity, and inhibited tumor growth. Moreover, as a proof of concept for the clinical use of PPARγ agonists to decrease MnSOD expression, biopsies derived from breast cancer patients who had received synthetic PPARγ ligands as anti-diabetic therapy had significantly reduced MnSOD expression. Finally, we provide evidence to implicate peroxynitrite as the mechanism involved in the increased sensitivity to chemotherapy induced by MnSOD repression.

INNOVATION AND CONCLUSION: These data provide evidence to link increased MnSOD expression with the aggressive basal breast cancer, and underscore the judicious use of PPARγ ligands for specifically down-regulating MnSOD to increase the chemosensitivity of this subtype of breast carcinoma.

Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells

OBJECTIVE: The efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of hypoxia-inducible-factor 1α (HIF1A). Overexpression of the hypoxia-responsive gene class III β-tubulin (TUBB3) has been associated with docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel.

METHODS: The cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with methylation status at the HIF1A binding site.

RESULTS: Prostate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression.

CONCLUSION: These data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer patients.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO₂-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO₂-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO₂-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO₂-DDE (10μM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO₂-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO₂-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO₂-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO₂-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO₂-DDE induced endocrine disruption in Leydig cells.

INVESTIGATING THE ROLE OF NOX NADPH OXIDASE SIGNALLING IN CELL MIGRATION AND ANGIOGENESIS OF ENDOTHELIAL COLONY-FORMING CELLS

Introduction. Endothelial colony-forming cells (ECFCs) hold great cytotherapeutic potential for ischaemic disease. Emerging evidence supports a key role for NADPH oxidases in underlying angiogenic processes of these and other endothelial cells. Aims. To study the influence of Nox NADPH oxidases on the pro-angiogenic function of ECFCs. Methods. Human ECFCs isolated from umbilical cord blood were treated with pro-oxidant PMA and assessed in vitro, both under basal conditions and after siRNA knockdown of Nox4, a key endothelial NADPH oxidase isoform, alongside primary mature human aortic endothelial cells (HAoECs) for comparison, using an established scratch-wound assay as the functional end-point. Results. PMA (500nM for 8h) increased cell migration (control 18.6±2.8, PMA 32.7±6.6% wound closure; n=6, P<0.05) in a superoxide-dependent manner, as indicated by attenuation of this effect in the presence of PEG-SOD. Although HAoEC migration in response to PMA also tended to increase, this did not reach statistical significance. Notably, cell migration at 16h was reduced by Nox4 knockdown in ECFCs (control siRNA 53.4±3.5, Nox4 siRNA 35.1±4.9% closure; n=3, P<0.05), but not in HAoECs, whilst the pro-migratory effect of PMA in ECFCs was potentiated after Nox4 knockdown (control siRNA 53.4±3.5, +PMA 61.5±3.2% closure; n=3, P=NS; Nox4 siRNA 35.1±4.9, +PMA 53.0±4.9% closure; n=3, P<0.05). Conclusion. ECFC migration is enhanced by low concentrations of superoxide, to a greater extent compared to mature endothelial cells, and appears to be at least partly dependent upon NADPH oxidase, including a specific role for Nox4. Although, the precise contribution of endothelial Nox NADPH oxidases isoforms remains to be determined, it is clear that these findings may have significant implications for potential ECFC-based therapies for ischaemic disease, which is associated with an oxidative microenvironment.