Levels In Conditioned Media Research Articles

The granulocyte colony-stimulating factor (G-CSF) upregulates metalloproteinase-2 and VEGF through PI3K/Akt and Erk1/2 activation in human trophoblast Swan 71 cells

IntroductionAlthough the expression of the granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in placental tissues suggests that the cytokine could play a role in placental development, the relevance of G-CSF:G-CSFR interaction in trophoblast cells remains to be studied. Thus, the possible functional role of G-CSF was examined in a human trophoblast cell line (Swan 71 cells). Methods and resultsThe expression of G-CSFR was detected by immunocytochemistry and Western blot assays. G-CSF treatment exerted neither a proliferative nor a protective effect on H2O2-mediated cell death in trophoblast cells. Gelatin zymography of supernatants collected from G-CSF-treated cells showed an increment of metalloproteinase-2 (MMP-2) activity. We also found higher MMP-2 and VEGF expression levels in conditioned medium from cells exposed to G-CSF. In addition, it was demonstrated that G-CSF induced the activation of PI3K/Akt and Erk1/2 pathways, which in turn activated NF-kB. By using selective pharmacological inhibitors, it was showed that these pathways are mediating the biological effects produced by G-CSF in Swan 71 cells. Discussion and conclusionWe have demonstrated for the first time that G-CSF increases MMP-2 activity and VEGF secretion in Swan 71 cells through activation of PI3K/Akt and Erk signaling pathways, both contributing to the translocation of NF-kB to the nucleus. These data suggest that G-CSF is involved in the regulation of trophoblast function, and should be considered as a locally produced cytokine probably contributing to embryo implantation and the development of a functional placenta.

Identification of noninvasive biomarkers for nephrotoxicity using HK-2 human kidney epithelial cells.

The kidney is an important site of xenobiotic-induced toxicity. Because the traditional markers of renal injury indicate only severe renal damage, new biomarkers are needed for a more sensitive and reliable evaluation of renal toxicity. This study was designed to identify in vitro noninvasive biomarkers for efficient assessment of nephrotoxicity by using cisplatin as a model of nephrotoxic compounds. To this end, a comparative proteomic analysis of conditioned media from HK-2 human kidney epithelial cells treated with cisplatin was performed. Here, we identified pyruvate kinase M1/M2 isoform M2 (PKM2) and eukaryotic translation elongation factor 1 gamma (EF-1γ) as potential biomarker candidates for evaluation of nephrotoxicity. PKM2 and EF-1γ were increased by cisplatin in a kidney cell-specific manner, most likely due to cisplatin-induced apoptosis. The increase of PKM2 and EF-1γ levels in conditioned media was also observed in the presence of other nephrotoxic agents with different cytotoxic mechanisms such as CdCl2, HgCl2, and cyclosporine A. Rats treated with cisplatin, CdCl2, or HgCl2 presented increased levels of PKM2 and EF-1γ in the urine and kidney tissue. Taken together, this study identified two noninvasive biomarker candidates, PKM2 and EF-1γ, by comparative proteomic analysis. These new biomarkers may offer an alternative to traditional renal markers for efficient evaluation of nephrotoxicity.

Islet-1 Overexpression in Human Mesenchymal Stem Cells Promotes Vascularization Through Monocyte Chemoattractant Protein-3

The LIM-homeobox transcription factor islet-1 (ISL1) has been proposed to mark a cardiovascular progenitor cell lineage that gives rise to cardiomyocytes, endothelial cells, and smooth muscle cells. The aim of this study was to investigate whether forced expression of ISL1 in human mesenchymal stem cells (hMSCs) influenced the differentiation capacity and angiogenic properties of hMSCs. The lentiviral vector, EF1α-ISL1, was constructed using the Multisite Gateway System and used to transduce hMSCs. We found that ISL1 overexpression did not alter the proliferation, migration, or survival of hMSCs or affect their ability to differentiate into osteoblasts, adipocytes, cardiomyocytes, or endotheliocytes. However, ISL1-hMSCs differentiated into smooth muscle cells more efficiently than control hMSCs. Furthermore, conditioned medium from ISL1-hMSCs greatly enhanced the survival, migration, and tube-formation ability of human umbilical vein endothelial cells (HUVECs) in vitro. In vivo angiogenesis assays also showed much more vascular-like structures in the group cotransplanted with ISL1-hMSCs and HUVECs than in the group cotransplanted with control hMSCs and HUVECs. Quantitative RT-PCR and antibody arrays detected monocyte chemoattractant protein-3 (MCP3) at a higher level in conditioned medium from ISL1-hMSCs cultures than in conditioned medium from control hMSCs. Neutralization assays showed that addition of an anti-MCP3 antibody to ISL1-hMSCs-conditioned medium efficiently abolished the angiogenesis-promoting effect of ISL1-hMSCs. Our data suggest that overexpression of ISL1 in hMSCs promotes angiogenesis in vitro and in vivo through increasing secretion of paracrine factors, smooth muscle differentiation ability, and enhancing the survival of HUVECs.

THU0553 Gliostatin Regulates Vascular Endothelial Growth-Factor Production in Human Fibroblast-Like Synoviocytes

BackgroundAngiogenesis, which is the growth of new capillaries from pre-existing blood vessels, is an essential feature of rheumatoid arthritis (RA). Neovascularisation might be triggered by several factors, such as cytokines,...

Proteolytic Activity Present in House-Dust-Mite Extracts Degrades ENA-78/CXCL5 and Reduces Neutrophil Migration.

Background. Bronchial smooth muscle cells (BSMC) are a major source of proinflammatory and proangiogenic cytokines and chemokines, including VEGF and CXC-chemokines. CXC-chemokines act primarily on neutrophils, mediating their recruitment to and activation at the site of inflammation. In humans, house-dust mite (HDM) allergens can cause asthmatic exacerbations and trigger an inflammatory response through protease-dependent mechanisms. Objective. We investigated the effect HDM extract on the release of pro-angiogenic and proinflammatory cytokines from BSMC. Methods. Human primary BSMC were stimulated with HDM extract in the absence or presence of fetal calf serum (FCS). Twenty angiogenic cytokines were detected by a specific antibody array and modified protein levels were confirmed by ELISA. Neutrophil migration was measured using a 96-well Boyden chamber. Results. ENA-78/CXCL5 protein levels in conditioned medium of BSMC stimulated with HDM extract were significantly reduced (n = 10, P < 0.05) but restored in the presence of 5% FCS. HDM extracts did not affect ENA-78/CXCL5 mRNA levels. Recombinant ENA-78/CXCL5 was degraded after incubation with HDM extracts (n = 7, P < 0.05) but restored after the addition of the serine protease AEBSF. Neutrophil migration towards recombinant ENA-78/CXCL5 was also reduced in the presence of HDM extract. Conclusion. HDM proteases degrade ENA-78/CXCL5. Thus exposure to HDM allergens may alter ENA-78/CXCL5 levels in the lungs and may affect angiogenesis and the inflammatory response in the airways of asthma patients.

RAGE/NF-κB pathway mediates lipopolysaccharide-induced inflammation in alveolar type I epithelial cells isolated from neonate rats.

Alveolar type I epithelial cells (AECIs) play an important role in the pathogenesis of acute lung injury. The receptor for advanced glycation end-products (RAGEs) is expressed at a high basal level in AECIs, and its soluble isoform is suggested as a marker of AECI injury. However, the molecular mechanism by which RAGE mediates inflammatory injury in AECIs remains elusive. In this study, we established lipopolysaccharide (LPS)-induced inflammation in AECIs isolated from neonate rats as the experimental model and investigated the role of RAGE/NF-κB signaling in mediating inflammatory response in AECIs. We found that LPS increased RAGE expression and the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in AECIs in a dose-dependent manner. Knockdown of RAGE significantly decreased TNF-α and IL-1β levels in conditioned medium of AECIs. Electrophoretic mobility shift assay (EMSA) showed that NF-κB activation was increased in AECIs treated by LPS. However, knockdown of RAGE inhibited both basic and LPS-induced NF-κB activity in AECIs. Finally, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced LPS-induced upregulation of RAGE expression at both protein and messenger RNA (mRNA) levels in AECIs. Our results suggest that RAGE mediates inflammatory response in AECIs via activating NF-κB, and RAGE/NF-κB pathway presents potential target for the prevention and therapy of acute lung injury.

MicroRNAs Regulate Human Adipocyte Lipolysis: Effects of miR-145 Are Linked to TNF-α

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α). Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d) modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.

Effect of urotensin II on apolipoprotein B 100 and apolipoprotein A-I expression in HepG2 cell line

Background:Increased apolipoprotein B100 (apo B) and decreased apolipoprotein A-I (apo A-I) production are important risk factors in atherosclerosis. Urotensin II (UII), as the most potent vasoconstrictor in human, is related with hypertension and probably atherosclerosis. Because of the relationship between the hypertension and lipoprotein metabolism in atherosclerosis, the aim of this study was to test the effect of urotensin II on apo B and apo A-I expression in hepatic (HepG2) cell line.Materials and Methods:HepG2 cells were treated with 10, 50, 100, and 200 nmol/L of urotensin II (n = 6). Relative apo B and apo A-I messenger RNA (mRNA) levels in conditioned media, normalized to glyceraldehyde-3-phosphate dehydrogenase, were measured with quantitative real-time polymerase chain reaction method. In addition, apo B and apo A-I levels were also estimated and compared with the controls using the western blotting method. Data were analyzed statistically by ANOVA and non-parametric tests.Results:The apo B mRNA levels were not increased significantly following the treatment with UII. However, apo B protein levels were increased significantly after the treatment with urotensin II, especially at 100 and 200 nmol/L. The apo A-I mRNA and protein levels in conditioned media also were not significantly changed. However, there was a significant decrease in apo A-I mRNA and protein levels at 200 nM UII.Conclusions:UII might increase apo B at protein level probably through participating factors in its synthesis and/ or stability/degradation. In addition, UII may have decreasing effect at more than 200 nM concentrations on apo A-I.

Thrombospondin-2 facilitates assembly of a type-I collagen-rich matrix in marrow stromal cells undergoing osteoblastic differentiation

We examined the effects of Thrombospondin-2 (TSP2) deficiency on assembly of collagenous extracellular matrix (ECM) by primary marrow-derived mesenchymal stromal cells (MSC) undergoing osteoblast differentiation in culture. After 30 d, wild-type cells had accumulated and mineralized a collagen-rich insoluble matrix, whereas the TSP2-null cultures contained markedly lower amounts of matrix collagen and displayed reduced mineral. Differences in matrix collagen were seen as early as day 9, at which time wild-type cultures contained more total collagen per cell than did TSP2-null cells. Collagen was unevenly distributed amongst different extracellular compartments in the two cell-types. Collagen levels in conditioned medium of wild-type cells were higher than those of TSP2-null cells, but were roughly equivalent in the acid-soluble, newly cross-linked matrixes. Conversely, the mature, cross-linked acid-insoluble matrix layer of wild-type cells contained about twice as much collagen as TSP2-null cell-derived matrix. Western blot analysis of type-I collagen in detergent-soluble and insoluble matrix fractions supported the premise that matrix collagen levels were reduced in TSP2-null MSC undergoing osteoblastic differentiation in vitro. Western blot and immunofluorescent analysis suggested that assembly of fibronectin into matrix was not affected by TSP2 deficiency. Instead, western blots of conditioned medium demonstrated a marked reduction in mature, fully processed type-I collagen in the absence of TSP2. Our data suggest that in the context of osteoblast differentiation, TSP2 promotes the assembly of a type-I collagen-rich matrix by facilitating pro-collagen processing.

Lower Cerebrospinal Fluid/Plasma Fibroblast Growth Factor 21 (FGF21) Ratios and Placental FGF21 Production in Gestational Diabetes

ObjectivesCirculating Fibroblast Growth Factor 21 (FGF21) levels are increased in insulin resistant states such as obesity, type 2 diabetes mellitus and gestational diabetes mellitus (GDM). In addition, GDM is associated with serious maternal and fetal complications. We sought to study human cerebrospinal fluid (CSF) and corresponding circulating FGF21 levels in women with gestational diabetes mellitus (GDM) and in age and BMI matched control subjects. We also assessed FGF21 secretion from GDM and control human placental explants.DesignCSF and corresponding plasma FGF21 levels of 24 women were measured by ELISA [12 GDM (age: 26–47 years, BMI: 24.3–36.3 kg/m2) and 12 controls (age: 22–40 years, BMI: 30.1–37.0 kg/m2)]. FGF21 levels in conditioned media were secretion from GDM and control human placental explants were also measured by ELISA.ResultsGlucose, HOMA-IR and circulating NEFA levels were significantly higher in women with GDM compared to control subjects. Plasma FGF21 levels were significantly higher in women with GDM compared to control subjects [234.3 (150.2–352.7) vs. 115.5 (60.5–188.7) pg/ml; P<0.05]. However, there was no significant difference in CSF FGF21 levels in women with GDM compared to control subjects. Interestingly, CSF/Plasma FGF21 ratio was significantly lower in women with GDM compared to control subjects [0.4 (0.3–0.6) vs. 0.8 (0.5–1.6); P<0.05]. FGF21 secretion into conditioned media was significantly lower in human placental explants from women with GDM compared to control subjects (P<0.05).ConclusionsThe central actions of FGF21 in GDM subjects maybe pivotal in the pathogenesis of insulin resistance in GDM subjects. The significance of FGF21 produced by the placenta remains uncharted and maybe crucial in our understanding of the patho-physiology of GDM and its associated maternal and fetal complications. Future research should seek to elucidate these points.

Herbal compound “Songyou Yin” attenuates hepatoma cell invasiveness and metastasis through downregulation of cytokines secreted by activated hepatic stellate cells

BackgroundActivated hepatic stellate cells (aHSCs) play an important role in the progression of hepatocellular carcinoma (HCC). Here, we determined if cytokines secreted in response to the herbal compound “Songyou Yin” (SYY) treatment of aHSCs could influence invasiveness and metastatic capabilities of hepatoma cells.MethodsPrimary rat hepatic stellate cells (HSCs) were isolated, activated, divided into SYY treated and untreated (nSYY) groups, and conditioned media (CM-SYY and CM-nSYY, respectively) were collected. The hepatoma cell line, McA-RH7777 was cultured for 4 weeks with SYY, CM-SYY, and CM-nSYY, designated McA-SYY, McA-SYYCM and McA-nSYYCM. The invasiveness and metastatic capabilities were evaluated using Matrigel invasion assay in vitro and pulmonary metastasis in vivo. Matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and vimentin protein levels in McA-SYYCM and McA-nSYYCM were evaluated by Western blot. Cytokine levels in conditioned media were tested using enzyme-linked immunosorbent assay (ELISA).ResultsMatrigel invasion assay indicated that the number of McA-SYYCM cells passing through the basement membrane was less than in McA-nSYYCM cells (P < 0.01). Similar results were also observed in vivo for lung metastasis. McA-SYYCM cells showed less pulmonary metastasis capabilities than McA-nSYYCM cells (P < 0.001). The reduced expression of MMP-2 and reversed epithelial to mesenchymal transition with E-cadherin upregulation, and N-cadherin and vimentin downregulation were also found in McA-SYYCM compared to McA-nSYYCM. Metastasis-promoting cytokines hepatocyte growth factor, interleukin-6, transforming growth factor-β1, and vascular endothelial growth factor were markedly decreased in CM-SYY compared to CM-nSYY.ConclusionsSYY attenuates hepatoma cell invasiveness and metastasis capabilities through downregulating cytokines secreted by activated hepatic stellate cells.

Association of the Thrombomodulin Gene c.1418C&gt;T Polymorphism With Thrombomodulin Levels and With Venous Thrombosis Risk

To investigate the association of the THBD c.1418C>T polymorphism, which encodes for the replacement of Ala455 by Val in thrombomodulin (TM), with venous thromboembolism (VTE), plasma soluble TM, and activated protein C levels. In addition, human umbilical vein endothelial cells (HUVEC) isolated from 100 umbilical cords were used to analyze the relation between this polymorphism and THBD mRNA and TM protein expression. The THBD c.1418C>T polymorphism was genotyped in 1173 patients with VTE and 1262 control subjects. Levels of soluble TM and activated protein C were measured in 414 patients with VTE (not on oral anticoagulants) and 451 controls. HUVECs were genotyped for the polymorphism and analyzed for THBD mRNA and TM protein expression and for the ability to enhance protein C activation by thrombin. The 1418T allele frequency was lower in patients than in controls (P<0.001), and its presence was associated with a reduced VTE risk, reduced soluble TM levels, and increased circulating activated protein C levels (P<0.001). In cultured HUVEC, the 1418T allele did not influence THBD expression but was associated with increased TM in cell lysates, increased rate of protein C activation, and reduced soluble TM levels in conditioned medium. The THBD 1418T allele is associated with lower soluble TM, both in plasma and in HUVEC-conditioned medium, and with an increase in functional membrane-bound TM in HUVEC, which could explain the increased activated protein C levels and the reduced VTE risk observed in individuals carrying this allele.

Adipocytes modulate vascular smooth muscle cells migration potential through their secretions

Impairment of vascular smooth muscle cells (VSMC) is recognized as a predisposition factor for atherosclerosis development. We hypothesize that the metabolic syndrome has a direct impact on VSMC migration and phenotypic switching, which may increase the incidence of atherosclerotic events. Aortic VSMC were extracted from 10 weeks old C57BL6 mice and incubated for 24 hr in adipocytes conditioned cell culture medium. Adipocytes were extracted from diabetic C57BL6 male mice fed with either a vegetal or an animal High-Fat-Diet (HFD) for 20 weeks. Migration of VSMC in response to conditioned media stimulations was significantly modulated compared to control. The most extended effects on VSMC were triggered by adipocytes from mice fed with animal HFD. These effects were concurrent with increased leptin concentrations and decreased adiponectin levels in conditioned media. A significant up-regulation of CD36 mRNA level was found in VSMC treated with adipocytes from HFD-fed mice. In conclusion, we have shown that the development of adipocyte-induced VSMC alterations is linked to diet fatty acid composition and the degree of metabolic alterations. The modulation of adipokine secretions in the adipose tissue that is linked to metabolic alterations may alter the physiology of VSMC and thus accelerate the development of metabolic-related vascular diseases.

Induction of the chemokine IL-8/Kc by the articular cartilage: Possible influence on osteoarthritis

PurposeIL-8 and its murine equivalent keratinocyte chemoattractant (Kc), chemokines produced by chondrocytes, contribute to the pathophysiology of osteoarthritis. However, the mechanisms leading to their production are poorly known. We aimed to investigate whether mechanical (compression), inflammatory (IL-1β) and metabolic (visfatin) stresses may induce the release of Kc when applied on murine cartilage. MethodsMouse cartilage explants were subjected to intermittent compression for 4, 6 and 24h. Primary cultures of immature murine articular chondrocytes were obtained by enzymatic digestion of articular cartilage from 6-days-old newborns mice. The effect of compression, IL-1β (10, 50, 100pg/mL) and of visfatin (5μg/mL) on the release of Kc was assessed by ELISA. IL-8 levels in conditioned media from human OA joint tissues (cartilage or synovium) were also assessed. ResultsIn comparison with non-compressed explants, loading increased Kc release of 3.2-, 1.9- and 2.0-fold at 4, 6 and 24h respectively (P<0.004, n=9). IL-1β triggered an increase of Kc release by primary cultured chondrocytes of 4.1-, 15.5- and 35.2-fold at 10, 50 and 100pg/mL of IL-1β respectively (P<0.05, n=4). Likewise, visfatin (5μg/mL) induced an increase in Kc release of 56.5±25.2 fold (P=0.002, n=6). IL-8 was released in conditioned media by synovium as well as by cartilage. ConclusionWe show for the first time that IL-8/Kc is highly responsive to mechanical, inflammatory and metabolic stresses, strengthening the hypothesis that IL-8/Kc could be added to the cytokines which may have a deleterious impact in osteoarthritis.

A Crucial Role of Activin A-Mediated Growth Hormone Suppression in Mouse and Human Heart Failure

Infusion of bone marrow-derived mononuclear cells (BMMNC) has been reported to ameliorate cardiac dysfunction after acute myocardial infarction. In this study, we investigated whether infusion of BMMNC is also effective for non-ischemic heart failure model mice and the underlying mechanisms. Intravenous infusion of BMMNC showed transient cardioprotective effects on animal models with dilated cardiomyopathy (DCM) without their engraftment in heart, suggesting that BMMNC infusion improves cardiac function via humoral factors rather than their differentiation into cardiomyocytes. Using conditioned media from sorted BMMNC, we found that the cardioprotective effects were mediated by growth hormone (GH) secreted from myeloid (Gr-1(+)) cells and the effects was partially mediated by signal transducer and activator of transcription 3 in cardiomyocytes. On the other hand, the GH expression in Gr-1(+) cells was significantly downregulated in DCM mice compared with that in healthy control, suggesting that the environmental cue in heart failure might suppress the Gr-1(+) cells function. Activin A was upregulated in the serum of DCM models and induced downregulation of GH levels in Gr-1(+) cells and serum. Furthermore, humoral factors upregulated in heart failure including angiotensin II upregulated activin A in peripheral blood mononuclear cells (PBMNC) via activation of NFκB. Similarly, serum activin A levels were also significantly higher in DCM patients with heart failure than in healthy subjects and the GH levels in conditioned medium from PBMNC of DCM patients were lower than that in healthy subjects. Inhibition of activin A increased serum GH levels and improved cardiac function of DCM model mice. These results suggest that activin A causes heart failure by suppressing GH activity and that inhibition of activin A might become a novel strategy for the treatment of heart failure.

Phenylpiperidine-type γ-secretase modulators target the transmembrane domain 1 of presenilin 1

Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.

Nicotinic acid induces apolipoprotein A-I gene expression in HepG2 and Caco-2 cell lines

The objective was to test the effect of nicotinic acid on apolipoprotein A-I (apo A-I) gene expression in hepatic (HepG2) and intestinal (Caco-2) cell lines. HepG2 and Caco-2 cells were treated with 0.1, 0.3, 1.0, 3.0, and 10 mmol/L of nicotinic acid; and apo A-I concentrations in conditioned media were measured with Western blots. Relative apo A-I messenger RNA (mRNA) levels, normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA, were measured with quantitative real-time polymerase chain reaction method. The nicotinic acid response element in the apo A-I promoter was identified using a series of apo A-I reporter plasmids containing deletion constructs of the promoter. In other experiments, HepG2 cells were also transfected with the apo A-I reporter plasmid and the hepatocyte nuclear factors 3α and β expression plasmids. The apo A-I levels in conditioned media from HepG2 cells, apo A-I mRNA levels, and apo A-I promoter activity increased significantly following treatment with 1.0, 3.0, and 10 mmol/L nicotinic acid. Nicotinic acid–induced promoter activity required a region of the apo A-I gene located between −170 and −186 base pairs. Exogenous overexpression of the hepatocyte nuclear factors 3α and β had no additive effect on apo A-I promoter. Apolipoprotein A-I concentrations in conditioned media and the apo A-I promoter activity were also significantly increased in Caco-2 intestinal cells. Nicotinic acid may increase apo A-I protein synthesis in the liver and small intestine. Induction of apo A-I gene by nicotinic acid requires a nicotinic acid responsive element in the apo A-I promoter.

Differential Secretomics of Streptococcus pyogenes Reveals a Novel Peroxide Regulator (PerR)-regulated Extracellular Virulence Factor Mitogen Factor3 (MF3)

Streptococcus pyogenes is a human pathogen that causes various diseases. Numerous virulence factors secreted by S. pyogenes are involved in pathogenesis. The peroxide regulator (PerR) is associated with the peroxide resistance response and pathogenesis, but little is known about the regulation of the secretome involved in virulence. To investigate how PerR regulates the expression of the S. pyogenes secretome involved in virulence, a perR deficient mutant was used for comparative secretomic analysis with a wild-type strain. The conditioned medium containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for two-dimensional gel electrophoresis analysis, where protease inhibitors were applied to avoid the degradation of extracellular proteins. Differentially expressed protein spots were identified by liquid chromatography electrospray ionization tandem MS. More than 330 protein spots were detected on each gel. We identified 25 unique up-regulated proteins and 13 unique down-regulated proteins that were directly or indirectly controlled by the PerR regulator. Among these identified proteins, mitogen factor 3 (MF3), was selected to verify virulence and the expression of gene products. The data showed that MF3 protein levels in conditioned medium, as measured by immunoblot analysis, correlated well with protein levels determined by two-dimensional gel electrophoresis analysis. We also demonstrated that PerR bound to the promoter region of the mf3 gene. The result of an infection model showed that virulence was attenuated in the mf3 deficient mutant. Additional growth data of the wild-type strain and the mf3 deficient mutant suggested that MF3 played a role in digestion of exogenous DNA for promoting growth. To summarize, we conclude that PerR can positively regulate the expression of the secreted protein MF3 that contributes to the virulence in S. pyogenes. The analysis of the PerR-regulated secretome provided key information for the elucidation of the host-pathogen interactions and might assist in the development of potential chemotherapeutic strategies to prevent or treat streptococcal diseases.

Hypoxia induces tumor and endothelial cell migration in a Semaphorin 3F- and VEGF-dependent manner via transcriptional repression of their common receptor Neuropilin 2

Neuropilin-2 (NRP2) is a receptor expressed by tumor cells and endothelial cells (EC) that binds both semaphorin 3F (SEMA3F), a potent inhibitor of tumor angiogenesis and metastasis, and vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. It was found that glioblastoma and melanoma cells repressed NRP2 expression when maintained under hypoxic conditions and after treatment with the hypoxia-mimetic agent desferrioxamine (DFO), at both the mRNA and protein levels. Silencing of HIF1-α, the hypoxia-induced subunit of the hypoxia inducible factor (HIF), abrogated DFO-induced NRP2 repression. Conversely, ectopic expression of HIF1-α directly repressed NRP2 promoter activity and expression. NRP2 is the sole receptor for SEMA3F. Loss of NRP2 expression in tumor cells inhibited SEMA3F-dependent activities, such as inactivation of RhoA, depolymerization of F-actin, and inhibition of tumor cell migration. On the other hand, loss of NRP2 expression in tumor cells increased VEGF protein levels in conditioned media, with no effects on VEGF mRNA levels. This increase in VEGF protein levels promoted paracrine activation of EC, including VEGF receptor-2 phosphorylation, and activation of downstream signaling proteins such as p44/42 MAPK and p38 MAPK. In addition, the elevated VEGF levels induced EC migration and sprouting, two key steps of tumor angiogenesis in vivo. It was concluded that hypoxia regulates VEGF and SEMA3F activities through transcriptional repression of their common receptor NRP2, providing a novel mechanism by which hypoxia induces tumor angiogenesis, growth and metastasis.

Decreased circulating soluble Tie2 levels in preeclampsia may result from inhibition of vascular endothelial growth factor (VEGF) signaling.

Recent studies have found dysregulation in circulating levels of a number of angiogenic factors and their soluble receptors in preeclampsia. In this study, we examined the mechanism of production of soluble Tie2 (sTie2) and its potential connection to the failure of vascular remodeling in preeclamptic pregnancies. Serum samples were collected prospectively from 41 pregnant subjects at five different time points throughout pregnancy. Five of these subjects developed preeclampsia. For a second study, serum and placental samples were collected at delivery from preeclamptic and gestational age-matched controls. We examined serum sTie2 levels, and angiopoietin 1, angiopoietin 2, and Tie2 mRNA expression and localization in placental samples from the central basal plate area. We also examined the effects of vascular endothelial growth factor (VEGF) and a matrix metalloproteinase (MMP) inhibitor on proteolytic shedding of Tie2 in uterine microvascular endothelial cells. Serum sTie2 levels were significantly lower in preeclamptic subjects starting at 24-28 wk of gestation and continued to be lower through the time of delivery. In culture experiments, VEGF treatment significantly increased sTie2 levels in conditioned media, whereas the MMP inhibitor completely blocked this increase, suggesting that VEGF-induced Tie2 release is MMP dependent. Our data suggest, for the first time, an interaction between VEGF and Tie2 in uterine endothelial cells and a potential mechanism for the decrease in circulating sTie2 levels in preeclampsia, likely through inhibition of VEGF signaling. Further studies on VEGF-Tie2 interactions during pregnancy should provide new insights into the mechanisms underlying the failure of vascular remodeling in preeclampsia and other pregnancy complications.