Arsenic Requires Sphingosine-1-Phosphate Type 1 Receptors to Induce Angiogenic Genes and Endothelial Cell Remodeling
2009; Elsevier BV; Volume: 174; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.081016
ISSN1525-2191
AutoresAdam C. Straub, Linda R. Klei, Donna B. Stolz, Aaron Barchowsky,
Tópico(s)Hormonal Regulation and Hypertension
ResumoArsenic in drinking water is a major public health concern as it increases risk and incidence of cardiovascular disease and cancer. Arsenic exposure affects multiple vascular beds, promoting liver sinusoidal capillarization and portal hypertension, ischemic heart disease, peripheral vascular disease, and tumor angiogenesis. While Rac1-GTPase and NADPH oxidase activities are essential for arsenic-stimulated endothelial cell signaling for angiogenesis or liver sinusoid capillarization, the mechanism for initiating these effects is unknown. We found that arsenic-stimulated cell signaling and angiogenic gene expression in human microvascular endothelial cells were Pertussis toxin sensitive, indicating a G-protein coupled signaling pathway. Incubating human microvascular endothelial cells with the sphingosine-1-phosphate type 1 receptor (S1P1) inhibitor VPC23019 or performing small interfering RNA knockdown of S1P1 blocked arsenic-stimulated HMVEC angiogenic gene expression and tube formation, but did not affect induction of either HMOX1 or IL8. Liver sinusoidal endothelial cells (LSECs) defenestrate and capillarize in response to aging and environmental oxidant stresses. We found that S1P1 was enriched on LSECs in vivo and in primary cell culture and that VPC23019 inhibited both sphingosine-1-phosphate-stimulated and arsenic-stimulated LSEC oxidant generation and defenestration. These studies identified novel roles for S1P1 in mediating arsenic stimulation of both angiogenesis and pathogenic LSEC capillarization, as well as demonstrating a role for S1P1 in mediating environmental responses in the liver vasculature, providing possible mechanistic insight into arsenic-induced vascular pathogenesis and disease. Arsenic in drinking water is a major public health concern as it increases risk and incidence of cardiovascular disease and cancer. Arsenic exposure affects multiple vascular beds, promoting liver sinusoidal capillarization and portal hypertension, ischemic heart disease, peripheral vascular disease, and tumor angiogenesis. While Rac1-GTPase and NADPH oxidase activities are essential for arsenic-stimulated endothelial cell signaling for angiogenesis or liver sinusoid capillarization, the mechanism for initiating these effects is unknown. We found that arsenic-stimulated cell signaling and angiogenic gene expression in human microvascular endothelial cells were Pertussis toxin sensitive, indicating a G-protein coupled signaling pathway. Incubating human microvascular endothelial cells with the sphingosine-1-phosphate type 1 receptor (S1P1) inhibitor VPC23019 or performing small interfering RNA knockdown of S1P1 blocked arsenic-stimulated HMVEC angiogenic gene expression and tube formation, but did not affect induction of either HMOX1 or IL8. Liver sinusoidal endothelial cells (LSECs) defenestrate and capillarize in response to aging and environmental oxidant stresses. We found that S1P1 was enriched on LSECs in vivo and in primary cell culture and that VPC23019 inhibited both sphingosine-1-phosphate-stimulated and arsenic-stimulated LSEC oxidant generation and defenestration. These studies identified novel roles for S1P1 in mediating arsenic stimulation of both angiogenesis and pathogenic LSEC capillarization, as well as demonstrating a role for S1P1 in mediating environmental responses in the liver vasculature, providing possible mechanistic insight into arsenic-induced vascular pathogenesis and disease. Exposure to arsenic in drinking water increases risk and incidence of a number of cardiovascular pathologies and diseases including acute myocardial infarctions,1Yuan Y Marshall G Ferreccio C Steinmaus C Selvin S Liaw J Bates MN Smith AH Acute myocardial infarction mortality in comparison with lung and bladder cancer mortality in arsenic-exposed region II of Chile from 1950 to 2000.Am J Epidemiol. 2007; 166: 1381-1391Crossref PubMed Scopus (172) Google Scholar cardiac ischemic disease,2Wang CH Hsiao CK Chen CL Hsu LI Chiou HY Chen SY Hsueh YM Wu MM Chen CJ A review of the epidemiologic literature on the role of environmental arsenic exposure and cardiovascular diseases.Toxicol Appl Pharmacol. 2007; 222: 315-326Crossref PubMed Scopus (200) Google Scholar peripheral vascular disease,2Wang CH Hsiao CK Chen CL Hsu LI Chiou HY Chen SY Hsueh YM Wu MM Chen CJ A review of the epidemiologic literature on the role of environmental arsenic exposure and cardiovascular diseases.Toxicol Appl Pharmacol. 2007; 222: 315-326Crossref PubMed Scopus (200) Google Scholar liver vascular diseases, and hypertension.3Chen Y Factor-Litvak P Howe GR Graziano JH Brandt-Rauf P Parvez F van Geen A Ahsan H Arsenic exposure from drinking water. Dietary intakes of B vitamins and folate, and risk of high blood pressure in Bangladesh: a population-based, cross-sectional study.Am J Epidemiol. 2007; 165: 541-552Crossref PubMed Scopus (112) Google Scholar, 4Kwok RK Mendola P Liu ZY Savitz DA Heiss G Ling HL Xia Y Lobdell D Zeng D Thorp Jr, JM Creason JP Mumford JL Drinking water arsenic exposure and blood pressure in healthy women of reproductive age in Inner Mongolia.China Toxicol Appl Pharmacol. 2007; 222: 337-343Crossref PubMed Scopus (65) Google Scholar Increased activity of vascular NADPH oxidase (Nox) enzymes that generate reactive oxygen species (ROS) often play a central role in the pathogenesis of these diseases,5Hordijk PL Regulation of NADPH oxidases: the role of Rac proteins.Circ Res. 2006; 98: 453-462Crossref PubMed Scopus (434) Google Scholar, 6Lee MY Griendling KK Redox signaling, vascular function, and hypertension.Antioxid Redox Signal. 2008; 10: 1045-1059Crossref PubMed Scopus (205) Google Scholar and arsenic rapidly stimulates both endothelial7Smith KR Klei LR Barchowsky A Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.Am J Physiol. 2001; 280: L442-L449Google Scholar, 8Qian Y Liu KJ Chen Y Flynn DC Castranova V Shi X Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization.J Biol Chem. 2005; 280: 3875-3884Crossref PubMed Scopus (75) Google Scholar and smooth muscle9Lynn S Gurr JR Lai HT Jan KY NADH oxidase activation is involved in arsenite-induced oxidative DNA damage in human vascular smooth muscle cells.Circ Res. 2000; 86: 514-519Crossref PubMed Scopus (275) Google Scholar Nox enzymes. Rac1 is an essential component of the arsenic-stimulated Nox complex7Smith KR Klei LR Barchowsky A Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.Am J Physiol. 2001; 280: L442-L449Google Scholar and is a key regulator of endothelial cell migration in angiogenesis.5Hordijk PL Regulation of NADPH oxidases: the role of Rac proteins.Circ Res. 2006; 98: 453-462Crossref PubMed Scopus (434) Google Scholar, 10Tan W Palmby TR Gavard J Amornphimoltham P Zheng Y Gutkind JS An essential role for Rac1 in endothelial cell function and vascular development.FASEB J. 2008; 22: 1829-1838Crossref PubMed Scopus (182) Google Scholar As with endogenous G-protein coupled receptor (GPCR)-mediated activation of Nox2-based oxidase, arsenic-stimulated oxidase activity requires Rac1- or Cdc42-GTPase activity and arsenic stimulates membrane translocation of Rac1 in both cultured endothelial cells7Smith KR Klei LR Barchowsky A Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.Am J Physiol. 2001; 280: L442-L449Google Scholar, 8Qian Y Liu KJ Chen Y Flynn DC Castranova V Shi X Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization.J Biol Chem. 2005; 280: 3875-3884Crossref PubMed Scopus (75) Google Scholar, 11Klei LR Barchowsky A Positive signaling interactions between arsenic and ethanol for angiogenic gene induction in human microvascular endothelial cells.Toxicol Sci. 2008; 102: 319-327Crossref PubMed Scopus (20) Google Scholar and in vivo.12Straub AC Stolz DB Ross MA Hernandez-Zavala A Soucy NV Klei LR Barchowsky A Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver.Hepatology. 2007; 45: 205-212Crossref PubMed Scopus (83) Google Scholar However, no GPCR has been identified that responds to arsenic and the mechanism(s) through which arsenic stimulates this Rac1-mediated signaling have not been identified. The present studies investigate arsenic-stimulated cell signaling through GPCR in endothelial cells from two vascular beds that produce different pathogenic responses when exposed to arsenic. The rationale for using two disparate endothelial phenotypes was to address whether arsenic activates heterogenic endothelium through common pathways. The first model was human microvascular cells (HMVEC) that produce angiogenic responses to arsenic.11Klei LR Barchowsky A Positive signaling interactions between arsenic and ethanol for angiogenic gene induction in human microvascular endothelial cells.Toxicol Sci. 2008; 102: 319-327Crossref PubMed Scopus (20) Google Scholar, 13Basu P Ghosh RN Grove LE Klei L Barchowsky A Angiogenic potential of 3-nitro-4-hydroxy benzene arsonic acid (roxarsone).Environ Health Perspect. 2008; 116: 520-523Crossref PubMed Google Scholar Arsenic stimulates microvascular angiogenesis in several in vivo models, and low environmental concentrations of arsenic enhance tumor growth.14Soucy NV Ihnat MA Kamat CD Hess L Post MJ Klei LR Clark C Barchowsky A Arsenic stimulates angiogenesis and tumorigenesis in vivo.Toxicol Sci. 2003; 76: 271-279Crossref PubMed Scopus (119) Google Scholar, 15Soucy NV Mayka D Klei LR Nemec AA Bauer JA Barchowsky A Neovascularization and angiogenic gene expression following chronic arsenic exposure in mice.Cardiovasc Toxicol. 2005; 5: 29-42Crossref PubMed Google Scholar, 16Kamat CD Green DE Curilla S Warnke L Hamilton JW Sturup S Clark C Ihnat MA Role of HIF signaling on tumorigenesis in response to chronic low-dose arsenic administration.Toxicol Sci. 2005; 86: 248-257Crossref PubMed Scopus (66) Google Scholar, 17Liu B Pan SG Dong XS Qiao HQ Jiang HC Krissansen GW Sun XY Opposing effects of arsenic trioxide on hepatocellular carcinomas in mice.Cancer Science. 2006; 97: 675-681Crossref PubMed Scopus (100) Google Scholar The second model was mouse liver sinusoidal endothelial cells (LSECs). LSECs are highly specialized endothelial cells with fenestrations organized into sieve plates to provide a dynamic filtration system.18DeLeve LD Hepatic microvasculature in liver injury.Semin Liver Dis. 2007; 27: 390-400Crossref PubMed Scopus (125) Google Scholar, 19Elvevold K Smedsrod B Martinez I The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity.Am J Physiol Gastrointest Liver Physiol. 2008; 294: G391-G400Crossref PubMed Scopus (184) Google Scholar, 20Le Couteur DG Cogger VC McCuskey RS de Cabo R Smedsrod B Sorensen KK Warren A Fraser R Age-related changes in the liver sinusoidal endothelium: a mechanism for dyslipidemia.Ann NY Acad Sci. 2007; 1114: 79-87Crossref PubMed Scopus (45) Google Scholar This filtration facilitates exchange of lipoproteins, nutrients, and macromolecules between the blood stream and the hepatocytes. LSEC membranes contain a highly active scavenger receptor system that provides the major site of clearance for modified albumin, hyaluronin, and advance glycation end products from the blood.18DeLeve LD Hepatic microvasculature in liver injury.Semin Liver Dis. 2007; 27: 390-400Crossref PubMed Scopus (125) Google Scholar, 19Elvevold K Smedsrod B Martinez I The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity.Am J Physiol Gastrointest Liver Physiol. 2008; 294: G391-G400Crossref PubMed Scopus (184) Google Scholar, 20Le Couteur DG Cogger VC McCuskey RS de Cabo R Smedsrod B Sorensen KK Warren A Fraser R Age-related changes in the liver sinusoidal endothelium: a mechanism for dyslipidemia.Ann NY Acad Sci. 2007; 1114: 79-87Crossref PubMed Scopus (45) Google Scholar In aging and in response to various oxidizing environmental stimuli,18DeLeve LD Hepatic microvasculature in liver injury.Semin Liver Dis. 2007; 27: 390-400Crossref PubMed Scopus (125) Google Scholar, 19Elvevold K Smedsrod B Martinez I The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity.Am J Physiol Gastrointest Liver Physiol. 2008; 294: G391-G400Crossref PubMed Scopus (184) Google Scholar, 20Le Couteur DG Cogger VC McCuskey RS de Cabo R Smedsrod B Sorensen KK Warren A Fraser R Age-related changes in the liver sinusoidal endothelium: a mechanism for dyslipidemia.Ann NY Acad Sci. 2007; 1114: 79-87Crossref PubMed Scopus (45) Google Scholar including arsenic,12Straub AC Stolz DB Ross MA Hernandez-Zavala A Soucy NV Klei LR Barchowsky A Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver.Hepatology. 2007; 45: 205-212Crossref PubMed Scopus (83) Google Scholar, 21Straub AC Clark KA Ross MA Chandra AG Li S Gao X Pagano PJ Stolz DB Barchowsky A Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.J Clin Invest. 2008; 118: 3980-3989Crossref PubMed Scopus (101) Google Scholar, 22Straub AC Stolz DB Vin H Ross MA Soucy NV Klei LR Barchowsky A Low level arsenic promotes progressive inflammatory angiogenesis and liver blood vessel remodeling in mice.Toxicol Appl Pharmacol. 2007; 222: 327-336Crossref PubMed Scopus (58) Google Scholar the LSECs undergo capillarization, a maturation process in which the cells defenestrate, develop a basement membrane, and up-regulate expression of junctional platelet endothelial cell adhesion molecule (PECAM)-1.18DeLeve LD Hepatic microvasculature in liver injury.Semin Liver Dis. 2007; 27: 390-400Crossref PubMed Scopus (125) Google Scholar, 19Elvevold K Smedsrod B Martinez I The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity.Am J Physiol Gastrointest Liver Physiol. 2008; 294: G391-G400Crossref PubMed Scopus (184) Google Scholar, 20Le Couteur DG Cogger VC McCuskey RS de Cabo R Smedsrod B Sorensen KK Warren A Fraser R Age-related changes in the liver sinusoidal endothelium: a mechanism for dyslipidemia.Ann NY Acad Sci. 2007; 1114: 79-87Crossref PubMed Scopus (45) Google Scholar, 21Straub AC Clark KA Ross MA Chandra AG Li S Gao X Pagano PJ Stolz DB Barchowsky A Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.J Clin Invest. 2008; 118: 3980-3989Crossref PubMed Scopus (101) Google Scholar This process promotes the conversion of the discontinuous sinusoidal endothelium into a continuous lining with tight intercellular junctions and limited fenestrations. Capillarization has been associated with increased risk of systemic atherogenesis, as well as being a requisite precursor for hepatic fibrosis.18DeLeve LD Hepatic microvasculature in liver injury.Semin Liver Dis. 2007; 27: 390-400Crossref PubMed Scopus (125) Google Scholar, 19Elvevold K Smedsrod B Martinez I The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity.Am J Physiol Gastrointest Liver Physiol. 2008; 294: G391-G400Crossref PubMed Scopus (184) Google Scholar, 20Le Couteur DG Cogger VC McCuskey RS de Cabo R Smedsrod B Sorensen KK Warren A Fraser R Age-related changes in the liver sinusoidal endothelium: a mechanism for dyslipidemia.Ann NY Acad Sci. 2007; 1114: 79-87Crossref PubMed Scopus (45) Google Scholar Since we previously demonstrated that Rac1 mobilization, oxidase activation, oxidant generation, and oxidative signaling all occur within the first minutes of arsenic exposure in large vessel endothelial cells, the current studies examined the hypothesis that this signaling is receptor initiated. These studies provide novel demonstration that the S1P1 receptor is an integral component of pathogenic arsenic signaling that is common in different endothelial cell types. The functional outcome of this signaling, however, is dictated by the different phenotypes and physiological roles of the two cell types. In addition to identifying novel pathways for the vascular response to this environmental toxicant, these studies are also the first to indicate a pathophysiological role for S1P1 in LSEC capillarization. Animal exposures were performed in agreement with institutional guidelines for animal safety and welfare at the University of Pittsburgh. C57BL/6-Tac (Taconic, Germantown, NY) male mice weighing ∼25g were untreated or exposed to 100 μg/L of sodium arsenite in their drinking water for 2 weeks, as previously described.12Straub AC Stolz DB Ross MA Hernandez-Zavala A Soucy NV Klei LR Barchowsky A Arsenic stimulates sinusoidal endothelial cell capillarization and vessel remodeling in mouse liver.Hepatology. 2007; 45: 205-212Crossref PubMed Scopus (83) Google Scholar, 21Straub AC Clark KA Ross MA Chandra AG Li S Gao X Pagano PJ Stolz DB Barchowsky A Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.J Clin Invest. 2008; 118: 3980-3989Crossref PubMed Scopus (101) Google Scholar, 22Straub AC Stolz DB Vin H Ross MA Soucy NV Klei LR Barchowsky A Low level arsenic promotes progressive inflammatory angiogenesis and liver blood vessel remodeling in mice.Toxicol Appl Pharmacol. 2007; 222: 327-336Crossref PubMed Scopus (58) Google Scholar This level of arsenic is ten times above the regulated limit of human exposure and has been demonstrated to significantly increase risk of cardiovascular disease. HMVECs (Lonza, Walkersville, MD) were cultured essentially as according to the supplier's instructions at 5% CO2 in complete MCDB 131 medium (Invitrogen, Carlsbad, CA.) supplemented with EGM−2 MV SingleQuot (Lonza). At confluence, the growth factors were reduced by a 1:5 dilution of complete medium with basal MCDB 131, and all experiments were conducted 18 to 24 hours later. LSECs were isolated from the non-parenchymal cell fraction of collagenase digested C57BL/6 mouse livers and cultured on gelatin-coated coverslips, as previously described.21Straub AC Clark KA Ross MA Chandra AG Li S Gao X Pagano PJ Stolz DB Barchowsky A Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.J Clin Invest. 2008; 118: 3980-3989Crossref PubMed Scopus (101) Google Scholar, 23Seglen PO Preparation of isolated rat liver cells.Methods Cell Biol. 1976; 13: 29-83Crossref PubMed Scopus (5224) Google Scholar, 24Ross MA Sander CM Kleeb TB Watkins SC Stolz DB Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.Hepatology. 2001; 34: 1135-1148Crossref PubMed Scopus (140) Google ScholarEx vivo sodium arsenite (ThermoFisher Scientific, Lafayette, CO) exposures ranged from 1 to 5 μmol/L, which was previously shown not to be cytotoxic.25Barchowsky A Dudek EJ Treadwell MD Wetterhahn KE Arsenic induces oxidant stress and NF-kappaB activation in cultured aortic endothelial cells.Free Radic Biol Med. 1996; 21: 783-790Crossref PubMed Scopus (268) Google Scholar Sphingosine-1-phosphate (Cayman Chemical Co, Ann Arbor, MI) was added to HMVEC or LSEC at a final concentration of 1 μmol/L. For small interfering (si)RNA knockdown experiments, 80% confluent cells were transfected with 40 nmol/L ON-TARGETplus SMARTpool of 4 siRNA sequences to human S1PR1 or 40 nmol/L non-targeting siRNA pool (ThermoFisher Scientific) using Lipofectamine 2000 and Opti-MEM (Invitrogen) reagents. After 4 days, the cells were placed in reduced medium for 18 hours before being exposed to sodium arsenite (As[III]). Cells were incubated with Pertussis toxin or the Rac1-selective inhibitor NSC23766 overnight before addition of arsenic to allow sufficient time for ADP ribosylation of Gi or incorporation of inhibitor into the Rac1-GTPase guanine nucleotide exchange factor binding site,26Gao Y Dickerson JB Guo F Zheng J Zheng Y Rational design and characterization of a Rac GTPase-specific small molecule inhibitor.Proc Natl Acad Sci USA. 2004; 101: 7618-7623Crossref PubMed Scopus (925) Google Scholar respectively. Frozen liver sections and cultured cells were prepared for fluorescence imaging and scanning electron microscopy (SEM), as previously described.21Straub AC Clark KA Ross MA Chandra AG Li S Gao X Pagano PJ Stolz DB Barchowsky A Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.J Clin Invest. 2008; 118: 3980-3989Crossref PubMed Scopus (101) Google Scholar, 24Ross MA Sander CM Kleeb TB Watkins SC Stolz DB Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.Hepatology. 2001; 34: 1135-1148Crossref PubMed Scopus (140) Google Scholar Quantitative measure of porosity (percentage of open area of fenestrations) was performed by measuring open fenestration area relative to total cell area in five ×10,000 images from each coverslip of treated or non-treated cells, using MetaMorph v.7.0 software. The percent porosity calculated in the five images was averaged to give a single value per coverslip and all experiments were performed on three separate LSEC isolations with two coverslips from each isolation per treatment. Quantitative fluorescence analysis for immunostained PECAM-1 or hydroethidium was performed, as described.21Straub AC Clark KA Ross MA Chandra AG Li S Gao X Pagano PJ Stolz DB Barchowsky A Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.J Clin Invest. 2008; 118: 3980-3989Crossref PubMed Scopus (101) Google Scholar, 24Ross MA Sander CM Kleeb TB Watkins SC Stolz DB Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver.Hepatology. 2001; 34: 1135-1148Crossref PubMed Scopus (140) Google Scholar Three ×400 images from six individual coverslips were used to quantify the % of thresholded pixels and further normalized to the % of thresholded nuclei. Antibodies for imaging included: rat monoclonal anti-mouse PECAM-1/CD31 (Becton Dickenson); H-60 affinity purified polyclonal anti-S1P1 (Santa Cruz Biotechnology); and Goat anti-rat Alexia 594 (Invitrogen), Goat anti-rabbit Alexa 488 (Invitrogen), and DRAQ5 (Biostatus, Leicestershire, UK) were used to stain nuclei. Total mouse liver or cellular HMVEC RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) and 1 μg was reverse-transcribed with MMLV (Promega, Madison, WI). PCR for S1P receptor transcripts was performed for 30 cycles in an MJ Research PT-100 thermal cycler using previously published primers sets for human27Balthasar S Samulin J Ahlgren H Bergelin N Lundqvist M Toescu EC Eggo MC Tornquist K Sphingosine 1-phosphate receptor expression profile and regulation of migration in human thyroid cancer cells.Biochem J. 2006; 398: 547-556Crossref PubMed Scopus (62) Google Scholar or mouse28Skaznik-Wikiel ME Kaneko-Tarui T Kashiwagi A Pru JK Sphingosine-1-phosphate receptor expression and signaling correlate with uterine prostaglandin-endoperoxide synthase 2 expression and angiogenesis during early pregnancy.Biol Reprod. 2006; 74: 569-576Crossref PubMed Scopus (41) Google Scholar S1P receptors or the house keeping gene RPL13A11Klei LR Barchowsky A Positive signaling interactions between arsenic and ethanol for angiogenic gene induction in human microvascular endothelial cells.Toxicol Sci. 2008; 102: 319-327Crossref PubMed Scopus (20) Google Scholar (human) and Hypoxanthine guanine phosphoribosyl transferase (HPRT) (mouse). For quantitative real-time-PCR, reverse transcribed cDNA was carried forward into real time PCR with SYBR greene master mix (Invitrogen, Carlsbad, CA) using an Opticon II DNA engine equipped with a continuous fluorescence detection system (BioRad, Hercules, Ca). RPL13A transcript levels were used for normalization. Interleukin (IL)8, heme oxygenase (decycling)1 (HMOX1), and RPL13A primer sequences were as published.11Klei LR Barchowsky A Positive signaling interactions between arsenic and ethanol for angiogenic gene induction in human microvascular endothelial cells.Toxicol Sci. 2008; 102: 319-327Crossref PubMed Scopus (20) Google Scholar Hepatocyte growth factor (HGF) (PPH00163B detecting transcripts of NM 000601) primers were from SuperArray Bioscience Corp (Frederick, MD) and insulin-like growth factor-1 (IGF1) (HS_IGF1_1_Sg detecting transcripts of NM 000618) primers were from Qiagen (Valencia, CA). SuperArray human angiogenesis RT2Wang CH Hsiao CK Chen CL Hsu LI Chiou HY Chen SY Hsueh YM Wu MM Chen CJ A review of the epidemiologic literature on the role of environmental arsenic exposure and cardiovascular diseases.Toxicol Appl Pharmacol. 2007; 222: 315-326Crossref PubMed Scopus (200) Google Scholar Profiler PCR arrays (#PAHS-024) specific for the Opticon II format were used to detect changes in HMVEC angiogenic genes. HMVEC Rac1-GTPase activity was determined with a G-LISA Rac1 activation assay (Cytoskeleton, Denver, CO). Briefly, after treatments, cells were placed on ice and washed twice with ice-cold PBS. The cells were lysed and scraped before being snap frozen in liquid N2. Buffer blanks and constitutively active Rac1 were included as negative and positive controls, respectively. Samples for G-LISA's were performed in triplicate and substrate color absorbance was read at 490 nm in a microplate reader. Data are expressed as Rac1 signal absorbance in samples minus the buffer blank. Primary LSECs were loaded with 5 μmol/L dihydroethidium (Invitrogen) for 10 minutes before adding arsenic for 30 minutes. The cells were then fixed with 4% paraformaldehyde and stained with DRAQ5. Dihydroethidium is converted to fluorescent hydroethidium in the presence of superoxide and arsenic-stimulated hydroethidium fluorescence is completely blocked in LSEC treated with the superoxide scavenger Tempol.21Straub AC Clark KA Ross MA Chandra AG Li S Gao X Pagano PJ Stolz DB Barchowsky A Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase-generated superoxide.J Clin Invest. 2008; 118: 3980-3989Crossref PubMed Scopus (101) Google Scholar SDS-polyacrylamide gel electrophoresis separation of total liver, LSEC, or HMVEC proteins and immunoblotting were performed using NuPage Novex 4% to 12% Bis-Tris gels (Invitrogen), as previously published.11Klei LR Barchowsky A Positive signaling interactions between arsenic and ethanol for angiogenic gene induction in human microvascular endothelial cells.Toxicol Sci. 2008; 102: 319-327Crossref PubMed Scopus (20) Google Scholar Membranes were probed with H-60 anti-S1P1 and monoclonal anti-β-actin (Sigma) followed by goat or donkey anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Amersham Bioscience UK, Buckinghamshire, UK). In vitro endothelial cell tube formation assays in three dimensional Matrigel (Becton Dickinson) cultures (performed as described11Klei LR Barchowsky A Positive signaling interactions between arsenic and ethanol for angiogenic gene induction in human microvascular endothelial cells.Toxicol Sci. 2008; 102: 319-327Crossref PubMed Scopus (20) Google Scholar) were used to measure arsenic stimulation of migration and initiation of an angiogenic response. Branch points of rhodamine-phalloidin (Molecular Probes, Invitrogen, Carlsbad, CA) stained endothelial cell tubes were quantified in ×40 images using MetaMorph v.7.0 software. One- or two-way analysis of variance was used to identify significant differences (P < 0.05) between treatment groups and controls. The degree of significance between groups was then compared using Bonferroni's posttest for multiple comparisons. All statistics were performed using GraphPad Prism, v5.0 software (GraphPad Software, San Diego, CA). Data are presented as means ± SD or SEM of quantified values or fold relevant control. Arsenic stimulates rapid translocation of Rac1 to the membrane and oxidant generation in large vessel endothelial cells.7Smith KR Klei LR Barchowsky A Arsenite stimulates plasma membrane NADPH oxidase in vascular endothelial cells.Am J Physiol. 2001; 280: L442-L449Google Scholar, 29Barchowsky A Klei LR Dudek EJ Swartz HM James PE Stimulation of reactive oxygen, but not reactive nitrogen species, in vascular endothelial cells exposed to low levels of arsenite.Free Radic Biol Med. 1999; 27: 1405-1412Crossref PubMed Scopus (240) Google Scholar The data in Figure 1A confirm that arsenic exposure rapidly activates HMVEC Rac1 GTPase activity. Specificity of the assay for Rac1 activity is demonstrated by inhibition of the arsenic response in cells treated with NSC23766 (iRac). Ptx, a Gi/o-selective inhibitor, prevented arsenite-stimulated Rac1 activity (Figure 1B), implicating a Gi/o receptor in mediating the arsenite response. Note that neither inhibitor affected basal Rac1activity, since NSC23766 prevents Rac1 interaction with quanine exchange factors that increase basal activity26Gao Y Dickerson JB Guo F Zheng J Zheng Y Rational design and characterization of a Rac GTPase-specific small molecule inhibitor.Proc Natl Acad Sci USA. 2004; 101: 7618-7623Crossref PubMed Scopus (925) Google Scholar and Ptx prevents upstream signals that activate these same factors. Pre-treating HMVEC with either Ptx or NSC23766 prevented arsenic from increasing IL8 transcripts within a 4 hours period (Figure 1C), indicating a functional linkage pathway between arsenic stimulated Gi/o and gene induction. Interestingly, neither Ptx nor NSC23766 pre-treatments prevented arsenic from inducing stress responsive HMOX1 transcripts (Figure 1D). Quantitative PCR transcript arrays were used to investigate the extent of arsenic-stimulated, Ptx-inhibitable effects on inducible angiogenic gene transcripts. Arsenic exposure increased or repressed only 7 of the 84 transcripts contained on the array (Table 1). Ptx a
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