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• W2004384162 abstract "Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in vascular endothelium and inflamed tissues, yet its role in inflammation-associated cytokine-regulated angiogenesis and underlying mechanism remains largely unclear. We show that tumor necrosis factor α (TNFα) and interferon α (IFNα) stimulate PML expression while suppressing EC network formation and migration, two key events during angiogenesis. By a knockdown approach, we demonstrate that PML is indispensable for TNFα- and IFNα-mediated inhibition of EC network formation. We further demonstrate that signal transducer and activator of transcription 1 (STAT1) binds PML promoter and that is an important regulator of PML expression. Knockdown of STAT1 reduces endogenous PML and blocks TNFα- and IFNα-induced PML accumulation and relieves TNFα- and IFNα-mediated inhibition of EC network formation. Our data also indicate that PML regulates EC migration, in part, by modulating expression of downstream genes, such as negatively regulating integrin β1 (ITGB1). In addition, knockdown of STAT1 or PML alleviates TNFα- and IFNα-mediated inhibition of ITGB1 expression. Antibody blockade demonstrates that ITGB1 is functionally important for PML- and STAT1-regulated EC migration. Taken together, our data provide novel mechanistic insights that PML functions as a negative regulator in EC network formation and migration.Background: PML is highly expressed in endothelial cells (ECs), but its role in ECs remains largely unexplored.Results: PML is critical for TNFα- and IFNα-mediated inhibition of EC network formation and migration.Conclusion: PML is an angiogenesis inhibitor following inflammation-associated cytokine signaling.Significance: PML is a novel regulator of endothelial cell physiology. Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in vascular endothelium and inflamed tissues, yet its role in inflammation-associated cytokine-regulated angiogenesis and underlying mechanism remains largely unclear. We show that tumor necrosis factor α (TNFα) and interferon α (IFNα) stimulate PML expression while suppressing EC network formation and migration, two key events during angiogenesis. By a knockdown approach, we demonstrate that PML is indispensable for TNFα- and IFNα-mediated inhibition of EC network formation. We further demonstrate that signal transducer and activator of transcription 1 (STAT1) binds PML promoter and that is an important regulator of PML expression. Knockdown of STAT1 reduces endogenous PML and blocks TNFα- and IFNα-induced PML accumulation and relieves TNFα- and IFNα-mediated inhibition of EC network formation. Our data also indicate that PML regulates EC migration, in part, by modulating expression of downstream genes, such as negatively regulating integrin β1 (ITGB1). In addition, knockdown of STAT1 or PML alleviates TNFα- and IFNα-mediated inhibition of ITGB1 expression. Antibody blockade demonstrates that ITGB1 is functionally important for PML- and STAT1-regulated EC migration. Taken together, our data provide novel mechanistic insights that PML functions as a negative regulator in EC network formation and migration. Background: PML is highly expressed in endothelial cells (ECs), but its role in ECs remains largely unexplored. Results: PML is critical for TNFα- and IFNα-mediated inhibition of EC network formation and migration. Conclusion: PML is an angiogenesis inhibitor following inflammation-associated cytokine signaling. Significance: PML is a novel regulator of endothelial cell physiology. The promyelocytic leukemia protein (PML), 2The abbreviations used are: PMLpromyelocytic leukemia proteinECendothelial cellTNFtumor necrosis factorECMextracellular matrixHUVEChuman umbilical vein endothelial cell. initially identified as a fusion partner of retinoic acid receptor α (RARα) involved in acute promyelocytic leukemia (APL), regulates several cellular processes including proliferation, apoptosis, transcription, virus infection, and DNA damage signaling in response to extracellular stimuli (1Crowder C. Dahle Ø. Davis R.E. Gabrielsen O.S. Rudikoff S. PML mediates IFN-α-induced apoptosis in myeloma by regulating TRAIL induction.Blood. 2005; 105: 1280-1287Crossref PubMed Scopus (45) Google Scholar, 2Kakizuka A. Miller Jr., W.H. Umesono K. Warrell Jr., R.P. Frankel S.R. Murty V.V. Dmitrovsky E. Evans R.M. Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RARα with a novel putative transcription factor, PML.Cell. 1991; 66: 663-674Abstract Full Text PDF PubMed Scopus (1292) Google Scholar, 3de Thé H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. The PML-RARα fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR.Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1196) Google Scholar, 4Everett R.D. DNA viruses and viral proteins that interact with PML nuclear bodies.Oncogene. 2001; 20: 7266-7273Crossref PubMed Scopus (222) Google Scholar, 5Negorev D. Maul G.G. Cellular proteins localized at and interacting within ND10/PML nuclear bodies/PODs suggest functions of a nuclear depot.Oncogene. 2001; 20: 7234-7242Crossref PubMed Scopus (233) Google Scholar, 6Lehembre F. Müller S. Pandolfi P.P. Dejean A. Regulation of Pax3 transcriptional activity by SUMO-1-modified PML.Oncogene. 2001; 20: 1-9Crossref PubMed Scopus (93) Google Scholar, 7Bernardi R. Guernah I. Jin D. Grisendi S. Alimonti A. Teruya-Feldstein J. Cordon-Cardo C. Simon M.C. Rafii S. Pandolfi P.P. PML inhibits HIF-1α translation and neoangiogenesis through repression of mTOR.Nature. 2006; 442: 779-785Crossref PubMed Scopus (320) Google Scholar). PML is the core scaffold component of a proteinaceous nuclear structure called PML nuclear bodies (NBs) (8Terris B. Baldin V. Dubois S. Degott C. Flejou J.F. Hénin D. Dejean A. PML nuclear bodies are general targets for inflammation and cell proliferation.Cancer Res. 1995; 55: 1590-1597PubMed Google Scholar). The precise function of PML NBs is not clear, but they are believed nuclear depots that sequester other factors to control cellular processes such as transcription (9Gao C. Cheng X. Lam M. Liu Y. Liu Q. Chang K.S. Kao H.Y. Signal-dependent regulation of transcription by histone deacetylase 7 involves recruitment to promyelocytic leukemia protein nuclear bodies.Mol. Biol. Cell. 2008; 19: 3020-3027Crossref PubMed Google Scholar, 10Reineke E.L. Kao H.Y. Targeting promyelocytic leukemia protein: a means to regulating PML nuclear bodies.Int. J. Biol. Sci. 2009; 5: 366-376Crossref PubMed Scopus (33) Google Scholar). While it is known that PML is highly expressed in vascular endothelium and inflamed tissues (8Terris B. Baldin V. Dubois S. Degott C. Flejou J.F. Hénin D. Dejean A. PML nuclear bodies are general targets for inflammation and cell proliferation.Cancer Res. 1995; 55: 1590-1597PubMed Google Scholar), its role in endothelial cells (ECs) is largely unknown. promyelocytic leukemia protein endothelial cell tumor necrosis factor extracellular matrix human umbilical vein endothelial cell. The inflammation-associated cytokines tumor necrosis factor α (TNFα) and interferons (IFNs) exert their cellular effects through signaling cascades that involve extracellular receptors, and intracellular mediators (11Aggarwal B.B. Signaling pathways of the TNF superfamily: a double-edged sword.Nature Reviews. Immunology. 2003; 3: 745-756Crossref PubMed Scopus (2147) Google Scholar, 12Sen G.C. Viruses and interferons.Annu. Rev. Microbiol. 2001; 55: 255-281Crossref PubMed Scopus (785) Google Scholar, 13Locksley R.M. Killeen N. Lenardo M.J. The TNF and TNF receptor superfamilies: integrating mammalian biology.Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). TNFα is released by activated macrophage at sites of inflammation and binds to TNFα receptors on the local vascular endothelium triggering multiple responses in these cells, including up-regulation of leukocyte adhesion molecules and increased endothelial permeability. TNFα possesses pro-apoptotic and anti-viral activities and is capable of inhibiting tumorigenesis; however it is also able to stimulate cell survival. As such, intracellular responses to TNFα are controversial and long-debated (13Locksley R.M. Killeen N. Lenardo M.J. The TNF and TNF receptor superfamilies: integrating mammalian biology.Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar). Type I (IFNα and IFNβ) and type II (IFNγ) IFNs are induced in the innate and adaptive immune responses, respectively and are generally considered to be anti-viral and pro-apoptotic (12Sen G.C. Viruses and interferons.Annu. Rev. Microbiol. 2001; 55: 255-281Crossref PubMed Scopus (785) Google Scholar, 14Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Interferons at age 50: past, current and future impact on biomedicine.Nature Reviews. Drug Discovery. 2007; 6: 975-990Crossref PubMed Scopus (872) Google Scholar). IFNα, released by leukocyte from inflamed tissues, activates macrophage, which in turn promotes the release of TNFα. Both TNFα and IFNα affect gene transcription in local ECs; however, the molecular networks downstream of TNFα and IFNα in ECs are still not fully elucidated. Angiogenesis is a hallmark of tumor development and chronic inflammatory diseases (15Albini A. Tosetti F. Benelli R. Noonan D.M. Tumor inflammatory angiogenesis and its chemoprevention.Cancer Res. 2005; 65: 10637-10641Crossref PubMed Scopus (166) Google Scholar, 16Bergers G. Benjamin L.E. Tumorigenesis and the angiogenic switch.Nat. Rev. Cancer. 2003; 3: 401-410Crossref PubMed Scopus (2854) Google Scholar, 17Folkman J. Angiogenesis.Annu. Rev. Med. 2006; 57: 1-18Crossref PubMed Scopus (1145) Google Scholar, 18Medina J. Arroyo A.G. Sánchez-Madrid F. Moreno-Otero R. Angiogenesis in chronic inflammatory liver disease.Hepatology. 2004; 39: 1185-1195Crossref PubMed Scopus (209) Google Scholar). It is believed that cytokines, including TNFα, and IFNα, are important regulators of angiogenesis in local endothelium (13Locksley R.M. Killeen N. Lenardo M.J. The TNF and TNF receptor superfamilies: integrating mammalian biology.Cell. 2001; 104: 487-501Abstract Full Text Full Text PDF PubMed Scopus (3004) Google Scholar, 14Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Interferons at age 50: past, current and future impact on biomedicine.Nature Reviews. Drug Discovery. 2007; 6: 975-990Crossref PubMed Scopus (872) Google Scholar, 17Folkman J. Angiogenesis.Annu. Rev. Med. 2006; 57: 1-18Crossref PubMed Scopus (1145) Google Scholar). Angiogenesis is a complex process and represents a systemic morphological alteration that involves enzymatic degradation and remodeling of the extracellular matrix, migration, apoptosis, and proliferation of ECs (16Bergers G. Benjamin L.E. Tumorigenesis and the angiogenic switch.Nat. Rev. Cancer. 2003; 3: 401-410Crossref PubMed Scopus (2854) Google Scholar). IFNs elicit anti-angiogenic activity (14Borden E.C. Sen G.C. Uze G. Silverman R.H. Ransohoff R.M. Foster G.R. Stark G.R. Interferons at age 50: past, current and future impact on biomedicine.Nature Reviews. Drug Discovery. 2007; 6: 975-990Crossref PubMed Scopus (872) Google Scholar), whereas TNFα has pro- or anti-angiogenic activity probably depending on the dose, duration of treatment, and cell type (19Leibovich S.J. Polverini P.J. Shepard H.M. Wiseman D.M. Shively V. Nuseir N. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-α.Nature. 1987; 329: 630-632Crossref PubMed Scopus (1000) Google Scholar, 20Rosenbaum J.T. Howes Jr., E.L. Rubin R.M. Samples J.R. Ocular inflammatory effects of intravitreally-injected tumor necrosis factor.Am. J. Pathol. 1988; 133: 47-53PubMed Google Scholar, 21Sunderkötter C. Roth J. Sorg C. Immunohistochemical detection of bFGF and TNF-α in the course of inflammatory angiogenesis in the mouse cornea.Am. J. Pathol. 1990; 137: 511-515PubMed Google Scholar). Therefore, identification of downstream effectors that mediate TNFα and IFNα activity in ECs will help understand the mechanisms by which these two cytokines regulate physiological and pathological angiogenesis. We have recently demonstrated that TNFα potently induces PML and regulates the expression of the matrix-associated metalloproteinase MMP-10 in ECs (9Gao C. Cheng X. Lam M. Liu Y. Liu Q. Chang K.S. Kao H.Y. Signal-dependent regulation of transcription by histone deacetylase 7 involves recruitment to promyelocytic leukemia protein nuclear bodies.Mol. Biol. Cell. 2008; 19: 3020-3027Crossref PubMed Google Scholar). Our current study supports a model in which STAT1 and PML play a role in TNFα- and IFNα-mediated inhibition of EC network formation and migration. Human TNFα was purchased from Promega (G5241). Human IFNα (11200) and IFNγ (11500) were purchased from R&D systems. IKK inhibitor VII was purchased from EMD Millipore (401486). The Matrigel kit for in vitro EC network formation assays was purchased from Chemicon (ECM625). The commercial antibodies used in this manuscript are from Santa Cruz Biotechnology, α-PML (sc-996, sc-5621), α-STAT1 (sc-346), α-ITGB1 (sc-6622), α-Mouse IgG conjugated with HRP (sc-2005), α-goat IgG conjugated with HRP (sc-2033); from Upstate α-acetyl-histone H3 (α-AcH3, 06-599); from Sigma, α-β-actin (A5441), from Invitrogen, normal goat IgG (10200); Alexa Fluor 488 μm goat anti rabbit (A-11008), Alexa Fluor 594 μm goat anti mouse (A-11005); from Millipore, α-rabbit-IgG conjugated with HRP (12-348). Human umbilical vein endothelial cells (HUVECs, Lonza, C2519A) were maintained in endothelial cell growth medium-2 (EGM-2, Lonza, CC-4176). Human microvascular endothelial cells (HMVECs, Lonza, CC-2543) were maintained in microvascular endothelial cell growth medium-2 (EGM-2MV, Lonza, CC-4147). Cells of <5 passages were used in this study. For cytokine treatment, unless otherwise specified, conditions were TNFα (20 ng/ml), IFNα (1000 units/ml), or IFNγ (1000 units/ml) for 16 h. Non-targeting control (D-001810-01), luciferase (D-001210-02), PML (J-006547-05 and J-006547-07), and STAT1 (J-003543-06 and J-003543-08) siRNAs and transfection reagent DharmaFECT1 (T-2001) were purchased from Thermo Scientific. HUVECs were concurrently treated with TNFα (20 ng/ml) in the presence of vehicle, 100 nm, or 200 nm IKK inhibitor VII. Cells were collected, and aliquots of the cells were subjected to whole cell extract preparation, immunofluorescence microscopy, and total RNA preparation. Cells were harvested, and total RNA was extracted with a PrepEase kit (USB/Affymetrix), quantified by A260/A280 spectrometry. 2 μg of total RNAs were used for RT-PCR. The cDNA pool was generated from each RNA sample with Superscript3 Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. The specific cDNAs of interest and internal controls were quantified by real-time PCR using an iCycler (Bio-Rad) platform with 2×iQ SYBR Green Supermix (Bio-Rad) and appropriate primer sets. The PCR program ran for 40 cycles with three steps of 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 30 s. Melting curves were acquired after PCR to ensure the homogeneity of the PCR products. The relative quantities of genes of interest were normalized to an internal control (GAPDH or 18s rRNA) and depicted as mean ± S.D. from three independent experiments. The primer sequences are shown in supplemental Table S1. To prepare whole cell extracts, cells were harvested and lysed in RIPA lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in 1× PBS) supplemented with a protease inhibitor mixture (Roche). The extracts were fractionated by SDS-PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked in 10% nonfat milk in 1× PBS supplemented with 0.1% Tween-20 (PBST) for 1 h and followed by incubation with primary antibodies in PBST for 1 h. The membranes were rinsed and washed in PBST and incubated with HRP-conjugated secondary antibodies in PBST for 1 h. The membranes were incubated with chemiluminescent substrate for HRP (Thermo Sci. 34080) and exposed on autoradiography film (Denville Sci.). For quantification of band density, films were scanned, and the integrated density of each band was quantified with the “Analyze: Gels” tools in ImageJ software (v1.42a, NIH). The densities of proteins of interest were normalized to that of an internal control, and the first lane was set as 1 to reflect the fold change in the remaining lanes. HUVECs, plated on glass cover slips, were treated with or without TNFα and IFNα for 16 h, and the same protocol was followed for HUVECs transfected with siRNAs. The cells were fixed in 1% paraformaldehyde in 1× PBS for 30 min at room temperature, permeabilized in 1× PBS supplemented with 0.1% Triton X-100 and 10% goat serum for 10 min, washed three times with 1× PBS, and blocked in 1× PBS containing 10% goat serum and 0.1% Tween-20 for 1 h followed by incubation with primary antibodies for 1 h. After washing, Alexa Fluor secondary antibodies were added for 1 h in the dark. Cover slips were mounted on slides using Vectashield mounting medium with DAPI (Vector Laboratories), visualized and images captured on a Leica immunofluorescence microscopy. Unless specified, all images were taken under same microscope setting. The assays were performed following the manufacturer's protocol (Lonza ECM625). Under our experimental conditions, we did not observe significant differences in apoptosis or viability of HUVEC transfected with or without siRNAs against PML or a control siRNA (data not shown). Briefly, HUVECs or HMVECs were transfected with control siRNA or siRNAs against PML for 72 h, and followed by a 16–20 h treatment with TNFα (10 ng/ml), IFNα (103 units/ml), or IFNγ (103 units/ml). Subsequently, the cells were trypsinized and counted. Equal numbers of HUVECs were plated on matrix gel (Chemicon ECM625) pre-coated 96-well plate (1 × 105/well) or chamber system (2.5 × 105/chamber, Lab-Tek 4808). A fraction of the cells was plated for Western blotting to examine PML knockdown efficiency. After seeding the cells on the ECM, the images of network formation from randomly chosen fields (plate, n = 12; chamber, n = 8) were taken at 3, 8, and 20 h. The trends of change in network formation are similar for these time points. The images taken at 20 h are presented. The numbers of branch points were quantified and depicted as mean ± S.D. For statistics used in Figs. 1, 2, 3, and 4, unpaired two-tail t-tests were applied, and the p values were presented as *, p < 0.05; **, p < 0.01; ***, p < 0.001; #, p < 0.00001; and ns (not significant, p > 0.05).FIGURE 2Effects of TNFα, IFNα, and IFNγ on PML protein levels and network formation in HUVECs and HMVECs. HUVECs (A–C) and HMVECs (D–E) were pre-treated with vehicle control, TNFα (20 ng/ml), IFNα (103 units/ml), or IFNγ (103 units/ml) for 16 h followed by in vitro EC network formation assays and quantification of branch points, mean ± S.D. (n = 12). An aliquot of HUVECs (C) or HMVECs (E) was used for Western blotting analysis with α-PML and α-β-actin antibodies. Statistics: unpaired two-tail t-tests (***, p < 0.0001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3PML is essential for TNFα-, IFNα-, and IFNγ-mediated inhibition of EC network formation. A and B, HUVECs were transiently transfected with siControl or siPML for 72 h, and treated with TNFα (20 ng/ml), IFNα (103 units/ml), or IFNγ (103 units/ml) for 16 h. The cells were subsequently trypsinized and counted. Equal numbers of cells were plated on the matrix gel for in vitro EC network formation assays. Images shown were taken 20 h after seeding the cells on the extracellular matrix gel (A). Quantitation of branch points is shown as mean ± S.D. (n = 12) (B). C, an aliquot of HUVECs from A and B was plated for Western blotting to examine PML expression. D, similar to B, in vitro EC network formation assays in HMVECs. Statistics: unpaired two-tail t-tests (***, p < 0.0001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4STAT1 participates in the TNFα- and IFNα-induced PML expression and inhibition of EC network formation in HUVECs. A, effects of STAT1 knockdown on PML mRNA levels analyzed by qRT-PCR. B, effects of TNFα and IFNα on endogenous STAT1 and PML protein levels in HUVECs assayed by Western blotting. C, effects of STAT1 knockdown by two siRNAs on PML protein levels following TNFα and IFNα treatment assayed by Western blotting. B and C, Western blotting bands were quantified by Image J as described in “Experimental Procedures.” ND, not detectable. D, effects of STAT1 knockdown on PML NBs examined by immunofluorescence microscopy (DAPI, blue; STAT1, green; PML, red). The microscopic parameters were set identically for all images taken. E, association of STAT1 with the PML promoter following TNFα and IFNα treatment assayed by chromatin immunoprecipitation followed by quantification with qPCR. HUVECs treated with TNFα (20 ng/ml) or IFNα (103 units/ml) for 16 h followed by ChIP assays as described in “Experimental Procedures.” Putative STAT binding sites were predicted with Genomatix. TNFα and IFNα increase acetylation of histone H3 flanking STAT1 binding sites at the PML promoter. F, effects of knockdown of STAT1 on TNFα- and IFNα-mediated inhibition of EC network formation in HUVECs. Quantitation of branch points after in vitro EC network formation assays was as described in Fig. 1, mean ± S.D. (n = 8). Statistics: unpaired two-tail t-tests (**, p < 0.001; ***, p < 0.0001; ns, not significant).View Large Image Figure ViewerDownload Hi-res image Download (PPT) This protocol was adapted with modification as previously described (22Cheng X. Kao H.Y. G protein pathway suppressor 2 (GPS2) is a transcriptional corepressor important for estrogen receptor α-mediated transcriptional regulation.J. Biol. Chem. 2009; 284: 36395-36404Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Briefly, 1 × 106 HUVECs were used for each immunoprecipitation. Crosslinked nuclei were sonicated on a sonic dismembrator (Fisher 550α) with power setting 4 of 120 s total processing time in a 10s-pulse-50s-idle manner to shear the chromatin to around 600 bp. The retrieved DNA was quantified by qPCR. Each ChIP experiment had 2–3 biological repeats. The antibodies used were listed under “Reagents and Antibodies.” The input signal was set to 1, and others were normalized as percentage of input. The primer sequences are listed in supplemental Table S1. The assay was adapted from a previous protocol with modification (23Mottet D. Bellahcène A. Pirotte S. Waltregny D. Deroanne C. Lamour V. Lidereau R. Castronovo V. Histone deacetylase 7 silencing alters endothelial cell migration, a key step in angiogenesis.Circ. Res. 2007; 101: 1237-1246Crossref PubMed Scopus (154) Google Scholar). Confluent HUVECs plated in a 6-well plate, 16–20 h after treatment of TNFα, IFNα, or IFNγ and 72 h post-transfection of siRNA if applied, were scratched with a pipette tip to create wounds. Normal IgG or blocking antibody against integrin β1 (α-ITGB1, 10 μg/ml) was supplemented in media during the assay if specified. Randomly chosen fields (n = 16) were used for imaging, and the images were taken at identical locations at time 0 and overnight. The wound widths were measured by Photoshop (Adobe) software, normalized and represented as the percentage of wound measured at time 0 (mean ± S.D.). All statistics were performed with unpaired two-tailed t test. Boyden chambers with 8 μm pore size were purchased from Corning (3422). HUVECs were transfected with control or PML siRNAs for 72 h. The cells were trypsinized, counted and 1 × 105 cells/well were plated on 6.5 mm diameter inserts in the upper chamber of the apparatus. Migration was allowed to proceed overnight. For migration toward serum (10% FBS), the cells were starved in medium containing 1% FBS for 24 h and migrated toward medium supplemented 10% FBS placed in the lower chamber. For migration toward extracellular matrix (ECM), the bottom of the inserts was coated with ECM (Chemicon, ECM625), and both upper and lower chambers contained normal culture medium. After migration, the cells that had migrated through the inserts and attached to the underneath of the inserts were fixed in 4% paraformaldehyde for 10 min and stained in crystal violet for 15 min. The inserts were then air dried and subjected to cell counting under light microscopy with 40× magnification. The experiments were done in duplicate, and 8 random fields were counted from each insert. The unpaired two-tail t-tests were used to determine the significance of differences in migration between the different conditions. Human umbilical vein endothelial cells (HUVECs) were transfected with control siRNA or two different PML siRNA for 72 h, and total RNA was extracted by a PrepEase Kit (USB/Affymetrix). The total RNAs were sent to the Genomics Core at Cleveland Clinic Foundation for microarray with HumanRef-8_V2_0_R0_11223162_A (Illumina) chip. Each sample had technical duplicates. All statistical analysis was done in R/Bioconductor (24Team R.D.C. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria2009Google Scholar, 25Gentleman R.C. Carey V.J. Bates D.M. Bolstad B. Dettling M. Dudoit S. Ellis B. Gautier L. Ge Y. Gentry J. Hornik K. Hothorn T. Huber W. Iacus S. Irizarry R. Leisch F. Li C. Maechler M. Rossini A.J. Sawitzki G. Smith C. Smyth G. Tierney L. Yang J.Y. Zhang J. Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar). Data were analyzed in the R/Bioconductor environment (24Team R.D.C. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria2009Google Scholar, 25Gentleman R.C. Carey V.J. Bates D.M. Bolstad B. Dettling M. Dudoit S. Ellis B. Gautier L. Ge Y. Gentry J. Hornik K. Hothorn T. Huber W. Iacus S. Irizarry R. Leisch F. Li C. Maechler M. Rossini A.J. Sawitzki G. Smith C. Smyth G. Tierney L. Yang J.Y. Zhang J. Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar). Briefly, the raw data were exported by BeadStudio (Illumina, v3.2.3) with background subtracted and imported and preprocessed by lumi package (26Du P Kibbe W.A. Lin S.M. lumi: a pipeline for processing Illumina microarray.Bioinformatics. 2008; 24: 1547-1548Crossref PubMed Scopus (1621) Google Scholar, 27Du P Kibbe W.A. Lin S.M. nuID: a universal naming scheme of oligonucleotides for illumina, affymetrix, and other microarrays.Biol. Direct. 2007; 2: 16Crossref PubMed Scopus (70) Google Scholar). Data were transformed by the Variance-Stabilizing Transformation (VST) package (28Lin S.M. Du P Huber W. Kibbe W.A. Model-based variance-stabilizing transformation for Illumina microarray data.Nucleic Acids Res. 2008; 36: e11Crossref PubMed Scopus (402) Google Scholar) and then normalized by the Robust Spline Normalization (RSN) package (28Lin S.M. Du P Huber W. Kibbe W.A. Model-based variance-stabilizing transformation for Illumina microarray data.Nucleic Acids Res. 2008; 36: e11Crossref PubMed Scopus (402) Google Scholar). The significantly changed gene list was retrieved by a general linear model and empirical Bayes method through the Linear Models for Microarray Data (limma) package (29Smyth G.K. Bioinformatics and Computational Biology Solutions using R and Bioconductor. 2005; 420: 397-420Crossref Google Scholar). The false discovery rate (FDR) was adjusted by Benjamini and Hochberg's method. Changes in expression level of >2 fold and p < 0.01 were considered significant. Gene ontology analysis was done by a hypergeometric test through the GeneAnswers package (30Feng G. Du P Krett N.L. Tessel M. Rosen S. Kibbe W.A. Lin S.M. A collection of bioconductor methods to visualize gene-list annotations.BMC Res. Notes. 2010; 3: 10Crossref PubMed Scopus (47) Google Scholar). The microarray data are deposited in the Gene Expression Omnibus (GEO) database. The accession number of the data set is GSE29238. To examine the role of PML in EC network formation, we carried out PML knockdown followed by in vitro network formation assay to assess the ability of ECs to undergo angiogenesis. We found that that knocking down PML potentiated the in vitro angiogenesis of primary human umbilical vein endothelial cells (HUVECs) (Fig. 1, A and B). TNFα has been shown to have an ambiguous role in endothelial angiogenesis (20Rosenbaum J.T. Howes Jr., E.L. Rubin R.M. Samples J.R. Ocular inflammatory effects of intravitreally-injected tumor necrosis factor.Am. J. Pathol. 1988; 133: 47-53PubMed Google Scholar, 21Sunderkötter C. Roth J. Sorg C. Immunohistochemical detection of bFGF and TNF-α in the course of inflammatory angiogenesis in the mouse cornea.Am. J. Pathol. 1990; 137: 511-515PubMed Google Scholar), cell migration, and adhesion (21Sunderkötter C. Roth J. Sorg C. Immunohistochemical detection of bFGF and TNF-α in the course of inflammatory angiogenesis in the mouse cornea.Am. J. Pathol. 1990; 137: 511-515PubMed Google Scholar, 31Av" @default.
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• W2004384162 date "2012-07-01" @default.
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• W2004384162 title "Promyelocytic Leukemia Protein (PML) Regulates Endothelial Cell Network Formation and Migration in Response to Tumor Necrosis Factor α (TNFα) and Interferon α (IFNα)" @default.
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