RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells

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Prepublished online February 24, 2004; doi:10.1182/blood-2003-06-2144

RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells Maria Simona Aguzzi, Claudia Giampietri, Francesco De Marchis, Fabrizio Padula, Roberto Gaeta, Gianluca Ragone, Maurizio C Capogrossi and Antonio Facchiano

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RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells.

Maria Simona Aguzzi1, Claudia Giampietri2, Francesco De Marchis1, Fabrizio Padula2, Roberto Gaeta3,4, Gianluca Ragone5, Maurizio C. Capogrossi1, Antonio Facchiano1*.

1 Laboratorio Patologia Vascolare, Istituto Dermopatico dell’Immacolata, IDI, Rome, Italy 2 Dipartimento Istologia ed Embriologia Medica, University La Sapienza, Rome, Italy 3 Divisione di Cardiochirurgia, IRCCS “San Matteo”, Pavia, Italy 4 Cattedra di Cardiochirurgia, University of Messina, Messina, Italy 5 Laboratorio Oncogenesi Molecolare, Istituto Dermopatico dell’Immacolata, IDI, Rome, Italy

* to whom correspondence should be addressed.

Copyright (c) 2004 American Society of Hematology

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Abstract

Peptides containing the RGD-motif inhibit cell adhesion and exhibit a variety of other biological effects including anti-coagulant and anti-metastatic activities. The aim of the present study was to examine the anchorage-independent effects of an RGD-containing peptide (RGDS) on human umbilical vein endothelial cells (HUVEC). Assays were performed on HUVEC seeded onto collagen IV; under these experimental conditions RGDS did not exert anti-adhesive effects but significantly reduced FGF-2dependent chemotaxis after 4 h treatment and reduced proliferation after 24 h treatment. Experiments carried out with caspase-specific inhibitors indicated that the observed anti-chemotactic effects required caspase 8 and caspase 9 activation. RGDS activated both caspase 8 and caspase 9 after 4 h treatment and caspase 3 after 24 h treatment, and markedly enhanced HUVEC apoptosis by TUNEL/Hoechst staining and FACS analysis. Finally, confocal microscopy showed that RGDS localizes in the cytoplasm of live HUVEC within 4 h and in vitro experiments showed that RGDS directly interacts with recombinant caspases 8 and 9 in a specific way. In summary, these results indicate that RGDS directly binds and activates caspases 8 and 9, inhibits chemotaxis and induces apoptosis of HUVEC with a mechanism independent from its anti-adhesive effect.

Keywords: caspases activation; apoptosis, intra-cellular action of RGDS

Abbreviations: RGDS, Arg-Gly-Asp-Ser; RGES, Arg-Gly-Glu-Ser; ECM, extra-cellular matrix; RT, room temperature; PBS, phosphate buffer saline, pH 7.4; BSA, bovine serum albumin; FCS, Fetal Calf Serum; HUVEC, Human Umbilical Vein Endothelial Cells; ECM, extra-cellular matrix; VEGF, Vascular Endothelial Growth Factor; SDS, Sodium Dodecyl solphate; OD, optical density; PLL, polyL-lysine; propidium iodide, PI.

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Introduction

Integrins mediate cell-cell and cell–extra-cellular matrix (ECM) interactions1,2 and recognize several ECM proteins including fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, osteopontin, thrombospondin and collagen. Endothelial cell adhesion to ECM is mediated by 2 1,

3 1,

5 1,

6 1,

v 3 and

v 5

1 1,

integrins3 and such interaction triggers mechanical as well as

chemical signals4,5, which promote the assembly of actin stress fibers6 and modulate adherent cells growth and survival2,7. One of the major sites mediating integrins action is the Arg-Gly-Asp (RGD) motif, present in ECM proteins and in other proteins such as disintegrins8-9. Integrins interact with ECM in both a RGDdependent and RGD-independent manner10,11. Peptides containing the RGD motif have been shown to inhibit cell adhesion by competing for integrins/matrix interaction; further, these peptides exert a variety of biological effects including anti-inflammatory, anti-coagulant and anti-metastatic activities1216

. RGD-containing peptides and disintegrins have an anti-angiogenic effect due, at least in part, to

inhibition of

v 3

and

v 5

– dependent adhesion8,9 and prevent vascular lumen formation17. RGD

peptidomimetics have also been shown to inhibit neointima formation and lumen restenosis following coronary artery injury18-19. Previous studies indicated that integrins regulate cell viability in different cell types; antiintegrins monoclonal antibody or RGD peptides reduce cell growth by inhibiting integrin ligation, therefore detaching cells from ECM and inducing an anchorage-dependent apoptosis, named anoikis2028

. Hence, integrin-mediated apoptosis has been attributed to the onset of an anti-adhesive effect; in fact

anchorage loss of adherent cells is known to trigger different pro-apoptotic signals, either by altering integrin expression29-31, or by blocking integrin signaling32-35, or by inducing integrin degradation36,37. Recent evidences show that RGD-containing peptides may induce an anchorage-independent apoptosis of T-cells and cardiomyocytes, by entering into the cell and directly promoting caspase 3 activation38-40. We focused our interest on the anchorage-independent effects of the RGD motif, which are still poorly investigated. We set experimental conditions to avoid any RGDS anti-adhesive action and examined the effect on HUVEC chemotaxis and on the activity of initiator caspases, namely caspases 8 and 9.

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Materials and methods

Materials – Arg-Gly-Asp-Ser (RGDS) peptide was obtained from Bachem (Haupstrasse, CH). Trypsin-EDTA, basic Fibroblast Growth Factor (FGF-2), human plasma fibronectin, phosphate buffer saline (PBS), sodium pyruvate, MEM amino acids without L-glutamine, L-glutamine, penicillinstreptomycin, MEM non-essential amino acids, HEPES, RPMI medium were from Gibco (Paisley, Scotland). Human plasma vitronectin was obtained from Calbiochem (San Diego, CA, USA). Mouse collagen IV was from Becton Dickinson (Bedford, MA). Bovin serum albumin (BSA), RGES peptide, protease inhibitors and the FITC-conjugated phalloidin were from Sigma (St. Louis, MO, USA). General caspase inhibitor (Z-VAD-FMK), caspase 1 inhibitor (Z-WEHD-FMK), caspase 3 inhibitor (Z-DEVD-FMK), caspase 8 inhibitor (Z-IEDT-FMK) and caspase 9 inhibitor (Z-LEHD-FMK) were from R&D System Inc. (Minneapolis, MN) and Calbiochem. Caspases substrates FITC-VAD-FMK and Z-DEVD-R110 were from Promega Corporation (Southampton, UK). Substrate for caspase 1, 8 and 9, DEVD-AFC, AEVD-AFC and IETD-AFC were from Calbiochem. Polyclonal rabbit anti-human caspase 3 antibody was from Santa Cruz Biotechnology Inc. (Alexa, CA.), anti-human caspase 8 antibody was from Zymed Laboratories Inc. (San Francesco, CA) and anti-human caspase 9 antibody from Pharmingen (San Diego, CA). Amino-terminal-biotinylated-RGDS was purchased from SigmaGenosys (Cambridge, UK). Anti-

v 3

(clone LM609), anti-

v 5

(clone P1F6), anti-

2 1

(clone

BHA2.1) blocking antibodies and human caspases 8 and 9 active recombinant proteins were purchased from Chemicon International Inc. (Temecula, CA).

Cells culture – Human Umbilical Vein Endothelial Cells (HUVEC) (from Clonetics, Walkersville, MD) were maintained in EBM-2 medium (Clonetics, MD) supplemented with EGM-2 kit (Clonetics) containing fetal calf serum (FCS) (2%), hydrocortisone (0.04%), hFGF-B (0.4%), VEGF (0.1%), R3IGF-1 (0.1%), ascorbic acid (0.1%), hEGF (0.1%), GA-1000 (0.1%), heparin (0.1%) (complete medium), according to manufacturer’s instructions. For all experiments, cells were used up to the 6th passage and harvested at 80% confluence. For all experiments cells were seeded for 24 h, then starved overnight and then used. Cell adhesion assay- Cell adhesion was quantified as reported11. Ninety-six-well tissue culture plates were coated overnight at 4°C with ECM proteins (fibronectin or vitronectin or collagen IV, diluted in

4

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PBS, pH 7.4, to the final concentration of 50 µg/ml). Wells were then blocked for 1 h at 37 °C with 5 mg/ml BSA in EBM-2 medium and washed three times with PBS, pH 7.4, before cell seeding. HUVEC harvested by trypsin-EDTA were suspended in EBM-2 medium + FCS 10% with serial dilutions of either peptides (RGDS or RGES) ranging from 500 to 62.5 µg/ml, corresponding to 1.1 to 0.1 mM. Cells were incubated with peptides at 37 °C for 15 min before seeding, then 15000 cells per well were added, and the plate was incubated at 37 °C for 1 h. Non-adherent cells were discarded by washing three times, then adherent cells were fixed with 4% formaldehyde in PBS, pH 7.4, for 10 min at RT and stained with 0.5% toluidine blue (Merck KgaA, Darmstadt, Germany) in 4% formaldehyde for 10 min at RT. Plates were then rinsed extensively with water and stain was extracted by incubation with SDS 1% in PBS, pH 7.4, for 30 min at RT. Cell adhesion was then quantified as OD at 595 nm.

Immunofluorescence - HUVEC were seeded on coverslips coated with PLL, vitronectin or collagen IV (50 µg/ml in PBS, pH 7.4), and incubated at 37°C for 24 h; then complete medium was replaced with unsupplemented medium (EBM-2) overnight at 37 °C. After treatment with RGDS in EBM-2 0.1% BSA for 4 h, cells were fixed with 3% paraformaldehyde in PBS, pH 7.4, for 10 min, permeabilized with 0.1% Triton X-100 in PBS, pH 7.4, for 5 min at RT and blocked for 30 min with BSA 2% in PBS, pH 7.4, at RT41. Cells were stained with FITC-conjugated phalloidin (1:1000) in PBS, pH 7.4, containing BSA 1% for 1 h at RT. Coverslips were mounted with a fluorescent mounting medium (Dako Corporation, CA, USA) and cells were visualized using a Zeiss Axioplan fluorescence microscope. Images were acquired with a Zeiss Axiocam digital camera.

Proliferation assay - HUVEC proliferation was evaluated as previously reported42. Briefly, HUVEC (8x104 cells/well) in EBM-2 medium supplemented with EGM-2 kit, were allowed to grow onto 6wells plates coated with collagen IV (50 µg/ml) (Becton Dickinson, Bedford, MA) for 24 h at 37 °C in 5% CO2. Medium was then replaced with EBM-2 overnight. Subsequently, cells were treated with serial dilutions of RGDS (from 500 to 125 µg/ml) dissolved in EBM-2 medium + BSA 0.1 % containing basic Fibroblast Growth Factor (FGF-2) (10 ng/ml) for 24 h at 37°C. Then, cells were harvested by trypsin-EDTA and counted using hemacytometer.

Cell migration assay - FGF-2-induced HUVEC migration was measured in modified Boyden chambers as previously reported43. Briefly, 8 µm pore-size polycarbonate filters (Costar, Cambridge, 5

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MA) were coated with murine collagen type IV (50 µg/ml) for 1 h. HUVEC were incubated for 48 h in RPMI 15% FBS, 1% sodium pyruvate, 2% MEM amino acids without L-glutamine, 1% L-glutamine, 1% penicillin-streptomycin, 1% MEM non-essential amino acids. Cells were harvested by trypsinization, re-suspended in RPMI 25 mmol/L HEPES, 0.1 % BSA and 200 µl were added to the upper portion of the chambers at 1x106 cells/ml with serial dilutions of RGDS (from 500 to 125 µg/ml); the lower portion of the Boyden chamber contained FGF-2 (10 ng/ml) as chemoattractant. After 4 h at 37°C in 5% CO2, cells were fixed in 95% ethanol and stained with 0.5% toluidine blue in PBS, pH 7.4, for 10 min. The number of migrated cells was evaluated by counting 15 fields at 400 X magnification. In other experiments, cells were pre-treated for 20 min at 37 °C with caspases inhibitors, namely ZVAD-FMK (general caspase inhibitor), Z-DEVD-FMK (caspase 3 inhibitor), Z-WEHD-FMK (caspase 1 inhibitor), Z-IEDT-FMK (caspase 8 inhibitor) and Z-LEHD-FMK (caspase 9 inhibitor) (50 µM) before treatment with RGDS. In additional experiments HUVEC were pre-incubated with RGDS (500 µg/ml, 20 min, 37oC); then cells were washed with 2M NaCl 20 mM HEPES, pH 7.4 and twice with 2M NaCl 20 mM acetic acid, pH 7.4 to wash out RGDS free in the medium or bound to the membranes44 . Cells treated in this way were then used for migration on collagen IV-coated filters as reported above.

Caspase activity assay - Caspase activity was measured as follows: HUVEC seeded on collagen IV were incubated at 37°C for 24 h; then medium was replaced with EBM-2 overnight before treatment with RGDS 500 µg/ml in EBM-2 0.1% BSA for 4 h. Before harvesting by trypsinization, cells were incubated for 20 min at 37°C with the fluorescent generic caspase substrate (FITC-VAD-FMK 10 µM). Subsequently, cells were washed twice with PBS, pH 7.4, and fixed with 2% paraformaldehyde in PBS, pH 7.4, for 30 min at 4°C. Cell fluorescence was then measured at a Coulter Epics XL flow cytometer (Beckman Coulter), with automatic gating. Data were analyzed with the WinMDI software. Caspase 3/7 enzymatic activity was measured by monitoring the fluorescence of the Z-DEVD-R110 substrate according to the manufacturer’s instructions. HUVEC seeded on collagen IV were treated as described above and incubated with RGDS 500 µg/ml in EBM-2 0.1% BSA for 4 h and 24 h at 37°C. The substrate was then added, cells were harvested by trypsinization and re-suspended in PBS, pH 7.4. Fluorescence was then measured (485 nm excitation wavelength and 530 nm emission wavelength) and expressed as fold increase vs control.

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Caspase 1, caspase 8 and caspase 9 activity were measured as previously reported45,46. Briefly, cells seeded on collagen IV (as reported above) were incubated in the presence or in the absence of RGDS (as previously reported), cells were then harvested with trypsin, washed and re-suspended in caspase buffer (100 µl) (20 mM HEPES pH 7.6, 10 mM KCl, 1.5 mM MgCl2 , 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.1 mM PMSF) containing 50 µM of the fluorogenic substrate for caspase 1, 8 and 9, DEVD-AFC, AEVD-AFC and IETD-AFC, respectively. The suspension was then passed through a 211/2G needle, left on ice for 15 min and centrifuged at 25000xg for 15 min. The supernatant was collected and centrifuged at 100000xg at 4°C for 75 min. Protein concentration in the supernatant was determined by the micro-BCA method (Pierce, Tattenhall, Cheshire, UK). Caspase activity was allowed to proceed for 2 h at 37oC. Under these conditions caspase activity is proportional to the release of AFC from the substrate; fluorescence (emission at 505 nm and excitation at 400 nm) was then measured with a Perkin Elmer LS50 fluorometer (Perkins Elmer Instruments, Beaconsfield, UK). Caspases activation was then reported as fold increase vs control.

Caspases activation by western blotting - Caspases 3, 8 and 9 activation was also followed as function of procaspase cleaveage38. HUVEC were incubated at 37°C for 24 h, then medium was replaced with EBM-2 overnight at 37 °C, cells were then treated with RGDS 500 µg/ml in EBM-2 0.1% BSA for 4 h (caspase 3 activation was analyzed at 4, 16, and 24 h) at 37 °C. Cells were washed in PBS, pH 7.4, and lysed in buffer containing 150 mM NaCl, 25 mM HEPES pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA, EDTA, and phenylmethylsulphonyl fluoride, 10 µg/ml aprotinin, leupeptin, and pepstatin. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. Membranes were then blocked with 5% milk (Bio-Rad Laboratories) in TPBS (0.1 % Tween 20 in PBS, pH 7.4). After washing three times with TPBS, membranes were incubated with antibody to caspase 3 (1:200), to caspase 8 (1:250) and to caspase 9 (1:1000) for 1 h to visualize both pro-caspases and cleaved–caspases bands. For detection, secondary antibody goat anti rabbit conjugate to horseradish peroxidase (Pierce) was used, 1:5000 in 2% BSA in TPBS, followed by chemiluminescence (ECL; Amersham, Buckinghamshire, United Kingdom) and autoradiography. Bands were quantified using a calibrated imaging densitometer (GS 710; Bio-Rad) and analyzed using the “Quantity one” software (Bio-Rad). Apoptosis assays - 5x104 HUVEC in EBM-2 medium supplemented with EGM-2 kit were seeded onto coverslips coated with collagen IV as reported above. After 24 h at 37°C, medium was replaced with 7

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EBM-2 overnight and then cells were incubated with RGDS 500 µg/ml diluted in EBM-2 0.1% BSA for 24 h. Then cells were fixed with 3% paraformaldehyde for 10 min at RT, permeabilized with 0.1% Triton X-100, 0.1 % sodium citrate for 2 min on ice, washed twice with PBS, pH 7.4, and incubated for 1 h at 37°C in the dark with a TUNEL reaction mixture (Boehringer Mannheim Corporation, Indianapolis, IN), for in situ detection of cell death. After washing twice with PBS, pH 7.4, cells were incubated at RT with the Hoechst solution for 5 min. All Hoechst-positive nuclei as well as TUNELpositive nuclei were visualized using a Zeiss Axioplan fluorescence microscope. Then, apoptosis was expressed as % of fragmented Hoechst positive nuclei vs total Hoechst positive nuclei, and as % of TUNEL-positive nuclei vs total Hoechst positive nuclei and was reported as fold increase vs control. To further analyze cell death inducing effect of RGDS, FACS analysis was carried out on HUVEC grown as reported above and treated with FGF-2 (10 ng/ml) or BSA alone in the presence or the absence of RGDS (500 µg/ml). In additional experiments, RGDS-treated HUVEC were pre-treated for 20 min with blocking antibodies anti

v 3

(10 µg/ml),

v 5

(0.1 µg/ml) or

2 1

(10 µg/ml)

according to manufacturer’s instructions. Then propidium iodide (PI) staining (1µg/ml; Sigma Chemicals) was performed according to standard protocols on ethanol-fixed cells. Cell cycle analysis was performed within 72 h from the staining, on a Coulter Epics XL flow-cytometer (Beckman Coulter). Data were then analyzed with WinMDI software.

Confocal microscopy - HUVEC seeded on coverslips and grown as described above, were treated for 4 h with biotinylated-RGDS (500 µg/ml) at 4 °C and 37 °C. Then cells were fixed and permeabilized as described in the immunofluorescence paragraph, and blocked for 30 min with 10% goat serum in PBS, followed by incubation with fluorescein avidin (Vector Laboratories, Peterborough, UK) (1:40) in PBS, pH 7.4, for 1 h at RT. After washing in 0.3% Triton X-100 in PBS, HUVEC were incubated with PI at a final concentration of 5 µg/ml to visualize nuclei. Cells were then analyzed using a Zeiss LSM 510 Meta Confocal Microscope. Lasers' power, beam splitters, filter settings, pinhole diameters and scan mode were the same for all examined samples. Fields reported in the figure are representative of all examined fields.

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RGDS direct interaction with membranes, cytoplasm and caspases – A partial sub-fractionation to obtain membranes and non-membranes (here referred to as “cytoplasmic”) extracts was carried out as reported38,47 with some modification. Briefly, HUVEC pellet was re-suspended in lysis buffer (4 mM HEPES, pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), 5 mM EGTA, 25 µg/ml leupeptin, 5 µg/ml pepstatin, 40 mM

-glycerophosphate, 1 mM phenyl methylsulphonyl fluoride

(PMSF)) and kept for 25 min at 4 °C. After three times freeze-and-thaw, membranes were separated by centrifugation at 13000 rpm at 4 °C for 20 min. Membranes and the corresponding cytoplasmic extracts were spotted onto nitrocellulose, as previously reported48. In other experiments human caspases 8 and 9 active recombinant proteins (0 to 10 units) or control proteins (BSA, fibronectin, 10 µg) were spotted onto nitrocellulose and blocked with 5% milk in TPBS (0.1% Tween 20 in PBS). After washing three times with TPBS, nitrocellulose membranes were incubated for 4 h at RT with 1 mg/ml of aminoterminal-biotinylated RGDS (Sigma-Genosys, Cambridge, UK). Some experiments were carried out with 1 mg/ml biotinylated-RGDS in the presence of 10 mg/ml not-biotinylated RGDS diluted in TPBS, as specific competitor. After extensive washing with TPBS, nitrocellulose was incubated for 1 h at RT with an avidin/biotinylated horseradish peroxidase kit (Vecstatin ABC, from Vector) followed by chemiluminescence reaction and exposure to Kodak film (Eastman Kodak). Binding in the presence of labeled-RGDS was considered as “total” binding, while the one in the presence of an excess of unlabeled-RGDS was considered as “nonspecific” binding. Interaction was measured by densitometry (GS 710; Bio-Rad), analyzed using the “Quantity one” software (Bio-Rad) and expressed as % of total binding.

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Results

HUVEC adhesion - Preliminary experiments showed that HUVEC adhesion to vitronectin, fibronectin and collagen IV (50 µg/ml) is proportional to the number of seeded cells with the mid point set to 15000 cells/well. Therefore this number of cells was seeded either in the presence or in the absence of increasing concentrations of RGDS. In these experiments, RGDS exhibited a dosedependent inhibitory effect on HUVEC adhesion to fibronectin and vitronectin (61±4.3% and 84±6.8% inhibition, p