Supplemental Methods
Isolation of platelets
Human platelets were isolated as previously described.1 Briefly, venous blood was drawn from the antecubital vein of healthy volunteers and collected in acid citrate dextrose (ACD)-buffer. After centrifugation at 430g for 20 min, platelet-rich plasma (PRP) was removed, was added to Tyrodes-HEPES buffer (HEPES, 2.5 mM/L (Carl Roth GmbH, Karlsruhe, Germany), NaCl, 150 mM/L, KCl, 1 mM/L, NaHCO3, 2.5 mM /L, NaH2PO4, 0.36 mM/L, glucose 5.5 mM/L (Sigma, Steinheim, Germany), BSA 1 mg/ml, pH 6.5), and was centrifuged at 900g for 10 min. After removal of the supernatant, the remaining platelet pellet was resuspended in Tyrodes-HEPES buffer (pH 7.4 supplemented with CaCl2, 1 mM/L; MgCl2, 1 mM/L).
Flow Cytometry
To evaluate SDF-1 expression on resting and activated isolated platelets with ADP (20µM; Chrono-Par, Havertown, USA) or TRAP (25µM; Sigma, Steinheim, Germany) one-colour flow cytometry was applied as previously described 2. In brief, a conjugated monoclonal antibody was used to measure platelet SDF-1 surface expression (R&D Systems, Minneapolis, USA; clone 79014) or P-selectin (Immunotec, Marseille, France; clone CLB-Thromb/6). To determine SDF-1 expression on adherent platelets, isolated platelets (2x108/ml) were allowed to adhere to 96-well plates pre-coated with collagen type I (10 µg/ml; BD Biosciences, Bedford, USA) for 15-30 minutes. Unspecific adhesion was prevented by blocking with 2% bovine albumin serum (BSA; Sigma, Steinheim, Germany). After two gentle washing steps with tyrodes buffer, in situ staining was followed by a 30-minute incubation time. Subsequently, platelets were removed into a FACS tube and preceded to FACS measurement. Mouse IgG1-FITC (BD Biosciences, Bedford, USA) was used as a monoclonal immunoglobulin isotype control, while CD62P was used as a positive control.
To determine SDF-1 expression on human arterial endothelial cells, endothelial cells were cultivated in a 24-well plate till confluence. Where indicated, endothelial cells were activated with TNF-α (50 ng/ml) and INF-γ (20 ng/ml) or IL-1β (100 ng/ml) (the referred cytokines were purchased from Peprotech Inc., New Jersey, USA). In situ immunostaining was performed after removal of the supernatant. FITC-conjugated anti-SDF-1 monoclonal antibody, positive control anti-CD54 (ICAM-1; Immunotec, Marseille, France; clone 84H10) and isotype control mouse IgG1 were incubated for one hour with the cells in a dark environment. The cells were removed with Trypsin-EDTA (Invitrogen Corporation, Paisley, Scotland, UK) and were immediately processed to FACS.
To determine endothelial phenotype and function of CD34+ cell-derived endothelial progenitor cells we evaluated the expression of the marker CD146 (R&D Systems, Minneapolis, USA; clone 128018), CD54 (ICAM-1; ), CD144 (VE-Cadherin; AbD Serotec, Oxford, UK; polyclonal IgG), CD31 (Immunotech Beckman Coulter, Marseille, France; clone 5.6E), CD34 (BD Biosciences, San Jose, USA; clone 8G12), CD18 (Beckton Dickinson Immunocytometry Sustems, San Jose, USA; clone L130) and CD45 (Beckton Dickinson BD Biosciences, San Jose, USA; clone 2D1) on CD34+ cell-derived endothelial progenitor cells by flow cytometry, as described above. CD34+ cells and human arterial endothelial cells were used as negative and positive control, respectively.
Isolation and culture of human arterial endothelial cells
Human arterial endothelial cells (haECs) were isolated and passaged according to techniques described previously.3 HaECs were identified by immunocytochemical staining against the von Willebrand factor (Boehringer, Mannheim, Germany) and their characteristic “cobblestone” growth pattern with contact inhibition between cells. Routine stainings with the DNA dye DAPI (4’, 6-diamino-2-phenylindole-dihydrochloride; Boehringer, Mannheim, Germany) were used to exclude mycoplasm contaminations. Cultivation was carried out with a special medium composed of endothelial cell growth medium MV2 (PromoCell, Heidelberg, Germany) plus 10% FCS and 1% penicillin-streptomycin (Invitrogen, Karlsruhe, Germany).
Isolation and culture of human CD34+ cells
Human CD34+ cells were isolated either from human cord blood or bone marrow and cultured as previously described.4 Human mononuclear cells were obtained from human umbilical cord blood or bone marrow by density gradient centrifugation on Biocoll separation solution (Biochrom, Berlin, Germany) at 600g for 15min. CD34+ cells were enriched by immunoaffinity selection (CD34 Progenitor Cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. For cell culture, IMDM with Glutamax was used, supplemented with 5% heat-inactivated fetal calf serum, 100 U/ml penicillin-streptomycin, 1% MEM-vitamines and 1% non-essential amino acids (all purchased from Invitrogen, Karlsruhe, Germany). The isolated cells were ³ 95% positive for CD34+ cells as determined by flow cytometry after each isolation procedure. Both populations of CD34+ cells (originated from cord blood or bone marrow) presented with identical results in our in vitro experiments.
Adhesion assays under static and dynamic conditions
Evaluation of CD34+ cells adhesion to immobilized platelets under static and dynamic conditions (flow chamber) and to human arterial endothelial cells was performed as previously described.1 For static adhesion assays, isolated platelets (2x108/ml) were allowed to adhere to 96-well plates pre-coated with collagen type I (10µg/ml) for 2 hours to achieve a monolayer of immobilized platelets. Subsequently, CD34+ cells were added and incubated for 30 minutes. Unspecific adhesion was prevented by blocking with BSA (2%). After two gentle washing steps with PBS, residual adherent CD34+ cells were counted by direct phase contrast microscopy. As negative control, similar experiments were performed with CD34+ cells adherent to collagen (10µg/ml). Where indicated, adherent platelets or CD34+ cells were pre-incubated for 30 minutes with a blocking monoclonal antibody (mAb) to SDF-1 (20µg/ml) or with a neutralizing mAb to CXCR4 (R&D Systems; Minneapolis, USA; clone 44716;10µg/ml), respectively. As control, adherent platelets or CD34+ cells were pre-treated with isotype control IgG1 (R&D Systems; Minneapolis, USA; clone11711; 20 µg/ml) or IgG2b (R&D Systems; Minneapolis, USA; clone133303; 10µg/ml), respectively. As a further control mAb, anti-CD14 (R&D Systems; Minneapolis, USA; clone134603; 20µg/ml) was applied.
To evaluate cell adhesion to immobilized platelets or haECs under flow conditions, platelets were allowed to adhere to collagen-coated glass coverslips or haECs were cultivated till confluence in glass coverslips, respectively, and then used in a flow chamber (Oligene, Berlin, Germany), as previously described 1. Where indicated, adherent platelets or monolayers of haECs were pre-incubated for 30 min with an anti-SDF-1 mAb or with a control mAb (2D1) (20 µg/ml each) before perfusion was started. Moreover, where indicated, CD34+ cells were pre-incubated for 30 min with an anti-CXCR4 mAb or with a control IgG2b (10 µg/ml each) before perfusion was started. Furthermore, where indicated, haECs were activated with TNF-a (50 ng/ml) and INF-g (20 ng/ml) or IL-1b (100 ng/ml), or co-incubated with isolated platelets (2x108/ml) for 12 hours. Perfusion experiments were performed at shear rates of 2000 s-1 (high shear). All experiments were recorded in real time on video-CD and evaluated off-line as previously described.1
Carotid ligation in mice and intravital microscopy
To evaluate the effect of the platelet-derived SDF-1 on progenitor cell recruitment in vivo, the common carotid artery of wild-type C57BL/6J mice was injured by ligation and DCF stained CD34+ cells were intravenously injected and visualized using intravital fluorescence microscopy as previously described 5. Prior to the experiments, CD34+ cells were stained with 5-carboxyfluorescein diacetate succinimidyl ester (DCF) and incubated with anti-CXCR4 (20 µg/ml) or isotype control IgG2b (20 µg/ml) for 30 min. Alternatively, an infusion of anti-SDF-1 or isotype control IgG1 was taken place. Wild-type C57BL/6J mice (Charles River Laboratories) were anesthetized by intraperitoneal injection of a solution of midazolame (5 mg/kg body weight; Ratiopharm), medetomidine (0.5 mg/kg body weight; Pfizer) and fentanyl (0.05 mg/kg body weight, CuraMed/Pharam GmbH). Polyethylene catheters (Portex) were implanted into one jugular vein and fluorescent CD34+ cells (5x104/ml) were injected intraveneously. The common carotid artery was dissected free and ligated vigorously for 5 min to induce vascular injury. Before and after vascular injury, interaction of the fluorescent CD34+ cells with the injured vessel wall was visualized by in situ in vivo video microscopy of the common carotid artery using a Zeiss Axiotech microscope (20x water immersion objective, W 20x/0.5; Carl Zeiss MicroI maging, Inc.) with a 100-W HBO mercury lamp for epi-illumination. The number of adherent CD34+ cells was assessed by counting the cells that did not move or detach from the endothelial surface within 15 sec. Their number is given as cells/ mm2 endothelial surface.
Intestinal ischemia / Reperfusion model in mice
To evaluate the biological significance of platelet-derived SDF-1 in the regulation of peripheral homing of CD34+ cells in microvasculature, fluorescent CD34+ cells were infused before intestinal ischemia / reperfusion injury and visualized in the postischemic microcirculation by intravital fluorescence microscopy as previously described 6. Wild-type C57BL/6J mice (Charles River Laboratories) were anesthetized and were placed in a heating pad. After laparotomy, a segment of the jejunum was exteriorized and constantly superfused with 37o C Ringer’s lactate. Segmental jejunal ischemia was induced for 60 min by occluding the supplying vessels by ligation. After reperfusion the intestinal segment was exposed on a mechanical stage and CD34+ cell-platelet/endothelium interaction in the postischemic microvasculature were investigated by intravital microscopy. The number of adherent CD34+ cells was assessed by counting the cells that did not move or detach from the endothelial surface within 15 sec. Their number is given as cells/ mm2 endothelial surface.
Colony Forming Unit Assay
To analyze the effect of SDF-1 on platelet-induced CD34+ cell differentiation to endothelial progenitor cells, isolated platelets were co-incubated with CD34+ cells as previously described.4 CD34+ cells were seeded on human fibronectin (Becton Dickinson, Heidelberg, Germany), collagen or on immobilized platelets (2x108 cells/ml) in the presence of anti-SDF-1 or isotype control IgG1 or anti-CXCR4 or isotype control IgG2b. Subsequently, the cells were cultivated for several days in endothelial cell growth medium MV2 containing 5% heat-inactivated fetal calf serum, 5.0 ng/ml epidermal growth factor, 0.2 µg/ml hydrocortisone, 0.5 µg/ml vascular endothelial growth factor, 10 ng/ml basic fibroblast factor, 20 ng/ml R3 insulin-like growth factor-1, 1µg/ml ascorbic acid (PromoCell, Heidelberg, Germany). Endothelial progenitor cell colony-forming units were counted between days 5 and 10 (number of endothelial colonies). To determine the expression of the endothelial cell marker CD146 cells were washed and resuspended in PBS, incubated with polyglobin (Bayer Vital; Leverkusen, Germany) for 15 min, washed, and incubated with the FITC- labelled antibody to CD146 (clone 128018; R&D Systems) for 30 min at room temperature. After washing, cells were analyzed on a FACS-Calibur flow cytometer (Becton Dickinson).
Immunofluorescence microscopy
In order to test the differentiation of CD34+ cells to endothelial progenitor cells, rhodamine phalloidin (5 units/ml, detection of cytoskeletal actin) or a rabbit anti-human vWF monoclonal antibody (Dako Cytomation) and a secondary sheep anti-rabbit mAb (Sigma) were used. CD34+ cells were co-incubated with medium or platelets for 10 days on chamber slides and processed for immunofluorescence microscopy. Between each incubation step, cells were gently washed with PBS.
In order to evaluate the surface expression of SDF-1, isolated platelets were seeded on chamber slides and processed for confocal microscopy. Rhodamine phalloidin (5 units/ml, detection of cytoskeletal actin) (red) and anti-SDF-1-FITC (green) were applied for 30 min.
Reverse Transcription – Polymerase Chain Reaction
Upon differentiation of CD34+ cells to endothelial progenitor cell colonies, endothelial cells were further cultivated in culture flasks and analyzed for the expression of mRNA for eNOS, CD34, PECAM-1, tie-2, flk-1 and β-actin by RT-PCR as previously described 1, 7. Briefly, mRNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed using the Im PromII Reverse Transcription System (Promega, Mannheim, Germany) and aTaq Polymerase (Promega, Mannheim, Germany). Annealing temperature was 68,4°C for 30 seconds, extension at 72°C for 45 seconds, with a final 5 minute extension at 72° C. The primer sequences were as follows: eNOS forward 5`-GGA AAA GGC CAG GGC TCT GCT GGA GC-3`, reverse 5`-GAA CAC CAG CTC GCT CTC CCT AAG CTG-3`, CD34 forward 5`-CAA GTT AGT AGC AGC CAA GGA GAG GCG CTG-3`, reverse 5`-GTC TGA AAC ATT TCC AGG TGA CAG GCT AGG-3` , CD31 (PECAM-1) forward 5`-GTT GTA TGA GGT CCA GAT TAT CCT GTC-3`, reverse 5`-GCT GAG GAC ACT TGA ACT TCC GTG TAC TGC`; tie-2 forward 5`-GAC GTA GTA GGA CGA TGC TAA TGG AAA GTC-3`, reverse 5`-GTG TAC TTC TAG AAT ATC AGG TAC TTC ATG C-3`, flk-1 (vascular endothelial growth factor-1) forward 5`-CTT CTC TAG ACA GGC GCT GGG AGA AAG AAC-3`, reverse 5`-CAC GTT GAG ATT TGA AAT GGA CCC GAG ACA TG-3`, β-actin (internal control), forward 5`-ACC TTC AAC ACC CCA GCC ATG-3`, reverse 5`-GCT CGG TCA GGA TCT TCA TGA GG-3`.
Supplemental Methods’ References
1. Langer H, May AE, Daub K, Heinzmann U, Lang P, Schumm M, Vestweber D, Massberg S, Schonberger T, Pfisterer I, Hatzopoulos AK, Gawaz M. Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro. Circ Res. 2006; 98:e2-10.
2. Gawaz M, Neumann FJ, Schomig A. Evaluation of platelet membrane glycoproteins in coronary artery disease : consequences for diagnosis and therapy. Circulation. 1999; 99:E1-E11.
3. Gawaz M, Neumann FJ, Ott I, Schiessler A, Schomig A. Platelet function in acute myocardial infarction treated with direct angioplasty. Circulation. 1996; 93:229-237.
4. Daub K, Langer H, Seizer P, Stellos K, May AE, Goyal P, Bigalke B, Schonberger T, Geisler T, Siegel-Axel D, Oostendorp RA, Lindemann S, Gawaz M. Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells. Faseb J. 2006; 20:2559-2561.
5. Massberg S, Brand K, Gruner S, Page S, Muller E, Muller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B, Gawaz M. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med. 2002; 196:887-896.
6. Massberg S, Enders G, Leiderer R, Eisenmenger S, Vestweber D, Krombach F, Messmer K. Platelet-endothelial cell interactions during ischemia/reperfusion: the role of P-selectin. Blood. 1998; 92:507-515.
7. May A, Kalsch T, Massberg S, Herouy Y, Schmidt R, Gawaz M. Engagement of glycoprotein IIb/IIIa (alpha(IIb)beta3) on platelets upregulates CD40L and triggers CD40L-dependent matrix degradation by endothelial cells. Circulation. 2002; 106:2111-2117.