C57BL/6J mice were purchased from Charles River Laboratories. Transgenic C57BL/6J mice ubiquitously expressing enhanced GFP under the control of the chicken β-actin promoter and CMV enhancer were obtained from The Jackson Laboratory. P-selectin–deficient mice were generated and provided by A.L. Beaudet (Baylor College of Medicine, Houston, TX). GPIIb (αIIb
integrin)-deficient mice were generated as described previously (40
). All experimental procedures performed on animals met the requirements of the German legislation on protection of animals and were approved by the Government of Bavaria/Germany.
4RA10 (function-blocking anti–mouse PSGL-1) were raised as described previously (22
). Rat anti–mouse GPIbα mAb was obtained from Emfret Analytics. Function-blocking anti–mouse/human SDF-1α (clone 79014) was obtained from R&D Systems. Anti-α2β1 mAb (clone: BMA2.1) was obtained from Chemicon. mAb 7E3 (gift from B. Coller, The Rockefeller University, New York, NY) inhibits fibrinogen binding to GPIIb-IIIa.
Separation and labeling of CD34+ BM-PCs, KSL BM-PCs, and platelets.
For separation of BM-PCs, mice (C57BL/6J or GFP+
) were killed by an overdose of pentobarbital, and both femura were harvested from each animal. Single cell suspensions of the BM were obtained by flushing the femura with PBS using a 21-gauge needle. CD34+
BM-PCs were separated as described previously (42
). Primitive KSL BM-PCs were purified according to a previously published protocol (6
) (for details, see supplemental Materials and methods, available at http://www.jem.org/cgi/content/full/jem.20051772/DC1
). For IVM, CD34+
or KSL BM-PCs were fluorescently labeled with 5-carboxyfluorescein diacetate succinimidyl ester (DCF). For each experiment, 107
BM-PCs or 1.5 × 105
fluorescent KSL cells were infused intravenously. Mouse platelets were isolated from whole blood as described previously (12
). For IVM, platelets were tagged with rhodamine-6G or DCF as reported previously (12
BM-PC adhesion to purified proteins under flow conditions.
BM-PCs were isolated as described in the previous paragraph. BM-PC (2 × 104
/ml) adhesion to coverslips coated with BSA, vitronectin (Becton Dickinson), fibrinogen (Sigma-Aldrich), collagen (Becton Dickinson), or recombinant mouse P-selectin, generated as described previously (23
), was assessed in a transparent flow chamber at a wall shear rate of 1,000 s−1
as described elsewhere (22
). In a separate set of experiments, platelets (4 × 108
/ml) were allowed to adhere to collagen-coated coverslips before perfusion with BM-PCs. Where indicated, function-blocking anti–PSGL-1 mAb (5 μg/ml) was added to the BM-PC suspension.
Characterization of BM-PC adhesion receptors and determination of platelet-binding by flow cytometry.
BM-PCs (10 × 103
or KSL cells/ml) resuspended in PBS were incubated with fluorophore-labeled anti-CD41 (αIIb integrin, clone: MWReg 30), anti-CD49d (α4 integrin, clone: 9C10), anti-CD162 (PSGL-1, clone: 2PH1) (all from Becton Dickinson), anti-GPIbα (Xia.B2, Emfret) or anti-GPVI mAb (2G3, generated as described (22
)), or irrelevant isotype-matched control antibody for 30 min at room temperature and directly analyzed on a FACSCalibur (Becton Dickinson). In a separate set of experiments, CD34+
or KSL BM-PCs (2 × 104
/ml) were coincubated with 108
/ml resting mouse platelets or platelets activated with mouse thrombin (2 U/ml, Sigma-Aldrich) for 15 min. Where indicated function-blocking anti-CD11b (M1/70, BD Biosciences) or anti-PSGL-1 mAb (4RA10, 5 μg/ml) was added to the cell suspension. PBS-incubated BM-PCs in the absence or presence of thrombin served as controls. Thereafter, the samples were incubated with FITC-labeled anti–mouse CD41 (MW Reg30) for 30 min to detect platelets attached to BM-PCs at room temperature and directly analyzed.
Assessment of BM-PCs and platelet adhesion and BM-PC–platelet interaction in response to carotid injury.
Platelet and BM-PC (CD34+
) adhesion dynamics before and after vascular injury were monitored in vivo by use of IVM. Carotid injury was induced in anesthetized C57BL6/J, P-selectin–, or GPIIb-deficient mice as described previously (12
). Before and after vascular injury, differentially fluorescent-tagged cells (platelets and BM-PCs) were visualized using a Zeiss Axiotech microscope (20× water immersion objective, W 20×/0.5, Zeiss) with a 100W HBO mercury lamp for epi-illumination as reported previously (12
). Where indicated, C57BL6/J mice were pretreated with 2 mg/kg body weight RB40.34 (anti–mouse P-selectin), 4RA10 (anti–mouse PSGL-1), rat anti–mouse GPIbα, or anti–mouse/human SDF-1α (clone 79014) 5 min before intravital microscopy (n
= 4–6 per group). BM-PC adhesion to the carotid artery after infusion of irrelevant isotype-matched IgG served as control (n
For direct visualization of platelet–KSL cell interactions at the site of vascular lesion differentially fluorescent-tagged cells (rhodamine-tagged platelets and DCF-tagged KSL BM-PCs) were infused i.v. KSL binding to adherent platelets was analyzed 5 min after induction of carotid injury by real-time double fluorescence IVM using a BX51WI microscope (Olympus), equipped with an Olympus MT20 monochromator.
Assessment of SDF-1α expression by immunohistochemistry.
To determine the presence of SDF-1α in megakaryocytes and aggregated platelets, paraffin-embedded sections of mouse femura, injured mouse carotid arteries (WT or GPIIb-deficient, 0.5, 4, and 24 h after endothelial denudation), and a fresh thrombus isolated from an occluded human coronary artery were cut into 2-μm sections and incubated with anti–mouse SDF-1α (clone 79018; R&D Systems), or anti–human SDF-1α (polyclonal goat; R&D Systems), respectively, and stained using APAAP chemMATE or DAB chemMATE detection kits (both obtained from DakoCytomation). For immunofluorescence microscopy, cryostat sections of injured mouse carotid arteries (24 h after endothelial denudation) were incubated with anti–mouse CD41 mAb (MW Reg30; BD Biosciences), anti–mouse SDF-1α mAb (clone 79014; R&D Systems), Alexa488-labeled goat anti–rat IgG, Alexa594-labeled goat anti–mouse IgG (both obtained from Invitrogen), and DAPI (D1306; Invitrogen). Fluorescence images were obtained using a Leica fluorescence microscope.
Determination of platelet and megakaryocytic SDF-1α protein and mRNA expression.
Platelet SDF-1α release was determined in vitro by enzyme-linked immunosorbent assay (R&D Systems). In brief, mouse SDF-1α standard lysates obtained from isolated resting mouse platelets, whole blood, or mouse BM cells were incubated in SDF-1α mAb precoated microplates and reacted with a polyclonal anti–SDF-1α–horseradish peroxidase antibody (all obtained from R&D Systems). For detection a tetramethyl benzidine peroxidase substrate was used. The mouse melanoma cell line D5 served as a control. Absorbance was read at 450 nm and the background was corrected. SDF-1α concentrations were calculated with standards and are adjusted to 100 μg total protein.
To determine SDF-1α expression by the megakaryocytic lineage, we generated megakaryocytes from mouse CD34+
cells as reported previously (28
) (for details see supplemental Materials and methods). Megakaryocytic SDF-1α expression was assessed by ELISA as described in the previous paragraphs. In addition, megakaryocytic SDF-1α mRNA expression was determined using RT-PCR. The mRNA expression of GPIIb integrin and β-actin served as controls. The primer sequences and their corresponding product sizes and annealing temperatures are shown in the Table S1 (available at http://www.jem.org/cgi/content/full/jem.20051772/DC1
Assessment of SDF-1α release upon platelet activation.
To determine the effect of platelet activation on SDF-1α release, human platelets (0.5 × 109 cells/ml) were incubated with 2 U/ml α-thrombin or PBS for 30 min. To determine the effects of GPIIb receptor cross-linking, human platelets were incubated with 100 μg/ml of soluble fibrinogen (Sigma-Aldrich) in the presence or absence of 5 μg/ml of bivalent mAb antifibrinogen (2C2-G7; BD Biosciences), or with 5 μg/ml of mouse anti-GPIIb mAb 7E3 in the presence or absence of 5 μg/ml of anti–mouse IgG mAb (DakoCytomation). SDF-1α release into the supernatants of resting or activated platelets was determined by ELISA as described in previous paragraphs.
To analyze the surface exposure of SDF-1α in response to platelet activation, resting or thrombin-activated human platelets were incubated with mouse anti–SDF-1α mAb (R&D Systems) or irrelevant isotype-matched control antibody for 30 min at room temperature, followed by staining with FITC-labeled rabbit anti–mouse IgG antibody (Becton Dickinson). The samples were directly analyzed on a FACSCalibur (Becton Dickinson).
To define the distribution of SDF-1α in resting and activated platelets, human platelets (2 × 108 cells/ml) were stimulated with 2 U/ml α-thrombin, 5 μM ADP, or PBS for 60 min. The platelets were fixed and stained with FITC-labeled anti-CD41 (P2; Beckman Coulter), monoclonal (R&D Systems) or polyclonal anti–SDF-1α antibody (goat; R&D Systems), Alexa Fluor 488–tagged goat anti–mouse, Alexa 568–tagged rabbit anti–goat, Alexa Fluor 488–tagged rabbit anti–goat (all obtained from Invitrogen), or Rhodamine-Phalloidin (R415; Invitrogen) as indicated. Confocal immunofluorescence analysis was performed using a LSM510 META confocal laser microscope equipped with the LSM510 META software (Carl Zeiss MicroImaging, Inc.).
Incorporation of BM-PCs into the neointima.
GFP+ CD34+ BM-PCs or KSL GFP+ CD34+ BM-PCs were isolated from the BM of GFP transgenic mice as described in previous paragraphs and injected into C57BL/6J recipient animals (n = 5; 105 cells per experiment). We mechanically injured the carotid artery. After 5 d, the injured carotid arteries were excised. GFP fluorescence was detected on cryostat sections by standard fluorescence microscopy.
Recruitment of endogenous PCs to the injured carotid artery.
To determine recruitment of endogenous circulating PCs, carotid arteries of C57BL/6J (n = 3), P-selectin–, (n = 4) or GPIIb-deficient mice (n = 2) or in anti–SDF-1α mAb-treated wild-type mice (n = 2) were injured as described in previous paragraphs and c-Kit and Sca-1 mRNA expression was assessed by RT-PCR (see supplemental Materials and methods). The constitutively expressed β-actin transcript was amplified as an internal control to compare relative abundance of PCR products, and the relative expression of each mRNA was normalized to the expression of β-actin for semiquantification. To detect c-Kit positive paraffin-embedded sections of injured mouse, carotid arteries (24 h after endothelial denudation) were incubated with anti–mouse c-Kit mAb (2B8; BD Biosciences) and stained using APAAP chemMATE mouse detection kit (DakoCytomation).
Comparisons between group means were performed using one-way analysis of variance on Ranks. Data represent mean ± SEM. A value of P < 0.05 was regarded as significant.
Online supplemental material.
Online supplemental Materials and methods provides additional methodological information in the following categories: separation of BM-PCs; effects of anti-GPIbα mAb on platelet function; transfection of DAMI cells; scanning electron microscopy; generation of mouse megakaryocytes; in vitro PC migration; and recruitment of endogenous PCs. Table S1 contains primer sequences, corresponding product sizes, and annealing temperatures. Fig. S1 shows the effects of anti- GPIbα mAb on platelet function. Fig. S2 addresses BM-PC binding to recombinant P-selectin. Fig. S3 illustrates the subcellular localization of SDF-1α in megakaryocytes. Fig. S4 investigates the effect of platelet SDF-1α on PC migration. All online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20051772/DC1