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* Medizinische Klinik III, Eberhard Karls Universität Tübingen, Tübingen, Germany;
Institute for Prevention of Cardiovascular Diseases, University of Munich, Munich, Germany; and
Medizinische Klinik III, Klinikum rechts der Isar München, Technische Universität, Munich, Germany
1Correspondence: Medizinische Klinik III, Universitätsklinikum Tübingen, Otfried-Müller Str.10, 72076 Tübingen, Germany. E-mail: meinrad.gawaz{at}med.uni-tuebingen.de
ABSTRACT
Recruitment of human CD34+ progenitor cells toward vascular lesions and differentiation into vascular cells has been regarded as a critical initial step in atherosclerosis. Previously we found that adherent platelets represent potential mediators of progenitor cell homing besides their role in thrombus formation. On the other hand, foam cell formation represents a key process in atherosclerotic plaque formation. To investigate whether platelets are involved in progenitor cell recruitment and differentiation into endothelial cells and foam cells, we examined the interactions of platelets and CD34+ progenitor cells. Cocultivation experiments showed that human platelets recruit CD34+ progenitor cells via the specific adhesion receptors P-selectin/PSGL-1 and ß1- and ß2-integrins. Furthermore, platelets were found to induce differentiation of CD34+ progenitor cells into mature foam cells and endothelial cells. Platelet-induced foam cell generation could be prevented partially by HMG coenzyme A reductase inhibitors via reduction of matrix metalloproteinase-9 (MMP-9) secretion. Finally, agonists of peroxisome proliferator-activated receptor-
and -
attenuated platelet-induced foam cell generation and production of MMP-9. The present study describes a potentially important mechanism of platelet-induced foam cell formation and generation of endothelium in atherogenesis and atheroprogression. The understanding and modulation of these mechanisms may offer new treatment strategies for patients at high risk for atherosclerotic diseases.—Daub, K., Langer, H., Seizer, P., Stellos, K., May, A. E., Goyal, P., Bigalke, B., Schönberger, T., Geisler, T., Siegel-Axel, D., Oostendorp, R. A. J., Lindemann, S., Gawaz, M. Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells.
Key Words: atherothrombosis • vascular lesion • statins • stem cells • matrix metalloproteinases
BEYOND THEIR ROLE IN HEMOSTASIS and thrombosis, platelets play a critical role in atherogenesis (1)
. Previously we showed that activated platelets interact with endothelium and induce alterations of chemotactic and adhesive properties of endothelial cells, a critical initial step in atherogenesis (2)
. We recently demonstrated in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE-deficient mice before the development of manifest atherosclerotic lesions (3)
. Platelet adhesion to the endothelium coincided with inflammatory gene expression and preceded atherosclerotic plaque invasion by circulating polymorphonuclear cells (3)
. Prolonged blockade of platelet adhesion in ApoE-deficient mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation (3
, 4)
.
Endothelial progenitor cells (EPCs) are increasingly recognized to play a critical role in vascular repair mechanisms and atherogenesis (5)
. Platelets are the first circulating blood cells that interact and adhere to vascular lesions (6)
. Thus, platelets may be involved in recruitment of circulating progenitor cells toward the injured vessel wall. Recently, we found that platelets stimulate chemotaxis and migration of mouse embryonic endothelial progenitor cells in vitro (7)
. Further, murine embryonic EPCs adhered substantially to immobilized platelets under dynamic flow conditions mediated by adhesion receptors PSGL-1/P-selectin, CD18 (ß2-integrin), and VLA4 (4/ß1-integrin) (7)
. Moreover, activated platelets secrete the chemokine SDF-1 and support primary adhesion and migration of progenitor cells in vivo (23)
.
EPCs are a circulating, bone marrow-derived cell population of large nonleukocyte cells characterized by the expression of three cell surface markers: CD133, CD34, and vascular endothelial growth factor receptor-2 (VEGFR-2) (8)
. EPCs appear to participate in vascular repair and homeostasis. In response to cytokine stimulation and ischemia, these cells are mobilized from the bone marrow, home to the ischemic tissue, and contribute to neovascularization and angiogenesis (9)
. Furthermore, EPCs are regarded to have a key role in the maintenance of vascular integrity and to act as "repair" cells in response to endothelial injury (10)
. Recruitment of EPCs toward vascular lesions has been regarded as a critical initial step in atherosclerosis and a result of the actions of various cardiovascular risk factors (5)
. Current data suggest that a decrease in circulating EPCs leads not only to impaired angiogenesis, but also to the progression of atherosclerosis (11)
. Moreover, patients at risk for coronary artery disease have a decreased number of circulating EPCs with impaired activity (12
13
14)
. Thus, there is increasing evidence that bone marrow-derived progenitor cells play a critical role in vascular repair mechanisms at the site of vascular lesions.
The present study evaluates the effect of platelets on the recruitment and differentiation of human CD34+ progenitor cells and describes a potentially important mechanism of platelet-induced foam cell formation and generation of endothelium in vascular repair mechanisms and atherogenesis.
MATERIALS AND METHODS
Reagents
For adhesion experiments, the function-blocking monoclonal antibodies (mAbs) mouse anti-CD162 (PSGL-1, clone PL1) and mouse anti-CD49d (4-integrin, clone HP2/1) were purchased from Immunotech (Marseille, France). Moreover, mouse anti-human CD62P (clone G1/G1–4) and mouse anti-human CD18 (ß2 integrin, clone IB4) were bought from Ancell (Bayport, MN, USA). 2D1 (monoclonal antibody, Ab) raised in a rat recognizes an irrelevant human antigen (3)
. For immunofluorescence staining, the following antibodies and reagents were used: mouse anti-human CD68 (clone KP1, DakoCytomation, Hamburg, Germany) in combination with Alexa fluor anti-mouse IgG1 Ab (Invitrogen, Karlsruhe, Germany); a rabbit anti-human polyclonal antibody (pAb) directed against "von Willebrand factor" (vWF, Dako) combined with anti-rabbit IgG-Cy3 (Sigma, Taufkirchen, Germany). 4',6'-Diamidino-2-phenylidole (DAPI) (4', 6-diamidino-2-phenylindole dihydrochloride, Sigma) was used to stain cell nuclei. The fluorochrome celltrackertm orange CMTMR (Molecular Probes, Leiden, Netherlands) and Dil-AcLDL (lipoprotein low density, acetylated human, Bio Trend, Köln, Germany) were used to evaluate platelet and AcLDL phagocytosis. For flow cytometry, fluorescein isothiocyanate (FITC) -labeled antibodies to CD31 (clone 5.6; Beckman Coulter, Krefeld, Germany) and CD146 (clone 128018; R&D Systems, Wiesbaden, Germany) were used. Inhibitors of the HMG coenzyme A reductase (statins)—simvastatin and pravastatin—were both from Merck (Schwalbach, Germany); fluvastatin was from Novartis (Basel, Switzerland) and atorvastatin from Pfizer (Karlsruhe, Germany). The peroxisome proliferator-activated receptor (PPAR) - agonists fenofibrate and WY14643 were purchased from Sigma and from Biomol (Hamburg, Germany), respectively; the PPAR- agonists troglitazone and GW1929 were from Biomol and Alexis Biochemicals (Grünberg, Germany). Quinacrine dihydrochloride (mepacrine) was used from Sigma. The peptide matrix metalloproteinase (MMP) inhibitor GM6001 was from Calbiochem (San Diego, CA, USA).
Isolation of platelets
Human platelets were isolated as described before (7)
. Briefly, venous blood was drawn from healthy volunteers and collected in acid-citrate-dextrose (-chlorohydrin) buffer. After centrifugation at 430 g for 20 min, platelet-rich plasma was removed, added to Tyrodes-HEPES buffer [HEPES 2.5 mmol/L, NaCl 150 mmol/L, KCl 1 mmol/L, NaHCO3 2.5 mmol/L, NaH2PO4 0.36 mmol/L, glucose (Glc) 5.5 mmol/L, BSA 1 mg/ml, pH 6.5], and centrifugated at 900 g for 10 min. After removal of the supernatant, the resulting platelet pellet was resuspended in Tyrodes-HEPES buffer (pH 7.4). The platelet isolation procedure revealed a high purity of platelets without any measurable contamination of polymorphonuclear cells or monocytes as verified by the absence of CD14 (flow cytometry) and of myeloperoxidase (ELISA).
Isolation, culture, and coincubation of human CD34+ cells with isolated platelets
Human CD34+ cells were isolated from human cord blood and cultured as described before (15)
. Human cord blood was obtained from healthy women immediately after childbirth with the approval of the local Ethics Committee (Project-No. 76/2005). The isolated cells were > 98% positive for CD34+ as determined by flow cytometry after each isolation procedure.
Human mononuclear cells were obtained from human umbilical cord blood by density gradient centrifugation on Biocoll separation solution (Biochrom, Berlin, Germany) at 600 g for 15 min. CD34+ cells were enriched by immunoaffinity selection (CD34 Progenitor Cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Cells were subsequently incubated on 96-well plates coated with 0.2% gelatin. For cell culture, IMDM with Glutamax supplemented with 5% heat-inactivated fetal calf serum, penicillin (100 U/ml)/streptomycin (100 µg/ml), 1% MEM vitamines, and 1% nonessential amino acids, all purchased from Life Technologies, Inc. (Invitrogen), was used. CD34+ progenitor cells (50,000 cells) were cocultured with platelets (2x108/ml) in 96-well plates precoated with 0.2% gelatin at 37°C and 5% CO2 for 10 days. Developing foam cells were counted in six windows by phase contrast microscopy. To evaluate the effect of statins and PPAR agonists on platelet-dependent foam cell generation, CD34+ progenitor cells were cultured in the presence of platelets and of fluvastatin, simvastatin, atorvastatin, and pravastatin or PPAR agonists in the concentrations indicated.
Determination of MMP-9
matrix metalloproteinase 9 (MMP-9) activity was determined in supernatants by gelatin zymography as described before (16)
. SDS gels containing 10% gelatin were from Invitrogen. Equal amounts of supernatants were loaded onto the gels; after electrophoresis, renaturation, and further incubation of the gels for 12 h at 37°C, gelatinolytic activity of MMP-2 and MMP-9 was detected as transparent bands on the Comassie brilliant blue-stained gels.
Platelet adhesion to CD34+ progenitor cells under static conditions
To evaluate CD34+/platelet adhesion under static conditions, isolated platelets (2x108/ml) were allowed to adhere to 96-well plates coated with collagen type I (10 µg/ml) for 2 h. Subsequently, CD34+ progenitor cells were added and incubated for 1 h. Unspecific adhesion was prevented by blocking with BSA (2%). After two gentle washing steps with PBS, residual adherent CD34+ progenitor cells were counted by direct phase contrast microscopy. Where indicated, EPCs or immobilized platelets were preincubated for 30 min with mAbs against known EPCs surface adhesion receptors [anti-P-selectin glycoprotein ligand-1 (CD162), anti-4 (CD49d), and anti-ß2 integrins (CD18)] and against platelet P-selectin (anti-CD62P), respectively.
Colony forming assay and flow cytometry
CD34+ progenitor cells were seeded onto human fibronectin (Becton Dickinson, Heidelberg, Germany) or on immobilized platelets (2x108 cells/ml) and cultivated for several days in endothelial cell growth medium MV 2 containing 5% heat-inactivated fetal calf serum, 5.0 ng/ml epidermal growth factor (EGF), 0.2 µg/ml hydrocortisone, 0.5 µg/ml VEGF, 10 ng/ml basic fibroblast factor, 20 ng/ml R3 insulin-like growth factor (IGF) -1, 1 µg/ml ascorbic acid (PromoCell, Heidelberg, Germany). After 48 h, nonadherent cells were removed. Endothelial colony-forming units were counted between days 5 and 10 (number of colonies/106 cells). Cells were washed and resuspended in PBS, incubated with polyglobin (Bayer Vital, Leverkusen, Germany) for 15 min, washed, and incubated with FITC-labeled antibodies to CD31 (clone 5.6; Beckman Coulter) and CD146 (clone 128018; R&D Systems) for 30 min at room temperature. After washing, cells were analyzed on a FACSCanto flow cytometer (Becton Dickinson).
Immunofluorescence microscopy and cell staining procedures
Immunofluorescence
CD34+ cells were coincubated 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. CD34+ cells were fixed with 2% formaldehyde solution for 20 min. Afterward cells were washed with 2% glycine, permeabilized with 0.2% Triton-X100, and incubated with PBS containing a monoclonal mouse anti-human CD68 Ab (4.7 µg/ml) for 1 h. As secondary Ab, an Alexa Fluor anti-mouse IgG1 Ab (10 µg/ml) was added for another hour; finally, DAPI staining (3.3 µg/ml) was performed to visualize cell nuclei. Unspecific binding was prevented by BSA (3%, 1 h). Rhodamine phalloidin (5 U/ml, detection of cytoskeletal actin) was applied for 30 min. For labeling, a rabbit anti-human vWF Ab (DakoCytomation) and a secondary sheep anti-rabbit Ab (Sigma) were used. Samples were analyzed by standard and confocal immunofluorescence microscopy.
Naphtyl acetate esterase staining
Cover slides were fixed in formol vapor for 4 min, incubated for 2 h in the dark, and stained for 2 h in 0.2 mol/L phosphate buffer with 0.5% sodium nitrate solution, 0.5% pararosanilin solution, and 0.3% -naphtyl acetate. After that, cover slides were washed in Aqua dest, stained for 10 min in Mayers Hemalaun solution, and for 15 min in tap water. Naphtyl acetate esterase (NSE) is specific for monocytes, macrophages, megakaryocytic cells, and platelets.
May-Gruenwald staining
CD34+ cells coincubated with platelets for 10 days on cover slides were stained with May-Gruenwald solution (Merck) for 5 min. After removal of supernatants, cells were incubated with Giemsa solution for 20 min, followed by incubation with buffer after "Weise" pH 6.8 (Merck) for another 2 min. Cover slides were analyzed by standard microscopy.
Sudan red staining
Cells were washed with PBS before each incubation step, fixed with 2% formaldehyde solution (20 min), and incubated with 0.5% Sudan red (Sigma; 20 min). Nuclei were counterstained with hematoxyline solution (Sigma) for 5 min and analyzed by standard microscopy.
Platelet phagocytosis
To visualize phagocytosis of platelets by foam cells, platelets were labeled with fluorochrome celltrackertm orange CMTMR and coincubated with CD34+ cells for 7 days in chamber slides. For LDL binding on platelets, platelets were incubated with Dil-AcLDL (red) and Mepacrine (green) for 4 h. Cells were then analyzed by flow cytometry and by standard and confocal fluorescence microscopy.
Scanning and transmission electron microscopy (TEM)
For scanning electron microscopy (SEM), CD34+ cells were cultivated on coverslips in the absence or presence of platelets (2x108 cells/ml) for 10 days. Thereafter, the cells were washed twice with PBS, and the coverslips were fixed and examined using a field emission scanning electron microscope (JSM-6300F, Jeol Ltd., Tokyo, Japan). For TEM, CD34+ cells were coincubated with isolated platelets (2x108/ml) for 10 days in culture medium. Subsequently, cells were washed and fixed with glutaraldehyde (2.5%) and tannin (0.02%) in a sodium cacodylate buffer (pH 7.4) before EM was performed (3)
.
Data presentation and statistics
Comparisons between group means were performed using Student’s t test or ANOVA, where applicable. Data are presented as mean ± SD. P 
0.05 was considered statistically significant.
RESULTS
Human CD34+ cells adhere to immobilized platelets
Recently we showed that adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells (7)
. To determine the molecular requirements of adhesion of human adult CD34+ cells to platelets, we performed experiments with human CD34+ cells isolated from cord blood. Isolated platelets (2x108/ml) were allowed to adhere to collagen-coated 96-well plates and adhesion of CD34+ cells was evaluated under static conditions, as described (7)
. We found that human CD34+ cells adhere to immobilized platelets but not to immobilized collagen type I alone, which represents the major extracellular matrix component of the injured arterial wall (Fig. 1
). Adhesion of CD34+ cells to immobilized platelets that were activated by adherence to collagen was significantly attenuated in the presence of blocking mAbs anti-CD162 or anti-CD62P, indicating that the platelet P-selectin interacts with the EPCs P-selectin glycoprotein ligand-1. Moreover, preincubation of CD34+ with the blocking monoclonal antibodies to 4-integrin (CD49d) or to ß2-integrin (CD18) resulted in a significant decrease of adherent CD34+ to immobilized platelets suggesting that both ß1- and ß2-integrins located on the surface of EPCs are involved in the adhesion process between these two types of human cells (Fig. 1)
.
|
Platelets induce distinct morphological changes of human CD34+ cells and differentiate the subpopulations of these progenitor cells into foam and endothelial cells in vitro
Next we asked whether CD34+ cells differentiate in the presence of platelets. Human CD34+ progenitor cells were isolated from umbilical cord blood and coincubated (50,000 cells) with isolated human platelets (2x108/ml). After coincubation with platelets for 5–10 days, CD34+ cells underwent substantial morphological changes. About one-third of the cells showed a 3-fold increase in size with round morphology, high granularity, and a diameter of 
25 µm (Fig. 2
A). No change in morphology of CD34+ cells could be seen during the observation period when platelets were absent (Fig. 2A
). To further characterize the granular cells, we performed May-Gruenwald, NSE staining, and CD68 immunostaining of the cultured cells. May-Gruenwald staining revealed a nonsegmented nucleus surrounded with a large cytoplasm with enhanced granularity (Fig. 2B
). These cells were positive for NSE and CD68, indicating differentiation into the macrophage/monocytic lineage (Fig. 2B
). To further characterize the intracellular granules, Sudan red III staining was performed, which was found to be positive for these large cells (Fig. 2B
). This indicates that a subpopulation of human CD34+ cells transform into large granular and lipid-rich cells, a morphology typical for foam cells.
|
Since platelets play a critical part in the capture and subsequent differentiation of murine EPCs (7)
, in the present work we found a subgroup of cells that revealed a spindle-shaped morphology and were positive for vWF (Fig. 3
A). Moreover, these cells had a substantially developed cytoskeleton characteristic of endothelial cells, but not progenitor cells, as verified by phalloidin staining (Fig. 3A
). To further evaluate the role of platelets for differentiation of CD34+ cells into endothelial cells, we cultivated isolated CD34+ cells on immobilized platelets and on immobilized fibronectin or plastic in endothelial cell growth medium. After 5 days the morphology of initially round-shaped CD34+ cells on immobilized platelets turned into adherent spindle-shaped cells that were positive for vWF (Fig. 3B
). Further, CD34+ cells formed colonies on immobilized platelets similar to immobilized fibronectin, indicating differentiation into endothelial cells (Fig. 3B
). In contrast, virtually no colonies were formed on plastic (Fig. 3B
). The number of colonies formed on immobilized platelets was significantly higher compared with plastic wells (number of colonies/106 cells, mean±SD: 38.4±7.17 platelets vs. plastic 6.63±8.78, P0.05) (Fig. 3B
). Moreover, CD34+ cells cultivated on immobilized platelets were positive for the endothelial markers CD146 and CD31 (Fig. 3C
).
|
Phagocytosis of platelets is involved in foam cell generation derived from CD34+ progenitor cells
As shown above, platelets induced foam cell generation derived from a subpopulation of human CD34+ progenitor cells in vitro (Fig. 4
A, B). Phase contrast microscopy showed that these foam cells are surrounded by a platelet-free zone, indicating enhanced phagocytotic activity of these cells (Fig. 4A
, Supplemental Movie 1). TEM of the foam cells revealed the presence of multiple vesicles with phagocytosed platelets or platelet fragments (Fig. 4B
). Next we asked whether platelets are internalized into macrophages/foam cells. Therefore, platelets were labeled with cell tracker orange and added to the platelet/CD34+ coculture (Fig. 4C
). Internalization of platelets occurred rapidly, and after 24 h a substantial number of platelets were internalized by foam cells (Fig. 4C
).
|
Uptake of modified LDL by macrophages plays an important role in the formation of foam cells, the early step of atherosclerosis (17)
. Platelets bind LDL, which in turn leads to enhanced platelet responsiveness (18)
. Therefore, we asked whether platelets mediate LDL uptake into foam cells. To visualize LDL binding, platelets were incubated with the fluorochrome-conjugated acetylated LDL (Dil-AcLDL) (red) and with mepacrine (green), which specifically accumulates in dense granules of platelets. We found that platelets bound and internalized substantial amounts of Dil-AcLDL, as verified by fluorescence microscopy and flow cytometry (Fig. 5
A). When Dil-AcLDL-labeled platelets were added to the platelet/CD34+ coculture, a significant uptake of LDL-positive platelets into foam cells was observed (Fig. 5B
), whereas virtually no uptake of LDL-labeled platelets was observed in cells that did not differentiate into foam cells (Fig. 5B
).
|
Inhibitors of HMG coenzyme A reductase (statins) reduce platelet-induced foam cell generation
HMG coenzyme A reductase inhibitors (statins) are potent drugs for the prevention of atherosclerotic disease. It is increasingly being recognized that statins reduce atheroprogression beyond mere lipid-lowering effects (19)
. Therefore, we evaluated the effect of various statins on platelet-mediated foam cell generation in vitro. We found that all compounds tested, including pravastatin, simvastatin, fluvastatin, and atorvastatin, substantially reduced platelet-mediated foam cell formation (Fig. 6
A, B). Pravastatin, however, was less potent in inhibiting platelet-mediated foam cell formation than the other compounds tested (Fig. 6A, B
). Statins have been shown to reduce secretion of MMPs from vascular cells, implying an antiatherosclerotic pleiotropic effect of these compounds (20)
. To evaluate the effect of statins on secretion of MMPs in our cocultures, CD34+ cells were cocultured with platelets in the absence and presence of various statins. We found that all tested statins substantially reduced MMP-9 activity in the culture supernatant (Fig. 6C
). Again, as noted above for foam cell generation, the inhibitory effect of pravastatin was less pronounced than that of simvastatin, fluvastatin, or atorvastatin (Fig. 6C
).
|
Inhibition of platelet-mediated foam cell formation from CD34+ cells and secretion of matrix metalloproteinase-9 by PPAR agonists
Peroxisome proliferator-activated receptors are nuclear receptors that regulate lipid and Glc metabolism as well as cellular differentiation (17)
. Agonists of PPARs have been found to reduce foam cell generation both in vitro (21)
and in vivo (22)
. To investigate the potential importance of PPAR on platelet-mediated foam cell generation, we performed a systematic analysis of the effects of various PPAR- (fenofibrate, WY14643) and - (troglitazone, GW1929) agonists. Both types of PPAR agonists reduced platelet-mediated foam cell formation (Fig. 7
). A less pronounced reduction in foam cell generation was observed in the presence of PPAR- agonists compared with PPAR- agonists (Fig. 7)
. In concert with their effects on foam cell generation, PPAR- and PPAR- agonists inhibited MMP-9 activity of the cell culture supernatant (Fig. 7)
. However, peptide MMP inhibitor GM6001, applied in different concentrations (1 and 20 µg/ml), did not attenuate foam cell generation (data not shown).
|
DISCUSSION
In this study we show that platelets regulate recruitment and transformation of foam cells and endothelial cells out of CD34+ progenitor cells. The major findings of the present study are 1) collagen-adherent platelets recruit CD34+ progenitor cells via specific adhesion receptors, including P-selectin/PSGL-1, ß1-, and ß2-integrins; 2) collagen-adherent and thereby activated platelets induce transformation of CD34+ progenitor cells into mature foam cells and endothelial cells; 3) HMG coenzyme A reductase inhibitors reduce platelet-induced foam cell generation derived from CD34+ progenitor cells via reduction of secretion of MMP-9; and 4) agonists of PPAR- and - attenuate platelet-induced foam cell generation and production of MMP-9.
These findings imply that interaction of platelets with circulating progenitor cells is important for repair mechanisms at the site of vascular lesions. It is tempting to speculate that an altered balance of platelet-mediated transformation of CD34+ progenitor cells into macrophages/foam cells and endothelial cells may play a critical role in atherogenesis and atheroprogression. Understanding and modulating these mechanisms may be a promising way to treat patients at high risk for atherosclerotic diseases.
The maintenance of vascular integrity is crucial for preventing atheroprogression. Circulating progenitor cells have been shown to instigate new vessel formation via angiogenesis and neovascularization but also have the potential to provide ongoing vascular repair by homing to site of vascular damage (5)
. However, the mechanisms that recruit circulating progenitor cells toward vascular lesions and regulate repair mechanisms are not understood completely. Platelets are the first circulating blood cells that interact with the injured vessel wall; they accumulate within seconds to sites of vascular injury and release a variety of potent chemotactic factors (e.g., SDF-1) (23
, 24)
. Thus, platelets induce recruitment of circulating blood cells, including progenitor cells toward sites of vessel injury. Here we show that human EPCs—these are still >98% positive for CD34+—adhere to immobilized platelets via P-selectin/PSGL-1 and ß1- and ß2-integrins. In the present study we focused on the potential role of platelets in the CD34+ transformation. When adherent human CD34+ were cocultivated with platelets for 1 wk, we found that a substantial number of CD34+ cells (one-third) transform into foam cells.
Foam cell generation from macrophages with subsequent fatty streak formation plays a key role in early atherogenesis (17)
. Foam cell formation is thought to be induced by low density lipoproteins (LDL), including oxidized LDL (OxLDL) or minimally modified LDL (mmLDL) (17)
. LDL binds to and activates platelets (25)
. We found that modified LDL (Dil-AcLDL) is taken up by platelets and stored specifically in mepacrine-containing organelles, thus in dense granules. Moreover, we demonstrate that platelets labeled with Dil-AcLDL are rapidly internalized into foam cells, as shown by electron and fluorescence microscopy. Thus, platelets may be a major vehicle for LDL, and phagocytosis of platelet/LDL may be a critical step during foam cell generation. This conclusion is supported by recent findings showing substantial platelet phagocytosis of macrophages both in vitro and in vivo (26
, 27)
.
Anti-inflammatory pleiotropic effects of statins are increasingly recognized to play a central role in the antiatherosclerotic activities of these drug compounds (19)
. Statins lower the expression and function of (MMPs in atherogenic cells, including macrophages, a major source of MMPs in lesions (19)
. Platelets have been shown to induce secretion of MMP-9 by monocytes (28)
. We found that all statins tested reduced platelet-induced foam cell generation derived from CD34+ progenitor cells. Moreover, we found that the reduction of foam cell generation was paralleled by reduced secretion of MMP-9, a proteinase critically involved in foam cell generation and atheroprogression (20
, 29
, 30)
. PPAR- and - regulate the expression of MMPs (31)
. We demonstrate that PPAR- and - agonists reduce MMP-9 secretion and foam cell generation. Thus, we conclude that MMP-9 may play a critical part in platelet-mediated foam cell generation derived from progenitor cells. However, a MMP inhibitor did not attenuate foam cell generation.
We cannot at this time provide direct evidence that platelet-mediated recruitment and differentiation of progenitor cells into foam cells and endothelial cells play a critical physiological or pathophysiological role in humans. However, only recently we found that platelets recruit EPCs to vascular lesions in mice in vivo (23)
. Platelet-mediated progenitor cell recruitment may help repair mechanisms at endothelial lesions and may favor regeneration of vascular lesions. Under pathophysiological conditions, CD34+ may undergo transformation in the presence of activated platelets. The balance between platelet-mediated endothelial cell regeneration and foam cells generation derived from CD34+ EPCs may determine the degree and progression of atherosclerosis (Fig. 8
).
|
ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Heike Runge, Iris Schäfer, Sandra Hippauf, and Jadwiga Kwiatkowska. The study was supported by grants of the Deutsche Forschungsgemeinschaft (Graduiertenkolleg (GK 794) "Zellbiologische Mechanismen immunassoziierter Prozesse"), Karl und Lore Klein Stiftung, and Novartisstiftung to M.G. We are grateful to Professor Rastetter, Technische Universität München, for continuous scientific advice.
Received for publication April 13, 2006. Accepted for publication July 17, 2006.
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