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Platelets induce differentiation of human CD34⁺ progenitor cells into foam cells and endothelial cells

Karin Daub^*, Harald Langer^*, Peter Seizer^*, Konstantinos Stellos^*, Andreas May^*, Pankaj Goyal, Boris Bigalke^*, Tanja Schönberger^*, Tobias Geisler^*, Dorothea Siegel-Axel^*, Robert A. J. Oostendorp, Stephan Lindemann^* and Meinrad Gawaz^*,1

^* 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

SPECIFIC AIMS

Beyond their role in hemostasis and thrombosis, platelets play a critical role in atherogenesis. They interact with the endothelium and induce alterations of chemotactic and adhesive properties of endothelial cells, a critical step in atherogenesis. The aim of this study was to evaluate the effect of platelets on recruitment and differentiation of human CD34⁺ progenitor cells into foam cells and endothelial cells. We demonstrate a potentially important mechanism that regulates the balance of vascular repair mechanisms and atherogenesis.

PRINCIPAL FINDINGS

1. Human CD34⁺ cells adhere to immobilized platelets under static conditions
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. 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. Adhesion of CD34⁺ cells to immobilized platelets was significantly attenuated in the presence of blocking mAbs anti-CD162 or anti-CD62P, indicating that the platelet P-selectin interacts with the endothelial progenitor cell (EPC), P-selectin glycoprotein ligand-1. Moreover, preincubation of CD34⁺ with blocking monoclonal antibodies to ₄-integrin (CD49d) or to ßbeta;₂-integrin (CD18) resulted in a significant decrease of adherent CD34⁺ to immobilized platelets, suggesting that both ßbeta;₁- and ßbeta;₂-integrins located on the surface of EPCs are involved in the adhesion process between these two types of human cells (Fig. 1 A).

View larger version (134K): [in this window] [in a new window]   Figure 1. Cocultivation of CD34+ cells with platelets induces foam cell generation in vitro. CD34+ cells were coincubated with human platelets (2x108/ml) in 96-well plates for up to 10 days. After 5 days CD34+ cells underwent substantial morphological changes, with an increase in diameter to 25 µm and high granularity. In the absence of platelets, no change in the morphology of CD34+ cells could be observed. A significant phagocytosis of LDL-laden platelets by foam cells was noted, indicated by the platelet-free halo around the CD34+ cell (circle).

2. 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. 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. 1B ). No change in the morphology of CD34⁺ cells was observed during the period when platelets were absent. To further characterize the cells, May-Gruenwald staining revealed a nonsegmented nucleus surrounded by a large cytoplasm with enhanced granularity and NSE and CD68 staining, indicating differentiation into the macrophage/monocytic lineage (Fig. 1B ). Moreover, Sudan red III staining indicates that CD34⁺ cells transform into large granular and lipid-rich cells, comparable to foam cells.

In the present work, we found that CD34⁺ cells formed colonies on immobilized platelets similar to immobilized fibronectin, indicating transformation into endothelial cells. In contrast, virtually no colonies were formed on plastic (Fig. 1C ). The cells cultivated on immobilized platelets were positive for the endothelial markers CD146 and CD31, but also for von Willebrand factor and phalloidin.

3. Phagocytosis of platelets is involved in foam cell generation derived from CD34⁺ progenitor cells
As above, platelets induced foam cell generation derived from a subpopulation of human CD34⁺ progenitor cells in vitro. Foam cells are surrounded by a platelet-free zone, indicating enhanced phagocytotic activity of these cells. Transmission electron microscopy of the foam cells revealed the presence of multiple vesicles with phagocytosed platelets or platelet fragments. Moreover, platelets were labeled with a fluorochrome and added to the platelet/CD34⁺ coculture. Internalization of platelets occurred rapidly; after 24 h a substantial number of platelets were internalized by foam cells.

Uptake of modified LDL by macrophages plays an important role in the formation of foam cells, an early step of atherosclerosis. We were tempted to speculate that platelets bind LDL, so we asked whether platelets mediate LDL uptake into foam cells. We found that platelets bound and internalized substantial amounts of Dil-AcLDL, as verified by fluorescence microscopy and flow cytometry. When Dil-AcLDL-labeled platelets were added to the platelet/CD34⁺ coculture, a significant uptake of LDL-positive platelets into foam cells was observed, whereas virtually no uptake of LDL-labeled platelets was observed in cells that did not differentiate into foam cells (Fig. 1D ).

4. Inhibitors of HMG coenzyme A reductase (statins) reduce platelet-induced foam cell generation and promote formation of endothelial cells derived from CD34⁺ progenitor cells
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. 2 ). Pravastatin, however, was less potent in inhibiting platelet-mediated foam cell formation than the other compounds (Fig. 2A ). We also found that all tested statins substantially reduced MMP-9 activity in the culture supernatant. Again, as noted for foam cell generation, the inhibitory effect of pravastatin was less pronounced than that of simvastatin, fluvastatin, or atorvastatin.

View larger version (10K): [in this window] [in a new window]   Figure 2. Inhibition of platelet-mediated foam cell formation from CD34+ cells and secretion of matrix metalloproteinase-9 by statins and PPAR agonists. CD34+ cells were coincubated with freshly isolated platelets in 96-well plates for 10 days. Cells were incubated with different statins. Fluvastatin, atorvastatin, and simvastatin (1 µM+10 µM) resulted in complete inhibition of foam cell generation whereas pravastatin (10 µM) was less potent.

5. Inhibition of platelet-mediated foam cell formation from CD34⁺ cells and secretion of matrix metalloproteinase-9 by peroxisome proliferator-activated receptor (PPAR) agonists
To investigate the potential importance of PPAR on platelet-mediated foam cell generation, we systematically analyzed the effects of various PPAR- (fenofibrate, WY14643) and - (troglitazone, GW1929) agonists. Both types of PPAR agonists reduced platelet-mediated foam cell formation (Fig. 2B ). A less pronounced reduction in foam cell generation was observed in the presence of PPAR- agonists than with PPAR- agonists (Fig. 2B ). In concert with their effects on foam cell generation, PPAR- and PPAR- agonists inhibited MMP-9 activity of the cell culture supernatant.

CONCLUSIONS AND SIGNIFICANCE

In this study we show that platelets regulate recruitment and differentiation of CD34⁺ progenitor cells into foam cells and endothelial cells. The findings imply that interaction of platelets with circulating progenitor cells is important for repair mechanisms at the site of vascular lesions. An altered balance of platelet-mediated differentiation of CD34⁺ progenitor cells of macrophages/foam cells and endothelial cells may play a critical role for atherogenesis and atheroprogression. When adherent human EPCs (>98% positive for CD34) were cocultivated with platelets for 1 wk, we found that a significant number of CD34⁺ cells (one-third) differentiated into foam cells.

Foam cell formation from macrophages with subsequent fatty streak formation plays a key role in early atherogenesis and is thought to be induced by low density lipoproteins (LDL), including oxidized LDL or minimally modified LDL. LDL binds to and activates platelets. 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 demonstrated that platelets labeled with Dil-AcLDL are rapidly internalized into foam cells as shown by electron and fluorescence microscopy. Thus, it is tempting to speculate that platelets are a major vehicle for LDL and that phagocytosis of platelet/LDL is a critical step during foam cell generation.

Anti-inflammatory pleiotropic effects of statins are increasingly recognized to play a central role in the antiatherosclerotic activities of these drug compounds. Statins lower the expression and function of matrix metalloproteinases (MMPs) in atherogenetic cells, including macrophages, a major source of MMPs in lesions. Platelets have been shown to induce secretion of MMP-9 by monocytes. We found that all tested statins reduced platelet-induced foam cell generation derived from CD34⁺ progenitor cells. Moreover, we found that the reduction of foam cell generation was paralleled by a reduced secretion of MMP-9, a proteinase critically involved in foam cell generation and atheroprogression. We also show that agonists of the PPARs and (PPARs) regulate expression of MMPs and reduce MMP-9 secretion and foam cell generation. Thus, we conclude that MMP-9 plays a critical part in platelet-mediated foam cell generation derived from progenitor cells.

Currently, we cannot 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. Nevertheless, only recently we found that platelets recruit EPCs to vascular lesions in mice in vivo (Massberg et al., unpublished results). Platelet-mediated progenitor cell recruitment may help repair mechanisms at endothelial lesions and favor regeneration of vascular lesions. Under pathophysiological conditions, EPCs may undergo differentiation in the presence of activated platelets. The balance between platelet-mediated endothelial cell regeneration and foam cell generation derived from CD34⁺ EPCs may determine the degree and progression of atherosclerosis.

We show that platelets induce recruitment and differentiation of CD34⁺ progenitor cells into foam cells and endothelial cells. Thus, it is tempting to speculate that LDL binds to platelets and that phagocytosis of platelet/LDL is a critical step during foam cell generation. Foam cell formation from macrophages with subsequent fatty streak formation affects proatherogenic cells, which could be inhibited by HMG coenzyme A reductase inhibitors (statins) and agonists of PPAR- and -. Platelet-mediated progenitor cell recruitment and differentiation into endothelial cell may favor regeneration of vascular lesions and affect antiatherogenic properties.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic diagram. The balance between platelet-mediated endothelial cell regeneration and foam cell generation derived from CD34+ progenitor cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6265fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6265fjev1 20/14/2559    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Daub, K. Articles by Gawaz, M. Search for Related Content PubMed PubMed Citation Articles by Daub, K. Articles by Gawaz, M.