Stephan Lindemann

The evolving role of platelets in inflammation

Summary.  Platelets are small in size and simple in structure. Nevertheless, these anucleate cytoplasts utilize complex molecular systems to regulate a variety of biological functions. Here we review evolutionary paths, traditional roles, and previously unrecognized biological capacities of platelets that interface thrombosis with inflammation and potentially identify new roles in inflammatory diseases.

Platelet-Derived Stromal Cell–Derived Factor-1 Regulates Adhesion and Promotes Differentiation of Human CD34+ Cells to Endothelial Progenitor Cells

Background— Peripheral homing of progenitor cells in areas of diseased organs is critical for tissue regeneration. The chemokine stromal cell–derived factor-1 (SDF-1) regulates homing of CD34+ stem cells. We evaluated the role of platelet-derived SDF-1 in adhesion and differentiation of human CD34+ cells into endothelial progenitor cells. Methods and Results— Adherent platelets express substantial amounts of SDF-1 and recruit CD34+ cells in vitro and in vivo. A monoclonal antibody to SDF-1 or to its counterreceptor, CXCR4, inhibits stem cell adhesion on adherent platelets under high arterial shear in vitro and after carotid ligation in mice, as determined by intravital fluorescence microscopy. Platelets that adhere to human arterial endothelial cells enhance the adhesion of CD34+ cells on endothelium under flow conditions, a process that is inhibited by anti-SDF-1. During intestinal ischemia/reperfusion in mice, anti-SDF-1 and anti-CXCR4, but not isotype control antibodies, abolish the recruitment of CD34+ cells in microcirculation. Moreover, platelet-derived SDF-1 binding to CXCR4 receptor promotes platelet-induced differentiation of CD34+ cells into endothelial progenitor cells, as verified by colony-forming assays in vitro. Conclusions— These findings imply that platelet-derived SDF-1 regulates adhesion of stem cells in vitro and in vivo and promotes differentiation of CD34+ cells to endothelial progenitor cells. Because tissue regeneration depends on recruitment of progenitor cells to peripheral vasculature and their subsequent differentiation, platelet-derived SDF-1 may contribute to vascular and myocardial regeneration.

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

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 β1‐ and β2‐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 metallo‐proteinase‐9 (MMP‐9) secretion. Finally, agonists of peroxisome proliferator‐activated receptor‐α and ‐γ 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. FASEB J. 20, E1935–E1944 (2006)

Novel Anti-bacterial Activities of β-defensin 1 in Human Platelets: Suppression of Pathogen Growth and Signaling of Neutrophil Extracellular Trap Formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBDs are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.

Platelets, inflammation and atherosclerosis

Summary.  An expanding body of evidence continues to build on the role of platelets as initial actors in the development of atherosclerotic lesions. Platelets bind to leukocytes and endothelial cells, and initiate monocyte transformation into macrophages. Platelets internalize oxidized phospholipids and promote foam cell formation. Platelets also recruit progenitor cells to the scene that are able to differentiate into foam cells or endothelial cells depending on conditions. Platelets tip the scales in the initiation, development and total extent of atherosclerotic lesions.

Expression of stromal-cell-derived factor-1 on circulating platelets is increased in patients with acute coronary syndrome and correlates with the number of CD34+ progenitor cells

AIMS Previous experimental studies have suggested that platelet stromal-cell-derived factor-1 (SDF-1) regulates mobilization and recruitment of haematopoietic progenitor cells supporting revascularization in mice. However, there are no clinical data available regarding platelet-bound SDF-1 in patients with acute coronary syndrome (ACS). The objective of this study was to evaluate the platelet-surface expression of SDF-1 in patients with ACS. METHODS AND RESULTS Patients with ACS (n = 418) showed a significantly enhanced SDF-1 expression on admission compared with those with stable angina pectoris (SAP, n = 486) [SAP (mean fluorescence intensity (MFI) +/- SD): 13.48 +/- 5.27; ACS: 18.45 +/- 12.85; P < 0.001) independent of cardiovascular risk factors and medication. Enhanced platelet-bound SDF-1 expression was found in patients with reduced left ventricular ejection fraction (LVEF <55%) in comparison to patients with normal LVEF (P = 0.005). Platelet-bound SDF-1 expression positively correlated with the degree of platelet activation [CD62P: r = 0.325; glycoprotein VI (GPVI): r = 0.277; PAC-1: r = 0.501; P < 0.001 for all] and showed a significant, but slight association with plasma levels of SDF-1 (r = 0.084; P = 0.045). In a subgroup of patients with coronary artery disease, platelet-bound SDF-1, but not other platelet activation markers, significantly correlated with the number of circulating CD34(+) progenitor cells (r = 0.252; P = 0.002) or CD34(+)/CD133(+) endothelial progenitor cells (r = 0.352; P = 0.008). CONCLUSION Platelet-bound SDF-1 may play an important role in peripheral homing of circulating progenitor cells thus in tissue regeneration.

EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation

UNLABELLED Upon coincubation with platelets, CD34(+) progenitor cells have the potential to differentiate into foam cells, and thereby may promote the progression of atherosclerosis. The exact mechanism of MMP-regulation during the cellular differentiation process to foam cells is still unclear. Thus, we investigated the role of EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) during foam cell formation originating from both monocytes/macrophages and CD34(+) progenitor cells. METHODS AND RESULTS Differentiation of CD34(+) progenitor to foam cells was analyzed in a coculture model of progenitor cells and platelets. While CD34(+) cells did not express EMMPRIN or MT1-MMP, mature foam cells strongly expressed EMMPRIN, which was associated with MT1-MMP expression as well as MMP-9. Gene silencing of EMMPRIN by siRNA during the cell differentiation process hindered not only the upregulation of MMPs (MT1-MMP, MMP-9), but also the secretion of the cytokine M-CSF. During the differentiation process CyPA was substantially released into the supernatant. The presence of the CyPA inhibitor NIM811 significantly reduced MMP-9 secretion during the differentiation process. Similar results were obtained using the classical pathway of foam cell formation by coincubating human macrophages with AcLDL. Additionally, the presence of soluble EMMPRIN ligands (CyPA, recombinant EMMPRIN) further enhanced MMP-9 secretion by mature foam cells. Consistently, CyPA and EMMPRIN were found in atherosclerotic plaques of ApoE-deficient mice by immunohistochemistry. CONCLUSION EMMPRIN is upregulated during the differentiation process from CD34(+) progenitor cells to foam cells, whereas its ligand, CyPA, is released. The CyPA/EMMPRIN activation pathway may play a relevant role in promoting the vulnerability of atherosclerotic plaques.

Expression of platelet glycoprotein VI is associated with transient ischemic attack and stroke

Background and purpose:  Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation. We aimed to investigate GPVI in patients presenting with symptoms of acute cerebrovascular disease and to define GPVI as biomarker for acute stroke.

Prostacyclin inhibits adhesion of polymorphonuclear leukocytes to human vascular endothelial cells due to adhesion molecule independent regulatory mechanisms.

Abstract. Prostacyclin is an important endothelial mediator involved in the interaction of neutrophils (PMN) with the vessel wall. Many studies have shown the beneficial effects of prostacyclin in ischemia and reperfusion. However, no previous study has investigated the direct effects of the prostacyclin analogs iloprost (ILO) and alprostadil (PGE1) on the endothelial part of the adhesion process.Human umbilical vein endothelial cells (HUVECs) were grown to confluence, stimulated with 300 U/ml TNF-α and treated with increasing concentrations of ILO and PGE1. The cells were washed to remove TNF and the inhibitors and adhesion of fluorescence-green labeled PMN was determined microscopically. ICAM-1, VCAM-1 and E-selectin expression were measured by a cell-surface ELISA. The chemoattractant activity of the endothelial cell releasate was tested in a Boyden chamber.ILO and PGE1 reduced PMN-adhesion in a concentration-dependent manner (ILO: –54 ± 9 % at 0.5 μM, PGE1: –46 ± 10 % at 10 μM). However, the surface expression of ICAM-1, VCAM-1 and E-selectin remained unaltered. When the supernatant of iloprost/PGE1-treated cells was transferred onto cells that were activated, but not treated with ILO or PGE1, the reduction of PMN adhesion remains sustained. These data indicate that the inhibitory effect of ILO/ PGE1 treatment is achieved by a reduced chemoattractant potential. PAF-antagonists were able to block neutrophil adhesion and mimicked the effect of ILO, while exogenous PAF diminished the inhibitory effect of ILO concentration-dependently. This study demonstrates the beneficial effects of ILO and PGE1 on inflammatorily activated endothelial cells. These prostacyclin analogs inhibit PMN-adhesion despite maximal adhesion molecule expression by regulating the balance of - yet to be determined - endothelial-derived mediators.

PI3 kinase-dependent stimulation of platelet migration by stromal cell-derived factor 1 (SDF-1)

Platelets have been regarded as static cells that do not move once they adhere to a matrix. The present study explored, whether platelets are able to migrate. In contrast to the current opinion, we found that platelets were mobile, able to migrate over a surface, and transmigrate through a transwell membrane and endothelium toward a source of stromal cell-derived factor 1 (SDF-1). Platelet migration was stimulated by SDF-1, which led to the downstream activation and phosphorylation of Wiskott–Aldrich syndrome protein. SDF-1 signaling and subsequent platelet migration could be inhibited by CXCR4-receptor blocker AMD3100, pertussis toxin, inhibition of phosphoinositol 3-kinase (PI3 kinase) with LY294002 or wortmannin, and disruption of actin polymerization with cytochalasin B. The potential of platelets to migrate in an SDF-1-mediated fashion may redefine the role of platelets in the pathophysiology of vascular inflammation, subsequent atherosclerotic degeneration, and vascular regeneration.