Bjoern F. Kraemer

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.

Lessons to be learned from primary renal cell carcinomas: novel tumor antigens and HLA ligands for immunotherapy

The lack of sufficient well-defined tumor-associated antigens is still a drawback on the way to a cytotoxic T-lymphocyte-based immunotherapy of renal cell carcinoma (RCC). We are trying to define a larger number of such targets by a combined approach involving HLA ligand characterization by mass spectrometry and gene expression profiling by oligonucleotide microarrays. Here, we present the results of a large-scale analysis of 13 RCC specimens. We were able to identify more than 700 peptides, mostly from self-proteins without any evident tumor association. However, some HLA ligands derived from previously known tumor antigens in RCC. In addition, gene expression profiling of tumors and a set of healthy tissues revealed novel candidate RCC-associated antigens. For several of them, we were able to characterize HLA ligands after extraction from the tumor tissue. Apart from universal RCC antigens, some proteins seem to be appropriate candidates in individual patients only. This underlines the advantage of a personalized therapeutic approach. Further analyses will contribute additional HLA ligands to this repertoire of universal as well as patient-individual tumor antigens.

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.

Statins do not adversely affect post-interventional residual platelet aggregation and outcomes in patients undergoing coronary stenting treated by dual antiplatelet therapy

AIMS There are growing data suggesting a clinical relevance of residual platelet aggregation (RPA) in patients undergoing PCI. Drug-drug interaction of statins and clopidogrel has been controversially discussed in ex vivo studies and clinical trials. The aim of the present study was to investigate the effects of peri-procedural statin medication on the metabolization of aspirin and clopidogrel with regard to platelet aggregation and clinical outcome in patients undergoing coronary intervention. METHODS AND RESULTS Patients with coronary stenting for symptomatic coronary artery disease are routinely evaluated by platelet function analysis in a monocentre registry, and for the present study, a consecutive cohort of 1155 patients were analysed. About 87.7% of the patients were treated with statins at the time of platelet function analysis. Residual platelet activity assessed by adenosine diphosphate (20 micromol/L)-induced platelet aggregation was not significantly influenced by statin treatment. Nor the significant effects of CYP3A4-metabolization pathway on post-treatment aggregation were recorded, although there was even a trend to lower RPA values in patients treated with CYP3A4-metabolized statins. Further, in an inter-individual analysis comparing patients treated with CYP3A4- and non-CYP3A4-metabolized statins, no time-dependent difference of clopidogreĺs anti-aggregatory effects was observed. Clinical follow-up of major adverse events (myocardial infarction, ischaemic stroke, death) in 991 patients within 3 months revealed no significant adverse effects of statin treatment on clinical outcome. Instead, statin treatment was independently associated with lower incidence of composite events (HR 0.44, 95% confidence interval 0.23-0.83, P = 0.01). CONCLUSION Peri-procedural co-administration of statins does not increase the post-interventional RPA in cardiovascular patients treated with dual antiplatelet therapy and does not worsen the clinical prognosis of these patients.

SGK1 Sensitivity of Platelet Migration

Recent observations pointed to the ability of platelets to migrate and thus to invade the inflamed vascular wall. Platelet migration could be stimulated by stromal cell-derived factor-1 (SDF-1), an effect dependent on phosphatidylinositide-3-kinase (PI3K) and paralleled by activation and phosphorylation of Wiskott-Aldrich syndrome protein (WASP). Migration is inhibited by vinculin, which is similarly regulated by phosphorylation. PI3K-sensitive kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored whether SGK1 modifies WASP and vinculin phosphorylation in murine platelets and participates in the regulation of platelet migration. Platelets were isolated from gene-targeted mice lacking SGK1 (sgk1-/-) and from their wild type littermates (sgk1+/+). Platelet migration stimulated with SDF-1 was significantly less pronounced in sgk1-/-platelets than in sgk1+/+ platelets. Moreover, SDF-1 significantly induced WASP phosphorylation, an effect again reduced in platelets lacking SGK1. Phosphorylation of vinculin was significantly enhanced in sgk1-/-platelets and was significantly reduced following treatment of platelets with Ca2+ chelator BAPTA. Immunohistochemical analysis of in vivo experiments in intestinal vessels after vascular inflammation revealed that transmigration of platelets into inflamed vessel walls was significantly less pronounced in sgk1-/-than in sgk1+/+ mice. In conclusion, SGK1 is a powerful regulator of platelet migration.

High shear flow induces migration of adherent human platelets

Shear forces are generated in all parts of the vascular system and contribute directly and indirectly to vascular disease progression. Endothelial cells are able to adapt to flow conditions, and are known to polarize and migrate in response to shear forces. Platelets exposed to shear stress are activated and release bioactive molecules from their alpha granules. So far, platelets have been considered to be static cells that do not leave the site of tight adhesion. However, we have recently been able to demonstrate the capacity of platelets to migrate in response to stromal derived factor-1 (SDF-1). In this project, we have demonstrated that platelets accumulate in areas with a high concentration of SDF-1 under flow conditions and respond to high shear stress by cellular polarization, cytoskeletal reorganisation, and flow-directed migration. In this context, we have shown increased Wiskott-Aldrich Syndrome protein (WASP) phosphorylation and intracellular redistribution of focal adhesion kinase (FAK) under high-shear stress conditions. The effect of flow-induced platelet migration has not previously been recognized and offers a new role for platelets as mobile cells. Their migratory potential may enable platelets to cover intimal lesions and contribute to vascular repair.

Ion channels in the regulation of platelet migration

Platelets have been shown to migrate and thus to invade the vascular wall. Platelet migration is stimulated by SDF-1. In other cell types, migration is dependent on Ca(2+) entry via Ca(2+) channels. Ca(2+) influx is sensitive to cell membrane potential which is maintained by K(+) channel activity and/or Cl(-) channel activity. The present study explored the role of ion channels in the regulation of SDF-1 induced migration. Platelets were isolated from human volunteers as well as from gene targeted mice lacking the Ca(2+) activated K(+) channel SK4 (sk4(-/-)) and their wild type littermates (sk4(+/+)). According to confocal microscopy human platelets expressed the Ca(2+) channel Orai1 and the Ca(2+)-activated K(+) channel K(Ca)3.1 (SK4). SDF-1 (100 ng/ml) stimulated migration in human platelets, an effect blunted by Orai1 inhibitors 2-aminoethoxydiphenyl borate 2-APB (10 μM) and SKF-96365 (10 μM), by unspecific K(+) channel inhibitor TEA (30 mM), by SK4 specific K(+) channel blocker clotrimazole (10 μM), but not by Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid NPPB (100 μM). Significant stimulation of migration by SDF-1 was further observed in sk4(+/+) platelets but was virtually absent in sk4(-/-) platelets. In conclusion, platelet migration requires activity of the Ca(2+) channel Orai1 and of the Ca(2+) activated K(+) channel SK4, but not of NPPB-sensitive Cl(-) channels.

Persistent Troponin Elevation in a Patient with Cardiac Amyloidosis

A 79‐year‐old patient repeatedly presented with chest discomfort and dyspnea on exertion. With echocardiography a prominent left ventricular and septal hypertrophy was detected with reduced left ventricular function. Despite successful revascularization and excellent results after stenting, the patient showed persistently elevated troponin levels. To investigate the abnormal findings of persistent troponin elevation, septal hypertrophy, and heart failure we performed endomyocardial biopsies which showed widespread myocardial amyloidosis. Amyloid subtyping revealed transthyretin amyloidosis. This is the first case showing persistent troponin elevation in a patient with tranthyretin amyloidosis. Very few other cases have been published on the topic of cardiac amyloidosis and troponin elevation so far. Our case serves as an illustrating example in the differential diagnosis of nonischemic causes of persistent troponin elevation. It is important to consider cardiac amyloidosis in patients with troponin elevation and heart failure since the clinical management differs significantly from other causes of heart failure. Copyright