Konstantinos Stellos

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.

The Residual Platelet Aggregation after Deployment of Intracoronary Stent (PREDICT) score

Summary.  Background: Recent studies suggest a high interindividual variability of response to clopidogrel associated with adverse cardiovascular outcome. Different clinical factors are considered to influence a persistent residual platelet aggregation (RPA) despite conventional antiplatelet therapy.

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)

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.

Identification and Characterization of Hypoxia-Regulated Endothelial Circular RNA

RATIONALE Circular RNAs (circRNAs) are noncoding RNAs generated by back splicing. Back splicing has been considered a rare event, but recent studies suggest that circRNAs are widely expressed. However, the expression, regulation, and function of circRNAs in vascular cells is still unknown. OBJECTIVE Here, we characterize the expression, regulation, and function of circRNAs in endothelial cells. METHODS AND RESULTS Endothelial circRNAs were identified by computational analysis of ribo-minus RNA generated from human umbilical venous endothelial cells cultured under normoxic or hypoxic conditions. Selected circRNAs were biochemically characterized, and we found that the majority of them lacks polyadenylation, is resistant to RNase R digestion and localized to the cytoplasm. We further validated the hypoxia-induced circRNAs cZNF292, cAFF1, and cDENND4C, as well as the downregulated cTHSD1 by reverse transcription polymerase chain reaction in cultured endothelial cells. Cloning of cZNF292 validated the predicted back splicing of exon 4 to a new alternative exon 1A. Silencing of cZNF292 inhibited cZNF292 expression and reduced tube formation and spheroid sprouting of endothelial cells in vitro. The expression of pre-mRNA or mRNA of the host gene was not affected by silencing of cZNF292. No validated microRNA-binding sites for cZNF292 were detected in Argonaute high-throughput sequencing of RNA isolated by cross-linking and immunoprecipitation data sets, suggesting that cZNF292 does not act as a microRNA sponge. CONCLUSIONS We show that the majority of the selected endothelial circRNAs fulfill all criteria of bona fide circRNAs. The circRNA cZNF292 exhibits proangiogenic activities in vitro. These data suggest that endothelial circRNAs are regulated by hypoxia and have biological functions.

The Inflammatory Chemokine CXC Motif Ligand 16 Triggers Platelet Activation and Adhesion Via CXC Motif Receptor 6–Dependent Phosphatidylinositide 3-Kinase/Akt Signaling

Rationale: The recently discovered chemokine CXC motif ligand 16 (CXCL16) is highly expressed in atherosclerotic lesions and is a potential pathogenic mediator in coronary artery disease. Objective: The aim of this study was to test the role of CXCL16 on platelet activation and vascular adhesion, as well as the underlying mechanism and signaling pathway. Methods and Results: Reverse-transcriptase polymerase chain reaction, Western blotting, confocal microscopy, and flow cytometry revealed that CXCL16-specific receptor, CXC motif receptor 6, is highly expressed in platelets. According to flow cytometry and confocal microscopy, stimulation of platelets with CXCL16 induced platelet degranulation, integrin &agr;IIb&bgr;3 activation, and shape change. CXCL16 increased Akt phosphorylation (Thr308/Ser473), an effect abrogated by phosphatidylinositide 3-kinase inhibitors wortmannin (100 nmol/L) and LY294002 (25 µmol/L). The phosphatidylinositide 3-kinase inhibitors and Akt inhibitor SH-6 (20 µmol/L) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin &agr;IIb&bgr;3 activation, and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXC motif receptor 6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro after high arterial shear stress (2000−s) and to injured vascular wall in vivo after carotid ligation. CXCL16-induced stimulation of platelet adhesion again was prevented by phosphatidylinositide 3-kinase and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P2Y1 (MRS2179, 100 µmol/L) and especially P2Y12 (Cangrelor, 10 µmol/L) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotid ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXC motif receptor 6–dependent phosphatidylinositide 3-kinase/Akt signaling and paracrine activation, suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.Rationale: The recently discovered chemokine CXCL16 is highly expressed in atherosclerotic lesions and a potential pathogenic mediator in coronary artery disease. Objective: To test the role of CXCL16 on platelet activation and vascular adhesion as well as the underlying mechanism and signaling pathway. Methods and Results: RT-PCR, western blotting, confocal microscopy and flow cytometry revealed that CXCL16-specific receptor CXCR6 is highly expressed on platelets. According to flow cytometry and confocal microscopy stimulation of platelets with CXCL16 induced platelet degranulation, integrin α IIb β 3 activation and shape change. CXCL16 increased Akt phosphorylation (Thr 308 /Ser 473 ), an effect abrogated by phosphatidylinositide 3-kinase (PI3K) inhibitors wortmannin (100nM) and LY294002 (25 µM). The PI3K inhibitors and Akt inhibitor SH-6 (20 µM) further diminished CXCL16-induced platelet activation. CXCL16-mediated platelet degranulation, integrin α IIb β 3 activation and Akt phosphorylation were blunted in platelets lacking CXCL16-specific receptor CXCR6. CXCL16-induced platelet activation was abrogated in Akt1- or Akt2-deficient platelets. CXCL16 enhanced platelet adhesion to endothelium in vitro following high arterial shear stress (2000 -s ) and to injured vascular wall in vivo following carotis ligation. CXCL16-induced stimulation of platelet adhesion was again prevented by PI3K and Akt inhibitors. Apyrase and antagonists of platelet purinergic receptors P 2 Y 1 (MRS2179, 100µM) and especially P 2 Y 12 (Cangrelor, 10µM) blunted CXCL16-triggered platelet activation as well as CXCL16-induced platelet adhesion under high arterial shear stress in vitro and after carotis ligation in vivo. Conclusions: The inflammatory chemokine CXCL16 triggers platelet activation and adhesion via CXCR6-dependent PI3K/Akt signaling and paracrine activation suggesting a decisive role for CXCL16 in linking vascular inflammation and thrombo-occlusive diseases.

Higher BDNF serum levels predict slower cognitive decline in Alzheimer's disease patients

The neurotrophin brain-derived neurotrophic factor (BDNF) plays a critical role in neuronal survival, synaptic plasticity, and memory. Several recent studies have demonstrated altered BDNF serum levels in Alzheimers disease (AD) patients. However, the association of BDNF serum levels with the rate of cognitive decline in AD patients is still unclear. We demonstrate that BDNF serum levels are significantly decreased in AD patients with fast cognitive decline [decrease of Mini-Mental State Examination (MMSE) score >4/yr; n=12] compared to AD patients with slow cognitive decline (decrease of MMSE score ≤4/yr, n=28) and show a significant correlation with the rate of cognitive decline during 1 yr follow-up. These results suggest that higher BDNF serum levels are associated with a slower rate of cognitive decline in AD patients. Further longitudinal studies are necessary to elucidate the kinetics and the potential role of serum BDNF as a surrogate marker of disease progression in AD patients.

Platelets Recruit Human Dendritic Cells Via Mac-1/JAM-C Interaction and Modulate Dendritic Cell Function In Vitro

Objective—Thrombotic events and immunoinflammatory processes take place next to each other during vascular remodeling in atherosclerotic lesions. In this study we investigated the interaction of platelets with dendritic cells (DCs). Methods and Results—The rolling of DCs on platelets was mediated by PSGL-1. Firm adhesion of DCs was mediated through integrin &agr;M&bgr;2 (Mac-1). In vivo, adhesion of DCs to injured carotid arteries in mice was mediated by platelets. Pretreatment with soluble GPVI, which inhibits platelet adhesion to collagen, substantially reduced recruitment of DCs to the injured vessel wall. In addition, preincubation of DCs with sJAM-C significantly reduced their adhesion to platelets. Coincubation of DCs with platelets induced maturation of DCs, as shown by enhanced expression of CD83. In the presence of platelets, DC-induced lymphocyte proliferation was significantly enhanced. Moreover, coincubation of DCs with platelets resulted in platelet phagocytosis by DCs, as verified by different cell phagocytosis assays. Finally, platelet/DC interaction resulted in apoptosis of DCs mediated by a JAM-C–dependent mechanism. Conclusions—Recruitment of DCs by platelets, which is mediated via CD11b/CD18 (Mac-1) and platelet JAM-C, leads to DC activation and platelet phagocytosis. This process may be of importance for progression of atherosclerotic lesions.

Early but not late stent thrombosis is influenced by residual platelet aggregation in patients undergoing coronary interventions

AIMS Recent studies suggest a relevant association of post-interventional residual platelet aggregation (RPA) under therapy with oral platelet inhibitors and the occurrence of atherothrombotic events. The influence of post-interventional RPA on the incidence of stent thrombosis (ST) has not been sufficiently evaluated in consecutive unselected cohorts of percutaneous coronary intervention (PCI) patients. The aim of this observational study was to investigate the impact of RPA on the incidence of ST within 3 months in patients treated with dual antiplatelet therapy. METHODS AND RESULTS The study population included a consecutive cohort of 1019 patients treated with PCI [n = 741 bare-metal stent (BMS) and n = 278 drug-eluting stent (DES)] due to symptomatic coronary artery disease. Residual platelet activity was assessed by adenosine disphosphate (20 micromol/L)-induced PA after 600 mg clopidogrel loading dose. Maximum RPA was measured as peak of aggregation, final RPA was measured 5 min after addition of agonist. The primary endpoint was the occurrence of ST within 3 months defined according to academic research consortium (ARC) criteria. Final and maximum RPA were independent predictors of ST after 3 months. In secondary analysis, the observed effects were independently associated with early ST (HR 1.05, 95% CI 1.01-1.08 and HR 1.05, 95% CI 1.01-1.09, P < 0.01, respectively). However, incidence of 3-month late stent thrombosis (LAT) was not influenced by post-interventional RPA in multivariable analysis. CONCLUSION Post-interventional RPA is associated with the occurrence of early ST in patients treated with either BMS or DES; however, there is no predictive value of RPA for the incidence of 3-month LAT, suggesting the involvement of other possible mechanisms like discontinuation of antiplatelet therapy.

Impact of glycoprotein VI and platelet adhesion on atherosclerosis—A possible role of fibronectin

Glycoprotein VI (GPVI) mediates binding of platelets to subendothelial collagen during acute arterial thrombosis. GPVI interactions with the activated atherosclerotic vascular endothelium during early atherosclerosis, however, are not well understood. In ApoE-/- mice, platelet adhesion to atherosclerotic arteries was increased, as measured by intravital microscopy. This platelet adhesion was significantly inhibited by IV injection of GPVI-Fc (1 mg/kg body weight). Atherosclerosis in ApoE-/- mice was attenuated both after 7 and 10 weeks of treatment with the anti-GPVI antibody JAQ1 (2 mg/kg body weight i.p. twice weekly). Binding of GPVI-Fc (1 mg/kg IV) occurred to deeper layers, but also to the luminal site of plaques in atherosclerotic rabbits, but not to the vessel wall of healthy littermates. Gene transfer of GPVI-Fc to the carotid vascular wall significantly attenuated athero-progression and endothelial dysfunction in atherosclerotic rabbits in vivo. Specific binding of the soluble GPVI receptor (GPVI-Fc) to fibronectin was found in vitro to coated ELISA plates. Platelet adhesion to fibronectin was significantly inhibited both by GPVI-Fc and by the anti-GPVI antibody 5C4 ex vivo in flow chamber experiments. GPVI plays a role in platelet adhesion to atherosclerotic endothelium in the absence of plaque rupture. Inhibition of GPVI both via GPVI-Fc and anti-GPVI-antibodies results in protection against atherosclerosis in both cholesterol-fed rabbits and ApoE-/- mice. This novel mechanism of GPVI-mediated platelet adhesion-possibly via fibronectin-could relevantly contribute to platelet-triggered atheroprogression.