BACKGROUND AND PURPOSENADPH oxidases (NOXs) contribute to platelet activation by a largely unknown mechanism. Here, we studied the effect of the novel NOX inhibitor 2-acetylphenothiazine (2-APT) on human platelet functional responses and intracellular signaling pathways. EXPERIMENTAL APPROACHThe generation of superoxide ions was assessed by single cell imaging on adhering platelets using dihydroethidium (DHE), while other reactive oxygen species (ROS) were detected with 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2-DCFDA). Whole blood thrombus formation, washed platelet aggregation, integrin aIIbb3 inside-out signalling, Syk phosphorylation and PKC activation were analysed to understand the functional consequences of NOX inhibition by 2-APT in platelets. KEY RESULTSSuperoxide ion generation stimulated by platelet adhesion on collagen and fibrinogen was significantly inhibited by 2-APT in concentration-dependent manner (IC50 = 306 nM and 227 nM, respectively), whereas cumulative ROS accumulation was not affected by this pharmacological agent. 2-APT also abolished collagen-dependent whole blood thrombus formation and washed platelet aggregation in response to collagen but not thrombin. The activation of integrin aIIbb3 and PKC in response to the GPVI-specific agonist collagen-related peptide (CRP) was significantly reduced, whereas the same responses to thrombin were not significantly affected by 2-APT. Finally, Syk activation in response to collagen but not thrombin was inhibited by 2-APT. CONCLUSIONS AND IMPLICATIONSTaken together, our results suggest that 2-APT attenuates GPVI-specific signalling and is a novel inhibitor of collagen-induced platelet responses. Therefore, NOXs could represent a novel target for the discovery of anti-thrombotic drugs. Abbreviations2-APT, 2-acetylphenothiazine; CRP, collagen-related peptide; DCFDA, 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein

Abstract-Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin ␣v␤3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential. Key Words: angiogenesis Ⅲ endothelium Ⅲ progenitor cells Ⅲ proteomics Ⅲ vascular biology H uman endothelial progenitor cells (EPCs) are attracting considerable attention in cardiovascular research, 1,2 but multiple culture methods from peripheral blood mononuclear cells (PB-MNCs) have been described [3][4][5][6][7] and studied for their clinical relevance. 6,8 -12 EPCs are commonly identified by cell surface antigen expression of CD133, CD34, and the vascular endothelial growth factor receptor-2 (VEGFR-2) (KDR). 13 CD34 and VEGFR-2, however, are also expressed in hematopoietic stem cells 14 ; thus, EPCs cannot yet be unambiguously defined. One alternative approach to flow cytometry has used the colony-forming unit (CFU) assay as a surrogate marker for EPCs. 15 This method has been fundamental to many of the clinical studies published on EPCs to date, which predominantly reported low numbers of CFUs to be correlated to cardiovascular disease risk. Nonetheless, recent publications have cast doubts about the origin of CFUs by demonstrating that they may be clonally derived from the hematopoietic system, possess myeloid progenitor cell activity, and differentiate into phagocytic macrophages. 16 Thus, there is an urgent need to provide a mechanistic underpi...

Reactive oxygen species (ROS) are now appreciated to play several important roles in a number of biological processes and regulate cell physiology and function. ROS are a heterogeneous chemical class that includes radicals, such as superoxide ion (O2(•-)), hydroxyl radical (OH(•)) and nitric oxide (NO(•)), and non-radicals, such as hydrogen peroxide (H2O2), singlet oxygen ((1)O2), hypochlorous acid (HOCl), and peroxynitrite (NO3 (-)). In the cardiovascular system, besides playing a critical role in the development and progression of vasculopathies and other important pathologies such as congestive heart failure, atherosclerosis and thrombosis, ROS also regulate physiological processes. Evidence from a wealth of cardiovascular research studies suggests that ROS act as second messengers and play an essential role in vascular homeostasis by influencing discrete signal transduction pathways in various systems and cell types. They are produced throughout the vascular system, regulate differentiation and contractility of vascular smooth muscle cells, control vascular endothelial cell proliferation and migration, mediate platelet activation and haemostasis, and significantly contribute to the immune response. Our understanding of ROS chemistry and cell biology has evolved to the point of realizing that different ROS have distinct and important roles in cardiovascular physiology. This review will outline sources, functions and molecular mechanisms of action of different ROS in the cardiovascular system and will describe their emerging role in healthy cardiovascular physiology and homeostasis.

No abstract