IntroductionPlatelet activation is thought to be a key event in acute vascular thrombosis. Therefore, prevention of enhanced platelet activation is a major target of therapeutic strategies fighting cardiovascular and cerebrovascular diseases. [1][2][3] An important stimulus for physiologic platelet activation and thrombus formation is the contact of platelets with components of the subendothelial matrix, like collagen. 4 Although Marcus et al have shown as early as 1977 that platelets have the ability to release superoxide anions (O 2 Ϫ ), 5 it was only recently proposed that platelets stimulated by collagen produce reactive oxygen species (ROS) such as hydrogen peroxide, 6 hydroxyl radicals, 7 or O 2 Ϫ . 7,8 While O 2 Ϫ , a highly reactive radical, damages cells in high concentrations by reacting with proteins, lipids, and DNA, in low concentrations its continuous production, with similarity to second messengers, has been suggested to indirectly affect signal transduction processes. 9,10 Platelet agonists other than collagen, such as thrombin or ADP, do not seem to induce ROS formation during aggregation. 8 This difference raises the question whether O 2 Ϫ formation could serve a modulating function when thrombus formation is induced by collagen.The cellular source of platelet O 2 Ϫ is unclear. Growing evidence supports the assumption that platelet activation by collagen is specifically due to binding to the glycoprotein VI (GPVI)-receptor, 11,12 resulting in a cascade of tyrosine phosphorylation events ultimately leading to activation of phospholipase C␥ (PLC␥), 13 which is known to strongly activate protein kinase C (PKC) through production of diacylglycerol. 4 Recently, evidence for the existence of a neutrophil-type reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase in platelets that can be activated by PKC and is involved in O 2 Ϫ formation has been presented, [14][15][16] similar as in other O 2 Ϫ -generating systems, like the vascular endothelium. In endothelial cells, an NAD(P)H oxidase is the main source of O 2 Ϫ . 17 As O 2 Ϫ readily reacts with NO, this has been suggested to result in attenuated 18,19 and the role of O 2 Ϫ in the regulation of vascular tone has become a major focus of interest. 17 Moreover, antioxidants like N-acetylcysteine (NAC) have been shown to exert direct antiaggregatory effects. 20 Although these findings raise the possibility that platelet-derived O 2 Ϫ is involved in regulating platelet activation, evidence for a role of platelet-derived O 2 Ϫ in platelet function is rare. In a canine model of coronary arterial thrombosis, thrombus formation was regulated by intraplatelet redox state. 21 Leo and colleagues have shown that platelets subjected to anoxia/ reoxygenation are more reactive, due to an enhanced O 2 Ϫ generation. 14 However, so far it remains unclear whether an enhanced O 2 Ϫ production occurs also during direct platelet activation, such as with collagen, and how this could affect thrombus growth. Whereas Supported by a grant from the Friedrich-...
BES=biodegradable polymer biolimus-eluting stent, SES=durable polymer sirolimus-eluting stent, PCI=percutaneous coronary intervention. Note that we have no reliable data on the number of patients assessed for eligibility Baseline characteristics Biodegradable polymer BES (N=857) Durable polymer SES (N=850) Age (years) 64•6 (10•8) 64•5 (10•7) Male 643 (75•0%) 634 (74•6%) Diabetes mellitus 223 (26•0%) 191 (22•5%) Insulin requiring 81 (9•5%) 77 (9•1%) Hypertension 630 (73•5%) 618 (72•7%) Hypercholesterolemia 560 (65•3%) 580 (68•2%) Current smoker 206 (24•0%) 214 (25•2%) Family history of coronary artery disease 339 (39•6%) 374 (44•0%)
The safety benefit of the biodegradable polymer BES, compared with the durable polymer SES, was related to a significant reduction in very late ST (>1 year) and associated composite clinical outcomes. (Limus Eluted From A Durable Versus ERodable Stent Coating [LEADERS] trial; NCT00389220).
Delivery of antisense oligodesoxynucleotides (ODN) into primary cells is a specific strategy for research with therapeutic perspectives but transfection-associated difficulties. We established the technique of magnetofection to enhance ODN delivery at low toxicity and procedure time in vitro and in vivo. In vitro, target knockout was assessed at protein and mRNA levels and by measuring superoxide generation after antisense magnetofection against the p22(phox) subunit of endothelial NAD(P)H-oxidase. Under magnetic field guidance, low-dose magnetic particle-bound ODN were transfected to 84% human umbilical vein endothelial cells within 15 min followed by nuclear accumulation within 2 h, which required 24 h using standard methods. Antisense magnetofection against p22(phox) significantly decreased basal and prevented stimulated superoxide release due to loss of NAD(P)H-oxidase activity by mRNA knockout as assessed after 24 h. Knockout of endothelial phosphatase SHP-1 and connexin 37 proteins confirmed the method's efficiency. Transfection-associated toxicity was minimal. Twenty-four hours after injection of fluorescence-labeled ODN into femoral arteries of male mice, there was specific ODN uptake only into cremaster vessels exposed to magnetic fields during injection. Magnetofection is an ideal tool for delivery of functionally active ODN to difficult-to-transfect cells to study gene/protein function and a promising strategy for targeted ODN delivery in vivo.
Considering that chronic elevation of shear stress results in remodeling of the vasculature, we analyzed whether mechanical load could mediate basic fibroblast growth factor (bFGF) release and whether bFGF would act as mediator of shear stress-induced endothelial proliferation and differentiation. Supernatant media of shear stress-exposed endothelial cells (EC) contained significantly higher amounts of bFGF than medium from static cells. Released bFGF was fully intact with regard to its function as an inductor of proliferation and differentiation. Shear stress-conditioned media induced capillary-like structure formation, whereas static control medium did not. Likewise, only shear stress-conditioned medium induced proliferation of serum starved EC. Both capillary-like structure formation and proliferation could be inhibited by neutralization of bFGF or its receptor. The release of bFGF was subject to specific, integrin-mediated control, since inhibition of ␣ v  3 integrin prevented it, whereas inhibition of ␣ 5  1 integrin had no effect. We conclude that shear stress induces the release of bFGF from EC in a tightly controlled manner. The release is dependent on specific cell-matrix interactions via ␣ v  3 integrins. The effects on cell proliferation and differentiation suggest that release of bFGF is functionally significant and may represent a necessary initial step in adaptive remodeling processes induced by shear stress.Chronic physical exercise leads to increases in the diameter of existing vessels as well as the formation of new vessels (angiogenesis), thereby enhancing the total number of vessels in the skeletal muscle (1-6). It is believed that shear stress represents an important stimulus for these vascular events. However, little is known about the mechanism of mechanosensing (the initial step) or about potential mediators involved in this shear stress response. Some growth factors controlling angiogenesis have similar vascular effects as shear stress. They are involved in a regulated time sequence of cell migration, proliferation, differentiation, and apoptosis. Therefore, growth factors might be sufficient to induce remodeling processes per se (3,7,8).In particular the basic fibroblast growth factor (bFGF) 1 may play a pivotal role in vascular remodeling. Several investigators have reported a critical participation of this cytokine in new vessel formation (4, 5, 9 -13). The expression of bFGF is not affected at the transcription level by shear stress, hypoxia, or hypertension (14), all of which have been shown to stimulate angiogenesis (3, 6), but bFGF is stored at significant concentrations within the endothelium and could well be released in amounts sufficient to initiate angiogenesis after exposure to high shear stress. Therefore, we hypothesized that shear stress could serve as an adequate stimulus for the release of bFGF from endothelial cells and that bFGF could function as a mediator of shear stress-induced angiogenesis. Because many investigators are claiming that part of the mechano-sensing...
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