Objective-Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced by endothelial cells. In many vessels, they are an endothelium-derived hyperpolarizing factor (EDHF). However, it is unknown whether they act as an EDHF on platelets and whether this has functional consequences. Methods and Results-Flow cytometric measurement of platelet membrane potential using the fluorescent dye DiBac 4 showed a resting potential of Ϫ58Ϯ9 mV. Different EET regioisomers hyperpolarized platelets down to Ϫ69Ϯ2 mV, which was prevented by the non-specific potassium channel inhibitor charybdotoxin and by use of a blocker of calcium-activated potassium channels of large conductance (BK Ca channels), iberiotoxin. EETs inhibited platelet adhesion to endothelial cells under static and flow conditions. Exposure to EETs inhibited platelet P-selectin expression in response to ADP. Key Words: epoxyeicosatrienoic acids Ⅲ platelet adhesion Ⅲ membrane potential Ⅲ potassium channels Ⅲ EDHF I ntact endothelial cells continuously release autacoids such as nitric oxide (NO), prostacyclin (PGI 2 ), or adenosine and an endothelial-derived hyperpolarizing factor (EDHF), thereby controlling vascular tone and platelet activity. 1,2 Endothelial dysfunction and the associated activation of platelets are synergistic factors in the development of cardiovascular disorders. Both may precede atherosclerosis 3,4 and are associated with an enhanced risk of adverse cardiovascular events. 5 Little is known about the role of EDHF in the control of platelet function, although this factor may be less susceptible to mediators that deteriorate endothelial function such as reactive oxygen species.In several vascular beds, EDHF seems to be identical with epoxyeicosatrienoic acids (EETs), 6 which are products of cytochrome P450 enzymatic metabolism of arachidonic acid. 7 There are data indicating that EETs are released into the lumen of isolated vessels 8,9 or from endothelial cells in culture, 10 -12 so they could influence not only the adjacent smooth muscle cells but also circulating blood constituents like platelets. Although in 1986, years before these compounds have been postulated to represent an EDHF in the vasculature, Fitzpatrick et al observed inhibition of platelet aggregation by EETs, 13 it was not investigated whether the platelet-endothelium interaction was affected or whether platelet membrane potential has a role in this.In general, EETs could influence platelets by activation of calcium-activated potassium channels or by effects that are independent of the membrane potential, similar as described for endothelial cells. 14 Platelets not only contain voltageoperated potassium channels (K v channels) 15 but also calcium-activated potassium channels (K Ca channels), so they are potential targets of EETs. 16 In this study, we investigated whether EETs hyperpolarize platelets via K Ca channels and whether this has an effect on platelet activation parameters and platelet adhesion to the endothelium. MethodsFor a detailed Methods section, please see ht...

Anti-angiogenetic cancer therapy is a potential new form for treatment of solid tumours. The a v -integrins (a v b 3 , a v b 5 ) mediate the contact of activated endothelial cells to proteins of the extracellular matrix during tumour angiogenesis as a prerequisite for survival of endothelial cells. The aim of this study was to investigate the effects of application of a methylated cyclic RGDpeptide as an a v -integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. Experiments were performed in the dorsal skinfold preparation of Syrian Golden hamsters bearing the amelanotic hamster melanoma A-Mel-3. Animals were injected intraperitoneally with a methylated cyclic RGD-peptide every 12 h, the control group received an inactive peptide. Microcirculatory parameters of tumour angiogenesis including functional vessel density, red blood cell velocity, vessel diameter and leucocyte -endothelium interaction were analysed using intravital microscopy. In an additional study the effects on growth and metastasis of subcutaneous A-Mel-3 were quantified. Functional vessel density was markedly reduced on day 3 in treated animals compared to controls (37.2+12.1 vs 105.2+11.2 cm 71 ; mean+s.e.m.; P50.05) and increased subsequently in both groups. Red blood cell velocity at day 3 was below values of controls (0.026+0.01 vs 0.12+0.03 mm s 71; P50.05). No differences were observed in vessel diameters and leucocyteendothelium interaction was almost absent in both groups. Furthermore, growth and metastasis of subcutaneous tumours after administration of the cyclic RGD-peptide was significantly delayed in comparison to controls (P50.05). Inhibition of a vintegrins by a cyclic RGD-peptide resulted in significant reduction of functional vessel density, retardation of tumour growth and metastasis in vivo. Taken together, these results implicate RGD-peptides as agents which have anti-tumour and antimetastatic activity in vivo.

In HUVEC, SHP-1 counteracts basal and stimulated NAD(P)H-oxidase activity by negative regulation of PI3K-dependent Rac1 activation; SHP-1 thus seems to be an important part of endothelial antioxidative defense controlling the activity of the O2(*-)-producing NAD(P)H-oxidase.

Background-Selective inhibitors of cyclooxygenase-2 (Cox-2) are reported to cause cardiovascular side effects in patients at risk. However, direct proof of prothrombotic effects of these drugs is lacking. We investigated in the microcirculation in vivo whether selective inhibition of Cox-2 induces platelet activation. Methods and Results-The behavior of fluorescence-labeled human platelets was studied in hamster arterioles (dorsal skinfold chamber) by intravital microscopy. Transient platelet-vessel wall interactions (PVWIs), firm platelet adhesion to the vessel wall, and vessel occlusion after FeCl 3 -induced wall injury were analyzed as platelet activation parameters.In vitro experiments in human umbilical vein endothelial cells (HUVECs) were performed to assess specific effects of Cox-2 inhibition on platelet adhesion under shear stress (16 dyn/cm 2 ) and on endothelial release of 6-ketoprostaglandin (PG) F 1␣ . Selective inhibition of Cox-2 (NS-398, 0.5 mg/kg) increased platelet adhesion to the vessel wall in vivo (11.9Ϯ3.9 platelets/mm 2 ; controls, 1.4Ϯ1.4 platelets/mm 2 , PϽ0.05) and platelet adhesion after ADP stimulation in vitro. PVWIs were significantly enhanced in NS-398 -treated animals, which were reduced by platelet pretreatment with aspirin (5 mg/kg) or iloprost (1 nmol/L). Inhibition of Cox-2 reduced levels of 6-keto-PGF 1␣ in vivo and in HUVEC supernatants. Time to occlusion after vessel wall injury was significantly shortened by NS-398 (125.4Ϯ13.6 seconds in NS-398 -treated animals versus 270.8Ϯ46 seconds in controls; PϽ0.01). Conclusions-Selective inhibition of Cox-2 reduces 6-keto-PGF 1␣ endothelial release, increases PVWIs, and increases firm platelet adhesion in hamster arterioles. Moreover, it leads to faster occlusion of damaged microvessels. Thus, selective inhibition of Cox-2 may trigger thrombotic events by diminishing the antiplatelet properties of the endothelium.