Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin

BACKGROUND: Elevated plasma counts of endothelial microparticles (MP) have been demonstrated in various diseases with a vascular injury component. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells (HUVEC) stimulated by various agonists. MP-release by a topoisomerase I inhibitor camptothecin has been studied in detail. RESULTS: Overnight stimulation of HUVEC with either LPS or TNFα, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 μM camptothecin, documented by 60–70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding microparticles (~80,000 MP/10(3) cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95% of microparticles were 0.3–1 μm in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105. CONCLUSIONS: Camptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury

P38 mitogen activated protein kinase inhibitor improves platelet in vitro parameters and in vivo survival in a SCID mouse model of transfusion for platelets stored at cold or temperature cycled conditions for 14 days.

Platelets for transfusion are stored at room temperature (20-24°C) up to 7 days but decline in biochemical and morphological parameters during storage and can support bacterial proliferation. This decline is reduced with p38MAPK inhibitor, VX-702. Storage of platelets in the cold (4-6°C) can reduce bacterial proliferation but platelets get activated and have reduced circulation when transfused. Thermocycling (cold storage with brief periodic warm ups) reduces some of the effects of cold storage. We evaluated in vitro properties and in vivo circulation in SCID mouse model of human platelet transfusion of platelets stored in cold or thermocycled for 14 days with and without VX-702. Apheresis platelet units (N = 15) were each aliquoted into five storage bags and stored under different conditions: room temperature; cold temperature; thermocycled temperature; cold temperature with VX-702; thermocycled temperature with VX-702. Platelet in vitro parameters were evaluated at 1, 7 and 14 days. On day 14, platelets were infused into SCID mice to assess their retention in circulation by flow cytometry. VX-702 reduced negative platelet parameters associated with cold and thermocycled storage such as an increase in expression of activation markers CD62, CD63 and of phosphatidylserine (marker of apoptosis measured by Annexin binding) and lowered the rise in lactate (marker of increase in anaerobic metabolism). However, VX-702 did not inhibit agonist-induced platelet aggregation indicating that it does not interfere with platelet hemostatic function. In vivo, VX-702 improved initial recovery and area under the curve in circulation of human platelets infused into a mouse model that has been previously validated against a human platelet infusion clinical trial. In conclusion, inhibition of p38MAPK during 14-days platelet storage in cold or thermocycling conditions improved in vitro platelet parameters and platelet circulation in the mouse model indicating that VX-702 may improve cell physiology and clinical performance of human platelets stored in cold conditions

In Vitro and In Vivo Characterization of Ultraviolet Light C-Irradiated Human Platelets in a 2 Event Mouse Model of Transfusion

<div><p>UV-based pathogen reduction technologies have been developed in recent years to inactivate pathogens and contaminating leukocytes in platelet transfusion products in order to prevent transfusion-transmitted infections and alloimmunization. UVC-based technology differs from UVA or UVB-based technologies in that it uses a specific wavelength at 254 nm without the addition of any photosensitizers. Previously, it was reported that UVC irradiation induces platelet aggregation and activation. To understand if UVC-induced changes of platelet quality correlate with potential adverse events when these platelets are transfused into animals, we used a 2-event SCID mouse model in which the predisposing event was LPS treatment and the second event was infusion of UVC-irradiated platelets. We analyzed lung platelet accumulation, protein content in bronchoalveolar lavage fluid as an indication of lung injury, and macrophage inflammatory protein-2 (MIP-2) release in mice received UVC-irradiated or untreated control platelets. Our results showed UVC-irradiated platelets accumulated in lungs of the mice in a dose-dependent manner. High-doses of UVC-irradiated platelets were sequestered in the lungs to a similar level as we previously reported for UVB-irradiated platelets. Unlike UVB-platelets, UVC-platelets did not lead to lung injury or induce MIP-2 release. This could potentially be explained by our observation that although UVC treatment activated platelet surface αIIbβ3, it failed to activate platelet cells. It also suggests lung platelet accumulation and subsequent lung damage are due to different and separate mechanisms which require further investigation.</p> </div

In vivo lung accumulation of UVC-irradiated HPs in the 2-event SCID mouse model.

<p><i>A</i>) Mice were pretreated with an intraperitoneal injection of 3 mg/kg LPS 2 hours before intravenous infusion of untreated control or UVC-irradiated HPs at low (0.2 J/cm²) and high (1.2 J/cm²) doses, respectively. Shown are anti-human CD41 immunohistochemistry staining of lung frozen sections; <i>B</i>) Quantification of pixel intensity of anti-hCD41 staining of images shown in A. Mean ± SE, n=3; <i>C</i>) Lung histology from H&E staining of lung paraffin sections. Shown is a representative of three independent experiments.</p

UVC-irradiated HPs did not induce lung injury in the 2-event SCID mouse model.

<p>Mice were treated as described above for the 2-event SCID mouse model. Bronchoalveolar fluid (BALF) was collected 1 hour after platelet infusion and the total protein concentration in BALF was measured using BCA protein assay. Mean ± SE, n=5-10.</p

UVC-irradiated HPs failed to induce MIP-2 release in the 2-event SCID mouse model.

<p>3 hours after HPs infusion mice were euthanized. Blood and BALF were collected and plasma was subsequently isolated as described in Materials and Methods. The concentrations of MIP-2 in plasma (<i>A</i>) and BALF (<i>B</i>) were measured using ELISA. Mean ± SE, n=4-5. </p

UVC irradiation induced platelet aggregation and potentiated ADP induced platelet aggregation.

<p><i>A</i>) Human platelets (HPs) at 1 x 10⁶/µL were exposed to 0.2 (30 seconds), 0.4 (1 minutes), 0.8 (2 minutes), and 1.2 J/cm² (3 minutes) UVC illumination. Single platelet count was measured immediately after light exposure on a Cell-Dyn 3700 blood cell counter. Mean ± SE, n=8. <i>B</i>) in vitro aggregation assay was performed with untreated (Ctrl, open circle) or UVC-exposed platelets (0.2 J/cm², filled square). Y axis represents percent of maximal aggregation (%MA). Mean ± SE, n=3.</p

UVC-irradiated HPs showed reduced in vivo recovery in circulation of SCID mice.

<p>Approximately 1 x 10⁹ untreated (open circle) or UVC-irradiated HPs at a dose of 0.2 J/cm² (filled square) were infused into SCID mice. Blood sampling was subsequently performed at indicated time points and the presence of human platelets in circulation positive for anti-human CD41a staining was detected by flow cytometry. Mean ± SE, n=5.</p