Date: Monday, May 4, 2015
Session Time: 5:30pm-6:30pm
Presentation Time: 5:30pm-6:30pm
Location: Exhibit Hall E
LG3, a C-terminal fragment of perlecan, has been implicated in aggravation of renal allograft rejection and vascular injury. LG3 is known to be produced at least in part by apoptotic vascular cells. Here we aim at characterizing further the molecular pathways implicated in LG3 export.
Primary human umbilical vein endothelial cells (EC) or mouse aortic endothelial cells (mEC) were exposed to serum starvation (SS) and H2O2, two stimuli mimicking clinically relevant stressors of importance during harvesting, preservation and ischemia-reperfusion. SS increased apoptosis in EC and mEC but failed to induce necrosis, as evaluated by fluorescence microscopy with Hoechst/Propidium iodide (PI), FACS analysis with annexin V and PI staining and caspase-3 activation. H2O2 led to enhanced necrosis, but did not enhance apoptosis or caspase-3 activation. SS-induced cell death was reduced in presence of DEVD-FMK, a caspase-3 inhibitor, or in EC from caspase 3-/- mice. LG3 release was increased in medium conditioned by SS apoptotic EC and DEVD-FMK or caspase-3 -/- EC led to reduced production of LG3. H2O2 did not increase the release of LG3.
We then evaluated whether extracellular vesicles contribute to LG3 export. Extracellular vesicles (EV) were detected by small particle flow cytometry, purified by sequential centrifugation and analyzed by WB and electron microscopy. Both SS and H2O2 triggered secretion of EV ranging in size from 30nm to 5 ¯o;m. EV purified from H2O2-treated EC showed increased levels of the exosome markers CD63 and MFGE8 but reduced levels of LG3. Electron microscopy with immunogold labelling demonstrated the presence of LG3 in EV ranging in size from 30 to 100 nm but lacking the characteristic cup shape morphology of exosomes.
These results demonstrate that LG3 is released by apoptotic cells downstream of caspase-3 activation through EV that differ from classic exosomes. Necrosis induced by oxidative stress does not lead to LG3 release, highlighting further the specific importance of apoptosis in LG3 export.
To cite this abstract in AMA style:Beillevaire D, Turgeon J, Gingras D, Boilard é, Dieudé M, Hébert M-J. The Immunogenic Perlecan Fragment LG3 Is Released Through Apoptotic Extracellular Vesicles Downstream of Caspase-3 Activation [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/the-immunogenic-perlecan-fragment-lg3-is-released-through-apoptotic-extracellular-vesicles-downstream-of-caspase-3-activation/. Accessed January 25, 2021.
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