Abstract
The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair.
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Abbreviations
- 2D-LC-MS/MS:
-
two-dimensional liquid chromatography and tandem mass spectrometry
- CTGF:
-
connective tissue growth factor
- DMSO:
-
dimethylsulfoxide
- EC:
-
endothelial cells
- ERK 1/2:
-
extracellular signal-regulated kinases 1/2
- FPLC:
-
fast protein liquid chromatography
- HO:
-
Hoechst 33342 (2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2.5′-bi-1H-benzimidazole)
- HUVEC:
-
human umbilical vascular endothelial cells
- LDH:
-
lactate dehydrogenase
- LG3:
-
C-terminal fragment of perlecan
- MMC:
-
mitomycin C
- MVB:
-
multivesicular bodies
- PARP:
-
poly(ADP-ribose) polymerase
- PI:
-
propidium iodide
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- siRNAs:
-
small interfering RNAs
- SN:
-
exosome-free supernatant
- SS:
-
serum starvation
- SSC:
-
serum-free media conditioned by endothelial cells
- TCTP:
-
translationally-controlled tumor protein
- TSG 101:
-
tumor susceptibility gene 101
- VSMC:
-
vascular smooth muscle cells
- LC3:
-
microtubule-associated proteins 1A and 1B, light chain 3
- mTOR:
-
mammalian target of rapamycin
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Acknowledgements
This work was supported by research grants from the Canadian Institutes of Health Research to MJH (MOP-15447) and the Canadian Institutes of Health Research to AVP (MOP-66980). MJH is the holder of the Shire Chair in Nephrology, Transplantation and Renal Regeneration of the University of Montreal. IS is the recipient of a training fellowship from the Canadian Institutes of Health Research. JFC is an FRSQ scholar. We thank the J-L Lévesque Foundation for renewed support, Nicolas Parent for help with JC-1 staining assay and D. Gingras for processing cells for electron microscopy.
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Sirois, I., Raymond, MA., Brassard, N. et al. Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication. Cell Death Differ 18, 549–562 (2011). https://doi.org/10.1038/cdd.2010.126
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DOI: https://doi.org/10.1038/cdd.2010.126
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