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. 2019 Aug;15(4):530-542.
doi: 10.1007/s12015-019-09891-6.

Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF

Marcin Wysoczynski  1   2 , Asif Pathan  3 , Joseph B Moore 4th  3 , Talha Farid  3 , Jae Kim  3 , Marjan Nasr  3 , Yi Kang  3 , Hong Li  3 , Roberto Bolli  4   5
Affiliations

Pro-Angiogenic Actions of CMC-Derived Extracellular Vesicles Rely on Selective Packaging of Angiopoietin 1 and 2, but Not FGF-2 and VEGF

Marcin Wysoczynski et al. Stem Cell Rev Rep. 2019 Aug.

Abstract

While the fundamental mechanism by which cardiac cell therapy mitigates ventricular dysfunction in the post ischemic heart remains poorly defined, donor cell paracrine signaling is presumed to be a chief contributor to the afforded benefits. Of the many bioactive molecules secreted by transplanted cells, extracellular vesicles (EVs) and their proteinaceous, nucleic acid, and lipid rich contents, comprise a heterogeneous assortment of prospective cardiotrophic factors-whose involvement in the activation of endogenous cardiac repair mechanism(s), including reducing fibrosis and promoting angiogenesis, have yet to be fully explained. In the current study we aimed to interrogate potential mechanisms by which cardiac mesenchymal stromal cell (CMC)-derived EVs contribute to the CMC pro-angiogenic paracrine signaling capacity in vitro. Vesicular transmission and biological activity of human CMC-derived EVs was evaluated in in vitro assays for human umbilical vein endothelial cell (HUVEC) function, including EV uptake, cell survival, migration, tube formation, and intracellular pathway activation. HUVECs incubated with EVs exhibited augmented cell migration, tube formation, and survival under peroxide exposure; findings which paralleled enhanced activation of the archetypal pro-survival/pro-angiogenic pathways, STAT3 and PI3K-AKT. Cytokine array analyses revealed preferential enrichment of a subset of prototypical angiogenic factors, Ang-1 and Ang-2, in CMC EVs. Interestingly, pharmacologic inhibition of Tie2 in HUVECs, the cognate receptors of angiopoietins, efficiently attenuated CMC-EV-induced HUVEC migration. Further, in additional assays a Tie2 kinase inhibitor exhibited specificity to inhibit Ang-1-, but not Ang-2-, induced HUVEC migration. Overall, these findings suggest that the pro-angiogenic activities of CMC EVs are principally mediated by Ang-1-Tie2 signaling.

Keywords: Angiogenesis; Angiopoietin; Cardiac Mesenchymal Cells; Endothelial Cells; Extracelluar Vesicles; Migration.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. CMC morphology and phenotype.
Brightfield microscopy image of human CMCs isolated from RAA; scale bar 100 μm (A); cell surface markers expression on CMCs (B); representative flow cytometry dot plots (C). Flow cytometric analysis was performed on CMCs isolated from three independent patents samples. Data are mean ± SEM.
Figure 2
Figure 2. Isolation and characterization of hCMC-derived EVs.
CMC-EV particle size distribution analysis with zeta-sizer (A); Western blot analysis of EV markers (B).
Figure 3
Figure 3. HUVECs bind and internalize CMC-derived EVs.
HUVECs were stimulated with PKH26 labeled EVs (50 μg/mL) or vehicle for up to 24 h. The EV-derived fluorescence was evaluated with flow cytometry and confocal microscopy. Representative flow cytometry dot plots of HUVECs stimulated with PKH26-labeled EVs of vehicle (A). Numerical representation of EV-uptake by HUVECs as a percentage of positive cells (B) or MFI measured in HUVECs (C). Representative fluorescent microscopy images of HUVECs stimulated with PKH26 labeled EVs (D). Data are mean ± SEM, n=4. *P<0.5 vs vehicle.
Figure 4
Figure 4. CMC-EVs activate intracellular pathways in HUVECs.
HUVECs were cultured in serum and growth factors free media to silence the intracellular signalling pathways. After 24 h cells were stimulated with escalating does of EVs for 10 min (A) or with 50 μg/mL of EVs for up to 30 min (B), and the cell lysates were evaluated for activation of MAPK, PI3K-AKT, and STAT pathways with Western blot. Representative Western blot images from three independent experiments are shown.
Figure 5
Figure 5. CMC-EVs induce angiogenesis in vitro.
Dose dependent increase of HUVECs migration (A) and tube formation (B) in response to CMC-EVs; CMC-EVs (100 μg/mL) improve HUVECs survival in response to increasing doses of H2O2 challenge (C); Data are mean ± SEM, n=3. P<0.5 vs vehicle control or EBM control.
Figure 6
Figure 6. Pro-angiogenic effect of CMC-EV is mediated via Ang-1-Tie2 signalling.
CMC-EVs induced HUVEC migration is inhibited with Tie2 kinase inhibitor, but not with AMD3100, Vetalanib, or FIIN1 (A); Tie2 kinase inhibitor specifically inhibit Ang-1 but not Ang-2 induced migration of HUVECs (B); Tie2 kinase inhibitor is ineffective in inhibition of HUVECs migration to endothelial growth medium (C). Data are mean ± SEM, n=3–4. *P<0.5 vs EVs (A) or vehicle control (B).
Figure 7
Figure 7. CMC-EVs are enriched in Ang-1 and Ang-2 but not in FGF-2 and VEGF.
CMC and CMC-EVs protein extracts where evaluated for Ang-1, Ang-2, FGF-2, and VEGF expression by ELISA. Data are mean ± SEM, n=5. *P<0.5 vs CMCs.
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