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. 2017 Oct 23;7(1):13794.
doi: 10.1038/s41598-017-14356-2.

Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs

Tek N Lamichhane  1 , Christopher A Leung  1 , Lampouguin Yenkoidiok Douti  1 , Steven M Jay  2   3   4
Affiliations

Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs

Tek N Lamichhane et al. Sci Rep. .

Abstract

Extracellular vesicles (EVs), such as exosomes, have been identified as regulators of vascular remodeling and have promise as therapeutics for vascularization applications. Towards development of EVs as therapeutics, it has been demonstrated that physiological stimuli of angiogenic phenotypes in EV-producing cells can enhance the potency of EVs for vascularization. The goal of this study was to assess whether ethanol, which induces angiogenic phenotypes in endothelial cells, could be employed to enhance endothelial-derived EV vascularization bioactivity. The results indicate that ethanol conditioning of endothelial cells increases the ability of endothelial EVs to induce a pro-vascularization response. This response is due in part to increased CD34 expression in recipient endothelial cells that may result from downregulation of microRNA-106b in EVs isolated from ethanol-conditioned producer endothelial cells. Further, ethanol-induced upregulation of long non-coding RNAs (lncRNAs) HOTAIR and MALAT1 in endothelial EVs was observed to play a significant role in mediating pro-angiogenic effects of these vesicles. Overall, these studies validate ethanol conditioning as a method to enhance the bioactivity of endothelial EVs via regulation of EV-associated microRNAs (miRNAs) and, especially, lncRNAs. Further, the results suggest that alcohol consumption may activate endothelial EVs towards a pro-vascularization phenotype, which could have implications for alcohol-induced tumor angiogenesis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Endothelial cell production of EVs in the presence of alcohol. (A) HUVEC survival was assessed with the indicated concentrations of ethanol (EtOH) included in growth medium (EGM2) (n = 3). (B) HUVEC EV size distribution in the presence or absence of the indicated concentrations of EtOH in the medium was assessed via NanoTracking Analysis (NTA) using a NanoSight LM10 (n = 3). Immunoblots for proteins associated with exosomes were performed for (C) HUVEC EVs and (D) HDMEC EVs isolated from producer cells cultured with the indicated EtOH concentrations. Blots shown are representative of three independent experiments. EV production rate by cells in medium containing the indicated EtOH concentrations was determined by NTA for (E) HUVEC and (F) HDMEC; n = 3, **P < 0.01, ***P < 0.001 compared to 0 mM EtOH condition.
Figure 2
Figure 2
Ethanol conditioning increases endothelial cell-derived EV vascularization bioactivity. Gap closure by (A) HUVECs and (B) HDMECs was assessed following stimulation by 100 µg/ml EVs isolated from the same producer cell type in medium with the indicated ethanol (EtOH) concentrations (n = 3, *P < 0.05, **P < 0.01 vs. 0 mM EtOH condition); HUVECs incubated in basal medium (EBM2, without growth factors) were used as the negative control (−) and HUVECs incubated in growth medium (EGM2, with growth factors) were used as positive controls (+). (C) Matrigel plugs injected into C57Bl/6 mice containing PBS as a negative control (−) or 100 µg HUVEC EVs from cells cultured with the indicated concentrations of EtOH in the media were removed 10 d after implantation and CD31+ cells were quantified using immunohistochemical staining (n = 6; **P < 0.01 compared to all other groups by one-way ANOVA). Data are presented as %CD31+ stained cells out of all cells counted for the gel sections from a given animal. (D) Representative gel images and immunohistochemistry sections from animals in the indicated groups are shown (n = 6).
Figure 3
Figure 3
CD34 is upregulated in endothelial cells (ECs) stimulated by EVs from ethanol (EtOH)-conditioned ECs. (A) Experimental schematic and representation of gene regulation profile of 92 genes associated with angiogenesis. Data shown represent mRNA levels in recipient HUVECs stimulated by 100 µg/ml EVs from producer HUVECs cultured in the presence vs. the absence of 100 mM EtOH for 24 h. (B) CD34 expression in recipient HUVECs and HDMECs following stimulation by 100 µg/ml EVs from producer ECs of the same type cultured in the presence or absence (control (Ctrl)) of 100 mM EtOH for 24 h was assessed by qPCR using distinct primers from those in the mRNA array (n = 3; *P < 0.05, **P < 0.01).
Figure 4
Figure 4
Endothelial cell receptor tyrosine kinase stimulation by EVs. (A) Phosphorylated RTK arrays were incubated for 30 min with lysates from recipient HUVECs stimulated by 100 µg/ml EVs from producer HUVECs cultured in the presence or absence of 100 mM ethanol (EtOH) for 24 h. Ovals indicate phospho-epidermal growth factor receptor (pEGFR) and rectangles indicate phosphor-insulin receptor (pIR). Blots shown are representative of three independent experiments. (B) The same conditions were used to generate separate immunoblots for pEGFR and pIR using different antibodies (n = 3; H1975 cells were used as a positive control for pEGFR (+)).
Figure 5
Figure 5
Ethanol (EtOH) regulates endothelial cell-derived EV microRNA content. (A) Luciferase expression in recipient HUVECs from a construct containing the 3′ untranslated region of CD34 was measured by bioluminescence imaging following stimulation by 100 µg/ml EVs from producer HUVECs cultured in the presence of the indicated concentrations of EtOH for 24 h (n = 4; **P < 0.01 vs. 0 mM EtOH condition, ##P < 0.01 vs. positive control (+) condition). Mock-transfected HUVEC not exposed to EVs were used as negative controls (−), while transfected HUVEC not exposed to EVs were used at the positive control (+). (B) Average whole miRNome array results comparing miR content of EVs derived from HUVEC cultured in the presence or absence of 100 mM EtOH for 24 h (n = 3). Red dots indicate upregulation of miRs and green dots indicate downregulation of miRs in HUVEC EVs based on producer cell EtOH exposure. Black dots indicate similar expression levels. (C) Expression levels of the indicated microRNAs (miRs) in endothelial cell EVs from producer cells cultured in the presence vs. absence of 100 mM EtOH for 24 h was determined by qPCR (n = 3). (D) Gap closure of HUVECs was assessed upon stimulated by EVs derived from HUVECs cultured in the absence (−EtOH) or presence (+EtOH) of 100 mM EtOH for 24 h following mock transfection or transfection by an antagomir to miR-106b (anti-miR-106b) or a scrambled oligo sequence (anti-miR-scr) (n = 3; ***P < 0.001, **P < 0.01 vs. EVs (−EtOH) group). HUVECs incubated in basal medium (EBM2, without growth factors) were used as the negative control (−) and HUVECs incubated in growth medium (EGM2, with growth factors) were used as positive controls (+).
Figure 6
Figure 6
Ethanol regulates endothelial cell-derived EV lncRNA content. (A) Expression levels of the indicated lncRNAs were assessed by qPCR in EVs from HUVECs cultured in the presence vs. absence of 100 mM EtOH for 24 h (n = 3; *P < 0.05). (B–D) HUVEC gap closure was assessed following 24 h stimulation by 100 µg/ml EVs from HUVECs cultured in the absence (−EtOH) or presence (+EtOH) of 100 mM EtOH for 24 h following transfection with a scrambled siRNA (scr) or siRNA specific to (B) HOTAIR, (C) MALAT1, or (D) both HOTAIR and MALAT1 (double transfection) (n = 4; ##P < 0.01 vs. – EtOH+ scr; **P < 0.01, ***P < 0.001 vs. +EtOH+ scr). HUVECs incubated in basal medium (EBM2, without growth factors) were used as the negative control (−) and HUVECs incubated in growth medium (EGM2, with growth factors) were used as positive controls (+).
Figure 7
Figure 7
Schematic of potential mechanism of alcohol effects on endothelial cell (EC) EVs. EtOH = ethanol, miRNA = microRNA, lncRNA = long non-coding RNA.
See this image and copyright information in PMC

References

    1. Andaloussi EL, Mager I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov. 2013;12:347–357. doi: 10.1038/nrd3978. - DOI - PubMed
    1. Lamichhane TN, et al. Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine. Tissue engineering. Part B, Reviews. 2015;21:45–54. doi: 10.1089/ten.teb.2014.0300. - DOI - PMC - PubMed
    1. Gyorgy B, Hung ME, Breakefield XO, Leonard JN. Therapeutic applications of extracellular vesicles: clinical promise and open questions. Annual review of pharmacology and toxicology. 2015;55:439–464. doi: 10.1146/annurev-pharmtox-010814-124630. - DOI - PMC - PubMed
    1. van Balkom, B. W. et al. Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells. Blood121, 3997–4006, S3991–3915, 10.1182/blood-2013-02-478925 (2013). - PubMed
    1. Deregibus MC, et al. Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood. 2007;110:2440–2448. doi: 10.1182/blood-2007-03-078709. - DOI - PubMed

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