Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Filters applied. Clear all
. 2018 Jun 28;7(1):1487250.
doi: 10.1080/20013078.2018.1487250. eCollection 2018.

Extracellular Vesicles From Endothelial Progenitor Cells Promote Thyroid Follicle Formation

Affiliations
Free PMC article

Extracellular Vesicles From Endothelial Progenitor Cells Promote Thyroid Follicle Formation

Jonathan Degosserie et al. J Extracell Vesicles. .
Free PMC article

Abstract

Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-α1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-α1.

Keywords: Thyroid; endothelial cells; extracellular vesicles; folliculogenesis; laminins.

Figures

Figure 1.
Figure 1.
EVs isolated from EPC-CM display exosomal properties. (A) Quantification and size distribution of EVs obtained by Nanoparticle Tracking analysis using a Zetaview (Particle Metrix). Results are presented as the number of particles per 106 producing cells. (B) Representative scanning electron microscopy image of the 150k pellet obtained from EPC-CM. Enlarged view of the boxed area is shown at the right panel. EVs are indicated by arrows. (C) Western blot analysis of calnexin (ER marker) and flotilin-1, CD63 and CD9 (EVs markers) in two EPCs and two EPC-EV lysates. (D) Western blot analysis of CD63 on eight fractions (F1–F8) resolved by floatation in iodixanol density gradient. Density of the eight fractions is shown in the upper graph.
Figure 2.
Figure 2.
EPC-EVs are taken up by embryonic thyroid progenitors and stimulate folliculogenesis. (A) Immunofluorescence for CD63 (red) of cultured embryonic thyroid progenitors without (left panel) or after incubation with PKH67-labeled EVs (green; right panel). Cell contours are delineated by dotted lines. Note clustering around cell. High magnifications of the boxed areas at left are shown at right in single channels for CD63 and PKH signals. (B) Immunofluorescence on sections of thyroid explants cultured without (control) or with EPC-EVs for E-cadherin (red) to define epithelial cells and ezrin (green) to identify their apical pole, thus lumen contour. (C) Folliculogenic index in control- and EPC-EVs-treated explants determined by quantification of ezrin+ open follicles (***p < 0.001; n = 5).
Figure 2.
Figure 2.
EPC-EVs are taken up by embryonic thyroid progenitors and stimulate folliculogenesis. (A) Immunofluorescence for CD63 (red) of cultured embryonic thyroid progenitors without (left panel) or after incubation with PKH67-labeled EVs (green; right panel). Cell contours are delineated by dotted lines. Note clustering around cell. High magnifications of the boxed areas at left are shown at right in single channels for CD63 and PKH signals. (B) Immunofluorescence on sections of thyroid explants cultured without (control) or with EPC-EVs for E-cadherin (red) to define epithelial cells and ezrin (green) to identify their apical pole, thus lumen contour. (C) Folliculogenic index in control- and EPC-EVs-treated explants determined by quantification of ezrin+ open follicles (***p < 0.001; n = 5).
Figure 3.
Figure 3.
Depletion of EVs in the EPC-CM abrogates folliculogenic activity. (A) Silver staining and western blot analysis using CD63 and laminin-α1 antibodies on equal volume of 10× EPC-CM, 10× EV-low EPC-CM and 20× EPC-EVs. (B) Quantification and size distribution of particles in EPC-CM, supernatant (EV-low EPC-CM) and 150k pellet from ultracentrifuged EPC-CM (EPC-EVs), obtained by Nanoparticle Tracking analysis using a Zetaview (Particle Metrix). (C) Immunofluorescence of whole thyroid explant cultured without (Control) or with EPC-EVs (20X concentration) or EV-low EPC-CM (10× concentration). Epithelial cells (E-cadherin) are visualised in red and their apical pole (ezrin) is labelled in green. (D) Quantification of the folliculogenic effect of EPC-EVs and EV-depleted EPC-CM (***p < 0.001; ** p < 0.01; six explants per condition were analysed in two independent experiments).
Figure 3.
Figure 3.
Depletion of EVs in the EPC-CM abrogates folliculogenic activity. (A) Silver staining and western blot analysis using CD63 and laminin-α1 antibodies on equal volume of 10× EPC-CM, 10× EV-low EPC-CM and 20× EPC-EVs. (B) Quantification and size distribution of particles in EPC-CM, supernatant (EV-low EPC-CM) and 150k pellet from ultracentrifuged EPC-CM (EPC-EVs), obtained by Nanoparticle Tracking analysis using a Zetaview (Particle Metrix). (C) Immunofluorescence of whole thyroid explant cultured without (Control) or with EPC-EVs (20X concentration) or EV-low EPC-CM (10× concentration). Epithelial cells (E-cadherin) are visualised in red and their apical pole (ezrin) is labelled in green. (D) Quantification of the folliculogenic effect of EPC-EVs and EV-depleted EPC-CM (***p < 0.001; ** p < 0.01; six explants per condition were analysed in two independent experiments).
Figure 4.
Figure 4.
Knockdown of laminin-α1 in EPC abrogates folliculogenic activity of the 150k pellet. (A) Western blot analysis of laminin-α1, CD63, flotilin-1 and CD9 on EPC-EVs, shα1-EVs and GipZ-EVs lysates, as compared to parental cell line lysates. (B) Quantification of laminin-α1 knockdown in infected cell lysates using GAPDH as a control (*p < 0.05; n = 4). (C) Immunofluorescence of thyroid explant sections after culture without (control) or with EPC-EVs, shα1-EVS or GipZ-EVs as indicated. Epithelial cells (E-cadherin) are labelled in red and their apical pole (ezrin), in green. (D) Quantification of the folliculogenic effect of EPC-EVs, shα1-EVs and GipZ-EVs (***p < 0.001; n = 5).
Figure 4.
Figure 4.
Knockdown of laminin-α1 in EPC abrogates folliculogenic activity of the 150k pellet. (A) Western blot analysis of laminin-α1, CD63, flotilin-1 and CD9 on EPC-EVs, shα1-EVs and GipZ-EVs lysates, as compared to parental cell line lysates. (B) Quantification of laminin-α1 knockdown in infected cell lysates using GAPDH as a control (*p < 0.05; n = 4). (C) Immunofluorescence of thyroid explant sections after culture without (control) or with EPC-EVs, shα1-EVS or GipZ-EVs as indicated. Epithelial cells (E-cadherin) are labelled in red and their apical pole (ezrin), in green. (D) Quantification of the folliculogenic effect of EPC-EVs, shα1-EVs and GipZ-EVs (***p < 0.001; n = 5).
Figure 5.
Figure 5.
Independent effect of EPC-EVs and EPC-laminins on thyroid folliculogenesis. (A) Western blot analysis of laminin-α1, CD63 and CD9 after separation of EPC-EVs by floatation on iodixanol gradient. (B) Immunofluorescence of whole thyroid explant cultured without (control) or with pooled fractions 1 to 4 (F1–F4), 5 to 8 (F5–F8) and 1 to 8 (F1–F8). Epithelial cells (E-cadherin) are labelled in red and their apical pole (ezrin) in green. A single section is shown for each condition. (C) Quantification of the folliculogenic effect of F1–F4, F5–F8 and F1–F8 (***p < 0.001; ** p < 0.01; n = 3).
Figure 5.
Figure 5.
Independent effect of EPC-EVs and EPC-laminins on thyroid folliculogenesis. (A) Western blot analysis of laminin-α1, CD63 and CD9 after separation of EPC-EVs by floatation on iodixanol gradient. (B) Immunofluorescence of whole thyroid explant cultured without (control) or with pooled fractions 1 to 4 (F1–F4), 5 to 8 (F5–F8) and 1 to 8 (F1–F8). Epithelial cells (E-cadherin) are labelled in red and their apical pole (ezrin) in green. A single section is shown for each condition. (C) Quantification of the folliculogenic effect of F1–F4, F5–F8 and F1–F8 (***p < 0.001; ** p < 0.01; n = 3).

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Fagman H, Andersson L, Nilsson M.The developing mouse thyroid: embryonic vessel contacts and parenchymal growth pattern during specification, budding, migration, and lobulation. Dev Dyn. 2006February;235(2):444–12. - PubMed
    1. Colin IM, Denef JF, Lengelé B, et al. Recent insights into the cell biology of thyroid angiofollicular units. Endocr Rev. 2013April;34(2):209–238. - PMC - PubMed
    1. Nilsson M, Fagman H. Development of the thyroid gland. Development. 2017June15;144(12):2123–2140. - PubMed
    1. Hick AC, Delmarcelle AS, Bouquet M, et al. Reciprocal epithelial: endothelialparacrine interactions during thyroid development govern follicular organization and C-cells differentiation. Dev Biol. 2013September1;381(1):227–240. - PubMed
    1. Villacorte M, Delmarcelle AS, Lernoux M, et al. Thyroid follicle development requires Smad1/5- and endothelial cell-dependent basement membrane assembly. Development. 2016June1;143(11):1958–1970. - PMC - PubMed

Grant support

This work was supported in part by grants from the Fonds pour la Recherche Scientifique (F.R.S–FNRS, Belgium), Université catholique de Louvain (Actions de Recherche concertées), The King Baudouin Foundation.

LinkOut - more resources

Feedback