Quantification of extracellular matrix composition and cell number.

<p>Total cell numbers, represented as DNA and total collagen secretion of collagen, represented as hydroxyproline (HYP) represented ≈ 50% of the native human aorta. The glycosaminoglycan (GAG) content was similar to native aorta.</p

HDL insudation in the engineered artery equivalent.

<p>25 µg/ml of HDL was circulated for 24 hours. The insudation of HDL (white) was assessed after cryosection of the tissue on the luminal side (A) and in the tissue (B) (blue: nuclei). HDL localization in the tissue was further analysed by confocal microscopy at 2.5 um (C) and 16 um (D) deep in the tissue. The arrow shows the intracellular vesicular localisation. The up-take of HDL (white) into HUVECs (E) and UCMFB (F) was analysed in regular cell culture after 24 hours incubation with 25 µg/ml. Bars represent 50 µm and L: Lumen.</p

Bioreactor and macrostructure of the engineered artery equivalent.

<p>A) Schematic view of the bioreactor set up. B) Macroscopic picture of the bioengineered artery demonstrated the presence of an open lumen after 5 weeks in culture as well as the formation of tissue on the luminal side of the graft. Bar represents 1 cm.</p

Insudation of LDL into the engineered artery.

<p>The insudation of LDL into the tissue (blue: nuclei) was analysed by confocal microscopy after incubation with 20 µg/ml of fluorescent LDL (white) for 24 hours and demonstrated the vesicular localization on the upper cell layer (A). The specificity of the signal was assessed by competition with 40-fold excess of non-labelled LDL (B). LDL localization in the tissue was further analysed by microscopy of tissue cryosections and demonstrated the time-dependent uptake and the sub-endothelial localization of the LDL (C: 2.5 hours and F: 24 hours). Zoom in of interesting regions shown with the arrows are presented in D and E for 2.5 hours and F and G for 24 hours. Bars represent 200 µm (A-C,F) and 20 µm (D-E, G-H) and L: Lumen.</p

Integrity and functionality of endothelial cells in the engineered artery.

<p>The endothelial integrity was analysed using Evan’s blue staining (0.5% for 10 minutes). In the absence of endothelial cell (A) the tissue appeared evenly stained in blue. Two weeks after endothelialization (B) the endothelial barrier retained the absorption of Evan’s blue and tissue appeared non-coloured. The integrity of the endothelium was further analysed by microscopy of cryosections for the expression of the tight junction protein (ZO)-1 (red) by endothelial cells (nuclei: blue). (C & D). Nitric oxide was measured using L-³H-arginine as the substrate and in presence of 10 nM acetylcholine (Ach) or 0.5 mg/ml HDL as the stimulator or 1 mM L-NAME as the inhibitor of endothelial nitric oxide synthase (eNOS). After 30 minutes L-³H-citruline was separated from L-³H-arginine by ion exchange column. Bar represents 100 (C) and 25 µm (D) and L: Lumen. *** p<0.001, ** p<0.01 and * p<0.05</p

Histological structure of an engineered artery equivalent.

<p>A) Haematoxylin-Sudan staining demonstrated the formation of tissue in and on the surface of a PGA/P4HB scaffold as well as the scaffold’s partial degradation. B) H&E staining revealed dense tissue formation composed of cells and extracellular matrix. C) The secretion of collagen was observed after Masson’s trichrome staining. The expression of α-smooth muscle actin (α-SMA) (D) confirmed the smooth muscle phenotype of the cells in the inner layer. Collagen IV positive staining (E) demonstrated the secretion of basement membrane and CD31 positive staining (F) confirmed the presence of an endothelial cell monolayer on the luminal side of the bioengineered artery equivalent. Bars represent 100 µm.</p

Endothelial monocyte adhesion and transmigration migration in the engineered artery.

<p>After pre-treatment in the absence (A, D) or the presence of 3 hours TNFα (10 ng/ml) (B, D) or 24 hours LDL (20 µg/ml) (C-I) 1×10⁶ fluorescently labelled monocytes (white) per ml were injected into the circulation loop and circulated for 24 hours. Tissues were analyzed by confocal microscopy and after cryosectionning. In addition, monocytes remaining in the circulation were counted. More monocytes (white arrows) adhered after pre-treatment with TNFα (B) or LDL (C) compared to the not stimulated (A). Less monocyte remained into the circulation after TNFα or LDL pre-treatment compared to the absence of stimuli (D). Monocytes adhesion and migration in the tissue was further analyzed by microscopy of cryosections after LDL pre-treatment. Microscopic observations demonstrated adhesion and migration of monocytes through the endothelium (dash line) (E, G) and accumulation of monocytes into the tissue (F, H-I). Bars represent 200 µm (A-C, E-F) and 20 µm (G-I).</p

Schematic model showing the links between the significant changes of muscle-specific attributes with the expression of ERV env genes, their receptors and muscle specific genes relating to cell fusion occurring <i>in vivo</i> (biopsies from cyclists at the pre- and post- competitive seasons) and <i>in vitro</i>.

<p>The top represents the muscle differentiation in cyclists from pre- (PRE) to post-competitive season (POST), whereas the bottom symbolizes the myoblast cultures proliferating in growth media (GM) or differentiating to myotubes in differentiation media (DM). Additionally, since SCs and myonuclei showed positive expression for protein kinase A activated pCREB-Ser133 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132099#pone.0132099.g004" target="_blank">Fig 4</a>) and treatment of primary myoblast cultures with the cAMP stimulator Forskolin did not promote myoblast cell fusion (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132099#pone.0132099.g006" target="_blank">Fig 6</a>), we predict that cAMP may be important for regulating SCs. SC = satellite cells; MP = muscle progenitors; MT = myotubes; PRE = pre-competition; POST = post-competition; GM = growth media; DM = differentiation media; arrow up = significantly up-regulated and arrow down = significantly down-regulated.</p

Immunofluorescence of human primary myoblasts in culture with GM and DM with or without anti-Syncytin-1 (-/+ Ab) for 4 days and analysis by confocal microscopy for nuclei (Hoechst 33342), F-actin (Phalloidin Alexa488), membrane (wheat germ agglutinin Alexa594) and the merge with all.

<p>Note the multinucleated myofibre in DM. DM composite of 96 z-stacks and GM of 17 z-stacks. Bars = 50μm.</p

Muscle cross-sections of cyclists after pre-competitive season show consecutive tissue sections with immuno-localization of MyHC-I and MyHC-IIA as well as the fusogenic ERVW-1 env protein Syncytin-1.

<p>For comparison of Syncytin-1 protein expression with muscle cells the far right picture shows a positive control of Syncytin-1 immunolocalization on normal third trimester placental tissues [left = extra villous trophoblasts (EVT); right = syncytiotrophoblast (SCT)]. The graph represents a semi-quantitative analysis of Syncytin-1 protein signal intensity measured using ImageJ. The Syncytin-1 expression was then correlated with the fiber types, including MyHC-I (set to 100%), MyHC-IIA and MyHC-IIX. Note that the upper panels show IHC and the lower panels show magnifications of the squares. Color code represents fiber type in the lower panels: red = MyHC-I, green = MyHC-IIA and difference of both is marked black = MyHC-IIX. Bars = 100μm. *** = statistically significant (p< 0.005).</p