Morphological analysis (A-L).

<p>Morphological at optical (A-E, C, I) and transmission electron microscopes (F, H, J-L) analyses of sections from <i>H</i>. <i>medicinalis</i> body wall. The body wall of untreated leeches results practically avascular (A) and the botryoidal tissue (b) appears as a solid chord of clustered cells (B). After 1 hour from MWCNTs treatment, numerous neo-vessels (v) are found among muscles (m) and under the epithelium (e) (C). Within the new cavities (c) lined by the botryoidal tissue (b), immunocompetent circulating precursors cells (arrowhead) are clearly distinguishable (D). After 3 hours from treatment, numerous fibroblasts are visible immersed in an abundant extracellular matrix ECM (E, F). After 1 (G, H) up to 5 weeks (I) from MWCNTs treatment, numerous vessels (v) and fibroblasts (arrowheads) are still visible in the body wall. (J-L) Detail of MWCNTs (arrowheads) freely dispersed in the cytoplasm of macrophage-like cells. Bars in A, C, G: 100μm; Bars in B, D-E, I: 10μm; Bar in F: 5μm; Bar in H: 2μm; Bar in J: 500nm; Bars in K-L: 200nm.</p

Acid phosphatase reaction (A-F) and anti CD68 immunofluorescence staining (G-L).

<p>Resident in untreated (A, G) and migrating macrophages-like cells in treated leeches (B-E) and (H-L), located under the epithelium (e) and among the muscle fibers (m), are positive for acid phosphatase reaction (arrowheads in A-F) and for anti-CD68 (G-L). (F) Quantitative evaluation of cell numbers. Column 1: number of cells in untreated sample, columns 2–10: number of cells in MWCNTs treated sample from 1h up to 5 weeks. *p<0.01. (J) Combined transmission and fluorescence images showing CD68⁺ cells (in red) in spatial association with MWCNTs aggregates (circled). Bars in A-E, G-I, K-L: 100μm; Bar in F, J: 10μm.</p

Determination of MWCNTs presence in tissues (A-D).

<p>(A) MWCNTs extracted by means of KOH digestion from exposed-animals tissues. (B-D) MWCNTs crude powder (used as control) raw (B), after sonication (C) and after KOH treatment (D). Bars in A-D: 500nm.</p

Anti-IL18 (A-F) and Thioflavine-T staining (G-L).

<p>(A-F) Localization of IL-18. Note the population of resident in untreated (A) and migrating (B- F) immune-responsive cells (arrowheads) located under the epithelium (e) and among the muscle (m). Nuclei are counterstained with DAPI (blue). (G-K) Thioflavin-T method. Amyloid material is stained in yellow (arrowheads). (L) Double-staining of Thioflavin-T (yellow) and macrophage markers CD68 (red) in a cryosection of 3 hours MWCNTs treated leech body wall. Bars in A-E, G-K: 100μm; EDS analysis.</p

Characterization of circulating precursors cells (A-D).

<p>Morphological (A) and immunohistochemical (B) analysis of cryosections from <i>H</i>. <i>medicinalis</i> body wall. The circulating precursors (arrowhead) visible in lumen of neo-vessels are highly positive for anti-CD45 antibody. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). (C-D) Western blot analysis. A protein extracts from the body wall of untreated (n.t) and MWCNTs treated leeches from 1 to 12 hours and from 1 to 5 weeks were probed with the anti-CD45 antibody. The housekeeping protein D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. In all samples, the anti-CD45 detected specific immunoreactive band 24 of about 145 kDa (C), according to the molecular weight ladder (kDa). CD45 protein was quantified by densitometry from three experiments. *P<0.05 compared with untreated leeches (D). Bars in A-B: 10μm.</p

Analysis of the phenotype of cells recruited into the matrigel sponges by MCP-1/CCL2.

<p>MCP-1/CCL2 induces the mobilization of cells from tissues surrounding the MG implant into the biopolymer that was recovered and processed for structural, ultrastructural immunocytochemical analysis or seeded for cell culture (A–O). After 1 week <i>in vivo</i> the matrigel implant contained granular cells that stained with Giemsa solution (A) that were ultrastructurally similar (B) to those localized in the extracellular matrix (ECM) in stimulated leeches (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001910#pone-0001910-g002" target="_blank">Fig. 2C</a>, arrowhead). These cells showed a cytoplasm filled with black granules (B, C). Again they were of a migratory phenotype with localized degradation of ECM (B, C arrowheads), and cathepsin B staining (D). Immunocytochemical characterization of cells infiltrating the MG supplemented with MCP-1/CCL2 (E–I). The cells expressed several markers typical of granulocytes: CD11c (E), CD14 (F), CD61 (G), CD68 (H); (I: negative control). After 1 week <i>in vivo</i> the MG was used to seed cell cultures (J–O). Phase-contrast images of granular cells obtained 3 days (J) and 1 week (K) post-seeding indicate increasing cell numbers (K). These Giemsa positive cells (L) also immuno-stained with a CD11c antibody (M), a marker of granulocytes, and with anti-BrdU (N) demonstrating that these cells attraversed S-phase in culture; O: negative control. After incubation with Dil-Ac-LDL, these cells also took up the fluorochrome-conjugated Ac-LDL (P). These results strongly indicate that the cells that migrated out of the matrigel <i>in vitro</i> consisted largely of cells maintaining a monocyte/macrophage function throughout <i>in vivo</i> and <i>in vitro</i> culture as indicated by expression of CD68 (Q, R). Bars: A, D, P–R 10 µm; B, C 1 µm; E–I 15 µm; J–O 20 µm</p

Culture of cells recruited into the matrigel sponges by VEGF.

<p>After 1 week <i>in vivo</i> the MG was removed and the cells infiltrating the matrigel sponge were plated out at a low density and allowed to form colonies over 24 hrs. (A) Phase contrast image of Matrigel extracted after 1 week <i>in vivo</i>. It is infiltrated by numerous rounded cells that stained with Giemsa solution (A1). (B) Phase contrast image of small colonies after 24 hrs from seeding formed by rounded cells which stained with Giemsa (C) incorporate BrdU in their nuclei (arrowheads in D). (E–G) Double labelling experiments showing the expression in these cells of the myogenic marker MyoD (green in E–G) and the endothelial markers FLK-1 (red in E), VE-cadherin (red in F), and CD34 (red in G). (H) TEM micrograph of precursors cells showing an undifferentiated phenotype with large nucleus (n) and scarce cytoplasm. (I) Phase contrast image of a single colony that was picked with a micropipette and clonally expanded for a total of 3 days in vitro. (J–L) Double labelling experiments of clonal colonies showing the expression of MyoD (green in J–L) and the endothelial markers FLK-1 (red in J), VE-cadherin (red in K) or CD34 (red in L). (M) Clonal expansion of single colonies in phase contrast after 8 days in vitro. Rounded (arrowhead) and spindle-shape cells (arrow) that stained with Giemsa solution (N) are visible. (O) Positivity for BrdU incorporation is detectable only in the nuclei (arrowhead) of the rounded cells. Double immunofluorescence experiments detect the expression of CD34 (red in P) and MyoD (green in P) in the cultured cells. These cells are desmin⁺ as well (red in Q). (R) TEM image showing thick filaments (arrowheads) in differentiating elongated cells. Phase contrast image (S) and Giemsa staining (T) of spindle-shape cells that dominate the clonal cultures after 20 days in vitro. The differentiating spindle shaped cells are BrdU⁻ (U), CD34⁺ (green in V) and skeletal MyHC⁺ (red in Z). (W) TEM photomicrograph showing a well differentiated muscle fibre with contractile material clearly organized into sarcomeres (arrowhead in W). Nuclei are counterstained with DAPI (blue in Q, V, Z). Bars in A–D: 100 µm; Bars in E–G: 20 µm Bars in I–L, V, Z 15 µm; Bars in M–Q, S–U: 10 µm; Bars in H, R, W: 400 nm.</p

Cells recruited by VEGF or by MCP-1 express different markers

<p>Cells infiltrating the matrigel (MG) supplemented with VEGF expressed only hematopoietic stem cells markers and are negative for myeloid lineage markers. On the contrary, cells that migrated into the matrigel supplemented with MCP-1/CCL2 expressed only myeloid lineage markers.</p

Histology of matrigel sponges <i>in vivo</i>.

<p>The cells infiltrating the gels without (A, D) and with the cytokines -VEGF (B, C) or MCP-1/CCL2 (E, F) after 48 h (A, B, E) and 1 week (D, C, F). The two cytokines selectively recruit different cellular types. Bars: A–F, 20 µm</p

Characterization of myoendothelial cells recruited into the matrigel sponges by VEGF.

<p>(A, B) Cryosections of MG, supplemented with VEGF and inoculated in leeches after an intramuscular Dil-Ac-LDL injection. After 1 week <i>in vivo</i> the matrigel implant contains rounded cells stained with Giemsa (A) and showing uptake of Dil-Ac-LDL (red in B). (C–I) Double labelling on serial cryosections of MG, supplemented with VEGF and inoculated in leeches. After 1 week <i>in vivo</i>, MG is infiltrated by cells expressing the MyoD muscle transcriptional factor (green in C–E and in the inserts) and the hematopoietic/endothelial precursor markers either CD34 (red in C and the insert), VE-cadherin (red in D and the insert), or Flk-1 (red in E and the insert). No staining is detected for the macrophage markers CD11c (red in F) CD68 (red in G) and CD14 (red in H). (I) Negative control where the primary antibodies were omitted and the secondary antibodies were applied together. After 15 days (J, K), MG is infiltrated by elongated Giemsa stained cells (J) that expressed desmin (green in K). After 1 month (L–N) groups of differentiated muscle fibres (L) are present in the MG sponge that are stained by the anti-skeletal MyHC A4.1025 antibody (green in M) and show the contractile material organized in sarcomeres with TEM (N). Nuclei are counterstained with DAPI (blue in B, K, M). (A1, J1, L1): cryosections of matrigel without VEGF injected as controls. Bars in A, A1, F-1: 100 µm; Bars in B–E: 50 µm; J, J1, K: 20 µm; Bars in L, L1, M: 10 µm; Bar in N: 2 µm.</p