Research ArticleENGINEERING

Macrophages of diverse phenotypes drive vascularization of engineered tissues

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Science Advances  01 May 2020:
Vol. 6, no. 18, eaay6391
DOI: 10.1126/sciadv.aay6391
  • Fig. 1 Overview of phenotype-induced changes in EC gene expression via Transwell culture with macrophages.

    (A) Primary human peripheral blood monocytes were isolated from four human donors, differentiated into macrophages, and polarized into M0, M1, M2a, M2c, or M2f phenotypes for 72 hours. (B) Gene expression of commonly associated M2 markers. (C) Levels of TGFB1 in cell culture media. Data in (B) and (C) represent means ± SEM with n = 3 to 5 technical replicates from one human donor. Differences were determined using one-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05 relative to M0, M1, and M2c phenotypes. (D) Primary HAMECs were Transwell cocultured with M0, M1, M2a, M2c, or M2f macrophages for 1 to 3 days. Changes in HAMEC gene expression were analyzed using a custom CodeSet from NanoString Technologies, inclusive of 97 genes related to angiogenesis. (E) PCA of HAMEC gene count data. To facilitate visualization, color-coded ellipses were drawn to include all samples for each phenotype. Open ellipses represent day 3 data; shaded ellipses represent day 1 data. (F) Gene counts for HAMEC treated with M0, M1, M2a, M2c, or M2f macrophages were z scored across rows and hierarchically clustered on the basis of Euclidean distance.

  • Fig. 2 Macrophage phenotypes differentially influence biological processes in ECs after 1 day of Transwell coculture.

    GO enrichment analysis was performed on lists of genes differentially regulated in ECs in response to macrophage phenotype. (A) Top five most highly enriched, nonredundant GO terms for each phenotype. (B) ssGSEA for gene sets related to specific processes of angiogenesis; analysis was not restricted to only differentially expressed genes. One-way ANOVA with Tukey’s post hoc analysis; *P < 0.05; **P < 0.01; ****P < 0.0001, # indicates P < 0.05 relative to the M0 control.

  • Fig. 3 Effects of macrophage phenotype on in vitro tissue vascularization dynamics.

    (A) In vitro engineered human blood vessels generated via coculture of HAMEC-dTom and MSCs on Gelfoam constructs. Vessel formation was visualized via live cell confocal imaging after 3 days, before addition of THP-1–derived M0, M1, or M2a macrophages. Changes in network morphology were observed 1 to 3 days after macrophage seeding via confocal microscopy. (B) Representative maximum-intensity projections of M0-, M1-, and M2a-seeded vessel networks illustrating interactions between macrophages (green) and vessels (red) 1 day after macrophage seeding. Scale bars, 50 μm. (C) Quantification of changes in network morphology via MATLAB analysis in 3D, in terms of the total number of vessels, total vessel length, and total number of branch points (junctions between two vessels). All data were normalized to day 3 baseline measurements and represent means ± SD, n = 3. Two-way ANOVA with Tukey’s post hoc analysis; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, and # indicates P < 0.05 relative to the M0 control. Data are representative of two independent experiments. (D) Representative maximum-intensity projections of engineered vascular grafts that were fixed and stained for the tip cell marker KDR (VEGFR2) via whole-mount immunohistochemistry. Scale bars, 100 μm, n = 3. Vessels shown in red; VEGFR2 staining shown in cyan. (E) Quantification of EC VEGFR2 staining, performed on maximum-intensity projections of 2 × 2 tile scans. Pixels colocalized with dTom and VEGFR2 expression were measured and normalized to the total number of dTom+ pixels in each image. Data represent means ± SEM, with n = 4 to 5. One-way ANOVA with Tukey’s post hoc analysis; *P < 0.05 and **P < 0.01.

  • Fig. 4 Macrophage interaction with host and engineered vasculature within the graft.

    (A) Representative image of an engineered vascular graft bearing GFP-expressing human blood vessels (green) after 14 days of in vitro culture. Scale bar, 1000 μm. (B) F4/80 immunostaining (cyan) of engineered vascular graft explanted 14 days after implantation, indicating interactions between engineered vessel network (green) and penetrating host vessels (red). Scale bar, 500 μm. (C to E) High-magnification images of macrophages (F4/80+, cyan) interacting with engineered (green) and host (magenta) vessels within graft marked with white arrows. Scale bars, 25 μm. (C) Macrophages form vessel-like structures; (D) macrophage adjacent to both engineered and host vessel; (E) macrophage bridging between vessel segments and wrapping around host vessel. (F) Quantification of the area of host vessels, engineered vessels, and F4/80+ cells in sectioned grafts explanted on days 1, 3, 7, and 14. Data represent means ± SD and were analyzed using one-way ANOVA with Tukey’s post hoc multiple comparisons test; *P < 0.05 for n = 6 to 8.

  • Fig. 5 Consequences of in vivo macrophage depletion on graft revascularization and integration with host vessels.

    (A) Timeline of clodronate- or PBS-loaded liposome administration, graft implantation, and retrieval. Representative images show engineered vessels (green) in graft before implantation and following retrieval. Host vessels shown in magenta and Rho-dextran perfusion in cyan. Scale bars, 1000 μm. (B) Flow cytometric analysis of whole-blood staining against the pan macrophage marker, murine F4/80, following the second administration of liposomes; n = 3 to 4. (C) Immunofluorescence staining and quantification of pan macrophage marker F4/80 (cyan) in grafts from macrophage-depleted (clodronate-liposome–treated) versus control (PBS-liposome–treated) mice 14 days after implantation. Scale bars, 100 μm. (D) Representative maximum-intensity projections of retrieved grafts 14 days after implantation in (i) control mice and (iii) macrophage-depleted mice. Scale bars, 1000 μm. High-magnification images of (ii) control and (iv) macrophage-depleted grafts. Scale bars, 100 μm. Host vessels shown in magenta, engineered vessels shown in green, and grafts perfused with Rho-dextran shown in cyan. (E) Quantification of total vessel length via AngioTool for engineered vessels and host vessels; engineered vessel perfusion of maximum-intensity projections analyzed using a custom MATLAB code. All data represent means ± SD and were analyzed using an unpaired t test with n = 3 to 4.

  • Fig. 6 Macrophage phenotype subsets during graft integration.

    Representative maximum-intensity projections of engineered vascular grafts bearing GFP-expressing vasculature (green) extracted on days 1, 3, 7, and 14 after implantation and sectioned. Graft sections were stained for pan macrophage marker F4/80 (cyan) and costained for either iNos [magenta, (A)] or Arg1 [magenta, (B)] to determine presence of macrophages expressing M1 (A) and M2 (B) markers. Scale bars, 50 μm. (C) Quantification of F4/80+iNos+ staining and F4/80+Arg1+ staining. Data represent means ± SD and were assessed via one-way ANOVA with Tukey’s post hoc analysis; n = 3 to 4.

Supplementary Materials

  • Supplementary Materials

    Macrophages of diverse phenotypes drive vascularization of engineered tissues

    P. L. Graney, S. Ben-Shaul, S. Landau, A. Bajpai, B. Singh, J. Eager, A. Cohen, S. Levenberg, K. L. Spiller

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