Research ArticleSURGICAL ADHESIONS

Surgical adhesions in mice are derived from mesothelial cells and can be targeted by antibodies against mesothelial markers

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Science Translational Medicine  28 Nov 2018:
Vol. 10, Issue 469, eaan6735
DOI: 10.1126/scitranslmed.aan6735
  • Fig. 1 Mouse surface mesothelium proliferates in response to the induction of adhesions.

    (A) Adhesions were induced in mice by surgical placement of ischemic buttons in the mouse peritoneum at t = 0 days. Ischemic buttons were analyzed for mesothelial proliferation 3 days after button induction. Adhesions were analyzed 7 days after button induction. (B) Images show H&E staining of ischemic buttons at 30 min and 1, 2, and 4 hours after button induction, followed by abrasion in the peritoneum (black arrowheads) (n = 2 mice per time point). (C) Immunofluorescence staining of ischemic buttons for PDPN and cytokeratin 19 (K19) at 0, 6, 12, and 24 hours after button induction and abrasion of the mouse peritoneum (n = 2 mice per time point). (D) Immunofluorescence staining of string adhesions between the liver (L) and peritoneum (P) for MSLN and PDPN 7 days after adhesion induction in mice. (E) Numbers of mesothelial cells (MSLN+) (top) and double-positive EdU+MSLN+ mesothelial cells (bottom) were counted in n = 20 high-powered fields (0.75 mm × 1 mm) in normal mouse peritoneal tissue versus peritoneal tissue with adhesions. (F) Immunofluorescence staining for MSLN and EdU (MSLN+EdU+ in white arrowheads) in normal mouse peritoneum (P) (top) and adhesions (ADH) between the peritoneum and large intestine (I). Scale bars, 100 μm. Values are presented as means ± SEM; ***P < 0.0005, ****P < 0.0001. Analyses were done with an unpaired t test.

  • Fig. 2 Lineage tracing and clonal analysis of mesothelial cells involved in adhesion formation in mice.

    (A) Whole-mount imaging of an ischemic button (IB) imaged in vivo 30 min after adhesion induction and treatment with the stain CFSE. (B) Immunofluorescence staining of PDPN, K19, and CFSE on the surface mesothelium surrounding a single ischemic button (single staining for PDPN, K19, and CFSE is shown in fig. S13). (C to E) Whole-mount imaging of an ischemic button 4 days after adhesion induction and treatment with CFSE stain. Adhesions (ADH) are indicated by white boxes and white arrowheads (n = 3). (F and G) Immunofluorescence staining of adhesions for PDPN and CFSE 7 days after adhesion induction (n = 5). (H) Immunofluorescence staining of adhesion sites for PDPN and ActinCreER; R26VT2/GK3 7 days after adhesion induction and CFSE injection. White arrowheads indicate cells originating from the same precursor. (I) Immunofluorescence staining for PDPN and ActinCreER; R26VT2/GK3 at adhesion sites 7 days after adhesion formation (n = 3). White arrowheads indicate a spindle-like phenotype. Scale bars, 100 μm unless otherwise indicated.

  • Fig. 3 Mesothelium is a necessary component of adhesions in mice.

    (A) Light sheet microscopy images after injection of anti-MSLN antibodies into the mouse peritoneum after adhesion induction in vivo. Autofluorescence (green) indicates the suture (S) and muscle fibers of the peritoneum (P). (B) Further analysis by light sheet microscopy shows longitudinal expansion of MSLN+ staining that is parallel to the muscle fibers. Volume rendering of 2D images (a virtual z-stack) as a 3D model was done using the MIP or blend mode. 2D representations are MIPs of 36-μm-thick sections from the 3D model. (C) CD47 expression in FPKM (fragments per kilobase of transcript per million mapped reads) at 6, 12, and 24 hours after adhesion formation in response to button induction in mice. (D) Phagocytosis of mesothelial cells treated with anti-CD47 antibody by macrophages. (E) Immunofluorescence staining of macrophages (red) and mesothelial cells (green) showing phagocytosis (denoted by white arrowheads) of mesothelial cells by macrophages. (F) Appearance of adhesions 2 weeks after button induction in mice. (G) Appearance of adhesions after treatment with anti-MSLN antibodies injected at 7, 10, or 13 days after adhesion formation. (H) Adhesion score after treatment of formed adhesions in mice with anti-CD47 antibody alone (n = 8), anti-MSLN antibody alone (n = 3), or a combination of anti-CD47 and anti-MSLN antibodies (n = 5) compared to control untreated mice injected with phosphate-buffered saline (PBS; n = 10). Antibodies were injected at 7, 10, and 13 days after induction of adhesions. Values are presented as means ± SEM; **P < 0.005, ****P < 0.0001. Statistical analyses were done with an unpaired t test. (I) Ischemic buttons (IB) from mice after antibody treatment showing immunofluorescence staining for collagen, fibronectin, CD31, F4/80, and MSLN.

  • Fig. 4 Mesothelial cells show a distinct transcriptional profile after adhesion induction in mice.

    (A) Surface mesothelium was isolated from ischemic buttons, and cells with a PDPN+LYVE1CD31CD45 surface phenotype were obtained by flow cytometry. DAPI, 4′,6-diamidino-2-phenylindole. (B) Heatmap of RNA expression in purified surface mesothelium immediately after button induction in mouse peritoneum (t = 0) and 6, 12, and 24 hours after button induction. Representative genes are shown above gene clusters. (C) Log fold changes in transcript expression were calculated 24 hours after induction of adhesions compared to controls and were plotted against gene identity. (D) Number of up-regulated and down-regulated genes from uninjured and injured mesothelium at 24 hours after button induction is shown versus the number of up-regulated or down-regulated genes in HSCs and their progeny after differentiation. Differentiated hematopoietic cells included multipotent progenitors (MPPs), common myeloid progenitors (CMPs), monocytes, natural killer (NK) cells, CD4+ and CD8+ T cells ± CD69 expression, AML stem cells (LSCs), AML progenitor cells (LPCs), and AML blasts. (E) Heatmap of gene sets up-regulated or down-regulated in mouse surface mesothelium 6, 12, and 24 hours after adhesion formation. NF-κB, nuclear factor κB; BMP, bone morphogenetic protein.

  • Fig. 5 Mouse mesothelial gene expression compared to fibroblast gene expression.

    (A) Expression of genes (indicated above graphs) in mouse mesothelial cells by RNA sequencing (FPKM) over 24 hours. (B) Immunofluorescence staining of adhesions 7 days after button induction for PDPN, MSLN, CD44, S100A4, and K19. Scale bars, 100 μm. ADH, adhesions; L, liver; P, peritoneal wall; I, intestine.

  • Fig. 6 Inhibition of HIF1α is sufficient to prevent adhesion formation in mice.

    (A) Mesothelial macrophage cocultures in vitro under normal oxygen conditions (normoxia) immunostained for PDPN or HIF1A expression. (B) Mesothelial macrophage cocultures in vitro under hypoxia conditions (5% O2 incubator) stained for PDPN or HIF1A expression. Scale bars, 250 μm. (C) Gross anatomy, immunofluorescence staining, and H&E staining of mouse adhesions 7 days after button induction and treatment with echinomycin (20 μg/kg) daily for 7 days. (D) Adhesion score after treatment of mouse adhesions with echinomycin or PX12, small-molecule inhibitors of HIF1α. (E) Numbers of double-positive mesothelial cells (MSLN+PDPN+) and triple-positive EdU+MSLN+ PDPN+ mesothelial cells were counted per high-powered field (0.75 mm × 1 mm) after treatment of mice with the HIF1α inhibitor echinomycin (n = 16 fields) and were compared to untreated control mice (n = 5 fields). (F) Expression measured by RNA sequencing (FPKM) of selected target genes during a 24-hour time course after adhesion induction and treatment of mice with the HIF1α inhibitor echinomycin (n = 2). Heatmap showing RNA sequencing of surface mesothelium immediately after echinomycin treatment and button induction and 6, 12, and 24 hours after echinomycin treatment and button induction. Scale bars, 100 μm unless otherwise noted. Values are presented as means ± SEM; **P < 0.005, ****P < 0.0001. Statistical analyses were done by an unpaired t test.

  • Fig. 7 Gene expression in human peritoneal adhesions.

    (A) H&E staining (left) of representative human adhesion tissue from n = 6 patients undergoing surgery showing mesothelial cells present within the adhesion. Trichrome staining (right) of representative human adhesion tissue from n = 6 patients undergoing surgery showing mesothelial cells within areas of fibrosis. (B) Immunofluorescence staining and in situ hybridization for PDPN, MSLN, S100A4, WT1, CD47, and UPK1B in human adhesion tissue isolated from patients undergoing surgery (n = 6). Scale bars, 100 μm unless otherwise noted.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/469/eaan6735/DC1

    Fig. S1. The surface mesothelium remains after gentle abrasion injury.

    Fig. S2. The surface mesothelium remains on buttons after placement.

    Fig. S3. PDPN and MSLN are exclusive to surface mesothelium.

    Fig. S4. The surface mesothelium buttons are MSLN+ after placement.

    Fig. S5. The surface mesothelium thickens in response to injury.

    Fig. S6. The surface mesothelium thickens in response to injury.

    Fig. S7. Chimerism in parabiotic mice.

    Fig. S8. Circulating cells do not contribute significantly to adhesions.

    Fig. S9. EdU+ cells are found in areas of tissue thickening.

    Fig. S10. Scanning electron microscopy of peritoneal buttons.

    Fig. S11. Scanning electron microscopy of peritoneal buttons.

    Fig. S12. Large block-face scanning electron microscopy of peritoneal buttons.

    Fig. S13. CFSE treatment labels only surface mesothelial cells.

    Fig. S14. CFSE-labeled mesothelial cells contribute to adhesions.

    Fig. S15. PDPN+ ActinCreER; R26VT2/GK3 cells give rise to adhesions.

    Fig. S16. In vitro mesothelial transition.

    Fig. S17. Anti-MSLN antibody specifically binds the surface mesothelium.

    Fig. S18. Antibodies against host IgG do not bind mesothelial cells.

    Fig. S19. Adhesion scoring characterized by surface area contact and molecular markers.

    Fig. S20. K19+PDPN+ mesothelium persists after antibody treatment.

    Fig. S21. Mesothelium persists after antibody treatment.

    Fig. S22. ECM genes are down-regulated after adhesion induction.

    Fig. S23. Validation of enriched genes via immunofluorescence.

    Fig. S24. Transcript levels of HIF1A stabilization proteins.

    Fig. S25. Mesothelial in vitro culture assay.

    Fig. S26. HIF1A and mesothelial gene targets after HIF1α inhibition.

    Fig. S27. HIF1A and mesothelial gene targets after HIF1α inhibition.

    Fig. S28. Echinomycin does not affect DNA replication.

    Fig. S29. A proposed mesothelial centric model of adhesion formation.

    Date file S1. Source data.

    Movie S1. 3D rendering of light sheet microscopy imaging of injured surface mesothelium.

    Movie S2. 3D rendering of light sheet microscopy imaging of injured surface mesothelium.

  • The PDF file includes:

    • Fig. S1. The surface mesothelium remains after gentle abrasion injury.
    • Fig. S2. The surface mesothelium remains on buttons after placement.
    • Fig. S3. PDPN and MSLN are exclusive to surface mesothelium.
    • Fig. S4. The surface mesothelium buttons are MSLN+ after placement.
    • Fig. S5. The surface mesothelium thickens in response to injury.
    • Fig. S6. The surface mesothelium thickens in response to injury.
    • Fig. S7. Chimerism in parabiotic mice.
    • Fig. S8. Circulating cells do not contribute significantly to adhesions.
    • Fig. S9. EdU+ cells are found in areas of tissue thickening.
    • Fig. S10. Scanning electron microscopy of peritoneal buttons.
    • Fig. S11. Scanning electron microscopy of peritoneal buttons.
    • Fig. S12. Large block-face scanning electron microscopy of peritoneal buttons.
    • Fig. S13. CFSE treatment labels only surface mesothelial cells.
    • Fig. S14. CFSE-labeled mesothelial cells contribute to adhesions.
    • Fig. S15. PDPN+ ActinCreER; R26VT2/GK3 cells give rise to adhesions.
    • Fig. S16. In vitro mesothelial transition.
    • Fig. S17. Anti-MSLN antibody specifically binds the surface mesothelium.
    • Fig. S18. Antibodies against host IgG do not bind mesothelial cells.
    • Fig. S19. Adhesion scoring characterized by surface area contact and molecular markers.
    • Fig. S20. K19+PDPN+ mesothelium persists after antibody treatment.
    • Fig. S21. Mesothelium persists after antibody treatment.
    • Fig. S22. ECM genes are down-regulated after adhesion induction.
    • Fig. S23. Validation of enriched genes via immunofluorescence.
    • Fig. S24. Transcript levels of HIF1A stabilization proteins.
    • Fig. S25. Mesothelial in vitro culture assay.
    • Fig. S26. HIF1A and mesothelial gene targets after HIF1α inhibition.
    • Fig. S27. HIF1A and mesothelial gene targets after HIF1α inhibition.
    • Fig. S28. Echinomycin does not affect DNA replication.
    • Fig. S29. A proposed mesothelial centric model of adhesion formation.
    • Legends for movies S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Date file S1 (Microsoft Excel format). Source data.
    • Movie S1 (.avi format). 3D rendering of light sheet microscopy imaging of injured surface mesothelium.
    • Movie S2 (.avi format). 3D rendering of light sheet microscopy imaging of injured surface mesothelium.

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