**3.2 Fibrin 15-42 protects the myocardium from Ischemic-Reperfusion (IR) injury**

A synthetic peptide of fibrin residues 15-42 has been implicated as a potential therapeutic agent to reduce tissue damage and scarring after a heart attack (Hirschfield & Pepys, 2003; Petzelbauer et al., 2005b; Roesner et al., 2007; Zacharowski et al., 2006; Zacharowski et al., 2007). Peptide 15-42 works by inhibiting leukocyte migration across the endothelium into heart tissue, which prevents excessive inflammation and tissue damage. Peptide 15-42 mediated reduction of tissue injury depends on its ability to bind to VE-cadherin. Peptide 15-42 competes with FDP (*e.g.*, the plasmin E domain of fibrin as depicted in **Fig. 4**) for binding to VE-cadherin to prevent transendothelial cell migration (TEM) of leukocytes during myocardial IR injury (Petzelbauer et al., 2005b; Roesner et al., 2007; Zacharowski et al., 2006; Zacharowski et al., 2007). These published reports demonstrate the physiologic efficacy of fibrin 15-42 for treating IR injury. *However, the molecular mechanisms induced by fibrin(ogen) 15-42 binding to VE-cadherin to mediate enhanced paracellular permeability and whether fibrinogen-induced cancer metastasis involves binding interactions with fibrin(ogen) 15-42 have not been previously studied*.

#### **3.3 Fibrin(ogen) 15-42 induces endothelial barrier permeability via VE-cadherin binding interactions**

In a recent report (Sahni et al., 2009), we sought to determine whether fibrin(ogen) 15-42 binding to VE-cadherin induced endothelial cell permeability, and whether fibrinogeninduced cancer metastasis involves binding interactions between VE-cadherin and fibrin(ogen) 15-42. Using transwell insert culture systems, we showed that Fg 15-42 and VEcadherin binding interactions promote endothelial cell barrier permeability (Sahni et al., 2009) (**Fig. 6**). Peptides containing or missing residues 15-17 critical for 15-42 binding to VEcadherin (Gorlatov & Medved, 2002) and neutralizing antibodies that bind to Fg 15-21 (T2G1) and VE-cadherin (BV9) (**Fig. 7A**) were used to induce or inhibit permeability. Fg induced dose-dependent permeability of human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (HMEC-1) (**Fig. 6**), but not epithelial cell barriers (as shown in Fig. 1 in ref (Sahni et al., 2009)), which could be inhibited by neutralizing antibodies against 15-21 (T2G1) and VE-cadherin (BV9) and synthetic peptides (not shown).

cadherin mediates homophilic cell-cell adhesion critical for the maintenance of barrier integrity of the endothelium. Disruption of VE-cadherin-mediated endothelial barrier function leads to altered vascular permeability found in a number of diseases including ischemia-reperfusion (IR) injury, inflammation, angiogenesis, and cancer growth and metastasis (discussed in (Sahni et al., 2009)). Exposure of 15-42 and binding by VE-cadherin is also required for endothelial capillary tube formation in fibrin gels (Bach et al., 1998a; Chalupowicz et al., 1995); portions of the third extracellular domain (EC3) of VE-cadherin constitute a fibrin 15-42 receptor (Bach et al., 1998b; Yakovlev & Medved, 2009). Newly exposed chain residues, 15-GHRP-18, play a critical role in fibrin monomer aggregation during polymerization and clot formation during secondary hemostasis (Mosesson, 2005). Furthermore, exposure of the 15-42 domain mediates heparin-dependent fibrin binding to endothelial cell surfaces (Odrljin et al., 1996a); promotes endothelial cell adhesion and spreading (Bunce et al., 1992); promotes the release of endothelial cell-specific markers of endothelial activation (Ribes et al., 1989); and stimulates proliferation of endothelial cells, fibroblasts and cancer cells (Rybarczyk et al., 2003; Sahni et al., 2008; Sporn et al., 1995).

**3.2 Fibrin 15-42 protects the myocardium from Ischemic-Reperfusion (IR) injury** 

*been previously studied*.

**interactions** 

A synthetic peptide of fibrin residues 15-42 has been implicated as a potential therapeutic agent to reduce tissue damage and scarring after a heart attack (Hirschfield & Pepys, 2003; Petzelbauer et al., 2005b; Roesner et al., 2007; Zacharowski et al., 2006; Zacharowski et al., 2007). Peptide 15-42 works by inhibiting leukocyte migration across the endothelium into heart tissue, which prevents excessive inflammation and tissue damage. Peptide 15-42 mediated reduction of tissue injury depends on its ability to bind to VE-cadherin. Peptide 15-42 competes with FDP (*e.g.*, the plasmin E domain of fibrin as depicted in **Fig. 4**) for binding to VE-cadherin to prevent transendothelial cell migration (TEM) of leukocytes during myocardial IR injury (Petzelbauer et al., 2005b; Roesner et al., 2007; Zacharowski et al., 2006; Zacharowski et al., 2007). These published reports demonstrate the physiologic efficacy of fibrin 15-42 for treating IR injury. *However, the molecular mechanisms induced by fibrin(ogen) 15-42 binding to VE-cadherin to mediate enhanced paracellular permeability and whether fibrinogen-induced cancer metastasis involves binding interactions with fibrin(ogen) 15-42 have not* 

**3.3 Fibrin(ogen) 15-42 induces endothelial barrier permeability via VE-cadherin binding** 

In a recent report (Sahni et al., 2009), we sought to determine whether fibrin(ogen) 15-42 binding to VE-cadherin induced endothelial cell permeability, and whether fibrinogeninduced cancer metastasis involves binding interactions between VE-cadherin and fibrin(ogen) 15-42. Using transwell insert culture systems, we showed that Fg 15-42 and VEcadherin binding interactions promote endothelial cell barrier permeability (Sahni et al., 2009) (**Fig. 6**). Peptides containing or missing residues 15-17 critical for 15-42 binding to VEcadherin (Gorlatov & Medved, 2002) and neutralizing antibodies that bind to Fg 15-21 (T2G1) and VE-cadherin (BV9) (**Fig. 7A**) were used to induce or inhibit permeability. Fg induced dose-dependent permeability of human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (HMEC-1) (**Fig. 6**), but not epithelial cell barriers (as shown in Fig. 1 in ref (Sahni et al., 2009)), which could be inhibited by neutralizing antibodies against 15-21 (T2G1) and VE-cadherin (BV9) and synthetic peptides (not shown). However, the neutralizing antibodies (T2G1 and BV9) did not completely inhibit Fg-induced permeability (**Fig. 7B**), suggesting that additional cell recognition domains on Fg participate in fibrin(ogen)-induced vascular permeability.

Fig. 6. Fg-induced EC permeability involves Fg 15-42 and VE-cadherin. Cells were grown to confluency on Millicell™ 24-well cell culture inserts. Panel 6A, HUVEC were left untreated (control) or treated for 15 min with increasing concentrations of Fg or VEGF as indicated. Panel 6B, HUVEC were treated with 30 nM of Fg plus 1 mg/ml FITC-Dextran for the times indicated. The FITC-Dextran flux to the bottom chamber was measured by fluorometry and the data presented as the mean relative FITC-Dextran Flux ± SEM. Data points were derived from 3 or more independent experiments with the total number of replicates per condition ranging from 6-13. (Reprinted from (Sahni et al., 2009) with permission). P-values can be found in ref (Sahni et al., 2009).

Fig. 7. Fg-induced EC permeability involves Fg 15-42 sequences and VE-cadherin. Panel 7A, schematics of the aminoterminus of the fibrin(ogen) B chain and the domain structure of VE-cadherin are depicted. The arrow denotes the thrombin cleavage site for release of FPB. The 18C6 epitope maps to FPB, the T2G1 epitope maps to 15-21 and the VE-cadherin binding site on fibrin maps to 15-42. The epitope of the VE-cadherin-specific monoclonal antibody BV9 maps to the third and fourth extracellular domains (EC3-EC4). The fibrin 15-42 binding site on VE cadherin maps to EC3 near the EC3-EC4 junction. TM, transmembrane domain. Panel 7B, all monoclonal antibodies used are IgG1 isotype murine antibodies and

The Role of Fibrin(ogen) in Transendothelial Cell Migration During Breast Cancer Metastasis 195

visualized by microscopy. VEGF was used as a positive control to induce endothelial cell permeability and TEM of breast cancer cells. The results indicated that TEM of both MCF-7 and MDA-MB-231 cells was increased in a Fg-concentration-dependent manner (see Fig. 3a of ref (Sahni et al., 2009)) and as visualized by immunofluorescence microscopy showing

To determine whether VE-cadherin and/or Fg 15-42 were involved in Fg-enhanced TEM of MDA-MB-231 cells, the assay was repeated in the presence of the neutralizing and control antibodies (as shown in Fig. 3c of ref (Sahni et al., 2009)). To determine whether Fg promoted gap formation between cells, confluent HUVEC were treated with 150 or 480 nM Fg or 100 Units/ml TNF-, a known inducer of endothelial permeability and gap formation, for 30 minutes then cells were fixed, permeabilized and immunostained with an anti-VEcadherin. Fg treatment induced gap formation between adjacent endothelial cells, and such treatment promoted the subcellular relocalization of VE-cadherin from the cell periphery as in control cells into the cytoplasm in Fg- and TNF--treated cells (**Fig. 8B**). Indirect evidence for Fg binding at endothelial cell-cell junctions was obtained by fluorescence microscopy. The data reveal that Fg binds to endothelial cell-cell junctions in a punctate pattern, consistent with cell surface receptor binding to the cell-cell adhesion receptor, VE-cadherin (**Fig. 8C, arrows**). Fg also assembles as part of the fibrillar subendothelial ECM (**Fig. 8C, arrowhead**). Taken together, the data in **Fig. 6-8** demonstrate that the VE-cadherin binding domain defined by residues 15-42 on the -chain of human Fg induces permeability of endothelial but not epithelial cell barriers and enhances TEM of malignant breast cancer cells by a VE-cadherin-dependent mechanism. In contrast, the basal level of TEM of nonmalignant breast epithelial cells was not enhanced by Fg treatment (Sahni et al., 2009).

MDA-MB-231 cells adhered to the bottom side of the transwell filter (**Fig. 8A**).

**3.5 Fibrinogen potentiates endothelial cell permeability at low doses of VEGF** 

Fig. 9. Fg enhances permeability induced by low concentrations of VEGF.

induced EC permeability (**Fig. 9**).

Both FGF-2 and VEGF bind to fibrin(ogen) at distinct sites with high affinity (Sahni & Francis, 2000; Sahni et al., 1998). Fg bound-FGF-2 potentiates endothelial cell proliferation over FGF-2 alone (Sahni et al., 2003; Sahni & Francis, 2004; Sahni et al., 2006; Sahni et al., 1999). Although Fg-bound VEGF remains active, it does not potentiate endothelial cell proliferation over VEGF alone (Sahni & Francis, 2000). Because Fg induces endothelial cell permeability through VE-cadherin binding interactions (Sahni et al., 2009) and VEGF binds to Fg (Sahni & Francis, 2000), we tested the hypothesis that Fg would potentiate VEGF-

nonimmune IgG1 was used for the control. Monoclonal antibodies were used at 3 nM in the absence of Fg, or with 0.3 nM or 30 nM Fg for 45 min. The data were plotted as the mean ± SEM of relative FITC-Dextran Flux and were obtained from three independent experiments with a total sample size of 6-9 per condition. (Reprinted from (Sahni et al., 2009) with permission). P-values can be found in ref (Sahni et al., 2009).
