Journal: Nature Medicine

Transcription

1 Journal: Nature Medicine Article Title: Authors Corresponding Author: PAI-1 mediates the anti-angiogenic and pro-fibrinolytic effects of 16K prolactin Khalid Bajou, Stephanie Herkenne, Victor L. Thijssen, Salvino D'Amico, Ngoc-Quynh-Nhu Nguyen, Ann Bouché, Sébastien Tabruyn, Mohammed Srahna, Jean-Yves Carabin, Olivier Nivelles, Cécile Paques, Ivo Cornelissen, Michelle Lion, Agnès Noel, Ann Gils, Stefan Vinckier, Paul J. Declerck, Arjan W. Griffioen, Mieke Dewerchin, Joseph A. Martial, Peter Carmeliet and Ingrid Struman Ingrid Struman Supplementary Item & Number (add rows as necessary) Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Title or Caption PAI-1 is required for 16K PRL to impair neovascularization Schematic outline of the PLA strategy for upa and 16K PRL. 16K PRL binds PAI-1, upar and upa PAI-1 and upar sirna reduces PAI-1 expression in HUVECs Inhibition of B16F10 tumor growth by 16K PRL is upa dependent 16K PRL inhibits PAI-1 s antiproteolytic activity in the presence of vitronectin. Supplementary Figure 7 Supplementary Table 1 16K PRL has no effect on clot lysis in the absence of PAI-1 and inhibits thromboplastin-induced pulmonary embolism PRL decreases reperfusion time and improves blood flow recovery in the carotid injury model.

2 FIGURE S1 FIGURE S1: (a-c) PAI-1 is required for 16K PRL to impair neovascularization. (a) Immunostaining for collagen type IV (red) and the proliferation marker Ki67 (green) of B16F10 tumors grown for 18 days in WT mice treated with CTL-Ad1. Nuclei are visualized by DAPI (blue). Arrows denote proliferative tumor cells; arrowheads proliferative ECs. Scale bar, 100 µm. (b,c) Percentage of Ki67 + tumor cells (b) or tumor endothelial cells (c) in subcutaneously implanted B16F10 tumors in WT or PAI-1 -/- mice treated with 16K PRL adenovirus (16K-Ad1) or control virus (CTL-Ad1) (mean±sem; N=7; *P=0.038). (d) INHIBITION OF 4T1 TUMOR GROWTH BY 16K PRL IS PAI-1 DEPENDENT Tumor weight of murine 4T1 breast carcinoma cells at 15 days after orthotopic implantation in Rag-1 -/- WT or Rag-1 -/- PAI-1 -/- mice. The mice were treated with CTL- Ad1 or 16K-Ad1 2 days prior and 6 days after tumor injection. The numbers between parentheses indicate the engraftment rate at the end of the experiment. (mean±sem; N=6-7; *P=0.049). (e) PAI-1 -/- MICE SHOW REDUCED NEOVASCULAR INGROWTH IN MATRIGEL PLUGS. Hemoglobin content in Matrigel plugs, 15 days after implantation into WT or PAI- 1 -/- mice treated with CTL-Ad1 (mean±sd; N=13-14; *P<0.001).

3 FIGURE S2 PAI-1 upar 16K upa FIGURE S2: Schematic outline of the PLA strategy for upa and 16K PRL. Proposed model of upar/upa/pai-1 and 16K PRL (16K) complex on the cell surface is shown. Primary antibodies for upa and 16K PRL raised in different species are used in the PLA assay (left), enabling juxtaposition of secondary, oligonucleotide-ligated antibodies (middle). Hybridization and ligation of connector oligos (middle) allows a rolling circle amplification detected by a cy3-probe (right).

4 FIGURE S3 FIGURE S3: 16K PRL binds PAI-1, upar and upa. (a,b) Western blotting (WB) for 16K PRL after immunoprecipitation (IP) of PAI-1 (a) or upar (b) from extracts of HUVECs incubated without (lanes 1 and 3) or with recombinant 16K PRL (lanes 2 and 4). Lane 5 shows recombinant 16K PRL loaded as positive control; lanes 1 and 2 show negative controls using mouse IgG as the IP control antibody. (c) Western blotting (WB) for upar after immunoprecipitation (IP) of 16K PRL from extracts of tumors grown in PAI-1 WT mice treated with 16K PRL adenovirus (16K-Ad1) or control virus (CTL-Ad1). (d,e) 16K PRL BINDS PAI-1/UPA COMPLEX. (d) Representative dose-response surface plasmon resonance sensorgrams of binding of PAI-1/uPA complex (250 nm - 2 µm, equimolar ratio) to immobilized 16K PRL. PAI-1/uPA complexes were incubated at equimolar ratio for 10 min at 37 C prior to loading onto a CM5 sensor chip (BIAcore). (e) Representative sensorgrams of binding of 500 nm of PAI-1 or 500 nm PAI-1 and upa to immobilized 16K PRL.

5 FIGURE S4 FIGURE S4: PAI-1 and upar sirna reduces PAI-1 expression in HUVECs. (a,b) qrt- PCR (a) and Western blot analysis (b) of PAI1 expression in HUVECs 48 hours (a) or 96 hours (b) after transfection with non-silencing sirna (CTL sirna) or with PAI-1 sirna. Ratio values in b represent the densitometric quantification of uparimmunoreactive bands normalized to the b-tubulin internal control and expressed as % of CTL sirna. (c) Western blotting for upar in HUVECs at 72 hours after transfection with control sirna (CTL sirna) or with upar sirna. The upper band corresponds to the glycosylated form. Ratio values represent the densitometric quantification of upar immunoreactive bands normalized to GAPDH internal control and expressed in % of CTL sirna). Data are mean±sd expressed relative to control; N=3; *P<0.05.

6 FIGURE S5 a b upar 16K PRL PAI-1 upa L R P FIGURE S5: (a) Inhibition of B16F10 tumor growth by 16K PRL is upa dependent. Growth curve of subcutaneously implanted B16F10 tumors in upa -/- mice treated with 16K PRL adenovirus (16K-Ad1) or control virus (CTL-Ad1) (mean±sem; N=5; P=NS). (b) Schematic representation of the 16K PRL complex with PAI-1,uPA, upar in interaction with LRP, proposed to mediate the anti-angiogenic activity of to 16K PRL CTL Ad1

7 16K PAI-1 PAI-1/16K PAI-1 PAI-1/Vn LPAI-1 LPAI-1/Vn FIGURE S6 a 16K PRL + IP: PAI-1, WB: 16K PRL b c FIGURE S6: 16K PRL inhibits PAI-1 s antiproteolytic activity in the presence of vitronectin. (a) Western blotting (WB) for 16K PRL after immunoprecipitation (IP) of wild type PAI-1 (PAI-1) or inactive latent PAI-1 (LPAI-1) from mixtures incubated in the presence or absence of vitronectin (Vn). Left panel: IP controls with only the indicated proteins. (b) Colorimetric determination of the proteolytic activity of upa on plasminogen (Plg) in the presence or absence of PAI-1, 16K PRL and vitronectin (Vn). Plasminogen activation was determined 4 hours after addition of a chromogenic substrate (mean±sd; N=3; *P=0.0016, **P=0.0001; a representative experiment of 3 repeat experiments is shown). (c) Representative dose-response surface plasmon resonance sensorgrams of binding of latent PAI-1 (250 nm - 2 µm) to immobilized 16K PRL.

8 FIGURE S7

9 FIGURE S7 FIGURE S7: 16K PRL has no effect on clot lysis in the absence of PAI-1 and inhibits thromboplastin-induced pulmonary embolism. (a) Lysis of human plasma clots by tpa (240 pm) in the absence of PAI-1, showing comparable clot lysis in the presence or absence of 16K PRL or buffer (CTL buffer). Plasma clots were prepared from pooled plasma collected from 6 healthy individuals. A representative experiment of 3 repeat experiments is shown. (b,c) Survival of BALB/c mice 15 min after thromboplastin injection to induce pulmonary embolism. Prior to embolism induction, the mice were treated with vehicle or recombinant 16K PRL (16K) for 5 min (b), or with control adenovirus (CTL-Ad1) or 16K PRL adenovirus (16K-Ad1) for 5 days (c). A contingency table listing the number and percentage of animals in each treatment/survival case is shown under the graphs.(d) Representative hematoxylin and eosin (H&E) staining of lungs from mice with induced thromboembolism, pretreated with CTL-Ad1 or 16K-Ad1. Thrombi are indicated by arrows. Scale bar, 200 µm. Quantification of emboli in lung sections is shown below (mean±sem; N=15; *p=0.001). (e) Survival of mice 15 min after thromboplastin injection in PAI- 1 WT or PAI-1 -/- mice. Prior to embolism induction, the mice were treated with CTL-Ad1 or 16K- Ad1 adenovirus for 5 days. A contingency is shown under the graph (f) Western blotting (WB) for 16K PRL after immunoprecipitation (IP) of endogenous PAI-1 from plasma of mice treated with recombinant 16K PRL or vehicle (left) or with 16K-Ad1 or CTL-Ad1 (right). (b,c,e) Fisher s exact test was performed to determine statistical differences between groups.

10 Table S1 Table S1. 16K PRL decreases reperfusion time and improves blood flow recovery in the carotid injury model. Five minutes before carotid injury, vehicle or 16K PRL (1.5 mg kg -1 ) was administered. Ten minutes after FeCl 3 injury-mediated occlusion, a bolus of unfractionated porcine heparin (200 U Kg -1 ) was administered followed by a continuous heparin infusion (70 U Kg -1 h -1 ). Ten minutes later, a continuous tpa infusion (100 µg kg -1 min -1 ) was started. Reperfusion time calculated as the time interval between tpa administration and blood flow restoration (return of carotid flow to 50% of baseline). A time value of 90 min was used for the conditions where no restoration of blood flow was observed (no lysis). Values per individual mouse are reported in the table along with overall flow recovery capacity (N=6; * P= vs vehicle in WT by Fisher s exact test).