S3 Role of PAI-1 in atherosclerosis

SUPPLEMENTARY INFORMATION FOR ON-LINE PUBLICATION

Supplement I: Diet composition

To accelerate lesion progression, mice were fed a cholesterol diet containing (wt/wt) 47 % sucrose, 1.25 % cholesterol, 20 % casein, 1 % corn oil, 5 % a-cellulose, 19 % butter, 0.5 % sodium cholate, 5 % mineral mix, 1 % vitamin mix, 1 % choline chloride, 0.3 % DL-methionine and 0.13 % a-tocopherol.

Supplement II: List of antibodies

Antigen /

Species

/ Source
Mouse PAI-1 / rabbit / Reference1
Human PAI-1 / rabbit / Reference1
Mouse Mac-3 / rat / Pharmingen, San Diego, USA
Mouse a-actin / rabbit / Sigma, Bornem, Belgium
Human COL2-3/4cshort / rabbit / References 2,3
Mouse tenascin / rat / Sigma, Bornem, Belgium
Human versican / mouse / USBiological, Swampscott, Massachusetts
Human fibronectin / rabbit / Sigma, Bornem, Belgium
Mouse fibrinogen / goat / Nordic Immunology, Turnhout, Belgium
Mouse MMP-9 / rabbit / Reference4
Mouse MMP-13 / goat / Reference5
Mouse plasminogen / rabbit / Reference6
Human platelet glycoprotein Iba chain (G28E5) / mouse / Reference7

Supplement III: Morphometric analysis

For determination of the cross-sectional plaque area, morphometric measurements were performed on 10 to 20 transversal sections, equally spaced (each 80 µm apart) throughout the abdominal and thoracic aorta, and averaged per aorta. Average plaque size was used to calculate the mean for each experimental group of 6 to 15 mice. Cross-sectional plaque area at the aortic root was measured on 5 sections (each 70 µm apart; starting at the level where the 3 aortic leaflets were visualized on the same section) and averaged per mouse. Cross-sectional plaque area at the carotid bifurcation was measured on 7 sections (each 70 mm apart, moving from the right common carotid artery into the bifurcation) and averaged per mouse. Cross-sectional plaque area in the brachiocephalic artery was measured on 5 sections (each 70 µm apart, starting at the level of the branching point of the brachiocephalic artery into the right common carotid and right subclavian arteries and moving into the brachiocephalic trunk) and averaged per mouse. Measurements of plaque areas positive after immunostaining or Sirius red staining was performed with a Leitz DMRXE microscope (Leica Imaging Systems Ltd, Cambridge, UK), a 3CCD colour video camera (model DXC-93OP; Sony, Tokyo, Japan) and a Leica Qwin software system (Leica Imaging Systems Ltd, Brussels, Belgium), by persons unaware of the genotype.

Supplement IV Bone marrow transplantation

ApoE-/-:PAI-1+/+ mice and their apoE-/-:PAI-1-/- littermates were further backcrossed for 3 generations in C57Bl/6 background, yielding mice with a 99.2% C57Bl/6 homology. All animals were housed in Thoran racks and provided sterilized food and water. Ten week old recipients were lethally irradiated at 16 hours before transplantation with 9.5 Gy at a rate of 200-300 cGy per minute using a linear accelerator. Bone marrow was isolated from 8 to 10 week old donors and 5 x 106 cells were injected in the tail vein of the recipient mice. One week after transplantation, mice were put on a cholesterol diet (see supplement I). Plaque analysis in the aortic root was performed after 20 weeks of diet. Leukocyte counts before and after transplantation were determined on blood samples taken from the retro-orbital plexus. Weight determined at the time of sacrifice in groups of 6 mice was not different between the four transplantation conditions (mean ± SEM of the weight: apoE-/-:PAI-1+/+ marrow in apoE-/-:PAI-1-/- recipients: 16.4 ± 1.7 g ; apoE-/-:PAI-1-/- marrow in apoE-/-:PAI-1+/+ recipients: 18.7 ± 0.9 g; apoE-/-:PAI-1-/- marrow in apoE-/-:PAI-1-/- recipients: 18.2 ± 1.4 g and apoE-/-:PAI-1+/+ marrow in apoE-/-:PAI-1+/+ recipients: 17.6 ± 1.2 g; p=NS). Leukocyte counts 8 weeks after transplantation were similar to those 1 week before transplantation in both transplanted genotypes (apoE-/-:PAI-1-/- before transplantation: 6.7 ± 0.6 x 109 cells/l versus apoE-/-:PAI-1-/- after transplantation: 6.0 ± 0.6 x 109 cells/l; p=NS; apoE-/-:PAI-1+/+ before transplantation: 7.4 ± 0.6 x 109 cells/l versus apoE-/-:PAI-1+/+ after transplantation: 6.4 ± 0.4 x 109 cells/l; p=NS).

Supplement V Adenoviral PAI-1 gene transfer

ApoE-/-:PAI-1-/- mice at advanced stages of atherosclerosis (16 weeks of diet) were injected with 1.3 x 109 plaque forming units (PFU) of either AdRR5 control virus or adenovirus expressing human PAI-1 (AdCMVPAI-1)1. As previously shown1, systemic virus injection preferentially targeted expression of human PAI-1 to the liver. Plasma antigen levels of human PAI-1 measured by ELISA1 at the time of sacrifice (day 6 after injection) were 1.1 ± 0.7 mg/ml in AdCMVPAI-1 treated mice versus undetectable (< 0.8 ng/ml, N=10; P<0.05) in AdRR5 treated mice. Aortas and livers were processed for measurement of TGF-ß1 and for PAI-1 immunostaining. Immunostaining with a mouse anti-human PAI-1 antibody revealed the presence of PAI-1 protein in plaques of all mice injected with AdCMVPAI-1 but not in AdRR5 treated mice (not shown). Plaque levels of latent and active TGF-ß1 were measured on whole protein extracts from plaque-rich areas using commercially available assays (R&D systems, Abingdon, UK).

Supplement VI Thrombosis model

For induction of thrombosis, mice were anaesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg), fixed on a heated operating table and intubated. A 2F venous catheter was inserted into the right jugular vein for injection of reagents and rose-bengal. The left carotid artery was carefully exposed and mounted on a transilluminator. The exposed artery was irradiated with green light (wavelength 540 nm) of a Xenon lamp (L4887, Hamamatsu Photonics, Hamamatsu, Japan) equipped with a heat-absorbing filter and a green filter. Irradiation was directed via a 3-mm diameter optic fibre attached to a manipulator. Just after the injection of rose-bengal (20 mg/kg) via the intravenous cathether, irradiation was started for 3 minutes. The analytical procedures for the quantitation of mural thrombi in the mouse carotid artery have been described.8 The total light intensity versus time curve was established over 40 minutes and thrombus formation was measured by comparing the area under the curve and expressed in arbitrary light units (A.U.).


REFERENCES IN SUPPLEMENT

1. Carmeliet P, Moons L, Lijnen R, Janssens S, Lupu F, Collen D, Gerard RD. Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice. Circulation. 1997;96:3180-91.

2. Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, Mitchell P, Hambor J, Diekmann O, Tschesche H, Chen J, Van Wart H, Poole AR. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest. 1997;99:1534-45.

3. Sukhova GK, Schonbeck U, Rabkin E, Schoen FJ, Poole AR, Billinghurst RC, Libby P. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation. 1999;99:2503-9.

4. Lijnen HR, Lupu F, Moons L, Carmeliet P, Goulding D, Collen D. Temporal and topographic matrix metalloproteinase expression after vascular injury in mice. Thromb Haemost. 1999;81:799-807.

5. Delaisse JM, Eeckhout Y, Neff L, Francois-Gillet C, Henriet P, Su Y, Vaes G, Baron R. (Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment. J Cell Sci. 1993;106:1071-82.

6. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, Dyspersin GD, Cleutjens JP, Shipley M, Angellilo A, Levi M, Nubetae O, Baker A, Keshet E, Lupu F, Herbert JM, Smits JF, Shapiro SD, Baes M, Borgers M, Collen D, Daemen MJ, Carmeliet P. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med. 1999;5:1135-42.

7. Nong Z, Hoylaerts M, Van Pelt N, Collen D, Janssens S. Nitric oxide inhalation inhibits platelet aggregation and platelet- mediated pulmonary thrombosis in rats. Circ Res. 1997;81:865-9.

8. Stockmans F, Stassen JM, Vermylen J, Hoylaerts MF, Nystrom A. A technique to investigate mural thrombus formation in small arteries and veins: I. Comparative morphometric and histological analysis. Ann Plast Surg. 1997;38:56-62.