Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth -- Data Supplement - Supporting Materials and Methods -- Proceedings of the National Academy of Sciences

Supporting information for Falkenberg et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.162236599

Supporting Materials and Methods
Cloning of Rabbit uPA.
We cloned a uPA cDNA from a rabbit kidney cDNA library (Stratagene). To generate a probe for library screening, we used nested PCR to amplify a uPA cDNA fragment from total library DNA. PCR primers were designed based on regions of high sequence identity in rat, bovine, porcine, and human uPA: 5´-GCAGCTGCCCAAAGAAATTC-3´ (sense) and 5´-GCCCCAGCTCACAATCCC-3´ (antisense). The reaction generated a 1032-bp fragment with 84–89% identity with uPA cDNA from the other species. This fragment was used as a template for a second PCR using nested primers: 5´-GAAACACAATTACTGCAGGAACC-3´ (sense) and 5´-GGCAGGCAGATGGTCTGTATGG-3´ (antisense). The 532-bp product of this PCR was labeled with ³²Pand used to screen the library. Purification of positive plaques yielded a 1.5-kb insert in pBluescript SK (pBSKrbtuPA). Sequencing of the insert confirmed a 1302-bp open reading frame, 84% identical to human uPA (GenBank accession no. AY122285). Adenoviral Vectors.
To construct an adenoviral vector that expresses rabbit uPA (AdrbtuPA), we removed the uPA cDNA from pBSKrbtuPA as a XhoI-NotI fragment and ligated it to XhoI-NotI-digested pCI (Promega) to yield pCIrbtuPA. The 2.9-kb BglII-ClaI fragment of pCIrbtuPA, containing the uPA expression cassette, was then ligated to the BglII-ClaI-digested adenoviral shuttle plasmid pD E1sp1A (Microbix Biosystems, Toronto). The ligation product, pD E1rbtuPA, was linearized by digestion with StuI and cotransfected into 293 cells along with the large ClaI fragment of dl327 (1). Recombinant plaques were identified by restriction digest of plaque DNA and by zymographic analysis of serum-free medium conditioned by 293 cells infected with either recombinant plaque lysate or (as a control) AdCMVNull, a virus that does not express a transgene (2). Viruses were propagated, titered, and stored as described (3). The absence of replication-competent virus was confirmed in all viral preparations by a PCR-based assay that is capable of detecting one E1A-containing genome per 1 ´ 10⁶ vector genomes (4). In Vitro
uPA Expression Assays. Expression of uPA by AdrbtuPA was confirmed by Northern analysis of transduced 293 cells and by zymography and plasminogen activator activity assay of conditioned media. The probe for northern analysis was a 1.2-kb ApaI fragment of pBSKrbtuPA that contains only uPA sequences. Casein-plasminogen zymography was performed on serum-free media conditioned by 293 cells infected with either AdrbtuPA or AdCMVNull. Zymography and plasminogen activator activity assay [using the plasmin substrate S-2390 (Chromogenix, Molndal, Sweden)] were done as described (5). Animals.
In vivo experiments were performed with specific-pathogen-free adult male New Zealand White rabbits (2.5–3.5 kg, Charles River Breeding Laboratories). For atherosclerosis studies, hypercholesterolemia was induced with a diet including 0.25% cholesterol and 3% soybean oil diet (Ziegler Brothers, Gardner, PA) (6). After 2 weeks, plasma cholesterol levels were measured weekly (Abbott Spectrum, Abbott Laboratories) and used to adjust individual diets (6). After 5 weeks of diet, approximately 80% of rabbits had plasma cholesterol of 400–700 mg/dl. Rabbits outside this range were withdrawn from the study. The preoperative diets were resumed after surgery. All animal protocols were approved by the Committee on Animal Research of the University of California, San Francisco. In Vivo
Gene Transfer. In vivo gene transfer to carotid artery endothelium was performed as described (6). For the dose–response study, viral stocks were diluted to 1 ´ 10⁸ to 4 ´ 10⁹ plaque-forming units (pfu)/ml (2^´ 10¹⁰ to 8 ´ 10¹¹ particles per ml). For the atherosclerosis study, to achieve equal exposure to viral particles in the AdrbtuPA and AdCMVNull groups, we used a final concentration of 8 ´ 10¹¹ particles per ml for both vectors. The validity of using particle titers to determine in vivo dosing of the vectors was supported by Southern analysis of arteries harvested 3 days after gene transfer, showing equal numbers of vector genomes in arteries receiving either AdrbtuPA or AdCMVNull (data not shown). We chose this vector concentration to ensure substantial uPA expression in all transduced arteries and to optimize generation of atherosclerotic lesions, which are produced by infusion of high concentrations of adenovirus into arteries of hypercholesterolemic rabbits (6). Intraluminal infusion of adenovirus to rabbit arteries at this concentration yields recombinant gene expression in approximately 30% of luminal endothelial cells, rare subendothelial smooth muscle cells, and rare adventitial fibroblasts (7, 8). Harvest of Arteries for uPA Expression Assays and Histology.
Arteries were harvested for expression studies at 1, 3, 7, and 14 days after gene transfer. These arteries were excised, rinsed with saline, and divided into four 5-mm rings. Two rings (each trimmed to 4 mm) were placed in cold M199 (GIBCO/BRL) for 1 h and used for a plasminogen activation assay. The two other rings were snap frozen in liquid nitrogen for northern analysis and direct uPA activity assay with the S-2444 substrate. Arteries were harvested for histochemical and morphometric analysis at 1, 2, 3, and 4 weeks after gene transfer. These vessels were perfusion-fixed at physiologic pressure, embedded in paraffin, sectioned, and stained as described (6). In Vivo
Expression of uPA mRNA and Protein. Northern analysis of carotid segments was performed essentially as described for rat arteries (4), using the rabbit uPA cDNA probe described above. Blots were also hybridized with a cDNA probe for glyceraldehyde phosphate dehydrogenase (GAPDH) (9). The abundance of vector-derived and endogenous uPA mRNA was measured with a phosphorimager.

We used two methods to measure uPA activity in arteries: an in situ plasminogen activation assay on freshly harvested, intact arterial segments, and a direct uPA activity assay performed on extracts of snap-frozen arteries. For the in situ plasminogen activation assay, freshly harvested artery segments were incubated at 37° C in M199 without phenol red (GIBCO/BRL), 0.7

mM Glu-plasminogen (American Diagnostica, Greenwich, CT), and the plasmin substrate S-2390 (0.85 mM, Chromogenix). OD₄₀₅ was measured over time and used to compare experimental and control arteries. To measure uPA activity in extracts, frozen artery rings were pulverized and suspended in 200 ml of buffer (75 mM acetic acid/75 mM KCl/225 mM NaCl/10 mM EDTA/100 mM arginine/0.25% Triton X-100, pH 4.2). The extract was homogenized with a Polytron (Brinkmann) and centrifuged. The supernatant was collected, and protein quantitated with the BCA assay (Pierce). Samples were diluted with extraction buffer to obtain equivalent protein concentrations. Samples (20 ml) were then added to 100 ml of assay buffer [50 mM Tris, pH 8.8/38 mM NaCl/0.1% BSA/10 mg/ml human plasmin (American Diagnostica)] and incubated at 37° C for 30 min to convert single- to two-chain uPA. Aprotinin (1.2 trypsin inhibitor units per ml, Sigma) was added (to inhibit plasmin), and the uPA-specific substrate S-2444 (12 mM, Chromogenix) was added to measure uPA activity. OD₄₀₅ was determined after an overnight incubation at 37° C. This assay gives a linear response to known concentrations of uPA over the range of OD₄₀₅ encountered in this study (data not shown). Morphometric Analysis.
Sections of arteries were stained with Movat’s pentachrome, and images of the sections were recorded with a digital SPOT camera (Diagnostic Instruments). An observer blinded to the identity of the specimens used computer-assisted planimetry to measure the intimal and medial areas, lumen circumference, and the lengths of the internal and external elastic laminae (IEL and EEL). Lumen area (assuming circular lumens) was calculated from lumen circumference (area = circumference² ¸ 4p). Mean wall (i.e., intimal + medial) thickness was determined by calculating the mean radius of the areas inside both the EEL and the lumen (radius = EEL or lumen circumference ¸ 2p ) and subtracting the latter. Measurements and calculations were made using four evenly spaced sections per artery, and the results from these four measurements were used to calculate mean values for each artery. In an initial study, 8–9 arteries per group were harvested at 1, 2, 3, and 4 weeks for both AdCMVNull and AdrbtuPA groups. Based on these initial results, we repeated the study with harvests at 1 and 4 weeks only (8–10 arteries per group). Measurements of Intimal Cellularity and Proliferation.
Intimal cell density was measured by counting all intimal nuclei in four high-power fields in each of four sections per artery (16 fields per artery). The mean intimal cell density of the artery was then calculated from these measurements and the number of intimal cells in an arterial section was calculated by multiplying the intimal area by the cell density. Intimal cell proliferation was measured by injecting rabbits with bromodeoxyuridine (BrdUrd; 100 mg s.c.) 1, 9, and 17 h before harvest and staining arterial sections for evidence of BrdUrd incorporation (10). BrdUrd-positive cells were counted in the intimas of four sections per artery. The proliferative index was calculated as BrdUrd-positive intimal cells/total intimal cells ´ 100. The proliferative index for each artery was calculated as the mean of the indices of the four sections. Assessment of Extracellular Matrix Components.
All Movat-stained sections from all four time points were examined by an observer blinded to treatment group. Initially, degree and pattern of collagen, proteoglycan, and elastin staining were assessed to determine whether the abundance or staining pattern of any of these matrix components appeared sufficiently variable to suggest an effect of uPA overexpression. Collagen and proteoglycan staining varied little among the arteries. However, there was a wide range of appearance of elastin. Some arteries had extensive elastic lamina fragmentation and loss, whereas others had largely intact elastic laminae. To test the hypothesis that elastic lamina fragmentation and loss were associated with uPA overexpression, sections from all arteries were examined and assigned to one of two groups: extensive elastic lamina fragmentation and loss present or absent. The vector treatments were then revealed, and the percentage of AdrbtuPA and AdCMVNull arteries in each group was calculated. Immunohistochemistry.
Macrophages were detected by immunostaining with the RAM-11 antibody (6). Fibrin(ogen) was detected by staining 1-week arteries with a mouse monoclonal antibody to human fibrin(ogen) (American Diagnostica #750). A thrombosed artery was used as a positive control. The specificity of the primary antibodies was confirmed by substituting isotype-matched antibodies. Bound antibodies were detected with biotinylated secondary antibody, streptavidin-peroxidase, and aminoethylcarbazole substrate. Immunohistochemical staining was quantified by analysis of slide images with image-processing tool kit (IPTK) filters (Reindeer Games, Raleigh, NC) and PHOTOSHOP 5.5 (Adobe Systems, Mountain View, CA). The percentage of intimal area staining for macrophages was calculated by dividing the stained area by the total intimal area of the same slide. This measurement was performed in four sections per artery, and the mean of the measurements used as a value for the artery. Statistical Methods.
Data are presented as mean ± SEM for normally distributed data or median for data not normally distributed. Group means were compared with t tests and medians were compared with the Mann–Whitney rank-sum test. The c ² test was used to determine whether elastic lamina fragmentation and loss were more common in the AdrbtuPA arteries.

1. Ginsberg, H. S., Lundholm-Beauchamp, U., Horswood, R. L., Pernis, B., Wold, W. S. M., Chanock, R. M. & Prince, G. A. (1989) Proc. Natl. Acad. Sci. USA 86, 3823–3827.

2. Wen, S., Schneider, D. B., Driscoll, R. M., Vassalli, G., Sassani, A. B. & Dichek, D. A. (2000) Arterioscler. Thromb. Vasc. Biol. 20, 1452–1458.

3. Lee, S. W., Trapnell, B. C., Rade, J. J., Virmani, R. & Dichek, D. A. (1993) Circ. Res. 73, 797–807.

4. DeYoung, M. B., Zamarron, C., Lin, A. P., Qiu, C., Driscoll, R. M. & Dichek, D. A. (1999) Hum. Gene Ther. 10, 1469–1478.

5. Dichek, D. A., Lee, S. W. & Nguyen, N. H. (1994) Blood 84, 504–516.

6. Schneider, D. B., Vassalli, G., Wen, S., Driscoll, R. M., Sassani, A. B., DeYoung, M. B., Linnemann, R., Virmani, R. & Dichek, D. A. (2000) Arterioscler. Thromb. Vasc. Biol. 20, 298–308.

7. Newman, K. D., Dunn, P. F., Owens, J. W., Schulick, A. H., Virmani, R., Sukhova, G., Libby, P. & Dichek, D. A. (1995) J. Clin. Invest. 96, 2955–2965.

8. Schneider, D. B., Sassani, A. B., Vassalli, G., Driscoll, R. M. & Dichek, D. A. (1999) J. Vasc. Surg. 29, 543–550.

9. Dong, G., Schulick, A. H., DeYoung, M. B. & Dichek, D. A. (1996) J. Biol. Chem. 271, 29969–29977.

10. Schulick, A. H., Taylor, A. J., Zuo, W., Qiu, C.-B., Dong, G., Woodward, R. N., Agah, R., Roberts, A. B., Virmani, R. & Dichek, D. A. (1998) Proc. Natl. Acad. Sci. USA 95, 6983–6988.