Proteases play essential roles in maintaining the integrity

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1 Comprehensive Transcriptome of Proteases and Protease Inhibitors in Vascular Cells Guo-Ping Shi, DSc; Gregory M. Dolganov, MD, PhD Background and Purpose Smooth muscle cells, endothelial cells, and macrophages are essential components of the vasculature, of which the homeostatic gene expression participate importantly in the maintenance of vascular wall integrity. The pathogenesis of vascular diseases, such as cerebral ischemia, atherosclerosis, and abdominal aortic aneurysms, often associates with inflammation and altered gene expression, including proteolytic enzymes that play multiple and important roles in extracellular matrix degradation, cell proliferation and migration, and latent enzyme or growth factor activation. Methods and Results Human saphenous vein smooth muscle cells, endothelial cells, and monocyte-derived macrophages from 3 independent donors were stimulated with interleukin 1, interferon, tumor necrosis factor, basic fibroblast growth factor, and vascular endothelial growth factor, 5 common proinflammatory mediators often found in diseased human microvessels and macrovessels. Quantitative real-time PCR was used to examine the mrna levels of 49 proteolytic enzymes and their inhibitors, selected from 4 protease families, in these vascular cells. Conclusions Although primary cultured cells from different donors may behave differently in response to these proinflammatory cytokines, data from this study revealed a broad view of vascular cell protease expression profiles under inflammatory conditions, critical to studies of inflammation-associated vascular tissue remodeling. (Stroke. 2006; 37: ) Key Words: cytokines inflammation protease inhibitors proteases Proteases play essential roles in maintaining the integrity of both microvessels and macrovessels. Altered protease expression often associates with disruption of vasculature homeostasis and with additional vascular wall rupture followed by hemorrhage or thrombosis. A spectrum of proteolytic enzymes, including serine, cysteine, and matrix metalloproteinases, mediate the pathological remodeling of vessel walls in cerebral infarction, atherosclerosis, and abdominal aortic aneurysms, 1,2 vascular inflammatory diseases associate with enhanced protease activities. Indeed, proinflammatory cytokines interferon, tumor necrosis factor (TNF), and interleukin 1, as well as angiogenic factors basic fibroblast growth factor and vascular endothelial growth factor, are abundant in cells immunoreactive for proteases in diseased vessels, 3,4 suggesting an interaction of such cellular mediators with vascular cells, including smooth muscle cells (SMCs), endothelial cells (ECs), and macrophages (M ). In this study, we used real-time PCR to examine the mrna expression profiles of 49 common proteases selected from 4 protease families and their inhibitors in primary cultured human vascular SMCs, ECs, and monocyte-derived M in response to 5 common proinflammatory mediators. Methods Cell Culture Human saphenous vein SMCs, ECs, and M from 3 independent donors were obtained, cultured, and stimulated with interleukin 1 (10 ng/ml, PeproTech, Rocky Hill, NJ), interferon (10 ng/ml, PeproTech), TNF- (10 ng/ml, PeproTech), basic fibroblast growth factor (10 ng/ml, PeproTech), or vascular endothelial growth factor (10 ng/ml, PeproTech) for 48 hours (SMCs and M ) or 24 hours (ECs) as described previously. 5 Cells were then harvested, and total RNAs were extracted with Trizol reagent according to the manufacturer s instructions (Stratagene). 5 Real-Time PCR We used real-time PCR to determine the expression of proteases and protease inhibitors in SMCs, ECs, and M before and after treatment with inflammatory cytokines. A total of 49 common proteases/ protease inhibitors were selected on the basis of their reported involvement in cardiovascular diseases and their availability in our database (available online at Genespecific primers for multiplex real-time PCR were designed for each gene of interest using Primer Express software (Perkin Elmer) and purchased from Biosearch Technologies (Novato), The specificity of each primer pair was validated using a set of appropriate positive and negative controls. To avoid any impact from genomic DNA or cdna contaminations, all of the RNA samples were digested with RNase-free DNase for 1 hour at 37 C followed by another round of Received October 19, 2005; accepted November 15, From the Department of Medicine (G.-P.S.), Brigham and Women s Hospital and Harvard Medical School, Boston, Mass, and Department of Medicine (G.M.D.), University of California, San Francisco, Calif. Correspondence to Guo-Ping Shi, DSc, Cardiovascular Medicine, NRB-7, 77 Avenue Louis Pasteur, Boston, MA gshi@rics.bwh. harvard.edu 2006 American Heart Association, Inc. Stroke is available at DOI: /01.STR e9 537

2 538 Stroke February 2006 TABLE 1. Relative Gene Copy Numbers (mean SE) of Protease and Protease Inhibitors in Human Vascular SMCs* Protease/Inhibitor Control Interleukin 1 Interferon TNF- Caspase Caspase Caspase Caspase Caspase Caspase Cathepsin B Cathepsin C Cathepsin K Cathepsin L Cathepsin S Casp Act Inh Cystatin A Cystatin SN Cystatin C Cystatin S MMP MMP MMP MMP MMP MMP MMP MMP MMP TIMP TIMP TIMP TIMP tpa upa Trypsin Trypsin PAI PAI Cathepsin D *Genes that showed 200 relative gene copy numbers in all treatments were not included. These include caspase 8, cystatins E/M and F, MMP-7 and 13, chymase 1, testisin, prostasin, cathepsin G, granzyme A, granzyme B, serpin 1, and elafin. Data are statistically significant relative to the controls by Mann Whitney test (P 0.05). Data are statistically insignificant (P 0.05) but have nearly 2-fold changes or more compared with control cells. purification with RNeasy Mini column (Qiagen). Total RNA concentration and quality was additionally assessed using the Agilent Bioanalyzer 2001 (Agilent Technologies). First-strand cdna synthesis was performed on total RNA using Powerscript reverse transcriptase (BD Biosciences) and random hexamers as described. 6 An equivalent of 10 ng of total RNA from the first-strand cdna synthesis reaction was used in 10 L of each direct TaqMan gene quantification in 384-well format. Universal Master Mix for real-

3 Shi and Dolganov Vascular Cell Proteases and Protease Inhibitors 539 TABLE 2. Relative Gene Copy Numbers (mean SE) of Protease and Protease Inhibitors in Human Vascular ECs* Protease/Inhibitor Control Control Interleukin 1 Interferon TNF- Caspase Caspase Caspase Caspase Caspase Caspase Cathepsin B Cathepsin C Cathepsin K Cathepsin L Cathepsin S Casp Act Inh Cystatin SN Cystatin C Cystatin S Cystatin F MMP MMP MMP MMP MMP MMP TIMP TIMP TIMP tpa upa Trypsin Trypsin Granzyme A PAI PAI Elafin undetectable Cathepsin D *Genes that showed 200 relative gene copy numbers in all treatments were not included. These include caspase 8, cystatins A and E/M, MMP-7, 9, 12, 13, and 17, TIMP-4, chymase 1, testisin, prostasin, cathepsin G, granzyme B, and serpin 1. Data are statistically significant relative to the controls by Mann Whitney test (P 0.05). Data are statistically insignificant (P 0.05) but have nearly 2-fold changes or more compared with control cells. time PCR was purchased from Invitrogen. Raw data from ABI Prizm7900 were processed and expressed as relative gene copy numbers as described previously. 6 Gene-expression analysis requires proper internal control genes for normalization. By using an endogenous control as a reference, quantification of an mrna target can be normalized for differences in the amount of total RNA added to each reaction. For this purpose, we used 6 human housekeeping genes: PPIA, GAPDH, PGK1, EEF1a, RPL13A, and ubiquitin. Moreover, using GeNorm, 7 we selected for each time 2 of the most stable housekeeping genes across all of the specimens and used their geomeans for normalization. Normalized data are presented in Tables 1, 2, and 3 as relative gene copy numbers.

4 540 Stroke February 2006 TABLE 3. Relative Gene Copy Numbers (mean SE) of Protease and Protease Inhibitors in Human Monocyte-Derived M * Protease/Inhibitor Control Interleukin 1 Interferon TNF- Caspase Caspase Caspase Caspase Caspase Caspase Cathepsin B Cathepsin C Cathepsin K Cathepsin L Cathepsin S Casp Act Inh Cystatin A Cystatin SN Cystatin C Cystatin F MMP MMP MMP MMP MMP MMP MMP TIMP TIMP TIMP TIMP upa Chymase Testisin Cathepsin G Granzyme A Granzyme B PAI Serpin Cathepsin D *Genes that showed 200 relative gene copy numbers in all treatments were not included. These include caspase 8, cystatins S and E/M, MMP-3, 11, 13, and 17, tpa, trypsin 1, trypsin 2, PAI-2, and elafin. Data are statistically significant relative to the controls by Mann Whitney test (P 0.05); Data are statistically insignificant (P 0.05) but have nearly 2-fold changes or more compared with control cells.

5 Shi and Dolganov Vascular Cell Proteases and Protease Inhibitors 541 Results and Discussion Inflammation-mediated protease expression is probably one of the most active fields in vascular research, and its regulation by different cytokines has been studied extensively. However, individual studies have focused on different protease classes or specific sets of inflammatory mediators, making comparison of data sets very difficult. Results from our real-time PCR show mean SE of 3 independent experiments with primary cultured human saphenous vein SMCs, ECs, and monocyte-derived M from 3 independent donors (Tables 1, 2, and 3). We compared data from each treatment with untreated control cells, and performed a nonparametric Mann Whitney test to examine the statistical significance (Tables 1, 2, and 3). Although we considered P values 0.05 significant, we often observed P values 0.05 with radically increased gene copy number and high standard errors, likely because of the relatively small sample sizes (n 3) and possible donor variations, such as age and gender, although we were unable to obtain detailed patient information because of the Hospital Ethics Policy. For example, TIMP-3 gene copy numbers in 3 nonstimulated SMCs were , , and , respectively. TNF- treatment reduced their gene copy numbers by 3- fold to , , and , respectively. However, both Mann Whitney test and paired ANOVA t test showed no statistical difference (P 0.13 and 0.08, respectively). Furthermore, primary cultured nonstimulated vascular SMCs and ECs likely behave differently from those that reside in healthy vessel walls. In this study, we considered untreated cells as resting cells, on the basis of several of our previous studies that demonstrated basal level expression of proteases, including cathepsins and matrix metalloproteinases, using the same cell types from the same bank. 3,5,8 Unlike SMCs or ECs, mouse and human M express high levels of proteases, such as cathepsins, including those directly isolated from lung alveolar lavage fluids from normal mice or healthy human volunteers (G.P. Shi, unpublished data, 2002). In this study, we used proinflammatory mediators to induce protease expression in human monocyte-derived M, which may have already been activated during differentiation, possibly leading to higher basal levels of protease expression in control M. Therefore, probability values in Tables 1 to 3 may serve only as comparative reference values for the effect of the cytokines and growth factors examined. Although regulation of cytokine-induced expression at the protein or activity levels of each protease and protease inhibitor is important, data from this study provide invaluable insight into the pathophysiological significance of each enzyme at the transcription level. Acknowledgments This study is supported by grants from the National Heart, Lung, and Blood Institute (HL60942 and HL67283, to G.-P.S., and HL66564, to G.M.D.). References 1. Lijnen HR. Plasmin and matrix metalloproteinases in vascular remodeling. Thromb Haemost. 2001;86: Liu J, Sukhova GK, Sun JS, Xu WH, Libby P, Shi GP. Lysosomal cysteine proteases in atherosclerosis. Arterioscler Thromb Vasc Biol. 2004;24: Shi GP, Sukhova GK, Grubb A, Ducharme A, Rhode LH, Lee RT, Ridker PM, Libby P, Chapman HA. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest. 1999;104: Kimura H, Gules I, Meguro T, Zhang JH. Cytotoxicity of cytokines in cerebral microvascular endothelial cell. Brain Res. 2003;990: Liu J, Sukhova GK, Yang JT, Sun J, Ma L, Ren A, Xu WH, Fu H, Dolganov GM, Hu C, Libby P, Shi GP. Cathepsin L expression and regulation in human abdominal aortic aneurysm, atherosclerosis, and vascular cells. Atherosclerosis. In press. 6. Dolganov GM, Woodruff PG, Novikov AA, Zhang Y, Ferrando RE, Szubin R, Fahy JV. A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na -K -Cl cotransporter (NKCC1) in asthmatic subjects. Genome Res. 2001;11: Vandesompele J, Speleman F, Van Roy N, Laureys G, Brinskchmidt C, Christiansen H, Lampert F, Lastowska M, Bown N, Pearson A, Nicholson JC, Ross F, Combaret V, Delattre O, Feuerstein BG, Plantaz D. Multicentre analysis of patterns of DNA gains and losses in 204 neuroblastoma tumors: how many genetic subgroups are there? Med Pediatr Oncol. 2001;36: Shi GP, Sukhova GK, Kuzuya M, Ye Q, Du J, Zhang Y, Pan JH, Lu ML, Cheng XW, Iguchi A, Perrey S, Lee AM, Chapman HA, Libby P. Deficiency of the cysteine protease cathepsin S impairs microvessel growth. Circ Res. 2003;92: