Proteases are required for a multitude of cellular processes including homeostatic tissue remodelling, invasion and angiogenesis. The physiological function of a cell or tissue is reflected by the set of proteases expressed, also termed degradome. The role of proteases in invasion and angiogenesis has been studied intensively, mostly in cancer. We aimed to compare the set of proteases required for physiological invasion versus physiological angiogenesis from cells deriving from the same organ, and to identify the proteases specific for each process. The human placenta comprises trophoblasts that invade the maternal uterus in a regulated, physiological manner, and it is the source of primary endothelial cells. We isolated the trophoblasts and endothelial cells and verified their invasive phenotype and angiogenic properties, respectively. We then performed gene expression analysis of the degradome, e.g. cysteine, metallo, serine, threonine and aspartic proteases, identified the differentially expressed proteases among the trophoblasts and endothelial cells, and clustered them hierarchically. The results revealed that the set of proteases in trophoblasts versus in endothelial cells overlaps, with a total of 69% in common. Nevertheless, 42% of the studied degradomes differed, with a fold change ≥2. For instance, metalloproteinases were predominantly expressed in trophoblasts, and 31% of the proteases were exclusively expressed in either trophoblasts or endothelial cells; this suggests particular roles for these proteases in either invasion or angiogenesis. Our data identify common and distinct proteases in cells capable of performing invasion and angiogenesis, and may provide basic information for the design of techniques to specifically investigate invasion or angiogenesis.

1.
Beristain, A.G., H. Zhu, P.C. Leung (2011) Regulated expression of ADAMTS-12 in human trophoblastic cells: a role for ADAMTS-12 in epithelial cell invasion? PLoS One 6: e18473.
2.
Bischof, P., M. Martelli, A. Campana, Y. Itoh, Y. Ogata, H. Nagase (1995) Importance of matrix metalloproteinases in human trophoblast invasion. Early Pregnancy 1: 263-269.
3.
Blaschitz, A., U. Weiss, G. Dohr, G. Desoye (2000) Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening. Placenta 21: 733-741.
4.
Brazma, A., P. Hingamp, J. Quackenbush, G. Sherlock, P. Spellman, C. Stoeckert, J. Aach, W. Ansorge, C.A. Ball, H.C. Causton, T. Gaasterland, P. Glenisson, F.C. Holstege, I.F. Kim, V. Markowitz, J.C. Matese, H. Parkinson, A. Robinson, U. Sarkans, S. Schulze-Kremer, J. Stewart, R. Taylor, J. Vilo, M. Vingron (2001) Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet 29: 365-371.
5.
Chan, H.S., S.J. Chang, T.Y. Wang, H.J. Ko, Y.C. Lin, K.T. Lin, K.M. Chang, Y.J. Chuang (2012) Serine protease PRSS23 is upregulated by estrogen receptor alpha and associated with proliferation of breast cancer cells. PLoS One 7: e30397.
6.
Chen, C.P., J.D. Aplin (2003) Placental extracellular matrix: gene expression, deposition by placental fibroblasts and the effect of oxygen. Placenta 24: 316-325.
7.
Chen, L.M., K.X. Chai (2002) Prostasin serine protease inhibits breast cancer invasiveness and is transcriptionally regulated by promoter DNA methylation. Int J Cancer 97: 323-329.
8.
Cohen, M., P. Bischof (2007) Factors regulating trophoblast invasion. Gynecol Obstet Invest 64: 126-130.
9.
Cohen, M., A. Meisser, P. Bischof (2006) Metalloproteinases and human placental invasiveness. Placenta 27: 783-793.
10.
Eccles, S.A. (2004) Parallels in invasion and angiogenesis provide pivotal points for therapeutic intervention. Int J Dev Biol 48: 583-598.
11.
Eck, S.M., P.J. Hoopes, B.L. Petrella, C.I. Coon, C.E. Brinckerhoff (2009) Matrix metalloproteinase-1 promotes breast cancer angiogenesis and osteolysis in a novel in vivo model. Breast Cancer Res Treat 116: 79-90.
12.
Fischer, A. (1946) Mechanism of the proteolytic activity of malignant tissue cells. Nature 157: 442.
13.
Fisher, S.J., T.Y. Cui, L. Zhang, L. Hartman, K. Grahl, G.Y. Zhang, J. Tarpey, C.H. Damsky (1989) Adhesive and degradative properties of human placental cytotrophoblast cells in vitro. J Cell Biol 109: 891-902.
14.
Fujiwara, H., T. Higuchi, Y. Sato, Y. Nishioka, B.X. Zeng, S. Yoshioka, K. Tatsumi, M. Ueda, M. Maeda (2005) Regulation of human extravillous trophoblast function by membrane-bound peptidases. Biochim Biophys Acta 1751: 26-32.
15.
Fukasawa, K., H. Fujii, Y. Saitoh, K. Koizumi, Y. Aozuka, K. Sekine, M. Yamada, I. Saiki, K. Nishikawa (2006) Aminopeptidase N (APN/CD13) is selectively expressed in vascular endothelial cells and plays multiple roles in angiogenesis. Cancer Lett 243: 135-143.
16.
Ghajar, C.M., S.C. George, A.J. Putnam (2008) Matrix metalloproteinase control of capillary morphogenesis. Crit Rev Eukaryot Gene Expr 18: 251-278.
17.
Hiden, U., N. Ghaffari-Tabrizi, M. Gauster, C. Tam-Amersdorfer, I. Cetin, M. Dieber-Rotheneder, U. Lang, G. Desoye (2013) Membrane-type matrix metalloproteinase 1 regulates trophoblast functions and is reduced in fetal growth restriction. Am J Pathol 182: 1563-1571.
18.
Im, E., A. Venkatakrishnan, A. Kazlauskas (2005) Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells. Mol Biol Cell 16: 3488-3500.
19.
Joyce, J.A., D. Hanahan (2004) Multiple roles for cysteine cathepsins in cancer. Cell Cycle 3: 1516-1619.
20.
Kessenbrock, K., V. Plaks, Z. Werb (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141: 52-67.
21.
Kim, H.J., Y.M. Kim, S. Lim, Y.K. Nam, J. Jeong, K.J. Lee (2009) Ubiquitin C-terminal hydrolase-L1 is a key regulator of tumor cell invasion and metastasis. Oncogene 28: 117-127.
22.
Lang, I., M.A. Pabst, U. Hiden, A. Blaschitz, G. Dohr, T. Hahn, G. Desoye (2003) Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells. Eur J Cell Biol 82: 163-173.
23.
Livak, K.J., T.D. Schmittgen (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25: 402-408.
24.
Lopez-Otin, C., C.M. Overall (2002) Protease degradomics: a new challenge for proteomics. Nat Rev Mol Cell Biol 3: 509-519.
25.
Ma, X.J., Y.Y. Fu, Y.X. Li, L.M. Chen, K. Chai, Y.L. Wang (2009) Prostasin inhibits cell invasion in human choriocarcinomal JEG-3 cells. Histochem Cell Biol 132: 639-646.
26.
Madri, J.A., D. Graesser (2000) Cell migration in the immune system: the evolving inter-related roles of adhesion molecules and proteinases. Dev Immunol 7: 103-116.
27.
Mayhew, T.M. (2002) Fetoplacental angiogenesis during gestation is biphasic, longitudinal and occurs by proliferation and remodelling of vascular endothelial cells. Placenta 23: 742-750.
28.
Miyakoshi, K., M.J. Murphy, R.R. Yeoman, S. Mitra, C.J. Dubay, J.D. Hennebold (2006) The identification of novel ovarian proteases through the use of genomic and bioinformatic methodologies. Biol Reprod 75: 823-835.
29.
Mohamed, M.M., B.F. Sloane (2006) Cysteine cathepsins: multifunctional enzymes in cancer. Nat Rev Cancer 6: 764-775.
30.
Moser, G., K. Orendi, M. Gauster, M. Siwetz, C. Helige, B. Huppertz (2011) The art of identification of extravillous trophoblast. Placenta 32: 197-199.
31.
Murphy, G., H. Nagase (2008) Progress in matrix metalloproteinase research. Mol Aspects Med 29: 290-308.
32.
Nissi, R., A. Talvensaari-Mattila, V. Kotila, M. Niinimaki, I. Jarvela, T. Turpeenniemi-Hujanen (2013) Circulating matrix metalloproteinase MMP-9 and MMP-2/TIMP-2 complex are associated with spontaneous early pregnancy failure. Reprod Biol Endocrinol 11: 2.
33.
Pepper, M.S. (2001) Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc Biol 21: 1104-1117.
34.
Perrin, B.J., A. Huttenlocher (2002) Calpain. Int J Biochem Cell Biol 34: 722-725.
35.
Pollheimer, J., V. Fock, M. Knofler (2014) Review: the ADAM metalloproteinases - novel regulators of trophoblast invasion? Placenta 35(suppl): S57-S63.
36.
Pop, C., G.S. Salvesen (2009) Human caspases: activation, specificity, and regulation. J Biol Chem 284: 21777-21781.
37.
Puente, X.S., L.M. Sanchez, C.M. Overall, C. Lopez-Otin (2003) Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 4: 544-558.
38.
Rakic, J.M., C. Maillard, M. Jost, K. Bajou, V. Masson, L. Devy, V. Lambert, J.M. Foidart, A. Noel (2003) Role of plasminogen activator-plasmin system in tumor angiogenesis. Cell Mol Life Sci 60: 463-473.
39.
Rangel, R., Y. Sun, L. Guzman-Rojas, M.G. Ozawa, J. Sun, R.J. Giordano, C.S. Van Pelt, P.T. Tinkey, R.R. Behringer, R.L. Sidman, W. Arap, R. Pasqualini (2007) Impaired angiogenesis in aminopeptidase N-null mice. Proc Natl Acad Sci USA 104: 4588-4593.
40.
Rawlings, N.D., A.J. Barrett, A. Bateman (2012) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 40: D343-D350.
41.
Rawlings, N.D., F.R. Morton, C.Y. Kok, J. Kong, A.J. Barrett (2008) MEROPS: the peptidase database. Nucleic Acids Res 36: D320-D325.
42.
Reed, M.J., A.C. Corsa, S.A. Kudravi, R.S. McCormick, W.T. Arthur (2000) A deficit in collagenase activity contributes to impaired migration of aged microvascular endothelial cells. J Cell Biochem 77: 116-126.
43.
Remacle, A.G., S.A. Shiryaev, E.S. Oh, P. Cieplak, A. Srinivasan, G. Wei, R.C. Liddington, B.I. Ratnikov, A. Parent, R. Desjardins, R. Day, J.W. Smith, M. Lebl, A.Y. Strongin (2008) Substrate cleavage analysis of furin and related proprotein convertases. A comparative study. J Biol Chem 283: 20897-20906.
44.
Robertson, D.M., D.C. Williams (1969) In vitro evidence of neutral collagenase activity in an invasive mammalian tumour. Nature 221: 259-260.
45.
Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, M. Seiki (1994) A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370: 61-65.
46.
Suhr, F., K. Brixius, W. Bloch (2009) Angiogenic and vascular modulation by extracellular matrix cleavage products. Curr Pharm Des 15: 389-410.
47.
van Goor, H., W.B. Melenhorst, A.J. Turner, S.T. Holgate (2009) Adamalysins in biology and disease. J Pathol 219: 277-286.
48.
van Hinsbergh, V.W., P. Koolwijk (2008) Endothelial sprouting and angiogenesis: matrix metalloproteinases in the lead. Cardiovasc Res 78: 203-212.
49.
Ward, C., D. Kuehn, R.E. Burden, J.A. Gormley, T.J. Jaquin, M. Gazdoiu, D. Small, R. Bicknell, J.A. Johnston, C.J. Scott, S.A. Olwill (2010) Antibody targeting of cathepsin S inhibits angiogenesis and synergistically enhances anti-VEGF. PLoS One 5: e12543.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.