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Kaur R, Deb PK, Diwan V, Saini B. Heparanase Inhibitors in Cancer Progression: Recent Advances. Curr Pharm Des 2021; 27:43-68. [PMID: 33185156 DOI: 10.2174/1381612826666201113105250] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2020] [Accepted: 08/25/2020] [Indexed: 11/22/2022]
Abstract
BACKGROUND An endo-β-glucuronidase enzyme, Heparanase (HPSE), degrades the side chains of polymeric heparan sulfate (HS), a glycosaminoglycan formed by alternate repetitive units of D-glucosamine and D-glucuronic acid/L-iduronic acid. HS is a major component of the extracellular matrix and basement membranes and has been implicated in processes of the tissue's integrity and functional state. The degradation of HS by HPSE enzyme leads to conditions like inflammation, angiogenesis, and metastasis. An elevated HPSE expression with a poor prognosis and its multiple roles in tumor growth and metastasis has attracted significant interest for its inhibition as a potential anti-neoplastic target. METHODS We reviewed the literature from journal publication websites and electronic databases such as Bentham, Science Direct, PubMed, Scopus, USFDA, etc., about HPSE, its structure, functions, and role in cancer. RESULTS The present review is focused on Heparanase inhibitors (HPIns) that have been isolated from natural resources or chemically synthesized as new therapeutics for metastatic tumors and chronic inflammatory diseases in recent years. The recent developments made in the HPSE structure and function are also discussed, which can lead to the future design of HPIns with more potency and specificity for the target. CONCLUSION HPIns can be a better target to be explored against various cancers.
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Affiliation(s)
- Rajwinder Kaur
- Chitkara College of Pharmacy, Chitkara University, Punjab, India
| | - Pran Kishore Deb
- Faculty of Pharmacy, Philadelphia University, Philadelphia, Jordan
| | - Vishal Diwan
- Faculty of Medicine, The University of Queensland, Queensland, Australia
| | - Balraj Saini
- Chitkara College of Pharmacy, Chitkara University, Punjab, India
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2
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Ghiselli G. Heparin Binding Proteins as Therapeutic Target: An Historical Account and Current Trends. MEDICINES (BASEL, SWITZERLAND) 2019; 6:E80. [PMID: 31362364 PMCID: PMC6789896 DOI: 10.3390/medicines6030080] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/16/2019] [Revised: 07/16/2019] [Accepted: 07/18/2019] [Indexed: 12/16/2022]
Abstract
The polyanionic nature and the ability to interact with proteins with different affinities are properties of sulfated glycosaminoglycans (GAGs) that determine their biological function. In designing drugs affecting the interaction of proteins with GAGs the challenge has been to generate agents with high binding specificity. The example to emulated has been a heparin-derived pentasaccharide that binds to antithrombin-III with high affinity. However, the portability of this model to other biological situations is questioned on several accounts. Because of their structural flexibility, oligosaccharides with different sulfation and uronic acid conformation can display the same binding proficiency to different proteins and produce comparable biological effects. This circumstance represents a formidable obstacle to the design of drugs based on the heparin scaffold. The conceptual framework discussed in this article is that through a direct intervention on the heparin-binding functionality of proteins is possible to achieve a high degree of action specificity. This objective is currently pursued through two strategies. The first makes use of small molecules for which in the text we provide examples from past and present literature concerning angiogenic factors and enzymes. The second approach entails the mutagenesis of the GAG-binding site of proteins as a means to generate a new class of biologics of therapeutic interest.
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Affiliation(s)
- Giancarlo Ghiselli
- Independent Researcher, 1326 Spruce Street Suite 706, Philadephia, PA 19107, USA.
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3
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The genetic mechanism of high prolificacy in small tail han sheep by comparative proteomics of ovaries in the follicular and luteal stages. J Proteomics 2019; 204:103394. [PMID: 31146049 DOI: 10.1016/j.jprot.2019.103394] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2019] [Revised: 05/21/2019] [Accepted: 05/25/2019] [Indexed: 12/16/2022]
Abstract
To investigate the genetic mechanism of sheep prolificacy, protein profiling of ovaries in the follicular and luteal phases was conducted. The tandem mass tag technique was used to analyze the proteomes of ovaries from STH sheep that did not have the FecB mutation in the bone morphogenetic protein receptor 1B gene. Parallel Reaction Monitoring (PRM) was operated to validate the target differentially abundant proteins (DAPs). The result showed, a total of 34,037 peptides were found, and 5074 proteins were identified. The screened DAPs strictly related to energy metabolism, hormone synthesis, ovarian function were significantly enriched in oxidative phosphorylation(COX7A, ND5, and UQCR10), ovarian steroidogenesis(StAR and HSD3B), taurine and hypotaurine metabolism(CSAD), glycosaminoglycan biosynthesis-heparin sulfate/heparin(GLCE), necroptosis(H2AX, AIFM1, and FTH1), protein digestion and absorption(COL4A1 and COL4A5) and glycosaminoglycan degradation(HYAL2 and HEXB) pathways. These analyses indicated that the reproductive performance of sheep is regulated through different pathways. In consequence, these findings are an important resource that can be used in future studies of the genetic mechanism of high fecundity traits in sheep, and these DAPs can be further investigated as candidate markers to predict prolificacy of sheep. SIGNIFICANCE: Litter size is an important quantitative trait, but the genetic mechanism of high-prolificacy is still unclear in sheep. Our study identified potential signaling pathways and differentially abundant proteins related to reproductive performance. These findings will facilitate a better revealing the mechanism and provide possible targets for molecular design breeding for the formation of polytocous traits in sheep.
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Mohan CD, Hari S, Preetham HD, Rangappa S, Barash U, Ilan N, Nayak SC, Gupta VK, Basappa, Vlodavsky I, Rangappa KS. Targeting Heparanase in Cancer: Inhibition by Synthetic, Chemically Modified, and Natural Compounds. iScience 2019; 15:360-390. [PMID: 31103854 PMCID: PMC6548846 DOI: 10.1016/j.isci.2019.04.034] [Citation(s) in RCA: 80] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Revised: 04/11/2019] [Accepted: 04/26/2019] [Indexed: 01/23/2023] Open
Abstract
Heparanase is an endoglycosidase involved in remodeling the extracellular matrix and thereby in regulating multiple cellular processes and biological activities. It cleaves heparan sulfate (HS) side chains of HS proteoglycans into smaller fragments and hence regulates tissue morphogenesis, differentiation, and homeostasis. Heparanase is overexpressed in various carcinomas, sarcomas, and hematological malignancies, and its upregulation correlates with increased tumor size, tumor angiogenesis, enhanced metastasis, and poor prognosis. In contrast, knockdown or inhibition of heparanase markedly attenuates tumor progression, further underscoring the potential of anti-heparanase therapy. Heparanase inhibitors were employed to interfere with tumor progression in preclinical studies, and selected heparin mimetics are being examined in clinical trials. However, despite tremendous efforts, the discovery of heparanase inhibitors with high clinical benefit and minimal adverse effects remains a therapeutic challenge. This review discusses the key roles of heparanase in cancer progression focusing on the status of natural, chemically modified, and synthetic heparanase inhibitors in various types of malignancies.
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Affiliation(s)
| | - Swetha Hari
- Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
| | - Habbanakuppe D Preetham
- Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
| | - Shobith Rangappa
- Adichunchanagiri Institute for Molecular Medicine, AIMS Campus, B. G. Nagar, Nagamangala Taluk, Mandya District 571448, India
| | - Uri Barash
- Technion Integrated Cancer Center (TICC), The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel
| | - Neta Ilan
- Technion Integrated Cancer Center (TICC), The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel
| | - S Chandra Nayak
- Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570006, India
| | - Vijai K Gupta
- Department of Chemistry and Biotechnology, ERA Chair of Green Chemistry, School of Science, Tallinn University of Technology, Tallinn, Estonia
| | - Basappa
- Department of Studies in Organic Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
| | - Israel Vlodavsky
- Technion Integrated Cancer Center (TICC), The Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.
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5
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Paluck SJ, Maynard HD. Structure Activity Relationship of Heparin Mimicking Polymer p(SS- co-PEGMA): Effect of Sulfonation and Polymer Size on FGF2-Receptor Binding. Polym Chem 2017; 8:4548-4556. [PMID: 29181096 PMCID: PMC5701782 DOI: 10.1039/c7py00861a] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Fibroblast growth factor-2 (FGF2) is a heparin binding protein that plays a role in a range of biological functions such as wound healing and bone regeneration. Heparin, a highly sulfated glycosaminoglycan, is required for FGF2 to bind to its receptor. Therefore, polymeric mimics of heparin are widely studied for their ability to manipulate FGF2-induced biological interactions. It is known that altering the degree of sulfonated monomer incorporation and size of heparin-mimicking polymers can affect protein-receptor binding. To elucidate the relationship between degree of sulfonation and receptor binding for the heparin-mimicking polymer, poly(styrene sulfonate-co-poly(ethylene glycol) methyl ether methacrylate) (p(SS-co-PEGMA)) a library was synthesized to contain nine polymers with degrees of sulfonation ranging from 0-100%. Kinetics of the polymerization was evaluated and reactivity ratios compared to literature results. These polymers were then tested for their ability to enhance FGF2 binding with its receptor as both covalent conjugates and as excipients. In a receptor based enzyme-linked immunosorbant assay (ELISA), as well as a cell-based study, the polymer with 81% SS incorporation enhanced receptor binding compared to FGF2 alone, and to a greater extent than the other polymers. Therefore, another library of polymers was prepared maintaining the degree of sulfonation at 81% and changing the size from 41 to 390 monomer repeat units. The polymers were again tested in receptor based ELISA and cell studies, and all of the different sizes performed similarly, except for degree of polymerization 295 and 390, which had reduced response in the cellular assay. These results provide important information for the use of pSS-co-PEGMA as a potential heparin-mimicking therapeutic.
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Affiliation(s)
- Samantha J Paluck
- Department of Chemistry and Biochemistry and California NanoSystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095-1569 United States
| | - Heather D Maynard
- Department of Chemistry and Biochemistry and California NanoSystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095-1569 United States
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6
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Paluck S, Nguyen TH, Maynard HD. Heparin-Mimicking Polymers: Synthesis and Biological Applications. Biomacromolecules 2016; 17:3417-3440. [PMID: 27739666 PMCID: PMC5111123 DOI: 10.1021/acs.biomac.6b01147] [Citation(s) in RCA: 140] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2016] [Revised: 09/26/2016] [Indexed: 12/13/2022]
Abstract
Heparin is a naturally occurring, highly sulfated polysaccharide that plays a critical role in a range of different biological processes. Therapeutically, it is mostly commonly used as an injectable solution as an anticoagulant for a variety of indications, although it has also been employed in other forms such as coatings on various biomedical devices. Due to the diverse functions of this polysaccharide in the body, including anticoagulation, tissue regeneration, anti-inflammation, and protein stabilization, and drawbacks of its use, analogous heparin-mimicking materials are also widely studied for therapeutic applications. This review focuses on one type of these materials, namely, synthetic heparin-mimicking polymers. Utilization of these polymers provides significant benefits compared to heparin, including enhancing therapeutic efficacy and reducing side effects as a result of fine-tuning heparin-binding motifs and other molecular characteristics. The major types of the various polymers are summarized, as well as their applications. Because development of a broader range of heparin-mimicking materials would further expand the impact of these polymers in the treatment of various diseases, future directions are also discussed.
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Affiliation(s)
- Samantha
J. Paluck
- Department of Chemistry and
Biochemistry and the California NanoSystems Institute, University of California−Los Angeles, 607 Charles E. Young Dr East, Los Angeles, California 90095, United States
| | - Thi H. Nguyen
- Department of Chemistry and
Biochemistry and the California NanoSystems Institute, University of California−Los Angeles, 607 Charles E. Young Dr East, Los Angeles, California 90095, United States
| | - Heather D. Maynard
- Department of Chemistry and
Biochemistry and the California NanoSystems Institute, University of California−Los Angeles, 607 Charles E. Young Dr East, Los Angeles, California 90095, United States
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7
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Öztürk E, Arlov Ø, Aksel S, Li L, Ornitz DM, Skjåk-Bræk G, Zenobi-Wong M. Sulfated hydrogel matrices direct mitogenicity and maintenance of chondrocyte phenotype through activation of FGF signaling. ADVANCED FUNCTIONAL MATERIALS 2016; 26:3649-3662. [PMID: 28919847 PMCID: PMC5597002 DOI: 10.1002/adfm.201600092] [Citation(s) in RCA: 65] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/26/2023]
Abstract
Deciphering the roles of chemical and physical features of the extracellular matrix (ECM) is vital for developing biomimetic materials with desired cellular responses in regenerative medicine. Here, we demonstrate that sulfation of biopolymers, mimicking the proteoglycans in native tissues, induces mitogenicity, chondrogenic phenotype, and suppresses catabolic activity of chondrocytes, a cell type that resides in a highly sulfated tissue. We show through tunable modification of alginate that increased sulfation of the microenvironment promotes FGF signaling-mediated proliferation of chondrocytes in a three-dimensional (3D) matrix independent of stiffness, swelling, and porosity. Furthermore, we show for the first time that a biomimetic hydrogel acts as a 3D signaling matrix to mediate a heparan sulfate/heparin-like interaction between FGF and its receptor leading to signaling cascades inducing cell proliferation, cartilage matrix production, and suppression of de-differentiation markers. Collectively, this study reveals important insights on mimicking the ECM to guide self-renewal of cells via manipulation of distinct signaling mechanisms.
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Affiliation(s)
- Ece Öztürk
- Cartilage Engineering+ Regeneration, ETH Zurich, Otto-Stern-Weg 7, 8093 Zurich, Switzerland
| | - Øystein Arlov
- Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7034 Trondheim, Norway
| | - Seda Aksel
- Department of Materials, Polymer Technology Laboratory, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093, Zurich, Switzerland
| | - Ling Li
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - David M. Ornitz
- Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA
| | - Gudmund Skjåk-Bræk
- Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7034 Trondheim, Norway
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8
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Ling L, Tan SK, Goh TH, Cheung E, Nurcombe V, van Wijnen AJ, Cool SM. Targeting the heparin-binding domain of fibroblast growth factor receptor 1 as a potential cancer therapy. Mol Cancer 2015. [PMID: 26201468 PMCID: PMC4511971 DOI: 10.1186/s12943-015-0391-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Background Aberrant activation of fibroblast growth factor receptors (FGFRs) deregulates cell proliferation and promotes cell survival, and may predispose to tumorigenesis. Therefore, selective inactivation of FGFRs is an important strategy for cancer therapy. Here as a proof-of-concept study, we developed a FGFR1 neutralizing antisera, IMB-R1, employing a novel strategy aimed at preventing the access of essential heparan sulfate (HS) co-receptors to the heparin-binding domain on FGFR1. Methods The mRNA and protein expression level of FGFR1 and other FGFRs were examined in several lines of breast cancer and osteosarcoma cells and corresponding normal cells using Taqman real-time quantitative PCR and Western blot analysis. The specificity of IMB-R1 against FGFR1 was assessed with various ELISA-based approaches and Receptor Tyrosine Kinase array. Proliferation assay and apoptosis analysis were performed to assess the effect of IMB-R1 on cancer cell growth and apoptosis, respectively, in comparison with known FGFR1 inhibitors. The IMB-R1 induced alteration of intracellular signaling and gene expression were analysed using Western blot and microarray approaches. Immunohistochemical staining of FGFR1 using IMB-R1 were carried out in different cancer tissues from clinical patients. Throughout the study, statistical differences were determined by Student’s t test where appropriate and reported when a p value was less than 0.05. Results We demonstrate that IMB-R1 is minimally cross-reactive for other FGFRs, and that it potently and specifically inhibits binding of heparin to FGFR1. Furthermore, IMB-R1 blocks the interaction of FGF2 with FGFR1, the kinase activity of FGFR1 and activation of intracellular FGFR signaling. Cancer cells treated with IMB-R1 displayed impaired FGF2 signaling, were unable to grow and instead underwent apoptosis. IMB-R1-induced cell death correlated with a disruption of antioxidative defense networks and increased expression of several tumor suppressors and apoptotic proteins, including p53. Immunostaining with IMB-R1 was stronger in human cancer tissues in which the FGFR1 gene is amplified. Conclusion Our study suggests that blocking HS interaction with the heparin-binding domains of FGFR1 inhibited cancer cell growth, which can be an attractive strategy to inactivate cancer-related heparin-binding proteins. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0391-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Ling Ling
- Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore
| | - Si Kee Tan
- Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, #02-01 Genome, Singapore, 138672, Singapore
| | - Ting Hwee Goh
- Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore
| | - Edwin Cheung
- Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), 60 Biopolis Street, #02-01 Genome, Singapore, 138672, Singapore.,Faculty of Health Sciences, University of Macau, E12 Avenida da Universidade, Taipa, Macau, China
| | - Victor Nurcombe
- Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore.,Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore
| | - Andre J van Wijnen
- Department of Orthopedic Surgery & Biochemistry and Molecular Biology, Mayo Clinic, 200 First Street SW, MedSci 3-69, Rochester, MN, 55905, USA.
| | - Simon M Cool
- Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore, 138648, Singapore. .,Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 119074, Singapore.
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Wang M, Lyu Z, Chen G, Wang H, Yuan Y, Ding K, Yu Q, Yuan L, Chen H. A new avenue to the synthesis of GAG-mimicking polymers highly promoting neural differentiation of embryonic stem cells. Chem Commun (Camb) 2015; 51:15434-7. [DOI: 10.1039/c5cc06944k] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
A new strategy for the fabrication of glycosaminoglycan (GAG) analogs with high bioactivities was proposed by copolymerizing the sulfonated unit and the glyco unit, ‘splitted’ from the sulfated saccharide building blocks of GAGs.
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Affiliation(s)
- Mengmeng Wang
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
| | - Zhonglin Lyu
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
| | - Gaojian Chen
- Center for Soft Condensed Matter Physics and Interdisciplinary Research
- Soochow University
- Suzhou 215006
- China
| | - Hongwei Wang
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
| | - Yuqi Yuan
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
| | - Kaiguo Ding
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
| | - Qian Yu
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
| | - Lin Yuan
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
| | - Hong Chen
- College of Chemistry
- Chemical Engineering and Materials Science
- Soochow University
- Suzhou 215123
- China
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Pu F, Wang E, Jiang H, Ren J. Identification of polyoxometalates as inhibitors of basic fibroblast growth factor. ACTA ACUST UNITED AC 2013; 9:113-20. [DOI: 10.1039/c2mb25389e] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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Salah Z, Uziely B, Jaber M, Maoz M, Cohen I, Hamburger T, Maly B, Peretz T, Bar-Shavit R. Regulation of human protease-activated receptor 1 (hPar1) gene expression in breast cancer by estrogen. FASEB J 2012; 26:2031-42. [PMID: 22291441 DOI: 10.1096/fj.11-194704] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
A pivotal role is attributed to the estrogen-receptor (ER) pathway in mediating the effect of estrogen in breast cancer progression. Yet the precise mechanisms of cancer development by estrogen remain poorly understood. Advancing tumor categorization a step forward, and identifying cellular gene fingerprints to accompany histopathological assessment may provide targets for therapy as well as vehicles for evaluating the response to treatment. We report here that in breast carcinoma, estrogen may induce tumor development by eliciting protease-activated receptor-1 (PAR(1)) gene expression. Induction of PAR(1) was shown by electrophoretic mobility shift assay, luciferase reporter gene driven by the hPar(1) promoter, and chromatin-immunoprecipitation analyses. Functional estrogen regulation of hPar1 in breast cancer was demonstrated by an endothelial tube-forming network. Notably, tissue-microarray analyses from an established cohort of women diagnosed with invasive breast carcinoma exhibited a significantly shorter disease-free (P=0.006) and overall (P=0.02) survival of patients that were positive for ER and PAR(1), compared to ER-positive but PAR(1)-negative patients. We propose that estrogen transcriptionally regulates hPar(1), culminating in an aggressive gene imprint in breast cancer. While ER(+) patients are traditionally treated with hormone therapy, the presence of PAR(1) identifies a group of patients that requires additional treatment, such as anti-PAR(1) biological vehicles or chemotherapy.
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Affiliation(s)
- Zaidoun Salah
- Sharett Institute of Oncology, Hadassah-Hebrew University Hospital, P.O. Box 12000, Jerusalem 91120, Israel
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12
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Carmel J, Arish A, Shoshany G, Baruch Y. Heparanase accelerates the proliferation of both hepatocytes and endothelial cells early after partial hepatectomy. Exp Mol Pathol 2012; 92:202-9. [PMID: 22305926 DOI: 10.1016/j.yexmp.2012.01.002] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2011] [Accepted: 01/17/2012] [Indexed: 02/07/2023]
Abstract
BACKGROUND AND AIMS Heparanase (HPSE) is an endo-β-D-glucuronidase, which cleaves heparan sulfate in the extracellular matrix (ECM) and has pro-angiogenic and pro-proliferative properties. The aim of this investigation was to study the effect of HPSE on hepatocytes and endothelial cells (EC) during liver regeneration. METHODS Following 70% hepatectomy (PHP), rats were injected daily with 1-50μg HPSE/rat. Liver samples were stained with H&E and anti-bromodeoxyuridine (BrdU) antibody. mRNAs of hepatocyte growth factor (HGF), stem cell factor, tumor necrosis factor (TNF)-α, interleukin(IL)-6, and cyclinD1 were tested by real-time qPCR. Matrix metalloproteinases (MMPs) were tested by gel zymography. RESULTS Compared to the saline control, HPSE increased hepatocyte proliferation 24h, 48h and 72h after PHP, with the maximal effect found at 24h with 50μg HPSE (40.9±2.5% vs. 8.6±4.3%, p<0.01 for BrdU staining; 5.5±0.9% vs. 0.8±0.5%, p<0.05 for mitosis). Proliferation of the sinusoidal and the portal vein radical ECs was also increased (p<0.05). HPSE caused a twofold increase in cyclinD1 mRNA (p<0.05) and in pro-MMP-9 levels (p<0.05). HPSE at all doses also caused significant reductions of TNF-α mRNA (p<0.05) and IL-6 mRNA, and no change in HGF mRNA. CONCLUSIONS HPSE enhances liver regeneration by inducing proliferation of hepatocytes and both sinusoidal and vascular ECs. Since the effect of HPSE on hepatocytes occurred earlier than that observed in ECs, this effect is not related to HPSE's effect on ECs. The mechanism of HPSE action is probably indirect and is mediated by HPSE-dependent ECM cleavage and the release of pre-existing enzymes.
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Affiliation(s)
- Julie Carmel
- Liver Unit, Rambam Health Care Campus and Bruce Rappaport Faculty of Medicine, Technion-Institute of Technology, Haifa, Israel
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Lerner I, Hermano E, Zcharia E, Rodkin D, Bulvik R, Doviner V, Rubinstein AM, Ishai-Michaeli R, Atzmon R, Sherman Y, Meirovitz A, Peretz T, Vlodavsky I, Elkin M. Heparanase powers a chronic inflammatory circuit that promotes colitis-associated tumorigenesis in mice. J Clin Invest 2011; 121:1709-21. [PMID: 21490396 DOI: 10.1172/jci43792] [Citation(s) in RCA: 160] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2010] [Accepted: 01/26/2011] [Indexed: 12/27/2022] Open
Abstract
Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Expression of the enzyme heparanase is clearly linked to colon carcinoma progression, but its role in UC is unknown. Here we demonstrate for what we believe to be the first time the importance of heparanase in sustaining the immune-epithelial crosstalk underlying colitis-associated tumorigenesis. Using histological specimens from UC patients and a mouse model of dextran sodium sulfate-induced colitis, we found that heparanase was constantly overexpressed and activated throughout the disease. We demonstrate, using heparanase-overexpressing transgenic mice, that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors. We found that highly coordinated interactions between the epithelial compartment (contributing heparanase) and mucosal macrophages preserved chronic inflammatory conditions and created a tumor-promoting microenvironment characterized by enhanced NF-κB signaling and induction of STAT3. Our results indicate that heparanase generates a vicious cycle that powers colitis and the associated tumorigenesis: heparanase, acting synergistically with the intestinal flora, stimulates macrophage activation, while macrophages induce production (via TNF-α-dependent mechanisms) and activation (via secretion of cathepsin L) of heparanase contributed by the colon epithelium. Thus, disruption of the heparanase-driven chronic inflammatory circuit is highly relevant to the design of therapeutic interventions in colitis and the associated cancer.
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Affiliation(s)
- Immanuel Lerner
- Sharett Institute, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
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Brafman DA, Chang CW, Fernandez A, Willert K, Varghese S, Chien S. Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces. Biomaterials 2010; 31:9135-44. [PMID: 20817292 PMCID: PMC2949524 DOI: 10.1016/j.biomaterials.2010.08.007] [Citation(s) in RCA: 138] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2010] [Accepted: 08/04/2010] [Indexed: 01/22/2023]
Abstract
Realization of the full potential of human pluripotent stem cells (hPSCs) in regenerative medicine requires the development of well-defined culture conditions for their long-term growth and directed differentiation. Current practices for maintaining hPSCs generally utilize empirically determined combinations of feeder cells and other animal-based products, which are expensive, difficult to isolate, subject to batch-to-batch variations, and unsuitable for cell-based therapies. Using a high-throughput screening approach, we identified several polymers that can support self-renewal of hPSCs. While most of these polymers provide support for only a short period of time, we identified a synthetic polymer poly(methyl vinyl ether-alt-maleic anhydride) (PMVE-alt-MA) that supported the long-term attachment, proliferation and self-renewal of HUES1, HUES9, and iPSCs. The hPSCs cultured on PMVE-alt-MA maintained their characteristic morphology, expressed high levels of markers of pluripotency, and retained a normal karyotype. Such cost-effective, polymer-based matrices that support long-term self-renewal and proliferation of hPSCs will not only help to accelerate the translational perspectives of hPSCs, but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation.
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Affiliation(s)
- David A Brafman
- Department of Bioengineering, University of California-San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0695, United States
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15
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Angiogenesis alteration by defibrotide: implications for its mechanism of action in severe hepatic veno-occlusive disease. Blood 2008; 112:4343-52. [DOI: 10.1182/blood-2008-04-149682] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Abstract
Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.
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Zhao Y, Tan T, Yokoi H, Tanaka M, Kinoshita T. Controlled release and interaction of protein using self-assembling peptide RATEA16 nanofiber hydrogels. ACTA ACUST UNITED AC 2008. [DOI: 10.1002/pola.22820] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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17
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Dias PF, Siqueira JM, Maraschin M, Ferreira AG, Gagliardi AR, Ribeiro-do-Valle RM. A polysaccharide isolated from the brown seaweed Sargassum stenophyllum exerts antivasculogenic effects evidenced by modified morphogenesis. Microvasc Res 2008; 75:34-44. [PMID: 17585952 DOI: 10.1016/j.mvr.2007.05.004] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2006] [Revised: 04/23/2007] [Accepted: 05/10/2007] [Indexed: 11/23/2022]
Abstract
A polysaccharide (Sarg) extracted from the brown marine alga Sargassum stenophyllum was studied for its antivasculogenic effects in both in vivo and in vitro assays, as well as for its capacity to modify embryonic morphogenetic processes endogenously regulated by bFGF, a well-known angiogenic stimulator. The antivasculogenic activity of Sarg (6-1500 microg/implant) was evaluated in a chick yolk sac membrane assay and the embryonic morphogenesis was measured as the percentage cephalic length. Sarg alone (96-1500 microg/implant) and co-administered with hydrocortisone (HC; 156 microg/implant) decreased the vitelline vessel number by 23-100% and 54-100% respectively. The polysaccharide potentiated the antivasculogenic effect of HC (42% inhibition). Basic fibroblast growth factor-stimulated vasculogenesis (141% of vessels as compared to control) was partially reversed by Sarg. The treatment with Sarg also decreased the percentage cephalic length of 3.5- and 4-day chick embryos (as cultured in vivo and in vitro, respectively), uncoupled from any impairment in the body shape or embryotoxic effect. Due to polyanionic characteristics of Sarg, which are similar to those seen in the heparin molecule, we suggest that this polysaccharide should modulate the activity of heparin-binding vascular growth factors (such as bFGF, which also acts as a morphogen) mimetically interfering with heparan sulfate proteoglycans during microvessel formation.
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Affiliation(s)
- Paulo Fernando Dias
- Department of Pharmacology, Biological Sciences Center (CCB), Block D, Federal University of Santa Catarina (UFSC), University Campus-Trindade, Florianópolis, 88.049-900, SC, Brazil.
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18
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Ma Q, Cornelli U, Hanin I, Jeske WP, Linhardt RJ, Walenga JM, Fareed J, Lee JM. Heparin oligosaccharides as potential therapeutic agents in senile dementia. Curr Pharm Des 2007; 13:1607-16. [PMID: 17504153 PMCID: PMC4114250 DOI: 10.2174/138161207780765918] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Heparin is a glycosaminoglycan mixture currently used in prophylaxis and treatment of thrombosis. Heparin possesses non-anticoagulant properties, including modulation of various proteases, interactions with fibroblast growth factors, and anti-inflammatory actions. Senile dementia of Alzheimer's type is accompanied by inflammatory responses contributing to irreversible changes in neuronal viability and brain function. Vascular factors are also involved in the pathogenesis of senile dementia. Inflammation, endogenous proteoglycans, and assembly of senile plagues and neurofibrillary tangles contribute directly and indirectly to further neuronal damage. Neuron salvage can be achieved by anti-inflammation and the competitive inhibition of proteoglycans accumulation. The complexity of the pathology of senile dementia provides numerous potential targets for therapeutic interventions designed to modulate inflammation and proteoglycan assembly. Heparin and related oligosaccharides are known to exhibit anti-inflammatory effects as well as inhibitory effects on proteoglycan assembly and may prove useful as neuroprotective agents.
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Affiliation(s)
- Qing Ma
- Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
| | - Umberto Cornelli
- Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
| | - Israel Hanin
- Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
| | - Walter P. Jeske
- Department of Pathology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
| | - Robert J. Linhardt
- Biotechnology Center, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
| | - Jeanine M. Walenga
- Department of Pathology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
| | - Jawed Fareed
- Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
- Department of Pathology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
| | - John M. Lee
- Department of Pharmacology and Experimental Therapeutics, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
- Department of Pathology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153
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19
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Fernández C, Hattan CM, Kerns RJ. Semi-synthetic heparin derivatives: chemical modifications of heparin beyond chain length, sulfate substitution pattern and N-sulfo/N-acetyl groups. Carbohydr Res 2006; 341:1253-65. [PMID: 16712822 DOI: 10.1016/j.carres.2006.04.014] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2006] [Revised: 04/04/2006] [Accepted: 04/09/2006] [Indexed: 11/24/2022]
Abstract
The glycosaminoglycan heparin is a polyanionic polysaccharide most recognized for its anticoagulant activity. Heparin binds to cationic regions in hundreds of prokaryotic and eukaryotic proteins, termed heparin-binding proteins. The endogenous ligand for many of these heparin-binding proteins is a structurally similar glycosaminoglycan, heparan sulfate (HS). Chemical and biosynthetic modifications of heparin and HS have been employed to discern specific sequences and charge-substitution patterns required for these polysaccharides to bind specific proteins, with the goal of understanding structural requirements for protein binding well enough to elucidate the function of the saccharide-protein interactions and/or to develop new or improved heparin-based pharmaceuticals. The most common modifications to heparin structure have been alteration of sulfate substitution patterns, carboxyl reduction, replacement N-sulfo groups with N-acetyl groups, and chain fragmentation. However, an accumulation of reports over the past 50 years describe semi-synthetic heparin derivatives obtained by incorporating aliphatic, aryl, and heteroaryl moieties into the heparin structure. A primary goal in many of these reports has been to identify heparin-derived structures as new or improved heparin-based therapeutics. Presented here is a perspective on the introduction of non-anionic structural motifs into heparin structure, with a focus on such modifications as a strategy to generate novel reduced-charge heparin-based bind-and-block antagonists of HS-protein interactions. The chemical methods employed to synthesize such derivatives, as well as other unique heparin conjugates, are reviewed.
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Affiliation(s)
- Cristina Fernández
- Division of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, IA 52242, USA
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20
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Strehblow C, Sperker W, Hevesi A, Garamvölgyi R, Petrasi Z, Shirazi M, Sylvén C, Weiss T, Lotan C, Pugatsch T, Ben-Sasson SA, Orlowski M, Glogar D, Gyöngyösi M. Paradoxical effects of aurintricarboxylic acid and RG-13577: acute thrombosis and in-stent stenosis in a passive-coated stent. J Endovasc Ther 2006; 13:94-103. [PMID: 16445329 DOI: 10.1583/05-1641.1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
PURPOSE To investigate if a platelet inhibitor (aurintricarboxylic acid [ATA]) and a heparin-mimicking antagonist (RG-13577) of basic fibroblast growth factor 2 (bFGF2) could be combined as a stable compound and attached to conventional bare metal stents to hinder thrombus formation and inflammatory reactions of stenting. METHODS Fifteen domestic pigs were stented with RG-13577/ATA-coated (n=6), ATA-coated (n=12), and bare metal stents (n=12) in the left anterior descending (LAD) and left circumflex (LCX) coronary arteries. All surviving pigs were evaluated with contrast angiography and intravascular ultrasonography (IVUS) after 4 weeks. Histological analysis of the stented arteries was performed after hematoxylin-eosin staining. Tissue factor (TF) staining and scanning electron microscopy (SEM) were performed in animals with acute stent thrombosis. RESULTS Five of the 6 animals receiving an RG-13577/ATA-coated stent experienced acute stent thrombosis, while no adverse events occurred in the animals of the other 2 groups. Follow-up angiography did not show significant in-stent stenosis in either bare or ATA-coated stents. However, histomorphometry revealed larger neointimal area (3.54+/-0.69 mm2 versus 1.82+/-0.27 mm2, p<0.05) and outward plaque area (1.56+/-0.34 mm2 versus 0.61+/-0.12 mm2, p<0.05) in ATA-coated stents. Three-dimensional IVUS analysis showed analogous results, with significantly larger neointimal volume and outward plaque volume in ATA-coated stents. There was a slight increase in TF staining around the stent struts, while SEM showed increased platelet adhesion and activity in RG-13577/ATA-coated stents versus the ATA-coated and bare metal stents. CONCLUSION RG-13577/ATA-coated stents lead to acute stent thrombosis. The ATA coating alone did not lead to acute events, but resulted in higher neointimal hyperplasia and expansive remodeling. These results underline the importance of preclinical studies before using new coated stents in human arteries.
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Affiliation(s)
- Christoph Strehblow
- Division of Cardiology, Department of Internal Medicine II, Medical University of Vienna, Austria, and Departmetn of Cardiology, Karolinska University Hospital at Huddinge, Stockholm, Sweden.
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21
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Dias PF, Siqueira JM, Vendruscolo LF, de Jesus Neiva T, Gagliardi AR, Maraschin M, Ribeiro-do-Valle RM. Antiangiogenic and antitumoral properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum. Cancer Chemother Pharmacol 2005; 56:436-46. [PMID: 15902462 DOI: 10.1007/s00280-004-0995-7] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2004] [Accepted: 11/29/2004] [Indexed: 11/29/2022]
Abstract
The potential antiangiogenic and antitumoral properties of SargA, a polysaccharide extracted from the brown marine alga Sargassum stenophyllum, were studied in assays carried out in chick embryos and mice. Gelfoam plugs containing SargA (2-1500 microg/plug) implanted in vivo into fertilized 6-day-old chicken eggs induced dose-related antiangiogenic activity in the chorioallantoic membrane (CAM). By day 8, the highest dose of SargA alone decreased the vessel number in the CAM by 64%, but coadministered with hydrocortisone (156 microg/plug, which alone caused 30% inhibition) failed to potentiate its antiangiogenic effect. Combined with basic fibroblast growth factor (50 ng/plug), SargA (1500 microg/plug) abolished angiogenesis stimulated by this factor in both chick embryo CAM and in subcutaneous (s.c.) Gelfoam plugs implanted in the dorsal skin of Swiss mice (measured as plug hemoglobin content). Repeated s.c. injections of SargA (1.5 or 150 microg per animal per day for 3 days) close to B16F10 melanoma cell tumors in the dorsal skin of mice markedly decreased tumor growth in a dose-related fashion (by 40% and 80% at 2 weeks after the first injection, respectively), without evident signs of toxicity. SargA caused graded inhibitions of migration and viability of cultured B16F10 cells and also displayed antithrombotic activity in human plasma (5 mg/ml increased thrombin time 2.5-fold relative to saline). Thus, SargA exhibits pronounced antiangiogenic as well as antitumoral properties. Although the latter action of SargA might be related to the inhibition of angiogenesis, the polysaccharide also exerts cytotoxic effects on tumor cells. Because of its chemical characteristics and polyanionic constituents, we postulate that the polysaccharide SargA might modulate the activity of heparin-binding angiogenic growth factors.
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Affiliation(s)
- Paulo Fernando Dias
- Departament of Pharmacology, Biological Sciences Center Block D, Federal University of Santa Catarina, University Campus - Trindade, Florianópolis, CEP 88.049-900, SC, Brazil
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22
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Chen KS, Zhang L, Tang L, Zhang YH, Gao DL, Yan L, Zhang L. Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells. World J Gastroenterol 2005; 11:4916-7. [PMID: 16097072 PMCID: PMC4398750 DOI: 10.3748/wjg.v11.i31.4916] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
AIM: To investigate the effects of anti-sense oligonucleotides (ASODNs) on mRNA expression of heparanase in human esophageal cancer EC9706 cells.
METHODS: One non-sense oligonucleotide (N-ODN) and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization.
RESULTS: The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs (P>0.05), but there was a significant difference between ASODNs and N-ODN or non-transfected group (ASODN1: 2.25±0.25, ASODN2: 2.21±0.23, ASODN3: 2.23±0.23, ASODN4: 2.25±0.24 vs N-ODN: 3.47±2.80 or non- transfected group: 3.51±2.93 respectively,P<0.05).
CONCLUSION: The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.
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Affiliation(s)
- Kui-Sheng Chen
- Department of Pathology, the First Affiliated Hospital, Zhengzhou University, Key Laboratory of Henan Tumor Pathology, Zhengzhou 450052, Henan Province, China.
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23
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Sandanaraj BS, Vutukuri DR, Simard JM, Klaikherd A, Hong R, Rotello VM, Thayumanavan S. Noncovalent modification of chymotrypsin surface using an amphiphilic polymer scaffold: implications in modulating protein function. J Am Chem Soc 2005; 127:10693-8. [PMID: 16045357 PMCID: PMC2596994 DOI: 10.1021/ja051947+] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
We report here on a new amphiphilic homopolymer that binds noncovalently to proteins. This polymer not only binds to the target protein chymotrypsin with submicromolar affinity but also stabilizes the native structure of the protein. Since the polymer-protein binding process is based on electrostatic interaction, the bound protein can be released from the polymer surface and reactivated either by increasing the ionic strength or by adding complementary cationic surfactants. The electrostatic binding of polymer to the protein results in a marked change in the substrate specificity of chymotrypsin.
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Affiliation(s)
- Britto S Sandanaraj
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
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24
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Sandanaraj BS, Vutukuri DR, Simard JM, Klaikherd A, Hong R, Rotello VM, Thayumanavan S. Noncovalent modification of chymotrypsin surface using an amphiphilic polymer scaffold: implications in modulating protein function. J Am Chem Soc 2005. [PMID: 16045357 DOI: 10.1021/ja051947] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
We report here on a new amphiphilic homopolymer that binds noncovalently to proteins. This polymer not only binds to the target protein chymotrypsin with submicromolar affinity but also stabilizes the native structure of the protein. Since the polymer-protein binding process is based on electrostatic interaction, the bound protein can be released from the polymer surface and reactivated either by increasing the ionic strength or by adding complementary cationic surfactants. The electrostatic binding of polymer to the protein results in a marked change in the substrate specificity of chymotrypsin.
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Affiliation(s)
- Britto S Sandanaraj
- Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
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25
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Maynard HD, Hubbell JA. Discovery of a sulfated tetrapeptide that binds to vascular endothelial growth factor. Acta Biomater 2005; 1:451-9. [PMID: 16701826 DOI: 10.1016/j.actbio.2005.04.004] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2005] [Revised: 04/05/2005] [Accepted: 04/07/2005] [Indexed: 11/17/2022]
Abstract
Molecules that mimic the sulfated glycosaminoglycan heparin and bind to heparin-binding growth factors would serve as important building blocks for synthetic biomaterials, e.g. to create a growth factor reservoir within a matrix. Peptide-based heparin mimetics would be particularly attractive, given the ease of peptide synthesis and modification. A sulfated tetrapeptide that fits this description and binds to vascular endothelial growth factor (VEGF) was discovered using a rationally-designed combinatorial approach. A approximately 6600 member library of tetrapeptides, designed to include heparin functionality, was synthesized by solid-phase Fmoc chemistry. The library was analyzed on-resin for VEGF binding using a fluorescence assay that employed a 7-amino-4-methylcoumarin-modified VEGF(165). The beads were ranked according to fluorescent signal and SY(SO(3))DY(SO(3)) was identified as the top binder. The binding affinity of the peptide for VEGF(165) was ascertained by surface plasmon resonance and compared with the heparin mimic suramin; the peptide binds to VEGF(165) 100-fold stronger than the sulfonated compound. These results suggest that the identified peptide may be useful in biomaterial applications where binding of VEGF is desired.
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Affiliation(s)
- Heather D Maynard
- Department of Materials Science and Institute for Biomedical Engineering, Swiss Federal Institute of Technology and University of Zurich, Switzerland
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26
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Wu Q, Wang J, Zhang L, Hong A, Ren J. Molecular Recognition of Basic Fibroblast Growth Factor by Polyoxometalates. Angew Chem Int Ed Engl 2005; 44:4048-52. [PMID: 15924284 DOI: 10.1002/anie.200500108] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Affiliation(s)
- Qiang Wu
- Subdivision of Biological Inorganic Chemistry, Key Laboratory of Rare Earth Chemistry and Physics, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China
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27
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Wu Q, Wang J, Zhang L, Hong A, Ren J. Molecular Recognition of Basic Fibroblast Growth Factor by Polyoxometalates. Angew Chem Int Ed Engl 2005. [DOI: 10.1002/ange.200500108] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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28
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Naggi A, Casu B, Perez M, Torri G, Cassinelli G, Penco S, Pisano C, Giannini G, Ishai-Michaeli R, Vlodavsky I. Modulation of the heparanase-inhibiting activity of heparin through selective desulfation, graded N-acetylation, and glycol splitting. J Biol Chem 2005; 280:12103-13. [PMID: 15647251 DOI: 10.1074/jbc.m414217200] [Citation(s) in RCA: 176] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Heparanase is an endo-beta-glucuronidase that cleaves heparan sulfate (HS) chains of heparan sulfate proteoglycans on cell surfaces and in the extracellular matrix (ECM). Heparanase, overexpressed by most cancer cells, facilitates extravasation of blood-borne tumor cells and causes release of growth factors sequestered by HS chains, thus accelerating tumor growth and metastasis. Inhibition of heparanase with HS mimics is a promising target for a novel strategy in cancer therapy. In this study, in vitro inhibition of recombinant heparanase was determined for heparin derivatives differing in degrees of 2-O- and 6-O-sulfation, N-acetylation, and glycol splitting of nonsulfated uronic acid residues. The contemporaneous presence of sulfate groups at O-2 of IdoA and at O-6 of GlcN was found to be non-essential for effective inhibition of heparanase activity provided that one of the two positions retains a high degree of sulfation. N-Desulfation/ N-acetylation involved a marked decrease in the inhibitory activity for degrees of N-acetylation higher than 50%, suggesting that at least one NSO3 group per disaccharide unit is involved in interaction with the enzyme. On the other hand, glycol splitting of preexisting or of both preexisting and chemically generated nonsulfated uronic acids dramatically increased the heparanase-inhibiting activity irrespective of the degree of N-acetylation. Indeed N-acetylated heparins in their glycol-split forms inhibited heparanase as effectively as the corresponding N-sulfated derivatives. Whereas heparin and N-acetylheparins containing unmodified D-glucuronic acid residues inhibited heparanase by acting, at least in part, as substrates, their glycol-split derivatives were no more susceptible to cleavage by heparanase. Glycol-split N-acetylheparins did not release basic fibroblast growth factor from ECM and failed to stimulate its mitogenic activity. The combination of high inhibition of heparanase and low release/potentiation of ECM-bound growth factor indicates that N-acetylated, glycol-split heparins are potential antiangiogenic and antimetastatic agents that are more effective than their counterparts with unmodified backbones.
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Affiliation(s)
- Annamaria Naggi
- G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo, 81, 20133 Milan, Italy
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29
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Calvitti M, Baroni T, Calastrini C, Lilli C, Caramelli E, Becchetti E, Carinci P, Vizzotto L, Stabellini G. Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development. J Histochem Cytochem 2004; 52:325-34. [PMID: 14966199 DOI: 10.1177/002215540405200303] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.
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Affiliation(s)
- M Calvitti
- Experimental Medicine and Biochemistry Science Department, University of Perugia, Italy
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30
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Guan R, Sun XL, Hou S, Wu P, Chaikof EL. A glycopolymer chaperone for fibroblast growth factor-2. Bioconjug Chem 2004; 15:145-51. [PMID: 14733594 DOI: 10.1021/bc034138t] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Mono- and disaccharide-containing glycopolymers were synthesized by cyanoxyl-mediated polymerization of acrylamide with acrylate-derivatized mono- and disaccharides. We demonstrate that a glycopolymer bearing pendant, fully sulfated lactose units effectively replaces heparin and heparan sulfate as a molecular chaperone for fibroblast growth factor-2 (FGF-2). Specifically, a compound was identified that protects FGF-2 from proteolytic, acid, and heat-induced degradation, while selectively promoting growth factor and receptor dimerization. Significantly, the capacity of this heparin-mimic to promote an FGF-2 specific proliferative cell response was confirmed and suggests potential applications for this compound and related derivatives in areas related to therapeutic angiogenesis.
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Affiliation(s)
- Ran Guan
- Departments of Surgery and Biomedical Engineering, Emory University School of Medicine, Atlanta, Georgia 30322
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31
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Zcharia E, Metzger S, Chajek-Shaul T, Aingorn H, Elkin M, Friedmann Y, Weinstein T, Li JP, Lindahl U, Vlodavsky I. Transgenic expression of mammalian heparanase uncovers physiological functions of heparan sulfate in tissue morphogenesis, vascularization, and feeding behavior. FASEB J 2004; 18:252-63. [PMID: 14769819 DOI: 10.1096/fj.03-0572com] [Citation(s) in RCA: 200] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
We have generated homozygous transgenic mice (hpa-tg) overexpressing human heparanase (endo-beta-D-glucuronidase) in all tissues and characterized the involvement of the enzyme in tissue morphogenesis, vascularization, and energy metabolism. Biochemical analysis of heparan sulfate (HS) isolated from newborn mice and adult tissues revealed a profound decrease in the size of HS chains derived from hpa-tg vs. control mice. Despite this, the mice appeared normal, were fertile, and exhibited a normal life span. A significant increase in the number of implanted embryos was noted in the hpa-tg vs. control mice. Overexpression of heparanase resulted in increased levels of urinary protein and creatinine, suggesting an effect on kidney function, reflected also by electron microscopy examination of the kidney tissue. The hpa-tg mice exhibited a reduced food consumption and body weight compared with control mice. The effect of heparanase on tissue remodeling and morphogenesis was best demonstrated by the phenotype of the hpa-tg mammary glands, showing excess branching and widening of ducts associated with enhanced neovascularization and disruption of the epithelial basement membrane. The hpa-tg mice exhibited an accelerated rate of hair growth, correlated with high expression of heparanase in hair follicle keratinocytes and increased vascularization. Altogether, characterization of the hpa-tg mice emphasizes the involvement of heparanase and HS in processes such as embryonic implantation, food consumption, tissue remodeling, and vascularization.
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Affiliation(s)
- Eyal Zcharia
- Department of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
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32
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Abstract
Novel reverse thermo-responsive (RTG) polymeric systems displaying superior rheological properties were generated by polymerization of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) segments. Two basic synthetic pathways were followed: (1) The bulk polymerization of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock (Pluronic(RTM) F127) (MW=12,600, 70wt% PEO) with hexamethylene diisocyanate (HDI) and (2) The covalent binding of poly(ethylene glycol) and poly(propylene glycol) chains, using phosgene as the connecting molecule. While in the former, the basic amphiphilic F127 repeating unit is known for its own RTG behavior, the latter polymers consist of segments unable of exhibiting reverse thermal gelation of their own. These new materials achieved viscosities at least 15 times higher than F127, at 37 degrees C. Dynamic light scattering measurements revealed that the microstructures formed by these novel polymers were markedly larger than those generated by PEO-PPO-PEO triblocks. While the size of Pluronic F127 micelles ranged from 15 to 20nm, the higher molecular weight amphiphiles generated much larger nanostructures (20-400nm). Finally, the ability of reverse thermo-sensitive gels to perform as drug delivery systems was exemplified by releasing an anti-restenosis model drug (RG-13577). A 30% P[F127](4) gel delivered the drug over 40 days, whereas a F127 gel having the same concentration released the drug over a 7 days period.
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Affiliation(s)
- Daniel Cohn
- School of Applied Science and Technology, Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel.
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33
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Abstract
Tumor angiogenesis is the proliferation of a network of blood vessels that penetrates into cancerous growths, supplying nutrients and oxygen and removing waste products. The process of angiogenesis plays an important role in many physiological and pathological conditions. Solid tumors depend on angiogenesis for growth and metastasis in a hostile environment. In the prevascular phase, the tumor is rarely larger than 2 to 3 mm3 and may contain a million or more cells. Up to this size, tumor cells can obtain the necessary oxygen and nutrient supplies required for growth and survival by simple passive diffusion. The properties of tumors to release and induce several angiogenic and anti-angiogenic factors which play crucial roles in regulating endothelial cell (EC) proliferation, migration, apoptosis or survival, cell-cell and cell-matrix adhesion through different intracellular signaling are thought to be the essential mechanisms during tumor-induced angiogenesis. Tumor angiogenesis actually starts with tumor cells releasing molecules that send signals to surrounding normal host tissue. This signaling activates certain genes in the host tissue that, in turn, make proteins to encourage growth of new blood vessels. In this review, we focus the mechanisms of tumor-induced angiogenesis, with an emphasis on the regulatory role of several angiogenic and anti-angiogenic agents during the angiogenic process in tumors. Advances in understanding the mechanisms of tumor angiogenesis have led to the development of several most effective anti-angiogenic and anti-metastatic therapeutic agents and also have provided several techniques for the regulation of cancer's angiogenic switch. The suggestion is made that standard cytotoxic chemotherapy and angiogenesis inhibitors used in combination may produce complementary therapeutic benefits in the treatment of cancer.
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Affiliation(s)
- Manoj Kumar Gupta
- Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei, China.
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Irony-Tur-Sinai M, Vlodavsky I, Ben-Sasson SA, Pinto F, Sicsic C, Brenner T. A synthetic heparin-mimicking polyanionic compound inhibits central nervous system inflammation. J Neurol Sci 2003; 206:49-57. [PMID: 12480085 DOI: 10.1016/s0022-510x(02)00318-0] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The immunomodulating capacity of heparin led us to test the effect of the synthetic heparin-mimicking and low anticoagulant compound RG-13577 on the course of experimental autoimmune encephalomyelitis (EAE) and central nervous system (CNS) inflammation. EAE was induced in SJL mice by inoculation with whole mouse spinal cord homogenate. RG-13577, delivered intraperitoneally, inhibited the clinical signs of acute EAE and markedly ameliorated inflammation in the spinal cord, primarily by inhibiting heparanase activity in lymphocytes and astrocytes and thus impairing lymphocyte traffic. RG-13577 treatment was effective when started on day of disease induction or day 7 after induction. The low molecular weight heparin, enoxaparin, tested under the same conditions, exerted only a minor insignificant inhibitory effect. RG-13577 also inhibited the tyrosine phosphorylation of several proteins, particularly Erk1 and Erk2 of the MAP kinase signaling pathways associated with inflammation and cell proliferation. RG-13577 blocked the activity of sPLA(2) and inhibited CNS PGE(2) production both in vivo and in vitro.
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Affiliation(s)
- Michal Irony-Tur-Sinai
- Laboratory of Neuroimmunology, Department of Neurology, Hadassah University Hospital and Hebrew University Medical School, P.O. Box 12000, 91120 Jerusalem, Israel
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Benezra M, Ishai-Michaeli R, Ben-Sasson SA, Vlodavsky I. Structure-activity relationships of heparin-mimicking compounds in induction of bFGF release from extracellular matrix and inhibition of smooth muscle cell proliferation and heparanase activity. J Cell Physiol 2002; 192:276-85. [PMID: 12124773 DOI: 10.1002/jcp.10136] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
A series of nine synthetic polyaromatic compounds were synthesized by polymerization of aromatic ring monomers with formaldehyde, which yield substantially ordered backbones with different functional anionic groups (hydroxyl and carboxyl) on the phenol ring. These compounds were tested for their heparin-mimicking activity: (1) inhibition of heparanase activity; (2) inhibition of SMC proliferation; and (3) release of bFGF from the ECM. We demonstrate that compounds that have two hydroxyl groups para and ortho to the carboxylic group and a carboxylic group at a distance of two carbons from the phenol ring inhibit heparanase activity and SMC proliferation, as well as induced an almost complete release of bFGF from ECM. Addition of a methyl group next to the carboxylic group led to a preferential inhibition of heparanase activity. Similar results were obtained with a compound that contains one hydroxyl group para to the carboxylic group and an ether group near the carboxylic group on the phenol ring. Preferential inhibition of SMC proliferation was best achieved when the position of the hydroxyl group is para and ortho to the carboxylic group and the carboxylic group is at a distance of one carbon from the phenol ring. On the other hand, for maximal release of bFGF from ECM, the position of the carboxylic group should be three carbons away from the phenol ring. These new heparin-mimicking compounds may have a potential use in inhibition of tumor metastasis, arteriosclerosis, and inflammation.
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Affiliation(s)
- Miriam Benezra
- Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel.
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36
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Matou S, Helley D, Chabut D, Bros A, Fischer AM. Effect of fucoidan on fibroblast growth factor-2-induced angiogenesis in vitro. Thromb Res 2002; 106:213-21. [PMID: 12297128 DOI: 10.1016/s0049-3848(02)00136-6] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Fucoidans are sulfated polysaccharides extracted from brown marine algae. A purified fucoidan fraction exhibits the same venous antithrombotic activity as heparin in rabbits, but with a lower anticoagulant effect. Because of its heparin-like structure, we postulated that fucoidan might modulate heparin-binding angiogenic growth factor activity. We thus studied its effect, at antithrombotic concentrations, on fibroblast growth factor (FGF)-2-induced proliferation and differentiation of human umbilical vein endothelial cells. The fucoidan effect on endothelial cell differentiation was evaluated by studying the expression of surface proteins (i.e. integrin, adhesion molecule) known to be modulated by FGF-2 and involved in angiogenesis, and by quantifying closed areas delimited by vascular tubes formed on reconstituted basement membrane. Fucoidan had no modulatory effect on the mitogenic activity of FGF-2, but significantly increased tubular structure density induced by FGF-2. Fucoidan alone increased alpha(6) integrin subunit expression with only partially organized tubular structure. In the presence of FGF-2, fucoidan enhanced alpha(6), beta(1) and PECAM-1 and inhibited alpha(v)beta(3) integrin expression. Heparin had no effect in these systems. The most striking effect of fucoidan was observed on alpha(6) expression and tube formation was abolished by monoclonal anti-alpha(6) antibodies. Fucoidan plus FGF-2 effect on alpha(6) expression was markedly decreased by monoclonal anti-FGF-2 antibodies, indicating that fucoidan acts mainly via FGF-2. These results show that, at antithrombotic concentrations, contrary to heparin, fucoidan can enhance vascular tube formation induced by FGF-2 with a modulation of the expression of surface proteins (mainly alpha(6)) involved in angiogenesis.
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Affiliation(s)
- Sabine Matou
- INSERM U428, Université Paris V, Hôpital Européen Georges Pompidou, 20 rue Leblanc, 75908 Paris Cédex 15, France
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37
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Deux JF, Prigent-Richard S, d'Angelo G, Feldman LJ, Puvion E, Logeart-Avramoglou D, Pellé A, Boudghène FP, Michel JB, Letourneur D. A chemically modified dextran inhibits smooth muscle cell growth in vitro and intimal in stent hyperplasia in vivo. J Vasc Surg 2002; 35:973-81. [PMID: 12021714 DOI: 10.1067/mva.2002.123093] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
PURPOSE Intimal smooth muscle cell (SMC) hyperplasia is a main component of the arterial wall response to injury. We have investigated the capacity of a water-soluble nonanticoagulant functionalized dextran (E9) in inhibition of SMC growth in vitro and in vivo. METHODS E9 was obtained with chemical substitutions with anionic and hydrophobic groups on the dextran backbone. SMC proliferation (cell counting, thymidine uptake, cell cycle analysis) was followed in culture in the presence of E9. Western blot analysis against phosphorylated mitogen-activated protein kinase (MAPK), extracellular signal-regulated protein kinase 1/2, and assessment of MAPK activity on serum-stimulated SMCs also were investigated. Binding/displacement experiments, electron microscopy, and cell fractionations were used to follow the binding and internalization of radiolabeled and fluorescentlabeled E9. New Zealand white rabbit iliac arteries were injured with balloon dilatation and stent deployment. Animals were treated for 14 days with saline solution or E9 (5 mg/kg injected subcutaneously, twice daily). Morphometric analyses were carried out in each group (n = 6 arteries, 18 sections). RESULTS Nonanticoagulant E9 inhibited SMC proliferation in vitro. Tyrosine phosphorylation of MAPK 1/2 and MAPK activity were inhibited with E9 within 5 minutes of incubation. The binding and rapid cytoplasmic internalization of the synthetic compound was evidenced, but, in contrast to heparin, we did not detect any nuclear localization of the antiproliferative E9. In the in vivo model, qualitative modifications of neointimal structure with a thinner fibrocellular neointima were noticed after E9 treatment. Morphometric analyses of stented arteries in E9-treated animals indicated an important reduction (P <.01) of intimal growth: 33% and 45% for intimal area and intima/media ratio, respectively. CONCLUSION Cytoplasmic internalization of the synthetic polysaccharide correlated to the SMC growth inhibition that involved the MAPK pathway. In vivo inhibition of intimal instent hyperplasia with this nonanticoagulant derived dextran is shown providing a new candidate for a potential selective treatment of SMC proliferation.
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Affiliation(s)
- Jean-François Deux
- INSERM ERIT-M 204, X Bichat Medical School, Bat INSERM 13, 46 Rue Henri Huchard, 75877 Paris Cedex 18, France
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38
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Letourneur D, Machy D, Pellé A, Marcon-Bachari E, D'Angelo G, Vogel M, Chaubet F, Michel JB. Heparin and non-heparin-like dextrans differentially modulate endothelial cell proliferation: in vitro evaluation with soluble and crosslinked polysaccharide matrices. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH 2002; 60:94-100. [PMID: 11835164 DOI: 10.1002/jbm.10072] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Proliferation of endothelial cells (ECs) is a cellular step of particular importance for implanted cardiovascular biomaterials. Heparin and some synthetic water-soluble non-anticoagulant polysaccharides derived from dextran and bearing anionic carboxymethyl and hydrophobic benzylamine groups were first investigated for their effects on EC proliferation in vitro. The results assessed by cell counting, 3H-thymidine uptake, and flow cytometry analysis, showed that the derivatized dextran-bearing hydrophobic groups stimulated the EC growth in the presence of serum, whereas native dextran or dextran-bearing anionic carboxymethyl groups were inactive and heparin was slightly inhibitory. Then, we showed that the derivatized dextran enhanced EC proliferation by potentiation of the mitogenic activities of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), two potent EC growth factors. In the presence of 2 nM of derivatized dextran, a 3-fold and 13-fold increase of 3H-thymidine uptake was obtained with VEGF and FGF-2, respectively. Finally, proliferation of ECs was investigated on crosslinked gels made of polysaccharides. It is of interest that EC proliferation was higher on gels containing the derivatized dextran than on plain hydrogels, and heparinized gels inhibited cell proliferation. From the obtained results, we propose that the synthetic non-heparin-like dextran may be of interest as a coating for the endothelialization of cardiovascular biomaterials.
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Schmidt A, Göpfert C, Vlodavsky I, Völker W, Buddecke E. Induction of a hypertrophic growth status of coronary smooth muscle cells is associated with an overexpression of TGF-beta. Eur J Cell Biol 2002; 81:138-44. [PMID: 11998865 DOI: 10.1078/0171-9335-00234] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Hypertrophy of vascular smooth muscle cells occurs during hypertension-induced remodelling of arteries and during development of arteriosclerosis and restenosis following angioplasty but the pathogenesis of the hypertrophic status is not yet fully understood. In a previous study we demonstrated that the synthetic non-sulfated, non-toxic heparin-mimicking compound RG-13577 is capable of inducing a cell cycle-arrested hypertrophic phenotype of coronary smooth muscle cells. In this study we clarify the mode of action of RG-13577 and demonstrate that the RG-13577-induced hypertrophy is associated with an increased expression of TGF-beta1 as indicated by an increase in TGF-beta1-specific protein and mRNA level. Furthermore we show that RG-13577-treated hypertrophic smooth muscle cells maintain full metabolic activity as indicated by a continuous de novo synthesis of protein and proteoglycans and that the RG-13577-induced growth arrest is caused not only by a higher expression of TGF-beta, but also by a reduced response of RG-treated cells to the mitogenic activity of bFGF, PDGF and EGF. The growth inhibitory activity of RG-13577 is reduced in the presence of neutralizing antibodies against TGF-beta. TGF-beta itself has anti-proliferative activity in serum-depleted medium. The RG-13577 effect is reversible since incubation of hypertrophic cells in RG-13577-free medium restores cell volume and [3H]thymidine incorporation to the values of untreated control cells within 4 days. We conclude, that the active metabolic status of RG-13577-treated cells in association with the overexpression of TGF-beta could promote repair processes of injured arteries after angioplasty without stimulating cell proliferation.
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Affiliation(s)
- Annette Schmidt
- Institute for Arteriosclerosis Research, Division of Molecular Cardiology, University of Münster, Germany.
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40
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Shargal Y, Viola N, Nagler A, Merin G, Schmidt A, Buddecke E, Ben-Sasson SA, Vlodavsky I. Inhibition of anastomotic intimal hyperplasia by a synthetic nonsulphated heparin-mimicking compound. Exp Clin Cardiol 2002; 7:73-9. [PMID: 19649227 PMCID: PMC2719179] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/28/2023]
Abstract
Despite extensive research in the design of endovascular catheters and advanced surgical techniques, stenosis recurs in a large percentage of patients undergoing angioplasty or anastomosis. Hence, neointimal hyperplasia, caused by migration and proliferation of vascular smooth muscle cells (SMC), remains a significant limitation to the relief of obstructive-occlusive vascular disease. It has been previously demonstrated that heparin displaces active basic fibroblast growth factor (bFGF) from the lumenal surface of blood vessels. Sequestration of the displaced bFGF by injured areas of the vessel wall is inhibited in the presence of a synthetic nonsulphated heparin-mimicking polyanionic compound (RG-13577). This compound also induces a phenotype transformation of coronary SMC into a metabolically active hypertropic status that could promote repair processes after balloon angioplasty while inhibiting cell proliferation. In this paper, the result of a continuous administration of compound RG-13577 both in the rat carotid catheter injury model and in a newly developed rat model of surgical arterial vascular injury (anastomosis) is reported: it causes a profound inhibition of intimal hyperplasia in both models. A combined treatment with heparin/heparan sulphate mimetics and halofuginone, a potent inhibitor of collagen synthesis, extracellular matrix deposition and SMC proliferation, is expected to inhibit restenosis through inhibition of both signals/activities induced by soluble molecules (ie, heparin-binding growth factors) and components of the extracellular matrix (ie, type I collagen).
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Affiliation(s)
| | - Nicola Viola
- Departments of Thoracic and Cardiovascular Surgery
| | | | - Gideon Merin
- Departments of Thoracic and Cardiovascular Surgery
| | - Annete Schmidt
- Institute for Arteriosclerosis Research, University of Muenster, Muenster, Germany
| | - Erick Buddecke
- Institute for Arteriosclerosis Research, University of Muenster, Muenster, Germany
| | - Shmuel A Ben-Sasson
- Department of Experimental Medicine, the Hebrew University-Hadassah Medical School, Jerusalem, Israel; and
| | - Israel Vlodavsky
- Oncology, Hadassah-University Hospital, Jerusalem, Israel
- Vascular and Tumor Biology Research Center, the Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel
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41
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Elkin M, Ilan N, Ishai-Michaeli R, Friedmann Y, Papo O, Pecker I, Vlodavsky I. Heparanase as mediator of angiogenesis: mode of action. FASEB J 2001; 15:1661-3. [PMID: 11427519 DOI: 10.1096/fj.00-0895fje] [Citation(s) in RCA: 237] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- M Elkin
- Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem 91120, Israel
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42
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Aviezer D, Seddon AP, Wildey MJ, Böhlen P, Yayon A. Development of a high throughput screening assay for inhibitors of fibroblast growth factor-receptor-heparin interactions. JOURNAL OF BIOMOLECULAR SCREENING 2001; 6:171-7. [PMID: 11689113 DOI: 10.1177/108705710100600307] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
High throughput screening (HTS) of large compound libraries for inhibitors of growth factors raises the requirement for simple yet reliable assays. Fibroblast growth factors (FGFs) play a pivotal role in the multistep pathway of malignant transformation, tumor progression, metastasis, and angiogenesis. FGF-2 (basic FGF) requires a cooperative interaction with heparin or heparan sulfate proteoglycans in order to form functional growth factor-receptor complexes that are essential for receptor binding and activation. We have developed a simple screening system, devised to identify molecules that modulate heparin-FGF-receptor interactions. The system is composed of a heparin matrix, FGF-2, and a FGF receptor-1 protein engineered by genetically fusing the extracellular domain of FGF receptor-1 to alkaline phosphatase (FRAP). The screen is conducted using 96-well plates to which heparin has been covalently attached. FGF-2 is then bound to the plates through heparin-FGF interactions, followed by the addition of FRAP and compounds to be screened for modulation of heparin-FGF, receptor-heparin, and receptor-FGF interactions. The endpoint of the assay is measured enzymatically using the alkaline phosphatase (AP)-catalyzed formation of a chromogenic product, which is directly proportional to the amount of FRAP present on the plates as a heparin-FGF-FRAP ternary complex. Reduced AP values relative to control, as measured by spectrophotometry, indicate inhibition of the formation of an active FGF-receptor-heparin complex. The simple and versatile nature of the assay makes it an attractive HTS system. The screen has identified several potent inhibitors of FGF-2 receptor binding and activation. Furthermore, secondary screening of the HTS-recognized compounds identified several compounds that have the capacity to block growth factor-mediated tumor progression and angiogenesis in vivo.
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Abstract
Leiomyomas are a significant problem in women's health. An understanding of the biology of these tumors and how their growth is regulated is emerging from in vitro studies using tissue specimens and cultured cells. These studies have clarified how the ovarian steroid hormones regulate growth of uterine SMCs and how the ovarian steroid ligand-receptor system has been altered in leiomyomas. Such information will allow investigators to identify steroid hormone antagonists and steroid hormone receptor modulators that may be useful for treatment of leiomyomas. We are now also developing a much better understanding of the growth factors that are produced by SMCs of leiomyoma tumors. These growth factors not only regulate the proliferation, apoptosis, and extra-cellular matrix production of the SMCs but also regulate proliferation and migration of vascular endothelial cells. Targeting these growth factors and their receptors can reduce leiomyoma growth through two different mechanisms. One targets the SMCs and the other targets the vascular system that supports the growth of the tumor. Another important lesson that can be learned from reading the scientific literature is that there are striking similarities between the biology of uterine leiomyomas and other pathologic diseases that involve mesenchymally derived cells. These include benign keloids, other fibrotic diseases such as pulmonary fibrosis, and vascular diseases such as atherosclerosis. Compounds that are developed to treat these conditions may also be beneficial for treatment of uterine leiomyomas. The next few years will undoubtedly yield many new drug discoveries for these diseases.
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Affiliation(s)
- R A Nowak
- University of Illinois, Urbana-Champaign, Illinois, USA
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44
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Zhang J, Riverst G, Zhu Y, Jacobson A, Peyers J, Grundstrom G, Burch P, Hussein S, Marolewski A, Herlihy W, Rusche J. Identification of inhibitors of heparin–growth factor interactions from combinatorial libraries of four-component condensation reactions. Bioorg Med Chem 2001; 9:825-36. [PMID: 11354665 DOI: 10.1016/s0968-0896(00)00317-5] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Chemical libraries based on four-component condensation (4CC) reactions of isocyanides were constructed to identify compounds capable of blocking heparin binding to vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). The reaction products in the synthesized libraries contain heparin mimetic functional groups such as carbohydrates, sulfonates, carboxylates, and hydroxy groups. These libraries have been screened for the inhibition of heparin binding to growth factors such as VEGF and bFGF. Single point screening at 5.0 microM of the 18,720 reaction products generated 26 candidates. The IC50S of these 26 compounds were determined using HPLC-purified products and 20 of the 26 showed significant inhibition of heparin binding to VEGF and/or bFGF. Eighteen of the 20 confirmed active compounds have a linear extended structure. Structures identified in this library revealed an initial relationship of structure and activity, thus providing direction for further investigation of this type of heparin mimetic libraries.
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Affiliation(s)
- J Zhang
- RepliGen Corporation, Needham, MA 02494, USA.
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45
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Hagedorn M, Zilberberg L, Lozano RM, Cuevas P, Canron X, Redondo-Horcajo M, Gimenez-Gallego G, Bikfalvi A. A short peptide domain of platelet factor 4 blocks angiogenic key events induced by FGF-2. FASEB J 2001; 15:550-2. [PMID: 11259363 DOI: 10.1096/fj.00-0285fje] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Platelet factor 4 (PF-4) is a CXC-chemokine with strong anti-angiogenic properties. We have shown previously that PF-4 inhibits angiogenesis by associating directly with fibroblast growth factor 2 (FGF-2), inhibiting its dimerization, and blocking FGF-2 binding to endothelial cells. We now have characterized a small peptide domain (PF-447-70) derived from the C-terminus of PF-4, which conserves anti-angiogenic effects of the parent protein. PF-447-70 inhibited internalization of 125I-FGF-2 by endothelial cells in a time-dependent manner. The peptide reduced FGF-2-stimulated cell migration to control levels in wounded monolayers of bovine capillary endothelial cells. PF-447-70 also reduced FGF-2 induced phosphorylation of MAP kinases ERK-1 and ERK-2, which are essential for migration and survival of endothelial cells. In a serum-free ex vivo angiogenesis assay, the peptide blocked microvessel outgrowth by 89%. A single amino acid substitution within PF-447-70 abolished all inhibitory activities. To simulate a real anti-angiogenic treatment situation, we administered PF-447-70 systemically to mice implanted subcutaneously with FGF-2 containing gelatin sponges with the result of sparse, scattered, and immature vessel growth. The small peptide fragment derived from the angio-inhibitory CXC-chemokine PF-4 might be used as a starting point to develop anti-angiogenic designer drugs for angiogenesis-dependent pathologies such as cancer, diabetic retinopathy, and rheumatoid arthritis.
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MESH Headings
- Amino Acid Sequence
- Animals
- Aorta
- Cell Division
- Cell Movement
- Cells, Cultured
- Culture Media, Serum-Free
- Culture Techniques
- Endothelium, Vascular/cytology
- Endothelium, Vascular/drug effects
- Enzyme Activation
- Fibroblast Growth Factor 2/metabolism
- Humans
- Mice
- Mitogen-Activated Protein Kinases/metabolism
- Models, Biological
- Molecular Sequence Data
- Neovascularization, Physiologic/drug effects
- Peptide Fragments/chemistry
- Peptide Fragments/genetics
- Peptide Fragments/pharmacology
- Platelet Factor 4/chemistry
- Platelet Factor 4/genetics
- Platelet Factor 4/pharmacology
- Protein Structure, Tertiary
- Rats
- Receptor Protein-Tyrosine Kinases/metabolism
- Receptor, Fibroblast Growth Factor, Type 2
- Receptors, Fibroblast Growth Factor/metabolism
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Affiliation(s)
- M Hagedorn
- Growth Factor and Cell Differentiation Laboratory, University Bordeaux I, 33405 Talence, France
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46
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Presta M, Rusnati M, Dell'Era P, Tanghetti E, Urbinati C, Giuliani R, Leali D. Examining new models for the study of autocrine and paracrine mechanisms of angiogenesis through FGF2-transfected endothelial and tumour cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2001; 476:7-34. [PMID: 10949652 DOI: 10.1007/978-1-4615-4221-6_2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumour neovascularization. Several growth factors and cytokines have been shown to stimulate endothelial cell proliferation in vitro and in vivo and among them FGF2 was one of the first to be characterised. FGF2 is a Mr 18,000 heparin-binding cationic polypeptide that induces proliferation, migration, and protease production in endothelial cells in culture and neovascularization in vivo. FGF2 interacts with endothelial cells through two distinct classes of receptors, the high affinity tyrosine-kinase receptors (FGFRs) and low affinity heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix. Besides experimental evidence for paracrine mode of action for FGF2, some observations raise the hypothesis that FGF2 may also play an autocrine role in endothelial cells. FGF2 may therefore represent a target for anti-angiogenic therapies. In order to assess the angiostatic potential of different classes of compounds, novel experimental models have been developed based on the autocrine and/or the paracrine capacity of FGF2.
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Affiliation(s)
- M Presta
- Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy
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47
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Abstract
Angiogenesis is a fundamental process in reproduction and wound healing. Under these conditions, neovascularization is tightly regulated. Unregulated angiogenesis may lead to several angiogenic diseases and is thought to be indispensable for solid tumor growth and metastasis. The construction of a vascular network requires different sequential steps including the release of proteases from "activated" endothelial cells with subsequent degradation of the basement membrane surrounding the existing vessel, migration of endothelial cells into the interstitial space, endothelial cell proliferation, and differentiation into mature blood vessels. These processes are mediated by a wide range of angiogenic inducers, including growth factors, chemokines, angiogenic enzymes, endothelial specific receptors, and adhesion molecules. Finally, when sufficient neovascularization has occurred, angiogenic factors are down-regulated or the local concentration of inhibitors increases. As a result, the endothelial cells become quiescent, and the vessels remain or regress if no longer needed. Thus, angiogenesis requires many interactions that must be tightly regulated in a spatial and temporal manner. Each of these processes presents possible targets for therapeutic intervention. Synthetic inhibitors of cell invasion (marimastat, Neovastat, AG-3340), adhesion (Vitaxin), or proliferation (TNP-470, thalidomide, Combretastatin A-4), or compounds that interfere with angiogenic growth factors (interferon-alpha, suramin, and analogues) or their receptors (SU6668, SU5416), as well as endogenous inhibitors of angiogenesis (endostatin, interleukin-12) are being evaluated in clinical trials against a variety of solid tumors. As basic knowledge about the control of angiogenesis and its role in tumor growth and metastasis increases, it may be possible in the future to develop specific anti-angiogenic agents that offer a potential therapy for cancer and angiogenic diseases.
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Affiliation(s)
- S Liekens
- Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000, Leuven, Belgium.
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48
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Benezra M, Vogel T, Ben-Sasson SA, Panet A, Sehayek E, Al-Haideiri M, Decklbaum RJ, Vlodavsky I. A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation. J Cell Biochem 2001. [DOI: 10.1002/1097-4644(20010401)81:1<114::aid-jcb1028>3.0.co;2-q] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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49
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Miao HQ, Lee P, Lin H, Soker S, Klagsbrun M. Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression. FASEB J 2000; 14:2532-9. [PMID: 11099472 DOI: 10.1096/fj.00-0250com] [Citation(s) in RCA: 230] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Neuropilin-1 (NRP1) is a VEGF(165) and semaphorin receptor expressed by vascular endothelial cells (EC) and tumor cells. The function of NRP1 in tumor cells is unknown. NRP1 was overexpressed in Dunning rat prostate carcinoma AT2.1 cells using a tetracycline-inducible promoter. Concomitant with increased NRP1 expression in response to a tetracycline homologue, doxycycline (Dox), basal cell motility, and VEGF(165) binding were increased three- to fourfold in vitro. However, induction of NRP1 did not affect tumor cell proliferation. When rats injected with AT2.1/NRP1 tumor cells were fed Dox, NRP1 synthesis was induced in vivo and AT2.1 cell tumor size was increased 2.5- to 7-fold in a 3-4 wk period compared to controls. The larger tumors with induced NRP1 expression were characterized by markedly increased microvessel density, increased proliferating EC, dilated blood vessels, and notably less tumor cell apoptosis compared to noninduced controls. It was concluded that NRP1 expression results in enlarged tumors associated with substantially enhanced tumor angiogenesis.
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Affiliation(s)
- H Q Miao
- Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA
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50
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Forsten KE, Fannon M, Nugent MA. Potential mechanisms for the regulation of growth factor binding by heparin. J Theor Biol 2000; 205:215-30. [PMID: 10873433 DOI: 10.1006/jtbi.2000.2064] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Heparin and heparan sulfate proteoglycans (HSPG) bind many soluble growth factors and this binding is now recognized as an important mechanism for modulation of cell activity. Fibroblast growth factor-2 (FGF-2) is one of the best characterized of the heparin-binding growth factors and it has been shown experimentally that heparin regulation of FGF-2 activity is dependent on the level of cell HSPG and the concentration of heparin. In this paper, we explore, using mathematical modeling, proposed mechanisms for heparin regulation and determine how they impact FGF receptor binding. We demonstrate that the experimentally observed receptor binding phenomena can be reproduced if cells (1) express heparin-binding cell surface molecules and if either (2) these heparin binding sites are FGFR and bind heparin and FGF-2-heparin complexes or (3) are surface molecules able to bind FGF-2 and couple with FGF-2 receptors to form high-affinity FGF-2-bound surface complexes. The ability of heparin to directly interact with the FGFR and bind FGF-2 in the absence of this coupling function was not sufficient to explain heparin activity. These findings have implications with regard to regulation of heparin-binding growth factors and could help guide the development of highly specific growth regulatory molecules through specific regulation by heparin and HSPG.
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Affiliation(s)
- K E Forsten
- Department of Chemical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0211, USA.
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