Role of matrix metalloproteinase (MMP)-1 and its activators in regulating capillary tube regression events : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2005]
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| Abstract: | ABSTRACT: Recent work has demonstrated that soluble matrix metalloproteinases (MMPs) are involved in tissue regression events both in vitro and in vivio. Specifically, it has been show that plasminogen/plasmin activate human endothelial cell (EC) derived MMP-1, which induces regression of newly formed capillary tubes during EC morphogenesis in three-dimensional collagen matrices. The goal of the research presented herein is to determine, at the molecular level, the central role human matrix metalloproteinase-1 (MMP-1) and its activators play in the regulation of capillary tube regression events. In chapter II, a novel, 384 micro-well regression assay is developed to effectively quantify the capillary tube regression response. Data are presented which demonstrate that six additional serine proteases are capable of activating proMMP-1 leading to capillary tube regression : plasma kallikrein, trypsin, neutrophil elastase, cathepsin G, tryptase and chymase. Time-lapse photography of this process indicates that formed capillary tubular networks undergo tube retraction and network regression prior to the onset of collagen gel contraction, making this system highly relevant to vascular regresssion events in vivo. Additionally, for the first time it is shown that proMMP-10 protein expression is markedly induced in ECs during capillary tube morphogenesis, and that proMMP-10 is activated in a similar manner by the serine proteases described above. Treatment of ECs with MMP-1 or MMP-10 siRNA markedly delayed capillary tube regression, whereas gelatinase A (MMP-2), gelatinase B (MMP-9), and stromelysin-1 (MMP-3) siRNA treated cell behaved similarly to controls and regressed normally. Increased expression of wild type MMP-1 or MMp-10 in ECs using recombinant adenoviral delivery markedly accelerated serine protease-induced capillary tube regression. Thus, MMP-10 contributes to the capillary tube regression response by its ability to directly activate proMMP-1. In chapter III, the central role of human MMP-1 as a key downstream mediator of vascular regression events is further established. Multiple, spontaneously active forms of MMP-1 were generated, which underwent auto-activation in the absence of serine proteases, Expression of these MMP-1 mutants using recombinant adenoviral delivery induced serine protease-indepedent capillary tube regression that was blocked by the addition of MMP, but not serine protease inhibitors. To evaluate the role of MMP-1 and MMP-10 as mediators of vascular regression in vivo, cells expressing wild type MMP-1 or MMP-10, or spontaneously active forms of MMP-1, were utilized to either inhibit formation or induce regression of VEGF-induced angiogenic fields using the chicken chorioallantoic membrane (CAM) model of angiogenesis. In both assays, wild-type or mutant MMP-1 blocked VEGF-induced angiogenesis and led to regression of early angiogenic responses. MMP-1-induced regression was enhanced by the co-delivery of MMP-10. These data clearly demonstrate that MMP-1 is the downstream mediator of human capillary tube regression events in vitro and is capable of inducing vascular regression in vivo. In chapter IV, the ability of the mouse equivalent of human MMP-1 (murine MMP-13) and human collagenase-3 (human MMP-13) to induce capillary tube regression in type I and type II collagen matrices is investigated. ECs underwent capillary tube formation (morphogenesis) and plasmin induced capillary tube regression in three-dimensional (3D) type II collagen matrices in a manner similar to matrices formed with type I collagen. ECs induced to express either murine or human MMP-13 via recombinant adenoviral delivery underwent capillary tube regression in an accelerated manner as compared to treatment with control virus. The addition of increasing doses of plasmingoren to culture media of ECs expressing these collagenases resulted in a dose-dependent capillary tube regression response that directly correlated with the degree of collagenase activation visible via Western blot analysis of conditioned media from these experiments. Importantly, this plasminogen/plasmin mediated activation of murine and human MMP-13 and the resulting capillary tube regression response were inhibited by the addition of both serine and metalloproteinase inhibitors. These results demonstrate that additional collagenases, both murine and human, are capable of inducing regression of EC capillary tube networks. Thus, multiple collagenases from different species are capable of inducing capillary tube networks, indicating this regression system is broadly relevant. In conclusion, this work demonstrates that a well regulated system of serine proteases and stromelysins (MMPs) exists to activate proMMP-1, which functions as a vascular regression factor in vitro and in vivo. These data also demonstrate that the collagenase-mediated regression of capillary networks is accomplished by multiple collagenases from various species. Elucidation of the mechanisms controlling capillary tube regression events is essential if attempts to treat tumor-associated blood vessels or disorders of increased vascularity are to become successful. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Graduate School of Biomedical Sciences of The Texas A&M University System Health Science Center in partial fulfillment for the requirements for the degree of Doctor of Philosophy May 2005." Approved as to style and content by: George E. Davis, Theresa W. Fossum, Gregg B. Wells, Michael J. Davis, Rajesh C. Miranda. |
| Physical Description: | xiv, 172 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 151-170). |