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The Nonproteolytic Intracellular Domain of Membrane-Type 1 Matrix Metalloproteinase Coordinately Modulates Abdominal Aortic Aneurysm and Atherosclerosis in Mice-Brief Report

Silvestro, Michele; Rivera, Cristobal F; Alebrahim, Dornazsadat; Vlahos, John; Pratama, Muhammad Yogi; Lu, Cuijie; Tang, Claudia; Harpel, Zander; Sleiman Tellaoui, Rayan; Zias, Ariadne L; Maldonado, Delphina J; Byrd, Devon; Attur, Mukundan; Mignatti, Paolo; Ramkhelawon, Bhama
BACKGROUND:MT1-MMP (membrane-type 1 matrix metalloproteinase, MMP-14) is a transmembrane-anchored protein with an extracellular proteinase domain and a cytoplasmic tail devoid of proteolytic functions but capable of mediating intracellular signaling that regulates tissue homeostasis. MT1-MMP extracellular proteolytic activity has been shown to regulate pathological remodeling in aortic aneurysm and atherosclerosis. However, the role of the nonproteolytic intracellular domain of MT1-MMP in vascular remodeling in abdominal aortic aneurysms (AAA) is unknown. METHODS:We generated a mutant mouse that harbors a point mutation (Y573D) in the MT1-MMP cytoplasmic domain that abrogates the MT1-MMP signaling function without affecting its proteolytic activity. These mice and their control wild-type littermates were subjected to experimental AAA modeled by angiotensin II infusion combined with PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression and high-cholesterol feeding. RESULTS:The mutant mice developed more severe AAA than the control mice, with concomitant generation of intraaneurysmal atherosclerotic lesions and dramatically increased macrophage infiltration and elastin degradation. Aortic lesion-associated and bone marrow-derived macrophages from the mutant mice exhibited an enhanced inflammatory state and expressed elevated levels of proinflammatory Netrin-1, a protein previously demonstrated to promote both atherosclerosis and AAA. CONCLUSIONS:Our findings show that the cytoplasmic domain of MT1-MMP safeguards from AAA and atherosclerotic plaque development through a proteolysis-independent signaling mechanism associated with Netrin-1 expression. This unexpected function of MT1-MMP unveils a novel mechanism of synchronous onset of AAA and atherogenesis and highlights its importance in the control of vascular wall homeostasis.
PMID: 36073351
ISSN: 1524-4636
CID: 5335022

Membrane-type 1 Matrix Metalloproteinase Modulates Tissue Homeostasis by a Non-proteolytic Mechanism

Attur, Mukundan; Lu, Cuijie; Zhang, Xiaodong; Han, Tianzhen; Alexandre, Cassidy; Valacca, Cristina; Zheng, Shuai; Meikle, Sarina; Dabovic, Branka Brukner; Tassone, Evelyne; Yang, Qing; Kolupaeva, Victoria; Yakar, Shoshana; Abramson, Steven; Mignatti, Paolo
Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with a short cytoplasmic tail, is a major effector of extracellular matrix remodeling. Genetic silencing of MT1-MMP in mouse (Mmp14
PMCID:7695985
PMID: 33294797
ISSN: 2589-0042
CID: 4735172

Periostin interaction with discoidin domain receptor-1 (DDR1) promotes cartilage degeneration

Han, Tianzhen; Mignatti, Paolo; Abramson, Steven B; Attur, Mukundan
Osteoarthritis (OA) is characterized by progressive loss of articular cartilage accompanied by the new bone formation and, often, a synovial proliferation that culminates in pain, loss of joint function, and disability. However, the cellular and molecular mechanisms of OA progression and the relative contributions of cartilage, bone, and synovium remain unclear. We recently found that the extracellular matrix (ECM) protein periostin (Postn, or osteoblast-specific factor, OSF-2) is expressed at high levels in human OA cartilage. Multiple groups have also reported elevated expression of Postn in several rodent models of OA. We have previously reported that in vitro Postn promotes collagen and proteoglycan degradation in human chondrocytes through AKT/β-catenin signaling and downstream activation of MMP-13 and ADAMTS4 expression. Here we show that Postn induces collagen and proteoglycan degradation in cartilage by signaling through discoidin domain receptor-1 (DDR1), a receptor tyrosine kinase. The genetic deficiency or pharmacological inhibition of DDR1 in mouse chondrocytes blocks Postn-induced MMP-13 expression. These data show that Postn is signaling though DDR1 is mechanistically involved in OA pathophysiology. Specific inhibitors of DDR1 may provide therapeutic opportunities to treat OA.
PMID: 32330138
ISSN: 1932-6203
CID: 4402442

Matrix Metalloproteinase Inhibitors in Cancer Therapy: Turning Past Failures Into Future Successes

Winer, Arthur; Adams, Sylvia; Mignatti, Paolo
The matrix metalloproteinases (MMP) are a family of proteolytic enzymes that degrade multiple components of the extracellular matrix. A large body of experimental and clinical evidence has implicated MMPs in tumor invasion, neoangiogenesis, and metastasis, and therefore they represent ideal pharmacologic targets for cancer therapy. From the 1990s to early 2000s, synthetic inhibitors of MMPs (MMPI) were studied in various cancer types. Unexpectedly, despite strongly promising preclinical data, all trials were unsuccessful in reducing tumor burden or improving overall survival; in addition, MMPIs had unforeseen, severe side effects. Two main reasons can explain the failure of MMPIs in clinical trials. It has now become apparent that some MMPs have antitumor effects; therefore, the broad-spectrum MMPIs used in the initial trials might block these MMPs and result in tumor progression. In addition, although MMPs are involved in the early stages of tumor progression, MMPIs were tested in patients with advanced disease, beyond the stage when these compounds could be effective. As more specific MMPIs are now available, MMP targeting could be reconsidered for cancer therapy; however, new trials should be designed to test their antimetastatic properties in early-stage tumors, and endpoints should focus on parameters other than decreasing metastatic tumor burden. Mol Cancer Ther; 1-9. ©2018 AACR.
PMCID:5984693
PMID: 29735645
ISSN: 1538-8514
CID: 3101502

Arrested Development: Infantile Hemangioma and the Stem Cell Teratogenic Hypothesis

Harbi, Shaghayegh; Park, Hannah; Gregory, Michael; Lopez, Peter; Chiriboga, Luis; Mignatti, Paolo
BACKGROUND: Early-life programming is defined by the adaptive changes made by the fetus in response to an adverse in utero environment. Infantile hemangioma (IH), a vascular anomaly, is the most common tumor of infancy. Here we take IH as the tumor model to propose the stem cell teratogenic hypothesis of tumorigenesis and the potential involvement of the immune system. OBJECTIVES: Teratogenic agents include chemicals, heavy metals, pathogens, and ionizing radiation. To investigate the etiology and pathogenesis of IH, we hypothesized that they result from a teratogenic mechanism. Immature, incompletely differentiated, dysregulated progenitor cells (multipotential stem cells) are arrested in development with vasculogenic, angiogenic, and tumorigenic potential due to exposure to teratogenic agents such as extrinsic factors that disrupt intrinsic factors via molecular mimicry. During the critical period of immunological tolerance, environmental exposure to immunotoxic agents may harness the teratogenic potential in the developing embryo or fetus and modify the early-life programming algorithm by altering normal fetal development, causing malformations, and inducing tumorigenesis. Specifically, exposure to environmental agents may interfere with physiological signaling pathways and contribute to the generation of IH, by several mechanisms. DISCUSSION: An adverse in utero environment no longer serves as a sustainable environment for proper embryogenesis and normal development. Targeted disruption of stem cells by extrinsic factors can alter the genetic program. CONCLUSIONS: This article offers new perspectives to stimulate discussion, explore novel experimental approaches (such as immunotoxicity/vasculotoxicity assays and novel isogenic models), and to address the questions raised to convert the hypotheses into nontoxic, noninvasive treatments.
PMID: 28520518
ISSN: 1557-8585
CID: 2562922

Infantile Hemangioma Originates From A Dysregulated But Not Fully Transformed Multipotent Stem Cell

Harbi, Shaghayegh; Wang, Rong; Gregory, Michael; Hanson, Nicole; Kobylarz, Keith; Ryan, Kamilah; Deng, Yan; Lopez, Peter; Chiriboga, Luis; Mignatti, Paolo
Infantile hemangioma (IH) is the most common tumor of infancy. Its cellular origin and biological signals for uncontrolled growth are poorly understood, and specific pharmacological treatment is unavailable. To understand the process of hemangioma-genesis we characterized the progenitor hemangioma-derived stem cell (HemSC) and its lineage and non-lineage derivatives. For this purpose we performed a high-throughput (HT) phenotypic and gene expression analysis of HemSCs, and analyzed HemSC-derived tumorspheres. We found that IH is characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis and associated signaling pathways. These results show that IH derives from a dysregulated stem cell that remains in an immature, arrested stage of development. The potential biomarkers we identified can afford the development of diagnostic tools and precision-medicine therapies to "rewire" or redirect cellular transitions at an early stage, such as signaling pathways or immune response modifiers.
PMCID:5081534
PMID: 27786256
ISSN: 2045-2322
CID: 2288792

INHIBITION OF BREAST CANCER METASTASIS BY PRESURGICAL TREATMENT WITH AN ORAL MATRIX METALLOPROTEINASE INHIBITOR: A PRECLINICAL PROOF-OF-PRINCIPLE STUDY

Winer, Arthur; Janosky, Maxwell; Harrison, Beth; Zhong, Judy; Moussai, Dariush; Siyah, Pinar; Schatz-Siemers, Nina; Zeng, Jennifer; Adams, Sylvia; Mignatti, Paolo
Breast cancer has the second highest death toll in women worldwide, despite significant progress in early diagnosis and treatments. The main cause of death is metastatic disease. Matrix metalloproteinases (MMPs) are required for the initial steps of metastasis, and have therefore been considered as ideal pharmacological targets for anti-metastatic therapy. However, clinical trials of MMP inhibitors were unsuccessful. These trials were conducted in patients with advanced disease, beyond the stage when these compounds could have been effective. We hypothesized that early treatment with a selective MMP inhibitor between the time of diagnosis and definitive surgery, the so-called "window-of-opportunity," can inhibit metastasis and thereby improve survival. To investigate our hypothesis we used the 4T1 mouse model of aggressive mammary carcinoma. We treated the animals with SD-7300, an oral inhibitor of MMP-2, -9 and -13, starting after the initial detection of the primary tumor. Seven days later the primary tumors were excised and analyzed for MMP activity, and the SD-7300 treatment was discontinued. After four weeks the animals were sacrificed and their lungs analyzed histologically for number of metastases and metastatic burden (metastases' area / lung section area). SD-7300 treatment inhibited 70-80% of tumor-associated MMP activity (P = 0.0003), reduced metastasis number and metastatic burden by 50-60% (P = 0.002; P = 0.0082, respectively), and increased survival (92% vs. 66.7%; P = 0.0409), relative to control vehicle. These results show that treatment of early invasive breast cancer with selective MMP inhibitors can lower the risk of recurrence and increase long-term disease-free survival.
PMCID:5050118
PMID: 27466357
ISSN: 1538-8514
CID: 2191612

Binding of periostin to discoidin domain receptor-1 (DDR1) promotes cartilage degeneration by inducing MMP-13 expression [Meeting Abstract]

Qing, Y; Mignatti, P; Ramme, A; Kirsch, T; Patel, J; Attur, M
Background/Purpose: We and others have previously shown that periostin (Postn) expression is dramatically elevated in cartilage and sub-chondral bone in OA patients and surgical models of OA (medial meniscectomy and anterior crucial ligament resection or PMX, partial meniscectomy) in rodents. In vitro Postn promotes collagen and proteoglycan degradation in human chondrocytes by upregulating MMP-13 and ADAMTS4 expression. Postn controls gene expression in bone cells by interacting with avb3 integrin. However, the nature of periostin receptor(s) in chondrocytes is unknown. DDR1, a collagen-binding receptor tyrosine kinase highly expressed in chondrocytes, controls MMP-13 expression during chondrogenesis. Therefore, we hypothesized that the effect of Postn on chondrocytes is mediated by DDR1 and Postn-deficient mice (Postn-/-) are protected from surgically-induced post-traumatic OA. Methods: (Postn-/-) mice were purchased from Jackson Laboratory (B6;129-Postntm1Jmol/J Stock No: 009067). We subjected 3- months old littermates (Postn+/+, Postn+/- and Postn-/-) to partial medial meniscectomy (PMX) or sham surgery, and harvested the knee joints 8 week post-surgery for histological assessment of OA progression. Human OA chondrocytes cultures were incubated in the presence or absence of the DDR1 inhibitor DDR1-IN-1 dihydrochloridein (100-500 nM) for 2 h before addition of Postn (1 mug/ml) or control vehicle to the culture medium. MMP-13 levels were determined by ELISA 24 h post stimulation. Results: We observed abundant expression of DDR1 mRNA in human chondrocytes and we found comparable levels of DDR1 in OA and normal cartilage. However, Postn expression was 3-4 times as high in OA than in normal cartilage. Pre-incubation of human cartilage explants or cultured chondrocytes with DDR1-IN-1 dihydrochloridein inhibited both constitutive and Postn-induced MMP-13 expression in a dose-dependent manner. In contrast, neutralizing antibody to alphavbeta3 integrin had no effect on Postn induction of MMP-13 expression. Co-immunoprecipitation experiments showed that Postn physically interacts with DDR1 in human chondrocytes. Furthermore, Postn-/- mice showed reduced PMX-induced cartilage degeneration and osteophyte formation, and both Postn+/- and Postn-/- mice had reduced subchondral bone thickening, relative to Postn+/+ mice. Conclusion: Postn-/- mice are protected from surgically-induced post-traumatic OA, showing that Postn promotes cartilage degeneration. DDR1 mediates the stimulatory effect of Postn on MMP-13 expression. Further studies are in progress to investigate the potential of periostin as a druggable target for the treatment of OA
EMBASE:613888787
ISSN: 2326-5205
CID: 2397892

Membrane-type 1 matrix metalloproteinase controls osteo-and chondrogenesis by a proteolysis-independent mechanism mediated by its cytoplasmic tail [Meeting Abstract]

Qing, Y; Attur, M; Kirsch, T; Lee, Y J; Yakar, S; Liu, Z; Abramson, S B; Mignatti, P
Background/Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, plays a key role in postnatal bone formation. The genetic deficiency of MT1-MMP in the mouse causes dwarfism, osteopenia and severe arthritis. Deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation in MSC. The phenotype of MT1-MMP-/- mice has been proposed to result from lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysis-independent signaling functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. Methods: We generated a mouse with the Y573D mutation in MT1-MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. Isolated BM-MSC were induced to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2 MSC were transfected with MT1-MMP cDNA and analyzed for Wnt signaling by luciferase reporter assays. Results: MT1-MMP Y573D mice had increased trabecular bone relative to wt littermates, marked thinning of articular cartilage with disorganized tissue architecture, clustering and cloning of chondrocytes, and pronounced decrease in bone marrow-associated and total body fat. We induced BM-MSC from wt and MT1-MMP Y573D littermates to differentiate into osteoblast and chondrocytes, and myeloid precursors into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers and strongly downregulated chondrocyte and osteoclast markers. These findings indicated that Wnt signaling is upregulated in MT1-MMP Y573D-expressing MSC. Therefore, we analyzed Wnt signaling. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cells were transfected with the empty vector (pcDNA) or with MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated Wnt signaling relative to wt MT1-MMP and MT1-MMP E240A. Conclusion: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway
EMBASE:613888758
ISSN: 2326-5205
CID: 2397902

Mitral Valve Prolapse is Associated with Altered Extracellular Matrix Gene Expression Patterns

Greenhouse, David G; Murphy, Alison; Mignatti, Paolo; Zavadil, Jiri; Galloway, Aubrey C; Balsam, Leora B
Mitral valve prolapse (MVP) is the leading indication for isolated mitral valve surgery in the United States. Disorganization of collagens and glycosaminoglycans in the valvular extracellular matrix (ECM) are histological hallmarks of MVP. We performed a transcriptome analysis to study the alterations in ECM-related gene expression in humans with sporadic MVP. Mitral valve specimens were obtained from individuals undergoing valve repair for MVP (n = 7 patients) and from non-beating heart-tissue donors (n = 3 controls). Purified RNA was subjected to whole-transcriptome microarray analysis. Microarray results were validated by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Gene ontology enrichment analysis was performed. 2,046 unique genes showed significant differential expression (false discovery rate <0.5%). After demonstrating appropriate sample clustering, microarray results were globally validated using a subset of 22 differentially expressed genes by RT-qPCR (Pearson's correlation r=0.65, p=0.001). Gene ontology enrichment analyses performed with ErmineJ and DAVID Bioinformatics Database demonstrated overrepresentation of ECM components (p<0.05). Functional annotation clustering calculated enrichment of ECM-related ontology groups (enrichment score = 4.1). ECM-related gene expression is significantly altered in MVP. Our study is consistent with the histologically observed alterations in collagen and mucopolysaccharide profiles of myxomatous mitral valves. Furthermore, whole-transcriptome analyses suggest dysregulation of multiple pathways, including TGF-beta signaling.
PMID: 27063507
ISSN: 1879-0038
CID: 2078232