Romana A. Nowak

Disrupting the EMMPRIN (CD147)–Cyclophilin A Interaction Reduces Infarct Size and Preserves Systolic Function After Myocardial Ischemia and Reperfusion

Objective—Inflammation and proteolysis crucially contribute to myocardial ischemia and reperfusion injury. The extracellular matrix metalloproteinase inducer EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) may be involved in both processes. The aim of the study was to characterize the role of the CD147 and CyPA interplay in myocardial ischemia/reperfusion (I/R) injury. Methods and Results—Immunohistochemistry showed enhanced expression of CD147 and CyPA in myocardial sections from human autopsies of patients who had died from acute myocardial infarction and from mice at 24 hours after I/R. At 24 hours and 7 days after I/R, the infarct size was reduced in CD147+/− mice vs CD147+/+ mice (C57Bl/6), in mice (C57Bl/6) treated with monoclonal antibody anti-CD147 vs control monoclonal antibody, and in CyPA−/− mice vs CyPA+/+ mice (129S6/SvEv), all of which are associated with reduced monocyte and neutrophil recruitment at 24 hours and with a preserved systolic function at 7 days. The combination of CyPA−/− mice with anti-CD147 treatment did not yield further protection compared with either inhibition strategy alone. In vitro, treatment with CyPA induced monocyte chemotaxis in a CD147- and phosphatidylinositol 3-kinase–dependent manner and induced monocyte rolling and adhesion to endothelium (human umbilical vein endothelial cells) under flow in a CD147-dependent manner. Conclusion—CD147 and its ligand CyPA are inflammatory mediators after myocardial ischemia and reperfusion and represent potential targets to prevent myocardial I/R injury.

Reactive Oxygen Species Mediate Mitogenic Growth Factor Signaling Pathways in Human Leiomyoma Smooth Muscle Cells

Abstract Uterine leiomyomas are benign uterine tumors characterized by extracellular matrix remodeling, increased collagen deposition, and increased smooth muscle cell (SMC) proliferation. The reactive oxygen species (ROS) producing NADPH oxidase complex has been shown to be involved in the signaling pathways of several growth factors, cytokines, and vasoactive agents that stimulate proliferation of a variety of cell types. Our objective was to test the hypothesis that ROS derived from NADPH oxidase is a necessary component of the MAP kinase mitogenic pathway activated by platelet derived growth factor (PDGF) and epidermal growth factor (EGF) in leiomyoma SMCs (LSMCs). Primary cell cultures of LSMCs were used as our experimental model. Our results showed that stimulation of these cells with PDGF or EGF caused a marked increase in intracellular ROS production and that the NADPH oxidase inhibitor, DPI, blocks ROS production. In addition, inhibition of ROS production by NADPH oxidase inhibitors blocked, in a dose-dependent manner, the EGF- and PDGF-induced increase in [3H]thymidine incorporation by LSMCs. Furthermore, an exogenous source of ROS, hydrogen peroxide, was sufficient to stimulate [3H]thymidine incorporation in LSMCs but did not affect COL1A2 and COL3A1 mRNA levels. Inhibition of the NADPH oxidase complex decreased PDGF-induced MAPK1/MAPK3 activation, whereas exogenous hydrogen peroxide induced MAPK1/MAPK3 activation. This article is the first report suggesting the presence of the NADPH oxidase system and its importance in mitogenic signaling pathways in LSMCs. The necessity of NADPH oxidase-derived ROS for EGF and PDGF signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.

Basigin-2 Is a Cell Surface Receptor for Soluble Basigin Ligand

The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand.

HMGA2 expression in uterine leiomyomata and myometrium: Quantitative analysis and tissue culture studies

The high mobility group gene, HMGA2, is frequently expressed in uterine leiomyomata (UL) with chromosomal rearrangements of 12q15. In contrast, HMGA2 expression has not been detected in karyotypically normal UL or in myometrium, but has been detected in these tissues after culture. To characterize further the expression pattern of HMGA2, we assessed HMGA2 expression by RT‐PCR followed by Southern blot hybridization, and by real‐time PCR in three tissue panels: (1) primary myometrial cultures, (2) uncultured tissue from 15 karyotypically normal samples consisting of eleven 46,XX UL and four matched myometrial specimens, and (3) uncultured tissue from ten UL with 12q15 rearrangements and three matched myometrial specimens. HMGA2 expression was detected in all samples from the three panels. The level of HMGA2 expression in karyotypically normal UL was similar to the level of expression in myometrium; however, it was significantly less than the level measured in UL with 12q15 rearrangements. This expression analysis by use of detection methods of different sensitivities underscores the importance of studies of HMGA2 expression in uncultured tissues and of careful interpretation of results from experiments on cultured cells. Moreover, detection of HMGA2 expression in myometrium and in UL without 12q15 rearrangements, tissues previously thought not to express HMGA2, suggests that HMGA2 expression is required in normal adult myometrial physiology.

Cytokines regulate matrix metalloproteinases in human uterine endometrial fibroblast cells through a mechanism that does not involve increases in extracellular matrix metalloproteinase inducer

Problem  Endometriosis is the presence of ectopic uterine endometrial tissue in the peritoneal cavity. Peritoneal fluid samples of women with endometriosis show elevated interleukin‐1 (IL‐1)β, tumor necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β1 levels, indicating that an altered immune system may play an important role in the pathogenesis of endometriosis. The invasion of ectopic endometrium into peritoneal mesothelium requires matrix metalloproteinases (MMPs) for tissue remodeling. Several MMPs are differentially expressed in human uterine endometrium with menstrual endometrium showing the highest level of expression. MMPs are stimulated by cytokines and also by the protein Extracellular Matrix Metalloproteinase Inducer (EMMPRIN).

Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids.

Anti-inflammatory effects of extracellular cyclosporins are exclusively mediated by CD147.

Leukocyte trafficking and recruitment is a critical process in host immune surveillance and in inflammatory diseases. Extracellular cyclophilins (eCyps) have been identified as a novel class of chemotactic mediators. The impact of eCyp/CD147 interactions for the recruitment of leukocytes during inflammation was analyzed using a structurally simplified cell-impermeable eCyp inhibitor. This compound was highly effective at inhibiting leukocyte migration toward CypA in vitro as well as in the recruitment of leukocytes during inflammation in a mouse model of experimentally induced peritonitis and delayed-type hypersensitivity reaction. By using CD147-/- mice in combination with the cell-impermeable eCyp inhibitor, we were able to show that the action of eCyps in inflammation is exclusively mediated by interaction with CD147. Our findings suggest that blocking eCyps may be an effective therapeutic target for reducing inflammatory diseases associated with leukocyte recruitment.

Basigin-Mediated Gene Expression Changes in Mouse Uterine Stromal Cells During Implantation

Implantation of mouse embryos is dependent on the proliferation and differentiation of uterine stromal cells in a process called decidualization. Decidualization both supports and limits the invasion of the implanting embryo and is regulated in part by the expression of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Molecules that alter the balance between MMP and TIMP expression could prevent implantation of the embryo. The membrane glycoprotein basigin (CD147/EMMPRIN), a known inducer of MMPs, is necessary for normal implantation in the mouse. The purpose of this study was to investigate the potential roles of basigin during implantation in the mouse. Using an in vitro stromal cell culture system, we found that recombinant human basigin protein (rBSG) increases MMP-3 and MMP-9 expression without altering TIMP-3 expression. Our results also showed rBSG induces expression of cytokines IL-1alpha/beta and leukocyte chemoattractants, CCL3, CCL20, CXCL2, and CXCL5. More importantly, rBSG significantly suppressed stromal cell decidualization as shown by the inhibition of alkaline phosphatase-2 expression and activity by rBSG. However, rBSG did not affect stromal cell proliferation. Taken together, our data indicate that basigin mediates gene expression changes in mouse uterine stromal cells and suggests that temporal and spatial regulation of basigin expression may be involved in the recruitment of leukocytes to the mouse uterus during early pregnancy.

Extracellular Matrix Collagen Alters Cell Proliferation and Cell Cycle Progression of Human Uterine Leiomyoma Smooth Muscle Cells

Uterine leiomyomas (ULs) are benign tumors occurring in the majority of reproductive aged women. Despite the high prevalence of these tumors, little is known about their etiology. A hallmark of ULs is the excessive deposition of extracellular matrix (ECM), primarily collagens. Collagens are known to modulate cell behavior and function singularly or through interactions with integrins and growth factor-mediated mitogenic pathways. To better understand the pathogenesis of ULs and the role of ECM collagens in their growth, we investigated the interaction of leiomyoma smooth muscle cells (LSMCs) with two different forms of collagen, non-polymerized collagen (monomeric) and polymerized collagen (fibrillar), in the absence or presence of platelet-derived growth factor (PDGF), an abundant growth factor in ULs. Primary cultures of human LSMCS from symptomatic patients were grown on these two different collagen matrices and their morphology, cytoskeletal organization, cellular proliferation, and signaling pathways were evaluated. Our results showed that LSMCs had distinct morphologies on the different collagen matrices and their basal as well as PDGF-stimulated proliferation varied on these matrices. These differences in proliferation were accompanied by changes in cell cycle progression and p21, an inhibitory cell cycle protein. In addition we found alterations in the phosphorylation of focal adhesion kinase, cytoskeletal reorganization, and activation of the mitogen activated protein kinase (MAPK) signaling pathway. In conclusion, our results demonstrate a direct effect of ECM on the proliferation of LSMCs through interplay between the collagen matrix and the PDGF-stimulated MAPK pathway. In addition, these findings will pave the way for identifying novel therapeutic approaches for ULs that target ECM proteins and their signaling pathways in ULs.

The Antifibrotic Drug Halofuginone Inhibits Proliferation and Collagen Production by Human Leiomyoma and Myometrial Smooth Muscle Cells

OBJECTIVE To investigate the effects of the antifibrotic drug halofuginone on extracellular matrix production, cell proliferation, and apoptosis of cultured myometrial and leiomyoma smooth muscle cells. DESIGN Comparative and controlled experimental research study. SETTING University research laboratory. PATIENT(S) Leiomyoma and myometrial tissues were obtained from eight different patients at the time of elective hysterectomy. MAIN OUTCOME MEASURE(S) The effects of halofuginone on cell proliferation were assessed by tritiated thymidine uptake assays and cell count assays. Effects on TGFbeta1, collagen type I, and collagen type III mRNA levels were assessed by quantitative real-time polymerase chain reaction. Effects on apoptosis were assayed using a chemiluminescent assay to measure changes in caspase 3 and 7. RESULT(S) Halofuginone inhibited cell proliferation of both leiomyoma and autologous myometrial cells in a dose-dependent manner by inhibiting DNA synthesis within 24 hours and later inducing apoptosis (as measured by increased caspase 3/7) by 48-72 hours. Halofuginone also significantly reduced collagen type I (alpha1) and collagen type III (alpha1) mRNA levels, as well as the profibrotic factor TGFbeta1 mRNA levels in both cell types. CONCLUSION(S) These results provide evidence to support the use of the antifibrotic drug halofuginone as a novel drug treatment for uterine leiomyomas.