Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells.

BACKGROUND: Extracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs. METHODS: Cell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins. RESULTS: We observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion. CONCLUSIONS: Our findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.

Small particles or vesicles released by cancer cells into their surroundings have the potential to stimulate the spread and growth of cancer cells. In this study, we focused on two specific molecules presented by these cancer cell-derived vesicles that could play a role in promoting the dissemination of cancer cells: a protein related to blood clotting and a protein on the cell surface.We found that large vesicles from bladder cancer cells that have the ability to spread had higher levels of these proteins than vesicles from nonspreading cancer cells. We also found that the former could make cancer cells move about more, produce more of a substance that helps cancer cells spread, and invade other tissues.To counteract the cancer-promoting actions of these vesicles, we examined the impact of combining a naturally occurring anticlotting protein that can be released by medications derived from heparin with an inhibitor targeting the cancer cell surface protein. We found that this combination stopped the vesicles from helping cancer cells move about more, produce more of the spreading substance, and invade other tissues.This approach of simultaneously targeting the two protein molecules present on cancer cell-derived vesicles might be a new way to treat bladder cancer.

Intervention with extracellular matrix metalloproteinase inducer in osteoclasts attenuates periodontitis-induced bone resorption.

Extracellular matrix metalloproteinase inducer (EMMPRIN) plays critical roles in the regulation of inflammation and bone metabolism. The roles of EMMPRIN signaling in osteoclasts are worthy of deep study. The present study aimed to investigate bone resorption in periodontitis through the intervention of EMMPRIN signaling. The distribution of EMMPRIN in human periodontitis was observed. RANKL-induced osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) were treated with EMMPRIN inhibitor in vitro. Rats with ligation-induced periodontitis were treated with EMMPRIN inhibitor and harvested for microcomputed tomography scanning, histologic observation, immunohistochemistry, and double immunofluorescence analysis. Positive expressions of EMMPRIN could be found in the CD68+-infiltrating cells. Downregulated EMMPRIN restrained osteoclast differentiation of BMMs in vitro, which also inhibited MMP-9 expression (*P < 0.05). In vivo, EMMPRIN inhibitor restrained ligation-induced bone resorption by decreasing tartrate-resistant acid phosphatase-positive osteoclasts. Both EMMPRIN-positive and MMP-9-positive osteoclasts were less common in the EMMPRIN inhibitor groups than in the control groups. Intervention of EMMPRIN signaling in osteoclasts could probably provide a potential therapeutic target for attenuating ligation-induced bone resorption.

Tofacitinib Regulates Endostatin via Effects on CD147 and Cathepsin S.

Angiogenesis is critical for rheumatoid arthritis (RA) progression. The effects of tofacitinib, a JAK-STAT inhibitor used for RA treatment, on angiogenesis in RA are unclear. We, therefore, evaluated the levels of angiogenic factors in two systems of a human co-culture of fibroblast (HT1080) and monocytic (U937) cell lines treated with tofacitinib and in serum samples from RA patients before and after six months of tofacitinib treatment. Tofacitinib reduced CD147 levels, matrix metalloproteinase-9 (MMP-9) activity, and angiogenic potential but increased endostatin levels and secreted proteasome 20S activity. In vitro, tofacitinib did not change CD147 mRNA but increased miR-146a-5p expression and reduced STAT3 phosphorylation. We recently showed that CD147 regulates the ability of MMP-9 and secreted proteasome 20S to cleave collagen XVIIIA into endostatin. We show here that tofacitinib-enhanced endostatin levels are mediated by CD147, as CD147-siRNA or an anti-CD147 antibody blocked proteasome 20S activity. The correlation between CD147 and different disease severity scores supported this role. Lastly, tofacitinib reduced endostatin' s degradation by inhibiting cathepsin S activity and recombinant cathepsin S reversed this in both systems. Thus, tofacitinib inhibits angiogenesis by reducing pro-angiogenic factors and enhancing the anti-angiogenic factor endostatin in a dual effect mediated partly through CD147 and partly through cathepsin S.

Pseudolaric Acid B Targets CD147 to Selectively Kill Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is an aggressive blood cancer. With low survival rates, new drug targets are needed to improve treatment regimens and patient outcomes. Pseudolaric acid B (PAB) is a plant-derived bioactive compound predicted to interact with cluster of differentiation 147 (CD147/BSG). CD147 is a transmembrane glycoprotein overexpressed in various malignancies with suggested roles in regulating cancer cell survival, proliferation, invasion, and apoptosis. However, the detailed function of PAB in AML remains unknown. In this study, AML cell lines and patient-derived cells were used to show that PAB selectively targeted AML (IC50: 1.59 ± 0.47 µM). Moreover, proliferation assays, flow cytometry, and immunoblotting confirmed that PAB targeting of CD147 resulted in AML cell apoptosis. Indeed, the genetic silencing of CD147 significantly suppressed AML cell growth and attenuated PAB activity. Overall, PAB imparts anti-AML activity through transmembrane glycoprotein CD147.

Replicative Endothelial Cell Senescence May Lead to Endothelial Dysfunction by Increasing the BH2/BH4 Ratio Induced by Oxidative Stress, Reducing BH4 Availability, and Decreasing the Expression of eNOS.

Vascular aging is associated with the development of cardiovascular complications, in which endothelial cell senescence (ES) may play a critical role. Nitric oxide (NO) prevents human ES through inhibition of oxidative stress, and inflammatory signaling by mechanisms yet to be elucidated. Endothelial cells undergo an irreversible growth arrest and alter their functional state after a finite number of divisions, a phenomenon called replicative senescence. We assessed the contribution of NO during replicative senescence of human aortic (HAEC) and coronary (CAEC) endothelial cells, in which accumulation of the senescence marker SA-ß-Gal was quantified by ß-galactosidase staining on cultured cells. We found a negative correlation in passaged cell cultures from P0 to P12, between a reduction in NO production with increased ES and the formation of reactive oxygen (ROS) and nitrogen (ONOO-) species, indicative of oxidative and nitrosative stress. The effect of ES was evidenced by reduced expression of endothelial Nitric Oxide Synthase (eNOS), Interleukin Linked Kinase (ILK), and Heat shock protein 90 (Hsp90), alongside a significant increase in the BH2/BH4 ratio, inducing the uncoupling of eNOS, favoring the production of superoxide and peroxynitrite species, and fostering an inflammatory environment, as confirmed by the levels of Cyclophilin A (CypA) and its receptor Extracellular Matrix Metalloprotease Inducer (EMMPRIN). NO prevents ES by preventing the uncoupling of eNOS, in which oxidation of BH4, which plays a key role in eNOS producing NO, may play a critical role in launching the release of free radical species, triggering an aging-related inflammatory response.

Elevated EMMPRIN Serum Levels Indicate Plaque Vulnerability in Patients With Asymptomatic High Grade Carotid Stenosis.

OBJECTIVE: Carotid atherosclerosis is an important cause of cerebral ischaemic stroke. Sonographic plaque characteristics are inappropriate for exact prediction of possible future ischaemic events. Additional markers are needed to predict the clinical outcome in high grade carotid stenosis. This study aimed to test extracellular matrix metalloproteinase inducer (EMMPRIN), due to its involvement in plaque formation and destabilisation, as a potential marker of high risk vulnerable plaques. METHODS: EMMPRIN was analysed in pre-operative serum samples from patients with symptomatic and asymptomatic carotid artery stenosis by a specific ELISA. Pre-operative duplex sonography classified the atherosclerotic plaque due to echogenicity. Histopathological analysis of vulnerable and non-vulnerable plaques was based on the American Heart Association (AHA) classification. RESULTS: The study included 265 patients undergoing carotid endarterectomy: 90 (m:f, 69:21) patients with symptomatic and 175 (m:f, 118:57) with asymptomatic disease. Analysis of circulating EMMPRIN revealed significantly higher levels in patients with echolucent plaques (4 480; IQR 3 745, 6 144 pg/mL) compared with echogenic plaques (4 159; IQR 3 418, 5 402 pg/mL; p = .025). Asymptomatic patients with vulnerable plaques had significantly higher levels of EMMPRIN (4 875; IQR 3 850, 7 016 pg/mL) compared with non-vulnerable plaques (4 109; IQR 3 433, 5 402 pg/mL; p < .001). In logistic regression analysis, duplex sonography combined with age, gender, and clinical risk factors predicted vulnerable plaques in asymptomatic patients with an AUC of 0.71 (95% CI 0.61 - 0.80). EMMPRIN significantly improved the AUC in asymptomatic patients (AUC 0.79; 95% CI 0.71 - 0.87; p = .014). CONCLUSION: Patients with high risk plaques according to ultrasound and histopathological characteristics demonstrated increased serum EMMPRIN levels. EMMPRIN on top of clinical risk factors, including age, gender, and duplex sonography may be used for pre-operative risk stratification in asymptomatic patients.

Identification of immunosuppressive factors in retinoblastoma cell secretomes and aqueous humor from patients.

The microenvironment of retinoblastoma, the solid malignancy of the developing retina, is immunosuppressive. To study the interactions between tumor-associated microglia/macrophages (TAMs) and tumor cells in retinoblastomas, we analyzed immunohistochemistry markers in 23 patient samples and characterized 105 secreted cytokines of 11 retinoblastoma cell models in culture. We detected profuse infiltration of CD163+ protumoral M2-like polarized TAMs in eyes enucleated due to cancer progression. Previous treatment of patients increased the number of TAMs but did not affect M2-like polarization. M2-like microglia/macrophages were almost absent in five eyes obtained from children enucleated due to nontumoral causes. CD8+ tumor-infiltrating lymphocytes (TILs) were moderately abundant in tumor eyes and very scarce in nontumoral ones. The expression of the immune checkpoint molecule PD-L1 was absent in 95% of the tumor samples, which is concordant with the finding of FOXP3+ Tregs infiltrating tumors. We confirmed the pathology results using single-cell transcriptome analysis of one tumor. We identified the cytokines extracellular matrix metalloproteinase inducer (EMMPRIN) and macrophage migration inhibitory factor (MIF), both with reported immunosuppressive activity, secreted at high levels in retinoblastoma primary cell cultures. Gene expression analysis of a large retinoblastoma cohort and single-cell transcriptome analysis confirmed that MIF and EMMPRIN were significantly upregulated in retinoblastomas, which led us to quantify both proteins by immunoassays in liquid biopsies (aqueous humor obtained from more than 20 retinoblastoma patients). We found a significant increase in the concentration of MIF and EMMPRIN in cancer patients, compared to 12 noncancer ones. Finally, we showed that macrophages derived from peripheral blood mononuclear cells increased the expression of markers of M2-like polarization upon exposure to retinoblastoma-conditioned medium or recombinant MIF. Overall, our findings suggest that retinoblastoma cell secretions induce the protumoral phenotype of this tumor. Our results might have clinical impact in the fields of biomarkers and treatment. © 2022 The Pathological Society of Great Britain and Ireland.

S-nitrosylation of EMMPRIN influences the migration of HSCs and MMP activity in liver fibrosis.

The mechanism of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of liver fibrosis has not been clarified. This study aims to investigate the role of EMMPRIN S-nitrosylation (SNO) in the regulation of hepatic stellate cell (HSC) migration and matrix metalloproteinase (MMP) activities in liver fibrosis. The results from the tissue microarrays and rat/mouse liver tissues suggest that EMMPRIN mRNA and protein levels in the fibrotic livers are lower than those in the corresponding normal control livers, but higher SNO level of EMMPRIN in fibrotic liver area was shown by immunohistochemistry, immunofluorescence staining, and biotin-switch assay conversely in vivo. Primary EMMPRIN comes from hepatocytes and liver sinus epithelial cells (LSECs) rather than quiescent HSCs. To mimic the uptake of extrinsic EMMPRIN, supernatants from mouse primary hepatocytes/293 cells transfected with EMMPRIN wild-type plasmids (WT) and EMMPRIN SNO site (cysteine 87) mutation plasmids (MUT) were collected and added to JS-1/LX2 cell medium. The MUT EMMPRIN diminishes SNO successfully, enhances the activities of MMP2 and MMP9, and subsequently increases HSC migration. In conclusion, SNO of EMMPRIN influences HSC migration and MMP activities in liver fibrosis. This finding may shed light on the possible regulatory mechanism of MMPs in ECM accumulation in liver fibrosis.

CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-ß1.

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-ß1 and proinflammatory interleukin (IL)-1ß are widely associated with fibrotic progression. TGF-ß1 induces myofibroblast differentiation, while IL-1ß induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-ß1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-ß1- and IL-1ß-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-ß1-induced fibroblast/myofibroblast differentiation and IL-1ß-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell-cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.

EMMPRIN Promotes the Expression of MMP-9 and Exacerbates Neurological Dysfunction in a Mouse Model of Intracerebral Hemorrhage.

Extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to be a vital inflammatory mediator in several neurological and neurodegenerative diseases. However, the role of EMMPRIN in intracerebral hemorrhage (ICH) remains unexplored. In this study, we aimed to exploit a highly selective monoclonal anti-EMMPRIN antibody to functionally inhibit EMMPRIN activity and thus that of MMPs as the downstream effector. To induce ICH pathology, adult C57BL/6 male mice were injected with collagenase type VII or saline as control into the right basal ganglia and were euthanized at different time points. The anti-EMMPRIN monoclonal antibody was intravenously injected once daily for 3 days to block the expression of EMMPRIN initiating at 4 h post-ICH. Western blot and immunofluorescence analysis results revealed that EMMPRIN expression was significantly increased surrounding the hematoma at 3 and 7 d time points after ICH when compared to the saline treated control group. EMMPRIN expression was co-localized with GFAP (astrocytes) and Iba1 (microglia) at 3 d time point post-ICH, but not in the control group mice. The co-localization of EMMPRIN with CD31 in endothelial cells occurred in both groups and was higher in the ICH brain. However, EMMPRIN expression was not detected in neurons from either group. The inhibition of EMMPRIN reduced the expression of MMP-9, the number of infiltrated neutrophils, the degree of brain injury and promoted neurological recovery after ICH. In conclusion, EMMPRIN could mediate the upregulation of MMP-9 and exacerbate neurological dysfunction in a mouse model of experimental ICH. Furthermore, blocking EMMPRIN reduced brain injury and subsequently promoted neurological recovery in ICH mice brains. These outcomes highlight that inhibition of EMMPRIN can be a potential therapeutic intervention strategy to regulate MMP-9's pathological roles during ICH.

Matrix Metalloproteinases and Their Inhibitors in Pulmonary Fibrosis: EMMPRIN/CD147 Comes into Play.

Pulmonary fibrosis (PF) is characterized by aberrant extracellular matrix (ECM) deposition, activation of fibroblasts to myofibroblasts and parenchymal disorganization, which have an impact on the biomechanical traits of the lung. In this context, the balance between matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs) is lost. Interestingly, several MMPs are overexpressed during PF and exhibit a clear profibrotic role (MMP-2, -3, -8, -11, -12 and -28), but a few are antifibrotic (MMP-19), have both profibrotic and antifibrotic capacity (MMP7), or execute an unclear (MMP-1, -9, -10, -13, -14) or unknown function. TIMPs are also overexpressed in PF; hence, the modulation and function of MMPs and TIMP are more complex than expected. EMMPRIN/CD147 (also known as basigin) is a transmembrane glycoprotein from the immunoglobulin superfamily (IgSF) that was first described to induce MMP activity in fibroblasts. It also interacts with other molecules to execute non-related MMP aactions well-described in cancer progression, migration, and invasion. Emerging evidence strongly suggests that CD147 plays a key role in PF not only by MMP induction but also by stimulating fibroblast myofibroblast transition. In this review, we study the structure and function of MMPs, TIMPs and CD147 in PF and their complex crosstalk between them.

Cyclophilin A Is Not Acetylated at Lysine-82 and Lysine-125 in Resting and Stimulated Platelets.

Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed-neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets.

Basigin Associates with Integrin in Order to Regulate Perineurial Glia and Drosophila Nervous System Morphology.

The Drosophila nervous system is ensheathed by a layer of outer glial cells, the perineurial glia, and a specialized extracellular matrix, the neural lamella. The function of perineurial glial cells and how they interact with the extracellular matrix are just beginning to be elucidated. Integrin-based focal adhesion complexes link the glial membrane to the extracellular matrix, but little is known about integrin's regulators in the glia. The transmembrane Ig domain protein Basigin/CD147/EMMPRIN is highly expressed in the perineurial glia surrounding the Drosophila larval nervous system. Here we show that Basigin associates with integrin at the focal adhesions to uphold the structure of the glia-extracellular matrix sheath. Knockdown of Basigin in perineurial glia using RNAi results in significant shortening of the ventral nerve cord, compression of the glia and extracellular matrix in the peripheral nerves, and reduction in larval locomotion. We determined that Basigin is expressed in close proximity to integrin at the glial membrane, and that expression of the extracellular integrin-binding domain of Basigin is sufficient to rescue peripheral glial compression. We also found that a reduction in expression of integrin at the membrane rescues the ventral nerve cord shortening, peripheral glial compression, and locomotor phenotypes, and that reduction in the integrin-binding protein Talin can partially rescue glial compression. These results identify Basigin as a potential negative regulator of integrin in the glia, supporting proper glial and extracellular matrix ensheathment of the nervous system.SIGNIFICANCE STATEMENT The glial cells and extracellular matrix play important roles in supporting and protecting the nervous system, but the interactions between these components have not been well characterized. Our study identified expression of a conserved Ig superfamily protein, Basigin, at the glial membrane of Drosophila where it associates with the integrin-based focal adhesion complexes to ensure proper ensheathment of the CNS and PNS. Loss of Basigin in the glia results in an overall compression of the nervous system due to integrin dysregulation, which causes locomotor defects in the animals. This underlies the importance of glia-matrix communication for structural and functional support of the nervous system.

Tet2 deficiency in immune cells exacerbates tumor progression by increasing angiogenesis in a lung cancer model.

Immune cells harboring somatic mutations reportedly infiltrate cancer tissues in patients with solid cancers and accompanying clonal hematopoiesis. Loss-of-function TET2 mutations are frequently observed in clonal hematopoiesis in solid cancers. Here, using a mouse lung cancer model, we evaluated the activity of Tet2-deficient immune cells in tumor tissues. Myeloid-specific Tet2 deficiency enhanced tumor growth in mice relative to that seen in controls. Single-cell sequencing analysis of immune cells infiltrating tumors showed relatively high expression of S100a8/S100a9 in Tet2-deficient myeloid subclusters. In turn, treatment with S100a8/S100a9 promoted Vegfa production by cancer cells, leading to a marked increase in the tumor vasculature in Tet2-deficient mice relative to controls. Finally, treatment of Tet2-deficient mice with an antibody against Emmprin, a known S100a8/S100a9 receptor, suppressed tumor growth. These data suggest that immune cells derived from TET2-mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis and may provide a novel therapeutic target for lung cancer patients with TET2-mutated clonal hematopoiesis.

Increased expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and MMP10, MMP23 in inflammatory bowel disease: Cross-sectional study.

It has been reported that EMMPRIN is involved in the regulation of immune response and the induction of MMPs production by fibroblasts. The aim of this study was to describe the intestinal gene expression and protein production of EMMPRIN, MMP23 and MMP10 in patients with ulcerative colitis (UC) and Crohn's disease (CD) and compared them with a control group. Gene expression of EMMPRIN, MMP10 and MMP23B was measured by RT-PCR. In order to determine EMMPRIN and MMP protein expression, colonic tissues were immunostained. The results of the study showed EMMPRIN gene expression was upregulated in rectal mucosa from active (a)UC versus aCD patients (P = .045), remission (r)CD group (P = .0009) and controls (P < .0001). We detected differences between rUC and aCD (P = .004), rCD (P < .0001) or control group (P < .0001). EMMPRIN showed a higher expression in mucosa (intraepithelial lymphocytes), submucosa and adventitia (endothelial cells) from aCD patients. MMP23 levels were increased in aUC and aCD compared to rUC and rCD and the control group (P = .0001). EMMPRIN+/MMP23+─expressing cells were localized mainly in mucosa, muscular and adventitia from active UC patients. MMP10 gene expression was increased in aUC versus CD patients and the control group (P = .0001). MMP10 gene expression is associated with inflammation in UC patients (P = .0001, r2  = .585). EMMPRIN+/MMP10+─producing cells were found mainly in all intestinal layers and perivascular inflammatory infiltrates from aUC patients. In conclusion, EMMPRIN, MMP23 and MMP10 were upregulated in patients with active UC versus remission UC , CD and control groups suggesting that, they are involved in the inflammatory process.

EMMPRIN expression is associated with metastatic progression in osteosarcoma.

BACKGROUND: Extracellular matrix metalloproteinase inducer (EMMPRIN), a cell-surface glycoprotein, is overexpressed in several cancer types. EMMPRIN induces a metastatic phenotype by triggering the production of matrix metalloproteinase proteins (MMPs) such as MMP1 and MMP2, and vascular endothelial growth factor (VEGF) in cancer cells and the surrounding stromal cells. The purpose of this study was to investigate the expression and role of EMMPRIN in osteosarcoma. METHODS: The level of EMMPRIN expression was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) in 6 tumor-derived osteosarcoma cell lines and compared with that in normal osteoblasts. To study the prognostic significance of EMMPRIN expression, immunohistochemistry was carried out in prechemotherapy biopsies of 54 patients. siRNA knockdown of EMMPRIN in SaOS-2 cells was conducted to explore the role of EMMPRIN. To study the role of EMMPRIN in tumor-stromal interaction in MMP production and invasion, co-culture of SaOS-2 cells with osteoblasts and fibroblasts was performed. Osteosarcoma 143B cells were injected into the tail vein of BALB/c mice and lung metastasis was analyzed. RESULTS: EMMRIN mRNA expression was significantly higher in 5 of 6 (83%) tumor-derived cells than in MG63 cells. 90% of specimens (50/54) stained positive for EMMPRIN by immunohistochemistry, and higher expression of EMMPRIN was associated with shorter metastasis-free survival (p = 0.023). Co-culture of SaOS-2 with osteoblasts resulted in increased production of pro-MMP2 and VEGF expression, which was inhibited by EMMPRIN-targeting siRNA. siRNA knockdown of EMMPRIN resulted in decreased invasion. EMMPRIN shRNA-transfected 143B cells showed decreased lung metastasis in vivo. CONCLUSIONS: Our data suggest that EMMPRIN acts as a mediator of osteosarcoma metastasis by regulating MMP and VEGF production in cancer cells as well as stromal cells. EMMPRIN could serve as a therapeutic target in osteosarcoma.

Interstitial collagenase MMP-1 and EMMPRIN in cell lines and in clinical specimens of cervical squamous cell carcinoma.

BACKGROUND: The aim of this study was to elucidate the features of the expression of matrix metalloproteinases inducer-EMMPRIN (EMN) and matrix metalloproteinase 1 (MMP-1) in cell lines and in clinical samples of cervical squamous cell carcinoma (SCC). MATERIAL AND METHODS: The study was carried out using RT-PCR, densitometry and immunohistochemical studies (IHC) on commercial cell lines Siha, Caski, transformed with HPV16; HeLa, and C33A transformed with HPV18, line C33A without HPV, and in clinical samples of SCC and morphologically normal tissue adjacent to the tumor. RESULTS: The data obtained indicate that the expression of mRNA EMN and MMP-1 occurs in all cell lines at different levels. HPV type and number of genes copies had no effect on expression degree both EMN and MMP-1. Gene expression of EMN and MMP-1 has been investigated in tumor and normal tissues. MMP-1 expression in tumor tissue in SCC, as a rule, has been significantly increased (2-6 times) compared to normal tissue. It was found in 90% of tumor samples. It is known, that MMP-1 promotes the development of invasive and metastatic processes. EMN expression was lower in the tumor tissue than in normal tissue in most cases. An increase in EMN expression was noted only in some cases of SCC. CONCLUSION: The data obtained indicate that MMP-1 can serve as a marker of the invasive potential of SCC. EMN, apparently, is not a major factor responsible for MMP-1 expression in SCC. Data are important for understanding the process of tumor development and may have prognostic value for the patient.

Ivabradine Induces Cardiac Protection against Myocardial Infarction by Preventing Cyclophilin-A Secretion in Pigs under Coronary Ischemia/Reperfusion.

In response to cardiac ischemia/reperfusion, proteolysis mediated by extracellular matrix metalloproteinase inducer (EMMPRIN) and its secreted ligand cyclophilin-A (CyPA) significantly contributes to cardiac injury and necrosis. Here, we aimed to investigate if, in addition to the effect on the funny current (I(f)), Ivabradine may also play a role against cardiac necrosis by reducing EMMPRIN/CyPA-mediated cardiac inflammation. In a porcine model of cardiac ischemia/reperfusion (IR), we found that administration of 0.3 mg/kg Ivabradine significantly improved cardiac function and reduced cardiac necrosis by day 7 after IR, detecting a significant increase in cardiac CyPA in the necrotic compared to the risk areas, which was inversely correlated with the levels of circulating CyPA detected in plasma samples from the same subjects. In testing whether Ivabradine may regulate the levels of CyPA, no changes in tissue CyPA were found in healthy pigs treated with 0.3 mg/kg Ivabradine, but interestingly, when analyzing the complex EMMPRIN/CyPA, rather high glycosylated EMMPRIN, which is required for EMMPRIN-mediated matrix metalloproteinase (MMP) activation and increased CyPA bonding to low-glycosylated forms of EMMPRIN were detected by day 7 after IR in pigs treated with Ivabradine. To study the mechanism by which Ivabradine may prevent secretion of CyPA, we first found that Ivabradine was time-dependent in inhibiting co-localization of CyPA with the granule exocytosis marker vesicle-associated membrane protein 1 (VAMP1). However, Ivabradine had no effect on mRNA expression nor in the proteasome and lysosome degradation of CyPA. In conclusion, our results point toward CyPA, its ligand EMMPRIN, and the complex CyPA/EMMPRIN as important targets of Ivabradine in cardiac protection against IR.

Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway.

Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte-monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.

Highly expressed tumoral emmprin and stromal CD73 predict a poor prognosis for external auditory canal carcinoma.

Squamous cell carcinoma of the external auditory canal (SCC-EAC) is rare and has a poor prognosis. The SCC-EAC cases with high-grade tumor budding (TB) or poorly differentiated clusters (PDCs) are associated with shorter survival than those with low-grade TB or PDCs. Extracellular matrix metalloproteinase inducer (emmprin) is a protein expressed in tumor cells that stimulates the production of MMP-2 by stromal fibroblasts to facilitate tumor invasion. Recently, we reported that emmprin forms a complex with CD73 to regulate MMP-2 production from fibroblasts in vitro. Here, we examined the association of emmprin and CD73 expression with TB or PDCs as well as with survival in 34 biopsy specimens of SCC-EAC patients. High tumoral emmprin expression was associated with high-grade TB, whereas high stromal CD73 expression was associated with high-grade PDCs. Furthermore, concurrent elevated expression of tumoral emmprin and stromal CD73 was determined to be an independent poor prognostic factor. In immunoprecipitation analyses, complex formation between emmprin and CD73 was demonstrated in vitro. Production of MMP-2 from fibroblasts was more abundant when cocultured with tumor cells than from fibroblasts cultured alone. Furthermore, MMP-2 production was reduced by the transfection of CD73 siRNA in fibroblasts cocultured with tumor cells. The colocalization of emmprin and CD73 was enhanced in not only the peripheral cells of the tumor cell clusters that interact with fibroblasts but also in the cells of intratumor clusters. Overall, this study provides novel insights into the roles of emmprin, CD73, and MMP-2 in tumor invasiveness.