Tissue Inhibitor Of Metalloproteinase-1 Secretion Research Articles

Remifentanil modulates the TLR4‑mediated MMP‑9/TIMP1 balance and NF‑κB/STAT3 signaling in LPS‑induced A549 cells.

Remifentanil is a widely used in general anesthetic that has been found to suppress the inflammatory response in aortic endothelial cells. Therefore, it was hypothesized that remifentanil can inhibit inflammatory dysfunction in lung epithelial cells to alleviate acute lung injury (ALI). The present study aimed to examine the effects of remifentanil on inflammatory injury, MMP-9/tissue inhibitor of metalloproteinase 1 (TIMP1) balance and the potential associated regulatory pathways in A549 cells. Lipopolysaccharide (LPS) was used to treat A549 cells to establish ALI models. The possible roles of different concentrations of remifentanil in cell viability was then determined by CCK-8 and Lactate dehydrogenase release assay. Apoptosis was assessed by flow cytometry analysis and western blotting. Inflammation and oxidative stress were measured by ELISA and corresponding kits respectively. Subsequently, the effects of remifentanil on Toll-like receptor 4 (TLR4) expression and the MMP-9/TIMP1 balance were assessed by western blotting and ELISA. In addition, the effects of remifentanil on NF-κB/STAT3 signaling were evaluated by measuring the protein expression levels of associated pathway components and the degree of NF-κB nuclear translocation using western blotting and immunofluorescence respectively. Remifentanil was found to increase cell viability whilst reducing apoptosis, inflammation and oxidative stress in the LPS-treated cells. In addition, TLR4 inhibitor CLI-095 suppressed MMP-9 expression and secretion while potentiating TIMP1 expression and secretion in LPS-challenged cells. Remifentanil treatment was able to modulate TLR4 to mediate LPS-induced MMP-9/TIMP1 imbalance and suppress the phosphorylation of NF-κB/STAT3 signaling components, in addition to inhibiting NF-κB nuclear translocation. Taken together, remifentanil downregulated TLR4 to reduce MMP-9/TIMP1 imbalance to inhibit inflammatory dysfunction in LPS-treated A549 cells, by regulating NF-κB/STAT3 signaling.

Secretory autophagy promotes Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1

BackgroundRab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1 (TIMP1), an inflammatory cytokine, under serum-depleted conditions which leads to suppression of lung cancer cell metastasis has been reported. Starvation is also a stimulus of autophagic activity. Herein, we reveal that starvation activates Rab37 and induces autophagy.MethodsWe used an overexpression/knockdown system to determine the relationship between autophagy and Rab37 in vitro and in vivo. The autophagy activity was detected by immunoblotting, transmission electron microscope, autophagosome purification, and immunofluorescence under the confocal microscope. Lung-to-lung metastasis mouse model was used to clarify the role of autophagy and Rab37 in lung cancer. Clinical lung cancer patient specimens and an online big database were analyzed.ResultsInitially, we demonstrated that active-form Rab37 increased LC3-II protein level (the marker of autophagosome) and TIMP1 secretion. Accordingly, silencing of Rab37 gene expression alleviated Rab37 and LC3-II levels as well as TIMP1 secretion, and induction of autophagy could not increase TIMP1 exocytosis under such conditions. Moreover, silencing the Atg5 or Atg7 gene of lung cancer cells harboring active-mutant Rab37 (Q89L) led to decreased autophagy activity and TIMP1 secretion. In the lung-to-lung metastasis mouse model, increased TIMP1 expression accompanied by amiodarone-induced autophagy led to decreased tumor nodules and cancer cell metastasis. These phenomena were reversed by silencing the Atg5 or Atg7 gene. Notably, increasing autophagy activity alone showed no effect on TIMP1 secretion under either Rab37 or Sec22b silencing conditions. We further detected colocalization of LC3 with either Rab37 or TIMP1, identified Rab37 and Sec22b proteins in the purified autophagosomes of the lung cancer cells harboring the active-form Rab37 gene, and confirmed that these proteins are involved in the secretion of TIMP1. We reveal that autophagic activity was significantly lower in the tumors compared to the non-tumor parts and was associated with the overall lung cancer patient survival rate.ConclusionsWe are the first to report that autophagy plays a promoting role in TIMP1 secretion and metastasis in a Rab37-dependent manner in lung cancer cells and the lung-to-lung mouse model.

Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) are localized in the nucleus of retinal Müller glial cells and modulated by cytokines and oxidative stress.

Matrix metalloproteinases (MMPs) are involved in the pathology of numerous inflammatory retinal degenerations, including retinitis pigmentosa (RP). Our previous work revealed that intravitreal injections with tissue inhibitor of metalloproteinases 1 (TIMP-1) reduce the progression of rod cell death and inhibit cone cell remodeling that involves reactive gliosis in retinal Müller glial cells (MGCs) in rodent models. The underlying cellular and molecular mechanisms of how TIMP-1 functions in the retina remain to be resolved; however, MGCs are involved in structural homeostasis, neuronal cell survival and death. In the present study, MMP-9 and TIMP-1 expression patterns were investigated in a human MGC line (MIO-M1) under inflammatory cytokine (IL-1β and TNF-α) and oxidative stress (H2O2) conditions. First, both IL-1β and TNF-α, but not H2O2, have a mild in vitro pro-survival effect on MIO-M1 cells. Treatment with either cytokine results in the imbalanced secretion of MMP-9 and TIMP-1. H2O2 treatment has little effect on their secretion. The investigation of their intracellular expression led to interesting observations. MMP-9 and TIMP-1 are both expressed, not only in the cytoplasm, but also inside the nucleus. None of the treatments alters the MMP-9 intracellular distribution pattern. In contrast to MMP-9, TIMP-1 is detected as speckles. Intracellular TIMP-1 aggregation forms in the cytoplasmic area with IL-1β treatment. With H2O2 treatments, the cell morphology changes from cobbles to spindle shapes and the nuclei become larger with increases in TIMP-1 speckles in an H2O2 dose-dependent manner. Two TIMP-1 cell surface receptors, low density lipoprotein receptor-related protein-1 (LRP-1) and cluster of differentiation 82 (CD82), are expressed within the nucleus of MIO-M1 cells. Overall, these observations suggest that intracellular TIMP-1 is a target of proinflammatory and oxidative insults in the MGCs. Given the importance of the roles for MGCs in the retina, the functional implication of nuclear TIMP-1 and MMP-9 in MGCs is discussed.

Silymarin modulates catabolic cytokine expression through Sirt1 and SOX9 in human articular chondrocytes

BackgroundSilymarin (SMN), a polyphenolic flavonoid, is involved in multiple bioactive functions including anti-inflammation. Pretreatment with SMN demonstrated chondroprotection against tumour necrosis factor-alpha (TNF-α) stimulation in a chondrocyte cell line. However, pre- and posttreatment with phytochemicals have varying effects on osteoarthritis (OA) chondrocytes, and the therapeutic potential of SMN after catabolic cytokine stimulation is not fully elucidated.MethodsThe cytotoxicity of SMN (12.5, 25, 50 and 100 μM) was evaluated in human primary chondrocytes. The chondrocytes were supplemented with SMN (25 and 50 μM) after interleukin-1beta (IL-1β) stimulation. The mRNA expression and protein production of catabolic/anabolic cytokines as well as extracellular matrix (ECM) components were evaluated.ResultsHigh-dose SMN (100 μM) impaired the mitochondrial activity in chondrocytes, and 50 μM SMN further caused cell death in IL-1β-stimulated cells. The addition of 25 μM SMN ameliorated cell senescence; downregulated the catabolic genes of inducible nitric oxide synthase, IL-1β, TNF-α, matrix metalloproteinase-3 (MMP-3), MMP-9 and MMP-13; upregulated the anabolic genes of tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagen type II alpha 1; and restored the expression of chondrogenic phenotype genes SOX9 and sirtuin-1 (Sirt1). In addition, the production of IL-1β, MMP-3 and MMP-9 decreased with an increase in TIMP-1 secretion. However, the mRNA levels of IL-6, IL-8 and IL-10 and protein production remained high. The addition of nicotinamide, a Sirt1 inhibitor, downregulated SOX9 and attenuated the therapeutic effects of SMN on IL-1β-stimulated chondrocytes.ConclusionSMN regulates the chondrocyte phenotype through Sirt1 and SOX9 to improve ECM homeostasis and may serve as a complementary therapy for early-stage knee OA.

Abstract 5091: TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction

Abstract Tumor associated fibroblasts (TAFs) are important regulators of tumor growth and resistance to therapies. We have recently shown that lung TAFs in vitro respond to the antifibrotic drug nintedanib in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). We also showed that the tumor-promoting effects of TAFs are driven by different mechanisms in ADC and SCC, which remain to be elucidated. Tissue inhibitor of metalloproteinases 1 (TIMP-1) has been associated with poor prognosis in lung cancer, its expression is downregulated by nintedanib, and our preliminary results reveal that its putative receptor, CD63, is overexpressed in ADC patients compared to SCC, supporting a selective crosstalk between TAFs and cancer cells in ADC through TIMP-1 and CD63. The aim of this study was to test this hypothesis using in vitro preclinical models. Primary fibroblasts were obtained with the patient informed consent, and using protocols approved by the Ethics Committee of the Hospital Clinic. ADC-TAFs and SCC-TAFs were stimulated with TGF-β1 in the presence or absence of nintedanib, and the TIMP-1 content in their conditioned medium was determined by ELISA. TIMP-1 was knocked-down in ADC-TAFs by siRNA, and the corresponding conditioned medium was used to stimulate growth and invasion of the high-CD63 ADC cell line, H1437. Likewise, CD63 expression in H1437 cells was reduced by siRNA. Our in vitro results showed that TIMP-1 secretion induced by TGF-β1 is significantly larger in ADC-TAFs compared to SCC-TAFs. Likewise, nintedanib elicited a higher downregulation of secreted TIMP-1 in ADC-TAFs compared to SCC-TAFs. Of note, TIMP-1 from ADC-TAFs was necessary to induce growth and invasion of H1437 cells. Likewise, knocking-down CD63 in H1437 ADC cells was sufficient to reduce the growth and invasion elicited by the conditioned medium of TGF-β1 activated ADC-TAFs. Collectively, our results unveil a novel stroma-carcinoma crosstalk driven by TIMP-1 and CD63 selectively in lung ADC, and support that such heterotypic crosstalk may underlie the aberrant tumor-promoting effects of ADC-TAFs that are selectively downregulated by nintedanib. Citation Format: Paula Duch, Marta Gabasa, Rafael Ikemori, Marselina Arshakyan, Frank Hillberg, Noemí Reguart, Derek Radisky, Jordi Alcaraz. TIMP-1 in tumor-associated fibroblasts drives tumor progression in lung adenocarcinoma through CD63 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5091.

VAMP8, a vesicle-SNARE required for RAB37-mediated exocytosis, possesses a tumor metastasis suppressor function

We previously identified a metastasis suppressor RAB37 small GTPase that regulated exocytosis of tissue inhibitor of metalloproteinases 1 (TIMP1) to suppress lung cancer metastasis. Here, we show that vesicle-associated membrane protein 8 (VAMP8), a v-SNARE (vesicle soluble N-ethylmaleimide-sensitive factor activating protein receptor), interacts with RAB37 and drives the secretion of TIMP1 to inhibit tumor metastases. Confocal and total internal reflection fluorescence microscopic images demonstrated that VAMP8 co-localized with RAB37 and facilitated trafficking of RAB37-TIMP1 vesicles. Reconstitution experiments using tail-vein injection and lung-to-lung metastasis in mice showed that VAMP8 was essential for RAB37-regulated vesicle trafficking of TIMP1 to suppress cancer metastasis. Lung cancer patients with low VAMP8 showed distant metastasis, poor overall survival and progression-free survival. Importantly, multivariate Cox regression analysis indicated that patients with low VAMP8/low RAB37 expression profile showed significantly high risk of death (hazard ratio = 3.42, P < 0.001) even after adjusting for tumor metastasis parameter. Our findings reveal that VAMP8 is a novel v-SNARE crucial for RAB37-mediated exocytic transport of TIMP1 to suppress lung tumor metastasis. VAMP8 possesses a tumor metastasis suppressor function with a prognostic value in lung cancer.

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts.

AIMTo investigate the inhibitory effect of astragaloside IV on the pathological functions of cancer-associated fibroblasts, and to explore the underlying mechanism.METHODSPaired gastric normal fibroblast (GNF) and gastric cancer-associated fibroblast (GCAF) cultures were established from resected tissues. GCAFs were treated with vehicle control or different concentrations of astragaloside IV. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs, and astragaloside IV-treated GCAFs, and used to culture BGC-823 human gastric cancer cells. Proliferation, migration and invasion capacities of BGC-823 cells were determined by MTT, wound healing, and Transwell invasion assays, respectively. The action mechanism of astragaloside IV was investigated by detecting the expression of microRNAs and the expression and secretion of the oncogenic factor, macrophage colony-stimulating factor (M-CSF), and the tumor suppressive factor, tissue inhibitor of metalloproteinase 2 (TIMP2), in different groups of GCAFs. The expression of the oncogenic pluripotency factors SOX2 and NANOG in BGC-823 cells cultured with different conditioned media was also examined.RESULTSGCAFs displayed higher capacities to induce BGC-823 cell proliferation, migration, and invasion than GNFs (P < 0.01). Astragaloside IV treatment strongly inhibited the proliferation-, migration- and invasion-promoting capacities of GCAFs (P < 0.05 for 10 μmol/L, P < 0.01 for 20 μmol/L and 40 μmol/L). Compared with GNFs, GCAFs expressed a lower level of microRNA-214 (P < 0.01) and a higher level of microRNA-301a (P < 0.01). Astragaloside IV treatment significantly up-regulated microRNA-214 expression (P < 0.01) and down-regulated microRNA-301a expression (P < 0.01) in GCAFs. Reestablishing the microRNA expression balance subsequently suppressed M-CSF production (P < 0.01) and secretion (P < 0.05), and elevated TIMP2 production (P < 0.01) and secretion (P < 0.05). Consequently, the ability of GCAFs to increase SOX2 and NANOG expression in BGC-823 cells was abolished by astragaloside IV.CONCLUSIONAstragaloside IV can inhibit the pathological functions of GCAFs by correcting their dysregulation of microRNA expression, and it is promisingly a potent therapeutic agent regulating tumor microenvironment.

Toxoplasma gondii infection shifts dendritic cells into an amoeboid rapid migration mode encompassing podosome dissolution, secretion of TIMP-1, and reduced proteolysis of extracellular matrix.

Dendritic cells (DCs) infected by Toxoplasma gondii rapidly acquire a hypermigratory phenotype that promotes systemic parasite dissemination by a "Trojan horse" mechanism in mice. Recent paradigms of leukocyte migration have identified the amoeboid migration mode of DCs as particularly suited for rapid locomotion in extracellular matrix and tissues. Here, we have developed a microscopy-based high-throughput approach to assess motility and matrix degradation by Toxoplasma-challenged murine and human DCs. DCs challenged with T.gondii exhibited dependency on metalloproteinase activity for hypermotility and transmigration but, strikingly, also dramatically reduced pericellular proteolysis. Toxoplasma-challenged DCs up-regulated expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) and their supernatants impaired matrix degradation by naïve DCs and by-stander DCs dose dependently. Gene silencing of TIMP-1 by short hairpin RNA restored matrix degradation activity in Toxoplasma-infected DCs. Additionally, dissolution of podosome structures in parasitised DCs coincided with abrogated matrix degradation. Toxoplasma lysates inhibited pericellular proteolysis in a MyD88-dependent fashion whereas abrogated proteolysis persevered in Toxoplasma-infected MyD88-deficient DCs. This indicated that both TLR/MyD88-dependent and TLR/MyD88-independent signalling pathways mediated podosome dissolution and the abrogated matrix degradation. We report that increased TIMP-1 secretion and cytoskeletal rearrangements encompassing podosome dissolution are features of Toxoplasma-induced hypermigration of DCs with an impact on matrix degradation. Jointly, the data highlight how an obligate intracellular parasite orchestrates key regulatory cellular processes consistent with non-proteolytic amoeboid migration of the vehicle cells that facilitate its dissemination.

Davallia bilabiata exhibits anti-angiogenic effect with modified MMP-2/TIMP-2 secretion and inhibited VEGF ligand/receptors expression in vascular endothelial cells

Ethnopharmacological relevanceDavallia bilabiata Hosokawa (D. bilabiata), also called GuSuiBu, is popularly used as a substitute for Drynaria fortunei J. Sm for rheumatoid and degenerative arthritis in traditional Chinese medicine. Little is known about the underlying mechanisms of anti-angiogenesis responsible for arthritis in D. bilabiata which needs to be elucidated. Aim of the studyThe present study is intended to investigate the anti-angiogenic effect of D. bilabiata associated with the modulation of matrix metalloproteinases (MMPs) and down regulation of vascular endothelial growth factor (VEGF) ligand/receptors both in vivo and in vitro. Materials and methodsWe investigated the potential anti-angiogenic effect of D. bilabiata by the in vivo neovascularization of chick chorioallantoic membranes (CAM) assay, and the in vitro migration and matrix-induced tube formation assay using human umbilical vascular endothelial cells (HUVECs). The expressions of MMP-2, TIMP-2, RECK and VEGF/VEGFR were analyzed by real-time RT-PCR or Western blot method. ResultsOne major compound from water extract of D. bilabiata was identified as Epicatechin 3-O-β-D-allopyranoside. D. bilabiata was confirmed to inhibit in vivo angiogenesis by CAM assay. D. bilabiata also exhibited in vitro anti-angiogenic and anti-regrowth effects as demonstrated by tube formation assay, transwell migration assay and wound healing assay. The mRNA expressions of MMP-2, and MMP-14 were decreased. On the contrary, tissue inhibitor of metalloproteinase-2 (TIMP-2), reversion-inducing cysteine-rich protein with kazal motifs (RECK) were increased by D. bilabiata. The extracellular MMP-2 activity was found to be reduced both in vitro and in vivo by D. bilabiata as determined by gelatin zymography. Results from western blot analysis and ELISA further demonstrated the decrease of MMP-2 and increase of TIMP-2 secretion after D. bilabiata treatment. The gene expressions of VEGF-A, -B, -C, -D and VEGFR-1, -2, -3 were all inhibited by D. bilabiata. ConclusionWe concluded that the anti-angiogenic effect of D. bilabiata was associated with the decreased MMP-2 activity mediated by the upregulation of TIMP-2 and RECK, and the suppression of VEGF/VEGFRs expression.

Aldosterone Induces Tissue Inhibitor of Metalloproteinases-1 Expression and Further Contributes to Collagen Accumulation: From Clinical to Bench Studies.

Aldosterone induces myocardial fibrosis. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a key factor of myocardial fibrosis. This study tested the hypothesis that aldosterone induces TIMP-1 expression and contributes to the fibrotic process. We prospectively enrolled 54 patients with primary aldosteronism, and measured plasma TIMP-1 and echocardiographic parameters. In the cell study, we investigated the possible molecular mechanism by which aldosterone induces TIMP-1 secretion and the effects on collagen accumulation. In the animal study, we measured serum TIMP-1 levels, cardiac TIMP-1 levels, and cardiac structure in an aldosterone infusion mouse model using implantation of aldosterone pellets. In patients with primary aldosteronism, plasma TIMP-1 was correlated with 24-hour urinary aldosterone, left ventricular mass, and impairment of left ventricular diastolic function. In human cardiac fibroblasts, TIMP-1 protein and mRNA expressions were significantly increased by aldosterone through the glucocorticoid receptor/PI3K/Akt/nuclear factor-κB pathway. TIMP-1 small-interfering RNA significantly reduced aldosterone-induced collagen accumulation, and aldosterone did not alter the levels of collagen1a1 or matrix metalloproteinase-1 mRNA. The aldosterone-induced TIMP-1 expression was inversely related to matrix metalloproteinase-1 activity. Furthermore, in the animal model, the serum and cardiac levels of TIMP-1 were significantly elevated in the mice that received aldosterone infusion. This elevation was blocked by RU-486 but not by eplerenone, suggesting that the effect was through glucocorticoid receptors. In a long-term aldosterone infusion model, serum TIMP-1 was associated with serum aldosterone level, cardiac structure, and fibrosis. In conclusion, aldosterone induced TIMP-1 expression in vivo and in vitro. This increased TIMP-1 expression resulted in enhanced collagen accumulation via the suppression of matrix metalloproteinase-1 activity.

Adiponectin Reduces Hepatic Stellate Cell Migration by Promoting Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Secretion

Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin.

CD97 inhibits cell migration in human fibrosarcoma cells by modulating TIMP-2/MT1- MMP/MMP-2 activity--role of GPS autoproteolysis and functional cooperation between the N- and C-terminal fragments.

CD97 is a tumor-associated adhesion-class G-protein-coupled receptor involved in modulating cell migration. Adhesion-class G-protein-coupled receptors are characterized by proteolytic cleavage at a G-protein-coupled receptor proteolysis site (GPS) into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), which remain associated noncovalently. The molecular mechanism and the role of GPS proteolysis in CD97-modulated cell migration are not completely understood. We report here that CD97 expression in HT1080 fibrosarcoma cells enhanced tissue inhibitor of metalloproteinase-2 secretion, leading to reduced membrane type 1 matrix metalloproteinase and matrix metalloproteinase 2 activities. This, in turn, impaired cell migration and invasion in vitro and lung macrometastasis in vivo. CD97 expression also upregulated the expression of integrins, promoting cell adhesion. Importantly, these cellular functions absolutely required the presence of both the NTF and the CTF of CD97, confirming functional cooperation between the two receptor subunits. CD97 gene knockdown reversed these phenotypic changes. We conclude that GPS proteolysis and the functional interplay between the NTF and the CTF are indispensible for CD97 to inhibit HT1080 cell migration by suppressing matrix metalloproteinase activity.

Human Adipose-Derived Stromal/Stem Cells Protect Against STZ-Induced Hyperglycemia: Analysis of hASC-Derived Paracrine Effectors

Adipose-derived stromal/stem cells (ASCs) ameliorate hyperglycemia in rodent models of islet transplantation and autoimmune diabetes, yet the precise human ASC (hASC)-derived factors responsible for these effects remain largely unexplored. Here, we show that systemic administration of hASCs improved glucose tolerance, preserved β cell mass, and increased β cell proliferation in streptozotocin-treated nonobese diabetic/severe combined immunodeficient mice. Coculture experiments combining mouse or human islets with hASCs demonstrated that islet viability and function were improved by hASCs following prolonged culture or treatment with proinflammatory cytokines. Analysis of hASC-derived factors revealed vascular endothelial growth factor and tissue inhibitor of metalloproteinase 1 (TIMP-1) to be highly abundant factors secreted by hASCs. Notably, TIMP-1 secretion increased in the presence of islet stress from cytokine treatment, while TIMP-1 blockade was able to abrogate in vitro prosurvival effects of hASCs. Following systemic administration by tail vein injection, hASCs were detected in the pancreas and human TIMP-1 was increased in the serum of injected mice, while recombinant TIMP-1 increased viability in INS-1 cells treated with interleukin-1beta, interferon-gamma, and tumor necrosis factor alpha. In aggregate, our data support a model whereby factors secreted by hASCs, such as TIMP-1, are able to mitigate against β cell death in rodent and in vitro models of type 1 diabetes through a combination of local paracrine as well as systemic effects.

SAT0035 Toll-like receptor-mediated expression of profibrotic mediators by circulating monocytes in systemic sclerosis

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterised by fibrosis, vascular dysfunction and abnormal activation of immune cells including monocytes. It was suggested that monocytes along with fibroblasts play an...

Toll-like receptor-mediated, enhanced production of profibrotic TIMP-1 in monocytes from patients with systemic sclerosis: role of serum factors

ObjectivesTo investigate whether monocytes contribute to matrix deposition in systemic sclerosis (SSc) by production of tissue-inhibitor of metalloproteinase-1 (TIMP-1).MethodsMatrix metalloproteinase-1 (MMP-1) and TIMP-1 expression and secretion were measured by qRT-PCR...

Prospective evaluation of serum tissue inhibitor of metalloproteinase 1 and carbonic anhydrase IX in correlation to circulating tumor cells in patients with metastatic breast cancer

IntroductionCirculating tumor cells (CTCs) reflect aggressive tumor behavior by hematogenous tumor cell dissemination. The tissue inhibitor of metalloproteinase 1 (TIMP-1) plays a role in tissue invasion and is also involved in angiogenesis, abrogation of apoptosis and in chemoresistance. Carbonic anhydrase IX (CAIX) is a metalloenzyme involved in cell adhesion, growth and survival of tumor cells. The aim of the study was to investigate whether serum concentrations of TIMP-1 and CAIX are associated with the detection of CTC in metastatic breast cancer.MethodsBlood was obtained in a prospective multicenter setting from 253 patients with metastatic breast cancer at the time of disease progression. Serum TIMP-1 and CAIX were determined using commercial ELISA-kits (Oncogene Science). CTC were detected with the CellSearch™ system (Veridex).ResultsFive or more CTCs were detected in 122 patients out of 245 evaluable patients (49.8%). Out of 253 metastatic patients 70 (28%) had serum TIMP-1 levels above 454 ng/mL. Serum CAIX was elevated above 506 ng/mL in 90 (35%) patients. Both serum markers had prognostic significance. Median progression free survival (PFS) was 7.2 months with elevated TIMP-1 vs. 11.4 months with non-elevated levels (p < 0.01). OS was 11.5 vs. 19.1 months (p < 0.01). Median PFS was 7.5 months with elevated CAIX vs. 11.7 months with non-elevated levels (p < 0.01), overall survival (OS) was 13.4 months vs. 19.1 months (p < 0.01). In patients with five or more CTCs, serum levels were above the cut-off for CAIX in 47% vs. 25% in those with less than five CTCs (p = 0.01). For TIMP-1, 37% patients with five or more CTCs had elevated serum levels and 17% of patients with less than five CTCs (p = 0.01). Including TIMP-1, CAIX, CTC and established prognostic factors in the multivariate analysis, the presence of CTCs, the therapy line and elevated CAIX remained independent predictors of OS.ConclusionsElevated serum levels of the invasion markers TIMP-1 and CAIX in metastatic breast cancer are prognostic markers and are associated with the presence of CTCs. Whether increased secretion of TIMP-1 and/or CAIX might directly contribute to tumor cell dissemination remains to be elucidated in further investigations.Trial registrationCurrent Controlled Trials: ISRCTN59722891

Anti-angiogenic effect of Tanshinone IIA involves inhibition of matrix invasion and modification of MMP-2/TIMP-2 secretion in vascular endothelial cells

Tanshinone IIA (Tan IIA) is one of the major lipophilic components of Salvia miltiorrhiza Bunge reported to exhibit anti-carcinogenic effect. In the present study, we further evaluated the anti-angiogenic effect of Tan IIA using the chorioallantoic membrane (CAM) in chicken embryos and human umbilical vascular endothelial cells (HUVECs). Tan IIA was confirmed to inhibit in vivo angiogenesis by CAM assay. Tan IIA also exhibited in vitro anti-angiogenic effects as demonstrated by tube formation assay, transwell migration assay and TNF-α-induced matrix invasion assay. The mRNA expressions of matrix metalloproteinase-2, -3, -9, -14 (MMP-2, -3, -9, -14), tissue inhibitor of metalloproteinase-2 (TIMP-2) and reversion-inducing cysteine-rich protein with kazal motifs (RECK) were not affected by Tan IIA as analyzed by reverse transcription polymerase chain reaction (RT–PCR). However, the extracellular matrix metalloproteinase-2 (MMP-2) activity was found to be reduced dose-dependently by Tan IIA as determined by gelatin zymography. Results from western blot analysis and ELISA further demonstrated the dose-dependent decrease of MMP-2 and increase of TIMP-2 secretion from cytosol of vascular endothelial cells simultaneously after Tan IIA treatment. Together, the present study confirmed the anti-angiogenic effects of Tan IIA both in vivo and in vitro. Our results also demonstrated that Tan IIA could modulate the secretion of MMP-2 and TIMP-2 in an opposite way and resulted in the decreased MMP-2 activity of vascular endothelial cells.

Role of tissue inhibitor of metalloproteinases-1 in the development of autoimmune lymphoproliferation

Inherited defects decreasing function of the Fas death receptor cause autoimmune lymphoproliferative syndrome and its variant Dianzani's autoimmune lymphoproliferative disease. Analysis of the lymphocyte transcriptome from a patient with this latter condition detected striking over-expression of osteopontin and tissue inhibitor of metalloproteinases-1. Since previous work on osteopontin had detected increased serum levels in these patients, associated with variations of its gene, the aim of this work was to extend the analysis to tissue inhibitor of metalloproteinases-1. Tissue inhibitor of metalloproteinases-1 levels were evaluated in sera and culture supernatants from patients and controls by enzyme-linked immunosorbent assay. Activation- and Fas-induced cell death were induced, in vitro, using anti-CD3 and anti-Fas antibodies, respectively. Tissue inhibitor of metalloproteinases-1 levels were higher in sera from 32 patients (11 with autoimmune lymphoproliferative syndrome and 21 with Dianzani's autoimmune lymphoproliferative disease) than in 50 healthy controls (P<0.0001), unassociated with variations of the tissue inhibitor of metalloproteinases-1 gene. Both groups of patients also had increased serum levels of osteopontin. In vitro experiments showed that osteopontin increased tissue inhibitor of metalloproteinases-1 secretion by peripheral blood monocytes. Moreover, tissue inhibitor of metalloproteinases-1 significantly inhibited both Fas- and activation-induced cell death of lymphocytes. These data suggest that high osteopontin levels may support high tissue inhibitor of metalloproteinases-1 levels in autoimmune lymphoproliferative syndrome and Dianzani's autoimmune lymphoproliferative disease, and hence worsen the apoptotic defect in these diseases.

666 THE EFFECTS OF SCY-635 A NON-IMMUNOSUPPRESSIVE CYCLOSPORIN ANALOG ON STELLATE CELL PROLIFERATION, COLLAGEN SYNTHESIS, TIMP-1 AND COLLAGENASE PRODUCTION

Aims: The non-immunosuppressive cyclophilin inhibitor SCY-635 is a potent inhibitor of HCV-specific RNA replication in genotype 1 derived HCV replicons. Clinical studies indicate that acute administration of SCY-635 significantly reduces plasma viremia in patients with chronic hepatitis C virus infection. Recent data have shown that cyclosporin and nonimmunosuppressive analogues such as NIM-811 can suppress the proliferation of, and collagen production in, hepatic stellate cells (HSC) (Kohjima et al. Liver Int., 2007). To examine the potential effects of SCY-635 on liver fibrosis and apoptosis, we have investigated the effects of SCY-635 on cell growth, collagen synthesis, TIMP-1 and collagenase levels and TGF-b signaling in HSCs. Methods: Human HSC-derived LI-90 cells either resting or stimulated with TGFb1 were used for these studies. The levels of collagen, matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and levels of phosphorylation of p38 and Smad2 were evaluated by ELISA. Cell proliferation (BrdU incorporation) and apoptosis (ApoGlow) were measured. Apoptosis was evaluated using primary rat hepatocytes after treatment with Ad5IkB adenovirus followed by overnight incubation with TNF-α. Results: SCY-635 and cyclosporin (CsA) decreased the synthesis of collagen. At a clinically relevant concentration of 0.25 μM in non-stimulated or in TGFβ1 stimulated LI-90 cells, SCY-635 significantly reduced the collagen production by more than 70%. There was a parallel decrease in TIMP-1 secretion into the HSC medium and an increase in the production of MMP-1. SCY-635 inhibited proliferation without induction of apoptosis. Concentrations of SCY-635 ranging from 0.1 to 5 μM enhanced the phosphorylation of p38 and suppressed the phosphorylation of Smad2. SCY-635 inhibition of liver apoptosis was equipotent with CsA exhibiting IC50 values of 2.8 ± 0.3 and 3.6 ± 0.3 μM, respectively. Conclusions: These findings demonstrated that SCY-635 suppressed cell growth, reduced type I collagen synthesis and TIMP-1 secretion, and enhanced MMP-1 production suggesting that SCY-635 may exert an anti-fibrogenic effect. These effects may be mediated through the TGF-β signaling pathway. Taken together, these data suggest that SCY-635 may not only reduce the replication of HCV but also reduce the fibrosis associated with chronic hepatitis C virus infection.

Thrombospondin-1 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human tumor cells: Exploring the functional significance in tumor cell invasion

Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression.