Administration of IL-18BP by gene therapy reduces inflammation and prevents joint destruction by downregulation of MMP-9 in rat AIA Role of MMP-9 in bone and joint destruction in arthritis

Background and objectives: Interleukin-18 (IL-18) is a pleiotropic cytokine involved in rheumatoid arthritis (RA) pathogenesis. This studywas carried out to evaluate the efficicacy of interleukin-18 binding protein (IL-18BP) gene therapy in the rat adjuvant-induced arthritis (AIA) model and to decipher the mechanisms by which IL-18BP delivery lessens bone destruction. Materials and methods: Arthritis was induced in female Lewis rat by Mycobacterium butyricum and the mRNA expression of IL-18 and IL-18BP was determined in the joints. In a preventative study, rats were divided into an adenovirus producing IL-18BP-Fc (AdmIL-18BP-Fc) group (n=8) and an adenovirus producing green fluorescent protein (AdGFP) group (n=7). On day 8 after AIA induction, adenoviruses were injected. Clinical parameters were assessed. At day 18, during maximal arthritis, the rats were euthanized, ankles were collected, and X-rays were performed. mRNA and protein were extracted from joints for analyses by qRT-PCR, multiplex, Western blot, and zymography. Results: We observed a decrease in the [IL-18BP/IL-18] ratio from day 7 to day 45. Administration of AdmIL-18BPd-Fc decreased clinical parameters and prevented bone and joint destruction compared to AdGFP administration. IL-18BP delivery reduced the metalloproteinase 9 (MMP-9) levels by 33% (at protein level (Fig. 1B) and functional level (Fig. 1C) and the tartrate-resistant acid phosphatase (TRAP) level by 44% (Fig. 1D) in the joint homogenates from AdmIL-18BPd-Fc compared to AdGFP treated rats.However, no variationwas observed forMMP-2 at the protein level (Fig.1A) and functional level (Fig. 1C). Conclusions: In rat AIA, a decrease in the [IL-18BP/ IL-18] ratio was observed. IL-18BP delivery prevented joint and bone destruction by downregulating MMP-9 and TRAP, suggesting a potential benefit of a similar therapy in RA.

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins.

Intimal hyperplasia (IH) is the major cause of stenosis of vein grafts. Drugs such as statins prevent stenosis, but their systemic administration has limited effects. We developed a hyaluronic acid hydrogel matrix, which ensures a controlled release of atorvastatin (ATV) at the site of injury. The release kinetics demonstrated that 100% of ATV was released over 10 hours, independent of the loading concentration of the hydrogel. We investigated the effects of such a delivery on primary vascular smooth muscle cells isolated from human veins. ATV decreased the proliferation, migration, and passage of human smooth muscle cells (HSMCs) across a matrix barrier in a similar dose-dependent (5-10 µM) and time-dependent manner (24-72 hours), whether the drug was directly added to the culture medium or released from the hydrogel. Expression analysis of genes known to be involved in the development of IH demonstrated that the transcripts of both the gap junction protein connexin43 (Cx43) and plasminogen activator inhibitor-1 (PAI-1) were decreased after a 24-48-hour exposure to the hydrogel loaded with ATV, whereas the transcripts of the heme oxygenase (HO-1) and the inhibitor of tissue plasminogen activator were increased. At the protein level, Cx43, PAI-1, and metalloproteinase-9 expression were decreased, whereas HO-1 was upregulated in the presence of ATV. The data demonstrate that ATV released from a hydrogel has effects on HSMCs similar to the drug being freely dissolved in the environment.

Lipoxin A₄ prevents the progression of de novo and established endometriosis in a mouse model by attenuating prostaglandin E₂ production and estrogen signaling.

Endometriosis, a leading cause of pelvic pain and infertility, is characterized by ectopic growth of endometrial-like tissue and affects approximately 176 million women worldwide. The pathophysiology involves inflammatory and angiogenic mediators as well as estrogen-mediated signaling and novel, improved therapeutics targeting these pathways are necessary. The aim of this study was to investigate mechanisms leading to the establishment and progression of endometriosis as well as the effect of local treatment with Lipoxin A4 (LXA₄), an anti-inflammatory and pro-resolving lipid mediator that we have recently characterized as an estrogen receptor agonist. LXA₄ treatment significantly reduced endometriotic lesion size and downregulated the pro-inflammatory cytokines IL-1β and IL-6, as well as the angiogenic factor VEGF. LXA₄ also inhibited COX-2 expression in both endometriotic lesions and peritoneal fluid cells, resulting in attenuated peritoneal fluid Prostaglandin E₂ (PGE₂) levels. Besides its anti-inflammatory effects, LXA₄ differentially regulated the expression and activity of the matrix remodeling enzyme matrix metalloproteinase (MMP)-9 as well as modulating transforming growth factor (TGF)-β isoform expression within endometriotic lesions and in peritoneal fluid cells. We also report for first time that LXA₄ attenuated aromatase expression, estrogen signaling and estrogen-regulated genes implicated in cellular proliferation in a mouse model of disease. These effects were observed both when LXA₄ was administered prior to disease induction and during established disease. Collectively, our findings highlight potential targets for the treatment of endometriosis and suggest a pleotropic effect of LXA₄ on disease progression, by attenuating pro-inflammatory and angiogenic mediators, matrix remodeling enzymes, estrogen metabolism and signaling, as well as downstream proliferative pathways.

Functions of the extracellular matrix and matrix degrading proteases during tumor progression

Cell interactions with extracellular matrices are important to pathological changes that occur during cell transformation and tumorigenesis. Several extracellular matrix proteins including fibronectin, thrombospondin-1, laminin, SPARC, and osteopontin have been suggested to modulate tumor phenotype by affecting cell migration, survival, or angiogenesis. Likewise, proteases including the matrix metalloproteinases (MMPs) are understood to not only facilitate migration of cells by degradation of matrices, but also to affect tumor formation and growth. We have recently demonstrated an in vivo role for the RGD-containing protein, osteopontin, during tumor progression, and found evidence for distinct functions in the host versus the tumor cells. Because of the compartmentalization and temporal regulation of MMP expression, it is likely that MMPs may also function dually in host stroma and the tumor cell. In addition, an important function of proteases appears to be not only degradation, but also cleavage of matrix proteins to generate functionally distinct fragments based on receptor binding, biological activity, or regulation of growth factors.

Regulation of pituitary hormones and cell proliferation by components of the extracellular matrix

The extracellular matrix is a three-dimensional network of proteins, glycosaminoglycans and other macromolecules. It has a structural support function as well as a role in cell adhesion, migration, proliferation, differentiation, and survival. The extracellular matrix conveys signals through membrane receptors called integrins and plays an important role in pituitary physiology and tumorigenesis. There is a differential expression of extracellular matrix components and integrins during the pituitary development in the embryo and during tumorigenesis in the adult. Different extracellular matrix components regulate adrenocorticotropin at the level of the proopiomelanocortin gene transcription. The extracellular matrix also controls the proliferation of adrenocorticotropin-secreting tumor cells. On the other hand, laminin regulates the production of prolactin. Laminin has a dynamic pattern of expression during prolactinoma development with lower levels in the early pituitary hyperplasia and a strong reduction in fully grown prolactinomas. Therefore, the expression of extracellular matrix components plays a role in pituitary tumorigenesis. On the other hand, the remodeling of the extracellular matrix affects pituitary cell proliferation. Matrix metalloproteinase activity is very high in all types of human pituitary adenomas. Matrix metalloproteinase secreted by pituitary cells can release growth factors from the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. In summary, the differential expression of extracellular matrix components, integrins and matrix metalloproteinase contributes to the control of pituitary hormone production and cell proliferation during tumorigenesis.

Inhibition of STAT3 by RNA interference suppresses angiogenesis in colorectal carcinoma

In order to investigate signal transduction and activation of transcription 3 (STAT3) signaling on angiogenesis in colorectal carcinoma (CRC) after inhibiting STAT3 expression, we constructed the HT-29-shSTAT3 cell line by lentivirus-mediated RNAi. Cell growth was assessed with MTT and the cell cycle distribution by flow cytometry. CRC nude mouse models were established and tumor growth was monitored periodically. On day 30, all mice were killed and tumor tissues were removed. Microvessel density (MVD) was determined according to CD34-positive staining. The expression of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-2 (MMP2) and basic fibroblast growth factor (FGF2) was monitored by quantitative real-time PCR and Western blot analysis. Knockdown of STAT3 expression significantly inhibited cell growth in HT-29 cells, with a significantly higher proportion of cells at G0/G1 (P < 0.01). Consistently, in vivo data also demonstrated that tumor growth was significantly inhibited in mice injected with HT-29-shSTAT3 cells. MVD was 9.80 ± 3.02 in the HT-29-shSTAT3 group, significantly less than that of the control group (P < 0.01). mRNA and protein levels of VEGFA and MMP2 in the HT-29-shSTAT3 group were significantly lower than in the control group (P < 0.05), but no significant difference was observed in the mRNA or protein level of FGF2 (P > 0.05). Taken together, these results demonstrate that STAT3 signaling is important to the growth of CRC and promotes angiogenesis by regulating VEGFA and MMP2 expression.

Moderate Champagne consumption promotes an acute improvement in acute endothelial-independent vascular function in healthy human volunteers

Epidemiological studies have suggested an inverse correlation between red wine consumption and the incidence of CVD. However, Champagne wine has not been fully investigated for its cardioprotective potential. In order to assess whether acute and moderate Champagne wine consumption is capable of modulating vascular function, we performed a randomised, placebo-controlled, cross-over intervention trial. We show that consumption of Champagne wine, but not a control matched for alcohol, carbohydrate and fruit-derived acid content, induced an acute change in endothelium-independent vasodilatation at 4 and 8 h post-consumption. Although both Champagne wine and the control also induced an increase in endothelium-dependent vascular reactivity at 4 h, there was no significant difference between the vascular effects induced by Champagne or the control at any time point. These effects were accompanied by an acute decrease in the concentration of matrix metalloproteinase (MMP-9), a significant decrease in plasma levels of oxidising species and an increase in urinary excretion of a number of phenolic metabolites. In particular, the mean total excretion of hippuric acid, protocatechuic acid and isoferulic acid were all significantly greater following the Champagne wine intervention compared with the control intervention. Our data suggest that a daily moderate consumption of Champagne wine may improve vascular performance via the delivery of phenolic constituents capable of improving NO bioavailability and reducing matrix metalloproteinase activity.

TNF-alpha Promotes an Odontoblastic Phenotype in Dental Pulp Cells

Dental pulp cells can differentiate toward an odontoblastic phenotype to produce reparative dentin beneath caries lesions. However, the mechanisms involved in pulp cell differentiation under pro-inflammatory stimuli have not been well-explored. Thus, we hypothesized that the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) could be a mediator involved in dental pulp cell differentiation toward an odontoblastic phenotype. We observed that TNF-alpha-challenged pulp cells exhibited increased mineralization and early and increased expression of dentin phosphoprotein (DPP), dentin sialoprotein (DSP), dentin matrix protein-1, and osteocalcin during a phase of reduced matrix metalloproteinase (MMP) expression. We investigated whether these events were related and found that p38, a mitogen-activated protein kinase, differentially regulated MMP-1 and DSP/DPP expression and mediated mineralization upon TNF-alpha treatment. These findings indicate that TNF-alpha stimulates differentiation of dental pulp cells toward an odontoblastic phenotype via p38, while negatively regulating MMP-1 expression.

Role of MMP9 on Invadopodia Formation in Cells From Adenoid Cystic Carcinoma. Study by Laser Scanning Confocal Microscopy

Migration, invasion and protease activity are essential for tumor progression and metastasis. Metastatic cells rely on invadopodia to degrade and invade extracellular matrix (ECM). Invadopodia are membrane protrusions with enzymes required for ECM degradation. These protrusions contain cortactin and membrane type I matrix metalloproteinase (MT1-MMP) superimposed to areas of digested matrix. Here we characterized invadopodia in a cell line (CAC2) derived from human adenoid cystic carcinoma. We carried out fluorescent-substrate degradation assay to assess in situ protease activity of CAC2 cells. Digestion spots in fluorescent substrate appear as black areas in green background. Cells were cultured on Matrigel-gelatin-FITC and fixed after 1 h and 3 h. CAC2 cells were double labeled to actin and cortactin. Cells were also double stained to actin and MT1-MMR Samples were studied by laser scanning confocal microscopy. In all time points CAC2 cells showed actin, cortactin, and MT1-MMP colocalized with digestion spots in fluorescent substrate. We searched for other proteases involved in invadopodia activity. We have previously demonstrated that MMP9 influences adenoid cystic carcinoma behavior. This prompted us to investigate role played by MMP9 on invadopodia formation. CAC2 cells had MMP9 silenced by siRNA. After I h in fluorescent substrate, cells with silenced MMP9 showed clear decrease in matrix digestion compared with controls. No differences were found in cells with silenced MMP9 grown for 3 h on fluorescent substrate. Our results showed that CAC2 cells exhibit functional invadopodia containing cortactin and MT1-MMR Furthermore, MMP9 would be required in the initial steps of invadopodia formation. Microsc. Res. Tech. 73:99-108, 2010. (C) 2009 Wiley-Liss, Inc.

The Short-Term Effectiveness of Non-Surgical Treatment in Reducing Levels of Interleukin-1 beta and Proteases in Gingival Crevicular Fluid From Patients With Type 2 Diabetes Mellitus and Chronic Periodontitis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Calcaneal Tendon Regions Exhibit Different MMP-2 Activation After Vertical Jumping and Treadmill Running

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fluoxetine Inhibits Inflammatory Response and Bone Loss in a Rat Model of Ligature-Induced Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Proteinase-activated receptor-2 (PAR2) agonist causes periodontitis in rats

Proteinase-activated receptor-2 (PAR2) is a G-protein-coupled receptor that mediates cellular responses to extracellular proteinases. Since PAR2 is expressed by oral epithelial cells, osteoblasts, and gingival fibroblasts, where its activation releases interleukin-8, we hypothesized that PAR2 activation may participate in periodontal disease in vivo. We investigated the role of PAR2 activation in periodontal disease in rats. Radiographic and enzymatic (myeloperoxidase) analysis revealed that topical application of PAR2 agonist causes periodontitis but also exacerbates existing periodontitis, leading to significant alveolar bone loss and gingival granulocyte infiltration. Inhibition of matrix metalloproteinase (MMP) and cyclo-oxygenase (COX) decreased PAR2 agonist-induced periodontitis. More specifically, the overexpression of COX-1, COX-2, MMP-2, and MMP-9 in gingival tissues suggests that they are involved in PAR 2-induced periodontitis. In conclusion, PAR2 agonist causes periodontitis in rats through a mechanism involving prostaglandin release and MMP activation. Inhibition of PAR2 may represent a novel approach to modulate host response in periodontitis.

Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP > AP > DP > VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma. © 2006 International Federation for Cell Biology.

Effect of phosphoric acid on the degradation of human dentin matrix

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We acid-etched experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices. © 2013 International & American Associations for Dental Research.