Modulation Of Matrix Metalloproteinases Research Articles

Lipoteichoic acid from Staphylococcus aureus increases matrix metalloproteinase 9 expression in RAW 264.7 macrophages: Modulation by A2A and A2B adenosine receptors

Peptidoglycan (PEG) and lipoteichoic acid (LTA) are the main constituents of Gram-positive bacteria cell wall and are described to modulate immune functions. Increased levels of matrix metalloproteinases (MMPs) were described in endotoxemia, suggesting that they participate to tecidual damage, multiple organs failure and vascular disfunction. Staphylococcus aureus PEG is described to increase MMPs 2 and 9 levels in plasma from rat and MMP 9 secretion by human neutrophils, however, the effect of LTA on MMPs is unknown. In this work, was evaluated the modulation of MMPs 2 and 9 expression and secretion in RAW 264.7 macrophages by LTA from S. aureus. The role of A2A and A2B adenosine receptors was also investigated. LTA increased MMP 9 expression and secretion at 12 h of treatment. The modulation of MMP 9 secretion was dose dependent, with maximal effect above 1 μg/ml. The inhibitor of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway (U0126, 10 μM) prevented LTA stimulation of MMP 9 secretion; however, the inhibitors of p38 (SB203580, 10 μM) and Jun N-terminal kinase (JNK; SP600125, 10 μM) presented any effect. A2A and A2B adenosine receptors pharmacological blockade or gene knockdown resulted in exacerbated MMP 9 secretion, while an adenosine receptors agonist inhibited LTA-stimulated MMP 9 secretion. These results suggest that LTA increased MMP 9 secretion in macrophages could be involved in complications associated to S. aureus infections. Moreover, LTA modulation of MMP 9 is dependent on MEK/ERK pathway and is regulated by A2A and A2B adenosine receptors.

GALNT3, a gene associated with hyperphosphatemic familial tumoral calcinosis, is transcriptionally regulated by extracellular phosphate and modulates matrix metalloproteinase activity

GALNT3 encodes UDP-N-acetyl-alpha- d-galactosamine: polypeptide N-acetylgalactosaminyl-transferarase 3 (ppGalNacT3), a glycosyltransferase which has been suggested to prevent proteolysis of FGF23, a potent phosphaturic protein. Accordingly, loss-of-function mutations in GALNT3 cause hyperphosphatemic familial tumoral calcinosis (HFTC), a rare autosomal recessive disorder manifesting with increased kidney reabsorption of phosphate, resulting in severe hyperphosphatemia and widespread ectopic calcifications. Although these findings definitely attribute a role to ppGalNacT3 in the regulation of phosphate homeostasis, little is currently known about the factors regulating GALNT3 expression. In addition, the effect of decreased GALNT3 expression in peripheral tissues has not been explored so far. In the present study, we demonstrate that GALNT3 expression is under the regulation of a number of factors known to be associated with phosphate homeostasis, including inorganic phosphate itself, calcium and 1,25-dihydroxyvitamin D 3. In addition, we show that decreased GALNT3 expression in human skin fibroblasts leads to increased expression of FGF7 and of matrix metalloproteinases, which have been previously implicated in the pathogenesis of ectopic calcification. Thus, the present data suggest that ppGalNacT3 may play a role in peripheral tissues of potential relevance to the pathogenesis of disorders of phosphate metabolism.

Candidate biomarkers for the detection of coronary plaque destabilization and rupture

Identification of high-risk patients presenting with chest pain remains challenging. The use of a single biomarker or a panel of biomarkers to detect occult plaque destabilization or rupture without frank infarction would allow for appropriate triage of such patients. Current data suggest that plaque vulnerability is determined by the relationship between forces that increase the size of the lipid core and destabilize the overlying thin fibrous cap. A variety of interrelated pathways are imputed to play important roles in this process of plaque evolution, destabilization, and rupture. These mechanisms include increased systemic and local inflammation, increased oxidative stress, matrix metalloproteinase modulation, and hemodynamic variables related to altered shear stress. Select candidate biomarkers that either reflect or influence these underlying processes and may ultimately have clinical application are highlighted in this review, which focuses on emerging biomarkers to define and predict the risks associated with the complex nature of vulnerable plaque biology.

Inhibitory Effects of Short-Chain Fatty Acids on Matrix Metalloproteinase Secretion from Human Colonic Subepithelial Myofibroblasts

Short-chain fatty acids (SCFAs), such as acetate, propionate and butyrate, are the major by-product of bacterial fermentation of dietary fiber in the colon. In this report, we investigated how SCFAs modulate matrix metalloproteinase (MMP) secretion from human colonic subepithelial myofibroblasts (SEMFs). SEMFs were identified by expression of alpha-smooth muscle actin and vimentin. Cytokine-induced MMP-1 and MMP-3 levels were determined by enzyme-linked immunosorbent assay. Cytokine-induced MMP mRNA expression was analyzed by RT-PCR and real-time PCR methods. Acetate had no effect on MMP secretion. Propionate and butyrate significantly attenuated IL-1 beta- and TNF-alpha-induced MMP-1 and MMP-3 secretion. Similar responses were also observed at the mRNA levels. Propionate and butyrate did not modulate IL-1 beta- and TNF-alpha-induced activation of mitogen-activated protein kinases (MAPKs), which play a crucial role in MMP induction. Trichostatin A, a histone-deacetylase inhibitor, reduced IL-1 beta-induced MMP-1 and MMP-3 mRNA expression, and suppressed TNF-alpha-induced MMP-3 mRNA expression. SCFAs play an anti-inflammatory role through suppression of MMP secretion in the colon. Inhibitory effects of SCFAs on MMP secretion might be associated with their action of histone hyperacetylation.

Effect of statins in 'upstream therapy' of atrial fibrillation: better reliability with implantable cardiac monitors

The therapeutic potential to suppress atrial fibrillation (AF) is currently under evaluation for several non-antiarrhythmic drugs: angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, aldosterone receptor antagonists, anti-inflammatory and antioxidative drugs, polyunsaturated fatty acids, and HMG-CoA reductase inhibitors, i.e. statins. Statins have shown multiple (‘pleiotropic’) beneficial effects beyond lowering of cholesterol: anti-inflammatory, antioxidant, and antifibrotic effects, modulation of matrix metalloproteinases, interaction with peroxisome receptors that regulate proliferation, and with endothelial nitric oxide synthase, protecting atrial myocardium during ischaemia.1 For these reasons, statins may be able to decelerate or even reverse structural remodelling in patients with AF. Additionally, pre-treatment with high-dose atorvastatin prevented electrical remodelling in a pericarditis dog model,2 and high-dose simvastatin attenuated downregulation of calcium channel subunits, preventing shortening of atrial refractoriness, a central element of electrical remodelling in AF.3 However, this sounds familiar: positive effects on intracellular calcium handling and atrial electrical remodelling have also been reported in animal studies with verapamil,4,5 but this drug disappointed in clinical trials, being unable to prevent atrial electrical remodelling and recurrence of AF.6,7 For statins and AF suppression, there are not only in vitro and animal studies available but also results from 16 clinical trials.1 Nine of … *Corresponding author. Tel: +49 69 6301 7387, Fax: +49 69 6301 3813, Email: C.W.Israel{at}em.uni-frankfurt.de

VEGF Stimulates HDAC7 Phosphorylation and Cytoplasmic Accumulation Modulating Matrix Metalloproteinase Expression and Angiogenesis

Histone acetylation/deacetylation plays an important role in the control of gene expression, tissue growth, and development. In particular, histone deacetylases 7 (HDAC7), a member of class IIa HDACs, is crucial in maintaining vascular integrity. However, whether HDAC7 is involved in the processes of vascular endothelial signaling and angiogenesis remains unclear. Here, we investigated the role of HDAC7 in vascular endothelial growth factor (VEGF) signaling and angiogenesis. We show for the first time that VEGF stimulated phosphorylation of HDAC7 at the sites of Ser178, Ser344, and Ser479 in a dose- and time-dependent manner, which leads to the cytoplasmic accumulation of HDAC7. Using pharmacological inhibitors, siRNA, and adenoviruses carrying dominant-negative mutants, we found that phospholipase Cgamma/protein kinase C/protein kinase D1 (PKD1)-dependent signal pathway mediated HDAC7 phosphorylation and cytoplasmic accumulation by VEGF. Infection of ECs with adenoviruses encoding a mutant of HDAC7 specifically deficient in PKD1-dependent phosphorylation inhibited VEGF-induced angiogenic gene expression, including matrix metalloproteinases MT1-matrix metalloproteinase (MMP) and MMP10. Moreover, HDAC7 and its targeting genes were involved in VEGF-stimulated endothelial cell migration, tube formation, and microvessel sprouting. Our results demonstrate that VEGF stimulates PKD1-dependent HDAC7 phosphorylation and cytoplasmic accumulation in endothelial cells modulating gene expression and angiogenesis.

020 Stress-Induced Modulation of Matrix Metalloproteinases During Cutaneous Wound Repair

Chronic nonhealing wounds have been long associated with an imbalance between the degrading metalloproteinases (MMPs) and their inhibitors (TIMPs). Human and animal studies have shown psychological stress to impair healing and differentially regulate gene expression. We hypothesized that psychological stress-impaired healing is mediated by altered expression of MMP and TIMP. SKH-1 mice were subjected to restraint stress 3 days before and 5 days after placement of 3.5 mm biopsy wounds. The wounds were biopsied at various time points and analyzed for gene expression by real-time PCR. The wounds of stressed mice showed significantly delayed healing when compared to the wounds of controls through day 5 postwounding. There was a significantly higher expression of MMP-8 in the stressed groups on days 1 (172%, p < 0.0003) and 3 (204%, p < 0.0003) postwounding. Stress decreased MMP-9 expression on day 5 postwounding (56%, p < 0.02), but showed no significant difference to controls on the other days. TIMP-1, which is known to inhibit most activated MMPs, had significantly lower expression on day 5 postwounding (56%, p < 0.0003). TIMP-2, which is constitutively expressed, did not show any difference in expression between the stress and the control groups. During stress, increased MMP-8 (neutrophil collagenase) activity is in agreement with previous studies in our model that have demonstrated a increase in neutrophil recruitment on days 3 and 5 postwounding. Likewise, lower expression of TIMP-1 during stress is similar to findings in chronic wounds as opposed to healing surgical wounds. Hence, altered expression of the metalloproteinases and their inhibitors, may lead to increased damage early in healing and decreased repair later in stress-impaired wound healing. Supported by NIDCR-P50(DE13749), NIA-P01(AG16321)

Statins suppress MMP2 secretion via inactivation of RhoA/ROCK pathway in pulmonary vascular smooth muscles cells

Recent studies have shown that statins ameliorate hypoxia-induced pulmonary hypertension in animal models. Yet, the underlying molecular mechanisms have not been completely understood. Here, we investigated the hypothesis that statins regulate vascular remodeling by modulation of matrix metalloproteinases (MMP) secretion in primary cultured pulmonary artery smooth muscle cells (PASMCs). Angiotensin II induced a concentration-dependent MMP2 secretion. A 2.75 fold increase was achieved by 100 nM angiotensin II stimulation for 24 h in primary cultured PASMCs ( P < 0.01 versus control), which was reversed by silencing Ras homolog gene family member A (RhoA) or inhibition of its downstream Rho-associated kinase (ROCK). There was no effect of angiotensin II on the expression of tissue inhibitors of matrix metalloproteinases (TIMPs). Pre-exposure of cells to simvastatin concentration-dependently blocked angiotensin II-stimulated MMP2 release. MMP2 release was reduced to1.34 fold increase in the presence of 10 µM simvastatin ( P < 0.01 versus angiotensin II-stimulated cells). We further revealed that simvastatin suppression of RhoA activation mediated its inhibitory effect on angiotensin II-triggered MMP2 release. Similarly, simvastatin suppressed endothelin-1-induced MMP2 release through RhoA/ROCK pathway. These results indicated a novel statins-regulation of RhoA/ROCK signaling against pulmonary vascular remodeling, and suggest that statins could prove useful in targeting this pathway in pulmonary hypertension and other disease conditions.

Modulation of matrix metalloproteinases by ultraviolet radiation in the canine cornea

To determine whether ultraviolet (UV) radiation can modulate expression and regulation of matrix metalloproteinases (MMP) in the canine cornea and to examine the expression of MMPs in canine chronic superficial keratitis (CSK). Immunohistochemistry for MMP-2 and MMP-9 was performed on samples of CSK. In vitro, canine corneal epithelial cell (CEC) and stromal cell cultures were exposed to UV-irradiation. Following 2, 8 or 24 h, cells were harvested. MMP expression was examined by zymography, and RT-PCR was used to examine expression of Slug and Snail. CEC cultures treated with an EGFR inhibitor or a p38 inhibitor were UV-exposed and harvested 24 h later to examine expression of MMPs, Slug and Snail. Canine CSK had increased immunopositivity for both MMP-2 and MMP-9 compared to normal canine corneas. In vitro, CEC and stromal cell cultures exposed to UV showed generally increased expression of MMP-2, -9, Slug, and Snail; this response was dose and time dependent. Inhibition of the EGFR pathway did not prevent increased expression of MMP-2, -9, Slug or Snail in UV-exposed CEC; however, p38 inhibition did attenuate UV induction. We have found increased expression of MMPs in clinical samples of CSK compared to normal corneas. In addition, we have shown that there is a temporal association and dose dependency between UV exposure and production of MMPs, Slug, and Snail. These findings suggest that overexpression of MMPs due to UV-exposure may be linked to changes in the cornea that allow an influx of inflammatory cells and vascularization.

Interplay of angiotensin II and angiotensin(1–7) in the regulation of matrix metalloproteinases of human cardiocytes

Angiotensin II (Ang II) is a critical effector in the renin-angiotensin system (RAS), which modulates cardiovascular homeostasis, and the matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) related metabolism of extracellular matrix (ECM). Angiotensin(1-7) [Ang(1-7)] is another bioactive peptide in the RAS and is considered to have opposite effects to Ang II. However, the modulation of MMPs and TIMPs by Ang(1-7) is largely unclear in cardiocytes, and the antagonistic effects of Ang(1-7) on Ang II-mediated expression of MMPs and TIMPs have yet to be identified. In the present study, we examined the transcript expression of MMPs and TIMPs in human cardiac fibroblasts (HCF) and myocytes (HCM) after Ang II or Ang(1-7) stimulation, and analysed the antagonistic effects of Ang(1-7) to Ang II. The results show that Ang II decreased transcript expression of MMP-1, MMP-2, TIMP-1, TIMP-2 and TIMP-3, but upregulated MMP-9 expression in the HCF cells. Transcript expression of MMP-9 and TIMP-2 was downregulated by Ang(1-7) in the same cells. In the HCM cells, Ang II induced MMP-1 and MMP-9 overexpression but MMP-2 was downregulated. All of the examined MMPs and TIMPs, except MMP-9, were markedly decreased by Ang(1-7). In the studies of antagonistic effects of Ang(1-7) to Ang II, Ang(1-7) counteracted the effects of Ang II-mediated regulation on MMP-9 and TIMP-1 in the HCF cells compared with the control group. The regulations of all examined MMPs by Ang II were reversed to basal expression by Ang(1-7) in the HCM cells. Our results suggest that Ang(1-7) and Ang II have opposite and antagonistic effects on regulation of transcription of MMPs and TIMPs in primary cultures of human cardiocytes. These effects lead to increased ratios of MMPs to TIMPs after Ang II stimulation and decreased ratios of MMPs to TIMPs after Ang(1-7) stimulation; effects which may partly depend of the type of cardiac cells. These results suggest a potential role for Ang(1-7) in attenuatating cardiac damage in Ang II-induced ECM remodelling.

Rap1 promotes Prostate Cancer metastasis

Elucidating the mechanisms of prostate cancer (CaP) survival and metastasis are critical to the discovery of novel therapeutic targets. Increasing evidence demonstrates significant roles for integrins and matrix metalloproteinases (MMP) in CaP metastasis, making them attractive targets for therapy. The monomeric G protein, Rap1 activates integrins and has been implicated in cancer tumorgenesis. Rap1 signals to pathways involved in cell adhesion, migration, and survival, suggesting Rap1 may promote several processes associated with CaP metastasis. Thus, we sought to explore the functional significance of Rap1 activation in CaP by examining its impact on invasion and migration of CaP cells during metastasis.Results from in vitro assays demonstrate activation of Rap1 increases CaP cell migration toward bone‐secreted factors. Similarly, results show activation of Rap1 enhances CaP cell invasion, and may involve modulation of MMPs. Inhibition studies indicate that integrin activation is a mechanism of Rap1‐mediated CaP migration and invasion. Additional studies will explore whether Rap1 influences MMPs directly or through its subsequent activation of integrins. The in vivo impact of Rap1 in CaP is being evaluated in the mouse and preliminary results demonstrate activated Rap1 drives the rate and incidence of CaP metastasis.Support for CLB: Department of Defense PCRP #DAMD17‐0510560

Modulation of Keratinocyte-Derived MMP-9 by IL-13: A Possible Role for the Pathogenesis of Epidermal Inflammation

Skin inflammation and remodeling are important pathophysiological features of chronic eczematous skin diseases. Matrix metalloproteinases (MMP) have been described to influence tissue remodeling and to facilitate cell migration through their ability to proteolyse the extracellular matrix. The aim of this study was to investigate the influence of IL-13 on the modulation of MMPs in human primary keratinocytes (KCs). IL-13 stimulation of KCs induced the expression of MMP-9 but not of MMP-3 or MMP-2 at mRNA level. A major substrate of MMP-9 is the type IV collagen of the basement membrane. IL-13 induced the release of active MMP-9 in KCs as detected by an ELISA-based assay. Moreover, migration of lymphocytes cultured with IL-13-activated KC showed increased migration through a basement membrane equivalent. The MMP-9 expression was prominent near the basement membrane of IL-13-treated skin biopsies. Collagen type IV staining pointed to a loss of this major basement membrane constituent in IL-13-treated skin. Finally, we demonstrated the concomitant mRNA expression of MMP-9 and IL-13 in biopsies from lesional, acutely inflamed eczematous skin. Our results suggest that release of active MMP-9 by IL-13-stimulated KCs may play a crucial role in skin inflammation by facilitating migration of leukocytes into the epidermis.

Culture of Human A375 Melanoma Cells in the Presence of Fibronectin Causes Expression of MMP-9 and Activation of MMP-2 in Culture Supernatants

Interactions between tumor cell surface integrin receptors and extracellular matrix (ECM) ligands play an important role in tumor development, affecting cell survival, proliferation, and migration. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen-activated protein kinase (MAPK) pathways. It has been reported that integrins also regulate expression and function of matrix metalloproteinases (MMPs). In this present work, we cultured human A375 melanoma cells in the presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. A375 cells were cultured in serum-free culture medium (SFCM) in the presence of fibronectin (25 microg/0.75 ml), SFCM was collected and gelatin zymography was performed. Western blot and RT-PCR were performed with A375 cells cultured in the presence of fibronectin. Culture of A375 cells in the presence of fibronectin led to expression of MMP-9 and activation of MMP-2 within 2 h. When cells were treated with ERK inhibitor (PD98059) or PI-3K inhibitor (LY294002) and grown in the presence of fibronectin, MMP-9 expression and MMP-2 activation was inhibited. Tyrosine phosphorylation of FAK and ERK were increased in A375 cells grown in the presence of fibronectin. Increased MMP-9 mRNA expression and processing of MT1-MMP were also observed in A375 cells grown in the presence of fibronectin. Our findings indicate culture of A375 cells in SFCM in the presence of fibronectin perhaps generates a signaling cascade that leads to expression of MMP-9 and activation of MMP-2 in culture supernatants within 2 h. The signaling pathway activated is probably the FAK/ERK pathway.

Modulation of membrane type 1 matrix metalloproteinase by LPS and gamma interferon bound to extracellular matrix in intestinal crypt cells

Membrane type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane protein that participates in the processing and degradation of cell surface proteins and the extracellular matrix (ECM). This enzyme regulates ECM turnover in wound repair, promotes cell migration and activates other MMPs, such as MMP-2, which is involved in angiogenesis, cell migration and tumoral metastasis. An increase in pro-inflammatory cytokine expression, such as gamma interferon (IFN-γ), has been associated with chronic wounds in inflammatory bowel diseases. However, the extent to which cytokines modulate MT1-MMP has not been totally defined. In this report, the effects of the bacterial lipopolysaccharide (LPS) and ECM-bound IFN-γ on MT1-MMP expression and MMP-2 activity were evaluated by Western blot, RT-PCR and zymography in isolated intestinal epithelial and cultured HT-29 cells. In the presence of LPS, ECM-bound IFN-γ, but not soluble IFN-γ, reduced the enterocyte MT1-MMP protein expression. In addition, the active form of MMP-2 was also decreased in the presence of both LPS and IFN-γ, indicating that lower MMP-2 activity accompanied the decrease in MT1-MMP expression. These results suggest the possibility that endotoxin and ECM-bound IFN-γ may affect matrix remodeling by modulating matrix metalloproteinase in enterocytes during wound healing.

Acetylcholine induces neurite outgrowth and modulates matrix metalloproteinase 2 and 9

The matrix metalloproteinases (MMPs), responsible for the degradation of extracellular matrix (ECM) proteins, may regulate brain cellular functions. Choline acetyltransferase (ChAT) transfected murine neuroblastoma cell line N18TG2, that synthesize acetylcholine and show enhancement of several neurospecific markers (i.e., sinapsin I, voltage gated Na + channels, high affinity choline uptake) and fiber outgrowth, were studied for the MMP regulation during neuronal differentiation. Zymography of N18TG2 culture medium revealed no gelatinolytic activity, whereas after carbachol treatment of cells both MMP-9 and activated MMP-2 forms were detected. ChAT-transfected clone culture medium contains three MMP forms at 230, 92, and 66 kDa. Carbachol treatment increased MMP-2 and MMP-9 gene expression in N18TG2 cells and higher levels for both genes were also observed in ChAT transfected cells. The data are consistent with the hypothesis that acetylcholine brings about the activation of an autocrine loop modulating MMP expression.

Inhibitory Effect of Polyunsaturated Fatty Acids on MMP-9 Release from Microglial Cells—Implications for Complementary Multiple Sclerosis Treatment

We investigated whether polyunsaturated fatty acids (PUFA), which might be a useful complementary therapy among patients with multiple sclerosis (MS), are able to modulate matrix metalloproteinase (MMP) production in microglial cultures. MMPs are myelinotoxic factors. Primary cultures of rat microglia were treated with different doses of omega-3 (omega-3) PUFA or purified fish oil, containing a mixture of omega-3 and omega-6 PUFA, and simultaneously activated by exposure to lipopolysaccharide (LPS). Culture supernatants were subjected to zymography and Western blot analysis for the assessment of MMP-2 and MMP-9 levels. Increased amounts of MMP-9, but not of the constitutively expressed MMP-2, were observed in supernatants from LPS-treated microglia in comparison with non-treated control cells. The treatment with both omega-3 PUFA and fish oil dose-dependently inhibited the LPS-induced production of MMP-9. Our results suggest that a low fat diet supplemented with omega-3 PUFA may become recommended for the well being of MS patients under therapy.

Interleukin-10 is not a critical regulator of infarct healing and left ventricular remodeling

Interleukin-10 (IL-10) exerts potent anti-inflammatory actions and modulates matrix metalloproteinase expression. We hypothesized that endogenous IL-10 may regulate infarct healing and left ventricular remodeling by promoting resolution of the post-infarction inflammatory response and by modulating extracellular matrix metabolism. IL-10 null and wildtype (WT) mice underwent reperfused infarction protocols. We compared the healing response and remodeling-associated parameters between IL-10-/-and WT infarcts. In addition, we studied the effects of IL-10 on inflammatory gene synthesis by stimulated murine cardiac fibroblasts. Infarcted IL-10-/-mice exhibited comparable mortality rates with WT animals. Although IL-10-/-mice had higher peak tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein (MCP)-1/CCL2 mRNA levels in the infarcted heart than WT mice, both groups demonstrated timely repression of pro-inflammatory cytokine and chemokine mRNA synthesis after 24 h of reperfusion and exhibited a similar time course of resolution of the neutrophil infiltrate. IL-10 gene disruption did not alter fibrous tissue deposition and dilative remodeling of the infarcted heart. Pre-incubation with IL-10 did not modulate the pro-inflammatory phenotype of TNF-alpha-stimulated cardiac fibroblasts, failing to inhibit chemokine mRNA synthesis. In contrast, transforming growth factor (TGF)-beta1 pre-incubation suppressed interferon-gamma-inducible protein (IP)-10/CXCL10 synthesis by cardiac fibroblasts exposed to TNF-alpha. IL-10 signaling plays a non-critical role in suppression of inflammatory mediators, resolution of the inflammatory response, and fibrous tissue deposition following myocardial infarction. This may be due to the relative selectivity of IL-10-mediated anti-inflammatory actions, with respect to cell type and stimulus. Resolution of post-infarction inflammation is likely to involve multiple overlapping regulatory mechanisms controlling various pro-inflammatory pathways activated in the infarcted myocardium.

MMP-2 and MMP-9 activity is regulated by estradiol and tamoxifen in cultured human breast cancer cells

Sex steroids play a dominant role in breast carcinogenesis by still largely unknown mechanisms. Matrix metalloproteinases (MMPs) have been extensively studied in the context of matrix biology but it is not known if sex steroids affect MMPs in breast cancer. MMPs degrade extracellular matrix components enabling tumor cell invasion and metastasis, but may also regulate the bioavailability of a variety of biologically active molecules such as anti-angiogenic fragments, which may be beneficial for the host. This study shows that estradiol and tamoxifen regulate MMP-2 and MMP-9 as well as TIMP-1 and TIMP-2 in ER + PR + human breast cancer cells. The main finding was a significant effect of tamoxifen exposure, which increased intracellular and secreted protein levels whereas estradiol induced a significant decrease. The overall net effect of these alterations resulted in increased MMP-2/MMP-9 activity by tamoxifen treatment, which also significantly increased extracellular endostatin levels. We conclude that estradiol and tamoxifen have the ability to modulate MMP-2/MMP-9 activity, and endostatin levels in human breast cancer in vitro. The results suggest a possible role of MMP modulation associated with a generation of anti-angiogenic fragments in the therapeutic effect of tamoxifen in breast cancer.

Low‐level laser irradiation modulates matrix metalloproteinase activity and gene expression in porcine aortic smooth muscle cells

The vascular extracellular matrix is maintained by a dynamic balance between matrix synthesis and degradation. This equilibrium is disrupted in arterial pathologies such as abdominal aortic aneurysm. Low-level laser irradiation (LLLI) promotes wound healing. However, its effect on smooth muscle cells (SMCs), a central player in these responses, has not been established. The current study was designed to determine the effects of LLLI on arterial SMC proliferation, inflammatory markers, and matrix proteins. Porcine primary aortic SMCs were irradiated with a 780 nm laser diode (1 and 2 J/cm(2)). Trypan blue exclusion assay, immunofluorescent staining for collagen I and III, Sircol assay, gelatin zymography, and RT-PCR were used to monitor proliferation; collagen trihelix formation; collagen synthesis; matrix metalloproteinase-2 (MMP-2) activity, and gene expression of MMP-1, MMP-2, tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, and IL-1-beta, respectively. LLLI-increased SMC proliferation by 16 and 22% (1 and 2 J/cm(2), respectively) compared to non-irradiated cells (P<0.01 and P<0.0005). Immediately after LLLI, trihelices of collagen I and III appeared as perinuclear fluorescent rings. Collagen synthesis was increased twofold (2 days after LLLI: 14.3+/-3.5 microg, non-irradiated control: 6.6+/-0.7 microg, and TGF-beta stimulated control: 7.1+/-1.2 microg, P<0.05), MMP-2 activity after LLLI was augmented (over non-irradiated control) by 66+/-18% (2 J/cm(2); P<0.05), and MMP-1 gene expression upregulated. However, TIMP-2 was upregulated, and MMP-2 gene expression downregulated. IL-1-beta gene expression was reduced. LLLI stimulates SMC proliferation, stimulates collagen synthesis, modulates the equilibrium between regulatory matrix remodeling enzymes, and inhibits pro-inflammatory IL-1-beta gene expression. These findings may be of therapeutic relevance for arterial diseases such as aneurysm where SMC depletion, weakened extracellular matrix, and an increase in pro-inflammatory markers are major pathologic components.

Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract

Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)-2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 microg/mL and MMP-9 at 500 microg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 microg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 microg/mL NM (P < 0.0001) and 1000 microg/mL NM (P < 0.0001), respectively. Our results suggest that the mixture of lysine, proline, arginine, ascorbic acid, and green tea extract has potential in the treatment of cervical cancer by inhibiting critical steps in cancer development and spread.