Fibrosis regression is induced by AdhMMP8 in a murine model of chronic kidney injury.

Adenoviral vector AdhMMP8 (human Metalloproteinase-8 cDNA) administration has been proven beneficial in various experimental models of liver injury improving liver function and decreasing fibrosis. In this study, we evaluated the potential therapeutic AdhMMP8 effect in a chronic kidney damage experimental model. Chronic injury was induced by orogastric adenine administration (100mg/kg/day) to Wistar rats for 4 weeks. AdhMMP8 (3x1011vp/kg) was administrated in renal vein during an induced-ligation-ischemic period to facilitate kidney transduction causing no-additional kidney injury as determined by histology and serum creatinine. Animals were sacrificed at 7- and 14-days post-Ad injection. Fibrosis, histopathological features, serum creatinine (sCr), BUN, and renal mRNA expression of αSMA, Col-1α, TGF-ß1, CTGF, BMP7, IL-1, TNFα, VEGF and PAX2 were analyzed. Interestingly, AdhMMP8 administration resulted in cognate human MMP8 protein detection in both kidneys, whereas hMMP8 mRNA was detected only in the left kidney. AdhMMP8 significantly reduced kidney tubule-interstitial fibrosis and glomerulosclerosis. Also, tubular atrophy and interstitial inflammation were clearly decreased rendering improved histopathology, and down regulation of profibrogenic genes expression. Functionally, sCr and BUN were positively modified. The results showed that AdhMMP8 decreased renal fibrosis, suggesting that MMP8 could be a possible therapeutic candidate for kidney fibrosis treatment.

Drupin, a cysteine protease from Ficus drupacea latex accelerates excision wound healing in mice.

Wound healing is a tightly regulated physiological process that restores tissue integrity after injury. Plant latex proteases (PLPs) are considered an integral part in herbal wound care as it interferes at different phases of the wound healing process. Although many studies have reported the involvement of PLPs in healing process, an in-depth investigation is required to understand the molecular mechanism. Hence, the effect of PLPs with fibrinolytic activity on wound healing was investigated systematically using mouse excision wound model. Among 29 latices from Ficus genus tested, Ficus drupacea exhibited potent fibrinolytic activity. Cysteine protease responsible for fibrinolysis was purified from the F. drupacea latex named it as drupin, tested for its wound healing efficacy. The accelerated wound healing was mediated by downregulation of matrix metalloprotease (MMP)-9 without altering MMP-8 expression. Besides, drupin enhanced the rate of collagen synthesis at the wound site by increasing arginase 1 activity. And also, drupin increased the expression of arginase 1 in macrophages and involved in cell proliferation, and migration via MAP kinase and PI3K/Akt pathways. Overall, the present study highlights the interference of drupin in wound healing by increased arginase 1 activity and collagen synthesis, and cell proliferation and migration.

Stem cell enriched lipotransfer reverses the effects of fibrosis in systemic sclerosis.

Oro-facial fibrosis in systemic sclerosis (Scleroderma;SSc) has a major impact on mouth function, facial appearance, and patient quality of life. Lipotransfer is a method of reconstruction that can be used in the treatment of oro-facial fibrosis. The effect of this treatment not only restores oro-facial volume but has also been found to reverse the effects of oro-facial fibrosis. Adipose derived stem cells (ADSCs) within the engrafted adipose tissue have been shown to be anti-fibrotic in SSc and are proposed as the mechanism of the anti-fibrotic effect of lipotransfer. A cohort of 62 SSc patients with oro-facial fibrosis were assessed before and after stem cell enriched lipotransfer treatment. Clinical evaluation included assessment of mouth function using a validated assessment tool (Mouth Handicap in Systemic Sclerosis Scale-MHISS), validated psychological measurements and pre and post-operative volumetric assessment. In addition, to understand the mechanism by which the anti-fibrotic effect of ADSCs occur, SSc derived fibroblasts and ADSCs from this cohort of patients were co-cultured in direct and indirect culture systems and compared to monoculture controls. Cell viability, DNA content, protein secretion of known fibrotic mediators including growth factor- ß1 (TGF ß-1) and connective tissue growth factor (CTGF) using ELISA analysis and fibrosis gene expression using a fibrosis pathway specific qPCR array were evaluated. Mouth function (MHISS) was significantly improved (6.85±5.07) (p<0.0001) after treatment. All psychological measures were significantly improved: DAS 24 (12.1±9.5) (p<0.0001); HADS-anxiety (2.8±3.2) (p<0.0001), HADS-depression (2.0±3.1) (p<0.0001); BFNE (2.9 ± 4.3) (p<0.0001); VAS (3.56±4.1) (p<0.0001). Multiple treatments further improved mouth function (p<0.05), DAS (p<0.0001) and VAS (p = 0.01) scores. SSc fibroblast viability and proliferation was significantly reduced in co-culture compared to monoculture via a paracrine effect over 14 days (p < 0.0001). Protein secretion of transforming growth factor (TGF-ß1) and connective tissue growth factor (CTGF) was significantly reduced in co-culture compared to monoculture (p < 0.0001). Multiple fibrosis associated genes were down regulated in SSc co-culture compared to monoculture after 14 days including Matrix metalloproteinase-8 (MMMP-8), Platelet derived growth factor-ß (PDGF-ß) and Integrin Subunit Beta 6 (ITG-ß6). Autologous stem cell enriched lipotransfer significantly improved the effects of oro-facial fibrosis in SSc in this open cohort study. Lipotransfer may reduce dermal fibrosis through the suppression of fibroblast proliferation and key regulators of fibrogenesis including TG-ß1 and CTGF. Our findings warrant further investigation in a randomised controlled trial.

Immunoexpression of MMP-8, MMP-9 and TIMP-2 in dilated cardiomyopathy.

Alteration of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) expression has been studied for various cardiac diseases, including dilated cardiomyopathy (DCM), with the significance of surrogate markers of extracellular matrix (ECM) remodeling. In this study, we determined the MMP-8, MMP-9 and TIMP-2 immunoexpression in the heart of patients diagnosed with DCM in relation to a histological composite score (HCS). The study included 40 cases of heart fragments that were processed by the usual paraffin inclusion technique, followed by a semi-quantitative evaluation of histopathological parameters, which summed, allowed the establishment of a HCS. Subsequently, the cases were immunohistochemically processed for MMP-8, MMP-9 and TIMP-2, followed by the semi-quantitative evaluation of their expression intensity. MMP-8 was identified only in myocardiocytes, while MMP-9 and TIMP-2 were present in both myocardiocytes and stroma, but with different intensity. The increasing intensity of MMP-8 and TIMP-2 immunoreactions was significantly associated with low HCS. In case of MMP-9, the immunostaining intensity analysis in relation to the HCS level revealed insignificant differences, but we found an association of increased and moderate intensity with low HCS. The imbalance between TIMPs and MMPs disrupts the ECM architecture and contributes to the remodeling process in DCM, aspect that can be used in the development of new clinical therapies.

Expression of cytokines and chemokines in mouse skin treated with sulfur mustard.

Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

MMP-1 and MMP-8 expression in giant-cell fibroma and inflammatory fibrous hyperplasia.

The aim of this study is to compare the immunoexpression of metalloproteinases 1 and 8 in giant-cell fibroma, inflammatory fibrous hyperplasia and normal mucosa. Twenty-two cases of giant-cell fibroma, inflammatory fibrous hyperplasia and oral mucosa (control) each were subjected to immunohistochemistry using anti-metalloproteinase-1 and anti-metalloproteinase-8 antibodies. Eight images of each case were captured and analysed through the a) application of a count grid to count the number of positive neutrophils, macrophages, lymphocytes, plasma cells, fibroblasts and blood vessels to obtain the percentage of staining and b) semi-automated segmentation quantifying the stained area in square micrometres. Statistical tests included ANOVA Two-way, Kruskal Wallis and Games-Howell, with a significance level of 5%. An increased percentage of metalloproteinase-1-immunopositive blood vessels were observed in giant-cell fibroma (26.6±22.4; p=0.02) and inflammatory fibrous hyperplasia (34.3±31.5; p=0.01) compared with the control group (19.6±9.2). No significant differences in inflammatory cells, fibroblasts and total area of metalloproteinase-1 and -8 were noted among the three groups. Metalloproteinase-1 apparently acts within the pathogenesis of giant-cell fibroma and inflammatory fibrous hyperplasia.

Effects of an early intervention using human amniotic epithelial cells in a COPD rat model.

The study aimed to investigate the effect of an early intervention using human amniotic epithelial cell (hAEC) in a rat model of chronic obstructive pulmonary disease (COPD). Twenty-four specific pathogen-free Wistar rats were randomized to the control, COPD, and COPD+hAEC groups. COPD was established by intratracheal LPS injection combined with smoke fumigation over 30days. On the first day of model establishment rats in the AEC group also received intratracheal instillation of 500,000 hAECs isolated from the placenta of healthy donors. The mean linear intercept (MLI) and mean alveolar number (MAN) were used to assess the degree of lung emphysema. IL-8 was measured using a radioimmunoassay, surfactant protein D (SP-D) was measured by ELISA, and matrix metalloproteinase (MMP)2 and MMP8 expression was assessed by PCR. Smoke fumigation combined to LPS injection successfully established a COPD rat model with significant emphysema and airway inflammation, elevated MLI and MAN, elevated systemic and lung tissue levels of IL-8 and SP-D (P<0.05), and high expression of MMP2 and MMP8. Rats in the COPD+hAEC group exhibited alleviated lung damage, MLI and MAN (P<0.05), reduced systemic and lung tissue levels of IL-8 and SP-D (P<0.05) and MMP2 and MMP8 expression (P<0.05). Early intervention using hAECs could delay disease progression in rats with COPD.

One week's holiday sun exposure induces expression of photoaging biomarkers.

INTRODUCTION: Skin aging is accompanied by the upregulation of the expression of various matrix metalloproteinases (MMPs). It was shown that exposure to ultraviolet radiation (UVR) may induce skin expression of MMPs and dysregulation of the transforming growth factor beta (TGF-ß)/Smad pathway. The aim of our study was to compare the effects of short holiday UVR exposure and lifetime UVR exposure, on the expression of MMP-8, TGF-ß1, and Smad2 in human skin biopsies. MATERIAL AND METHODS: Skin biopsies were taken from the outer upper arm of 15 elderly people with significant photoaging (mean age 64.1 years) (Group 1) and from 15 healthy young adult volunteers (mean age 24.1 y) who participated in a six-day sun holiday. Biopsies were taken twice: 24 hours before leaving for holiday (Group 2a) and 24 hours after returning (Group 2b). The expression of TGF-ß1, Smad2, and MMP-8 was examined by immunochemistry and measured semiquantitatively by two independent pathologists. RESULTS: The mean expression of TGF-ß1 in dermal fibroblasts and keratinocytes in Group 1 and Group 2b was significantly lower than in Group 2a (0.54% ± 0.44% and 0.48% ± 0.51% vs. 1.48% ± 0.72%, respectively). The percentage of Smad2 (+) cells in Group 1 and Group 2b was lower than in Group 2a (2.13% ± 1.39% and 1.81% ± 1.16% vs. 4.13% ± 1.58%, respectively). The MMP-8 expression in Group 2b was 1.36% ± 0.68% and was significantly higher than in Group 1 (0.34% ± 0.42%) and Group 2a in which the protein was not detected (p < 0.001). CONCLUSIONS: We conclude that the decrease in the expression of TGF-ß1 and Smad2 is a persistent biomarker of skin photoaging, while the increased expression of MMP-8 in keratinocytes can be regarded as a marker of acute sun exposure.

Visfatin Destabilizes Atherosclerotic Plaques in Apolipoprotein E-Deficient Mice.

OBJECTIVES: Although there is evidence that visfatin is associated with atherogenesis, the effect of visfatin on plaque stability has not yet been explored. METHODS: In vivo, vulnerable plaques were established by carotid collar placement in apolipoprotein E-deficient (ApoE-/-) mice, and lentivirus expressing visfatin (lenti-visfatin) was locally infused in the carotid artery. The lipid, macrophage, smooth muscle cell (SMC) and collagen levels were evaluated, and the vulnerability index was calculated. In vitro, RAW264.7 cells were stimulated with visfatin, and the MMPs expressions were assessed by western blot and immunofluorescence. And the mechanism that involved in visfatin-induced MMP-8 production was investigated. RESULTS: Transfection with lenti-visfatin significantly promoted the expression of visfatin which mainly expressed in macrophages in the plaque. Lenti-visfatin transfection significantly promoted the accumulation of lipids and macrophages, modulated the phenotypes of smooth muscle cells and decreased the collagen levels in the plaques, which significantly decreased the plaque stability. Simultaneously, transfection with lenti-visfatin significantly up-regulated the expression of MMP-8 in vivo, as well as MMP-1, MMP-2 and MMP-9. Recombinant visfatin dose- and time-dependently up-regulated the in vitro expression of MMP-8 in macrophages. Visfatin promoted the translocation of NF-κB, and inhibition of NF-κB significantly reduced visfatin-induced MMP-8 production. CONCLUSIONS: Visfatin increased MMP-8 expression, promoted collagen degradation and increased the plaques vulnerability index.

Reciprocal activation between MMP-8 and TGF-ß1 stimulates EMT and malignant progression of hepatocellular carcinoma.

The efficiency of surgery in hepatocellular carcinoma (HCC) is limited due to metastasis and recurrence, but the molecular mechanisms are unclear. Here, we show that MMP-8 and TGF-ß1 accumulate in highly invasive HCC cell lines and invasive HCC patient tissues. Upregulation of MMP-8 and TGF-ß1 correlated with changes in cellular epithelial-mesenchymal transition (EMT) phenotypes and HCC migration and invasion. The expression of TGF-ß1 was markedly restored by MMP-8 overexpression in TGF-ß1-depleted HCC cells mainly via the activation of PI3K/Akt/Rac1 pathway. Similarly, the expression of MMP-8 was restored by TGF-ß1 treatment in MMP-8-depleted HCC cells mainly through the activation of the same PI3K/Akt/Rac1 pathway. MMP-8 expression was significantly related to TGF-ß1 expression in HCC patient tissues, and high expression of MMP-8 or TGF-ß1 was significantly associated with TNM stage and HCC metastasis. Specifically, patients with high co-expression of MMP-8 and TGF-ß1 had a shorter time-to-recurrence than those with low co-expression. Therefore, the reciprocal positive interplay between MMP-8 and TGF-ß1 contributes to HCC invasion and metastasis by inducing EMT mainly through the PI3K/Akt/Rac1 pathway.

Adenoviral delivery of truncated MMP-8 fused with the hepatocyte growth factor mutant 1K1 ameliorates liver cirrhosis and promotes hepatocyte proliferation.

Liver cirrhosis is a chronic liver disease caused by chronic liver injury, which activates hepatic stellate cells (HSCs) and the secretion of extracellular matrix (ECM). Cirrhosis accounts for an extensive level of morbidity and mortality worldwide, largely due to lack of effective treatment options. In this study, we have constructed a fusion protein containing matrix metal-loproteinase 8 (MMP-8) and the human growth factor mutant 1K1 (designated cMMP8-1K1) and delivered it into hepatocytes and in vivo and in cell culture via intravenous injection of fusion protein-harboring adenovirus. In doing so, we found that the cMMP8-1K1 fusion protein promotes the proliferation of hepatocytes, likely resulting from the combined inhibition of type I collagen secretion and the degradation of the ECM in the HSCs. This fusion protein was also observed to ameliorate liver cirrhosis in our mouse model. These changes appear to be linked to changes in downstream gene expression. Taken together, these results suggest a possible strategy for the treatment of liver cirrhosis and additional work is warranted.

EFFECT OF RELATIVE GENE EXPRESSION ON PLAQUE VULNERABILITY IN PATIENTS WITH STABLE ANGINA AND PATIENTS WITH ACUTE CORONARY SYNDROME.

This study was conducted to investigate the effect of relative gene expression on plaque vulnerability in patients with either stable angina or acute coronary syndrome (ACS). A total of 30 patients with ACS, 28 patients with stable angina and 17 healthy volunteers were selected. High resolution ultrasound was used to detect carotid arterial intima-media thickness (IMT) and plaque score, Sandwich enzyme linked immunoassay to determine the change of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1. The three groups had no statistically significant difference in age, gender, total cholesterol, triglyceride, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol. MMP-9, TIMP-1, MMP-9/TIMP-1 and IMT, total plaque score, soft plaque score and hard plaque score of patients’ acute coronary syndrome were obviously higher than those with stable angina and normal people. It was also found that MMP-9 was in a positive correlation with IMT, total and soft plaques score, TIMP-1 was positively correlated with IMT as was MMP-9/TIMP-1. Regardless of age, IMT was in a positive correlation with MMP-9, TIMP-1 and MMP-9/TIMP-1 in partial correlation analysis. All these findings suggest that ACS patients have remarkably higher MMP-9, 1TIMP-1, MMP- 9/TIMP-1, IMT, total plaque score, soft plaque score and hard plaque score compared to patients with stable angina pectoris and healthy subjects (P<0.05) and there are positive correlations between MMP- 9, TIMP-1, 1MMP-8/TIMP-1, total plaque and soft plaque score.

Screening of differentially expressed genes between multiple trauma patients with and without sepsis.

The purpose of this study was to identify critical genes associated with septic multiple trauma by comparing peripheral whole blood samples from multiple trauma patients with and without sepsis. A microarray data set was downloaded from the Gene Expression Omnibus (GEO) database. This data set included 70 samples, 36 from multiple trauma patients with sepsis and 34 from multiple trauma patients without sepsis (as a control set). The data were preprocessed, and differentially expressed genes (DEGs) were then screened for using packages of the R language. Functional analysis of DEGs was performed with DAVID. Interaction networks were then established for the most up- and down-regulated genes using HitPredict. Pathway-enrichment analysis was conducted for genes in the networks using WebGestalt. Fifty-eight DEGs were identified. The expression levels of PLAU (down-regulated) and MMP8 (up-regulated) presented the largest fold-changes, and interaction networks were established for these genes. Further analysis revealed that PLAT (plasminogen activator, tissue) and SERPINF2 (serpin peptidase inhibitor, clade F, member 2), which interact with PLAU, play important roles in the pathway of the component and coagulation cascade. We hypothesize that PLAU is a major regulator of the component and coagulation cascade, and down-regulation of PLAU results in dysfunction of the pathway, causing sepsis.

Induction of IL-6 and MMP-8 in human periodontal fibroblasts by static tensile strain.

OBJECTIVES: Mechanical loading is a potential activator of inflammation and able to stimulate factors for periodontal and alveolar bone destruction. Aim of this study was to investigate the inflammatory response and synthesis of proteinases by human periodontal ligament fibroblast (HPdLF) dependent on different strengths of static tensile strain (STS). MATERIALS AND METHODS: HPdLFs were loaded with different STS strengths (1, 5, and 10 %) in vitro. Gene expressions of cyclooxygenase (COX)-2 and interleukin (IL)-6 were analyzed by quantitative real-time polymerase chain reaction. Production of IL-6, prostaglandin E2 (PGE2), matrix metalloproteinase (MMP)-8, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were measured by enzyme-linked immunosorbent assay. Receptor activator of nuclear factor-kappa ligand (RANKL) synthesis was detected by immunocytochemical staining. RESULTS: Ten percent STS led to an increased gene expression of IL-6 and COX-2 (34.4-fold) in HPdLF, and 1 and 5 % STS slightly reduced the gene expression of IL-6. Synthesis of IL-6 was significantly reduced by 1 % STS and stimulated by 10 % STS. Ten percent STS significantly induced PGE2 production. RANKL was not detectable at any strength of STS. MMP-8 synthesis showed significantly higher values only at 10 % STS, but TIMP-1 was stimulated by 5 and 10 % STS, resulting into highest TIMP-1/MMP-8 ratio at 5 % STS. CONCLUSIONS: High-strength STS is a potent inducer of periodontal inflammation and MMP-8, whereas low-strength STS shows an anti-inflammatory effect. Moderate-strength STS causes the highest TIMP-1/MMP-8 ratio, leading to appropriate conditions for reformation of the extracellular matrix. CLINICAL RELEVANCE: Furthermore, this study points out that the strength of force plays a pivotal role to achieve orthodontic tooth movement without inducing periodontal inflammation and to activate extracellular matrix regeneration.

Proteome analysis reveals antiangiogenic environments in chronic wounds of diabetes mellitus type 2 patients.

In contrast to normal healing wounds, chronic wounds commonly show disturbances in proteins regulating wound healing processes, particularly those involved in cell proliferation and protein degradation. Multidimensional protein identification technology MS/MS was conducted to investigate and compare the protein composition of chronic diabetic foot exudates to exudates from split-skin donor sites of burn victims otherwise healthy. Spectral counting revealed 188 proteins differentially expressed (more than twofold and p-value <0.05) in chronic wounds. Most were involved in biological processes including inflammation, angiogenesis, and cell mortality. Increased expression of the inflammatory response stimulating S100 proteins, predominantly S100A8 and S100A9 (almost tenfold), was identified. Matrix metalloproteinases (MMPs) MMP1, MMP2, and MMP8 were identified to be elevated in chronic wounds with significant impact on collagen degradation and tissue destruction. Further, proteins with antiangiogenic properties were found at higher expression levels in chronic wounds. Reduced angiogenesis leads to drastic shortage in nutrition supply and causes increased cell death, demonstrated by Annexin A5 exclusively found in chronic wound exudates. However, excessive nucleic and cytosolic material infers cell death occurring not only by apoptosis but also by necrosis. In conclusion, mass spectrometric investigation of exudates from chronic wounds demonstrated dramatic impairment in wound repair with excessive inflammation, antiangiogenic environment, and accelerated cell death.

Matrix metalloproteinase 8 (collagenase 2) induces the expression of interleukins 6 and 8 in breast cancer cells.

Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.

Cigarette smoke-induced collagen destruction; key to chronic neutrophilic airway inflammation?

BACKGROUND: Cigarette smoking induces inflammatory responses in all smokers and is the major risk factor for lung disease such as chronic obstructive pulmonary disease (COPD). In this progressive disease, chronic inflammation in the lung contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). The generation of this tripeptide is mediated by a multistep pathway involving matrix metalloproteases (MMPs) 8 and 9 and prolyl endopeptidase (PE). Here we investigated whether cigarette smoke extract (CSE) stimulates human PMNs to breakdown whole matrix collagen leading to the generation of the chemotactic collagen fragment N-ac-PGP. METHODOLOGY/PRINCIPAL FINDINGS: Incubating PMNs with CSE led to the release of chemo-attractant CXCL8 and proteases MMP8 and MMP9. PMNs constitutively expressed PE activity as well as PE protein. Incubating CSE-primed PMNs with collagen resulted in collagen breakdown and in N-ac-PGP generation. Incubation of PMNs with the tripeptide N-ac-PGP resulted in the release of CXCL8, MMP8 and MMP9. Moreover, we tested whether PMNs from COPD patients are different from PMNs from healthy donors. Here we show that the intracellular basal PE activity of PMNs from COPD patients increased 25-fold compared to PMNs from healthy donors. Immunohistological staining of human lung tissue for PE showed that besides neutrophils, macrophages and epithelial cells express PE. CONCLUSIONS: This study indicates that neutrophils activated by cigarette smoke extract can breakdown collagen into N-ac-PGP and that this collagen fragment itself can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. MMP-, PE- or PGP-inhibitors can serve as an attractive therapeutic target and may open new avenues towards effective treatment of COPD.

Elevated levels of a disintegrin and metalloproteinase 8 in gingival crevicular fluid of patients with periodontal diseases.

BACKGROUND: A disintegrin and metalloproteinase 8 (ADAM8) is involved in inflammation and is essential for osteoclastogenesis. Elevated ADAM8 levels are detected in human serum and other body fluids in several inflammatory conditions. Therefore, we hypothesized that ADAM8 levels are also raised in gingival crevicular fluid (GCF) of patients with periodontal diseases. METHODS: Forty-five patients with periodontal diseases (n = 15 for each group: the group of patients with gingivitis, the group with aggressive periodontitis [AgP], and the group with chronic periodontitis [CP]) and 15 volunteers who exhibited healthy gingiva were recruited. Four periodontal parameters, gingival index, plaque index, probing depth, and clinical attachment level, were recorded before GCF collection. The presence of ADAM8 in GCF was shown by immunoblotting using anti-human ADAM8 polyclonal antibody against its prodomain, and the ADAM8 levels were measured by an enzyme-linked immunosorbent assay. RESULTS: Four immunoreactive bands at 120, 70, 50, and <30 kDa were detected in the groups of patients with periodontitis, whose intensities were stronger than those in the group of patients with gingivitis, consistent with significantly greater ADAM8 levels in both groups of patients, with either CP or AgP, than those in the group of patients with gingivitis and in the group that was healthy (P <0.001). Moreover, the ADAM8 levels correlated significantly with the four periodontal parameters (P <0.001), indicating that ADAM8 levels are positively associated with the degree of periodontal tissue inflammation and destruction. CONCLUSIONS: The ADAM8 levels are elevated in the GCF of patients with periodontal diseases, including gingivitis, CP, and AgP, in comparison to control participants who are healthy, and they correlate with four clinical parameters that reflect the degree of disease severity.

Overexpression of matrix metalloproteinase-9 is correlated with carotid intraplaque hemorrhage in a swine model.

OBJECTIVE: Carotid intraplaque hemorrhage may result in rapid worsening of stenosis and thrombus formation leading to stroke in patients with carotid atherosclerosis. The purpose of this study was to assess the association of the lesional expression of matrix metalloproteinase (MMP)-9 with carotid plaque and intraplaque hemorrhage in a swine model. METHODS: Carotid atherosclerosis was induced in miniswine using a combination of partial ligation and a high cholesterol diet. The carotid artery and rete mirabile were obtained for histopathological and immunohistochemical studies at 3 months. Atherosclerotic changes were classified by Stary stage according to the American Heart Association and the features of vulnerable carotid plaque were assessed. The association of MMP-9 expression in the carotid plaque with intraplaque hemorrhage was analyzed. RESULTS: One hundred and ninety-one carotid segments from 10 carotid artery models were assessed. Among 139 segments with atherosclerotic changes, 102 had advanced plaque (Stary stage IV-VI). Atheroemboli were found in all 10 rete mirabili, confirming the presence of vulnerable ipsilateral carotid plaques. There was a trend to increased MMP-9 expression in the group with advanced plaque. Areas positive for MMP-9 were significantly greater in plaques with intraplaque hemorrhage than in those without intraplaque hemorrhage (11.84±1.22% vs 6.63±0.59%, p<0.001). CONCLUSIONS: Increased expression of MMP-9 is associated with intraplaque hemorrhage in a swine model of vulnerable carotid atherosclerosis.

Multiplex array proteomics detects increased MMP-8 in CSF after spinal cord injury.

INTRODUCTION: A variety of methods have been used to study inflammatory changes in the acutely injured spinal cord. Recently novel multiplex assays have been used in an attempt to overcome limitations in numbers of available targets studied in a single experiment. Other technical challenges in developing pre-clinical rodent models to investigate biomarkers in cerebrospinal fluid (CSF) include relatively small volumes of sample and low concentrations of target proteins. The primary objective of this study was to characterize the inflammatory profile present in CSF at a subacute time point in a clinically relevant rodent model of traumatic spinal cord injury (SCI). Our other aim was to test a microarray proteomics platform specifically for this application. METHODS: A 34 cytokine sandwich ELISA microarray was used to study inflammatory changes in CSF samples taken 12 days post-cervical SCI in adult rats. The difference between the median foreground signal and the median background signal was measured. Bonferroni and Benjamini-Hochburg multiple testing corrections were applied to limit the False Discovery Rate (FDR), and a linear mixed model was used to account for repeated measures in the array. RESULTS: We report a novel subacute SCI biomarker, elevated levels of matrix metalloproteinase-8 protein in CSF, and discuss application of statistical models designed for multiplex testing. CONCLUSIONS: Major advantages of this assay over conventional methods include high-throughput format, good sensitivity, and reduced sample consumption. This method can be useful for creating comprehensive inflammatory profiles, and biomarkers can be used in the clinic to assess injury severity and to objectively grade response to therapy.