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.

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.

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.

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.

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.

Bone substitution materials on the basis of BONITmatrix® up-regulate mRNA expression of IGF1 and Col1a1.

The aim of this study was to investigate the effects of BONITmatrix(®) and OSSA NOVA on the expression of growth factors and osteogenic differentiation. For this purpose, the mRNA expression of VEGF, IGF1, IGF2, collagen-1, collagen-2 and MMP8 was analysed in surgically created defects on the crania of adult male rats. Cranial samples were collected after implantation of BONITmatrix(®) or OSSA NOVA scaffolds for 4 weeks and determinations of gene expression were performed by quantitative RT-PCR. Real-time RT-PCR analyses showed a significantly higher expression of IGF1 in both groups treated with BONITmatrix(®) and OSSA NOVA compared to untreated controls, whereas type I collagen mRNA expression only increased in BONITmatrix(®) treated rats compared to controls. No changes in transcript expression of IGF2, VEGF, collagen-2 and MMP8 were detectable between the analysed groups. In conclusion, BONITmatrix(®) and OSSA NOVA stimulate the expression of growth factor IGF1, but only the granular dosage form is able to stimulate osteoblast differentiation.

Effect of estrogen and altered diet hardness on the expression of estrogen receptor alpha and matrix metalloproteinase-8 in rat condylar cartilage.

AIMS: To examine the effect of decreased estrogen level and altered diet hardness on condylar cartilage morphology of the rat temporomandibular joint (TMJ) and on the expression of condylar cartilage estrogen receptor alpha (ERa) and matrix metalloproteinase-8 (MMP-8). METHODS: A total of 36 female rats was divided into four groups: ovariectomized rats fed a normal diet, non-ovariectomized controls fed a normal diet, ovariectomized rats fed a soft diet, and non-ovariectomized controls fed a soft diet. Ovariectomy was performed at the age of 60 days. Seven days after the operation, the rats were sacrificed. Repeated measures ANOVA and Duncan's multiple comparison tests were used for statistical analysis. RESULTS: The ovariectomized rats had thicker cartilage layers than the controls, both in the normal diet and soft diet groups. The thinnest cartilage layers were found in the control rats fed with the soft diet. The thickness of the chondroblastic layer was significantly higher (P < .001) in the normal-diet rats than in the soft-diet rats in both ovariectomized and non-ovariectomized groups. The thickness of the proliferative layer was significantly higher (P < .001) in the ovariectomized soft-diet rats than in the soft-diet control rats. The proportional amount of ERa was statistically significantly higher (P < .001) in the condylar cartilage of the ovariectomized rats than in the non-ovariectomized control rats both in the normal- and soft-diet groups. The proportional amount of ERa was statistically significantly higher (P < .001) in the ovariectomized normal-diet rats than in the ovariectomized soft-diet rats. The proportional number of MMP-8-positive cells was statistically significantly higher (P < .001) in the condylar cartilage of ovariectomized rats fed the soft diet than in non-ovariectomized control rats fed the soft diet. Control rats fed with the normal diet had a higher proportional amount of MMP-8 positive cells than control rats fed with the soft diet (P < .05). CONCLUSION: The rat TMJ condylar cartilage is sensitive to changes in estrogen levels and altered diet hardness.

Salivary interleukin-6, matrix metalloproteinase-8, and osteoprotegerin in patients with periodontitis and diabetes.

BACKGROUND: Diabetes and periodontitis produce a protein discharge that can be reflected in saliva. This study evaluates the salivary concentrations of interleukin (IL)-6, matrix metalloproteinase (MMP)-8, and osteoprotegerin (OPG) in patients with periodontitis with type 2 diabetes. METHODS: Whole saliva samples were obtained from 90 subjects who were divided into four groups: healthy (control; n = 22), untreated periodontitis (UPD; n = 24), diabetes mellitus (DM; n = 20), and UPD + DM (n = 24) groups. Clinical and metabolic data were recorded. Salivary IL-6, MMP-8, and OPG concentrations were determined by a standard enzyme-linked immunosorbent assay. RESULTS: The UPD and UPD + DM groups exhibited higher salivary IL-6 than the control and DM groups (P <0.01). The salivary MMP-8 concentrations in all diseased groups (UPD, DM, and UPD + DM) were higher than in the control group (P <0.01). The salivary OPG concentrations in the DM group were higher than in the UPD and control groups (P <0.05). In the UPD + DM group, salivary IL-6 was correlated with glycated hemoglobin (HbA1c) levels (r = 0.60; P <0.05). The regression analysis indicated that the number of remaining teeth, clinical attachment level, and IL-6 might have influenced the HbA1c levels in patients with diabetes. CONCLUSIONS: Salivary IL-6 concentrations were elevated in patients with periodontitis with or without diabetes. Salivary MMP-8 and OPG concentrations were elevated regardless of periodontal inflammation in patients with diabetes. Therefore, periodontitis and diabetes are conditions that may interfere with protein expression and should be considered when using saliva for diagnoses.

Matrix metalloproteinase-26 is present more frequently in squamous cell carcinomas of immunosuppressed compared with immunocompetent patients.

BACKGROUND: Skin cancers are the most frequent malignancies in organ transplant recipients (OTRs). Squamous cell carcinomas (SCCs) occur 65-250 times more frequently in OTRs and tend to be aggressive in behavior. Because matrix metalloproteinases (MMPs) have a central role in tumorigenesis and invasion, we investigated the epithelial and stromal MMP and tissue inhibitor of MMP (TIMP) expression profile in SCCs of immunosuppressed (IS) compared with immunocompetent (IC) patients to determine if differences could explain the more aggressive behavior of SCCs in OTRs. METHODS: Matched pairs from 20 SCCs of IS and IC patients were studied using immunohistochemistry for MMP-1, MMP-7, MMP-8, MMP-9, MMP-13 and MMP-26 and TIMP-1 and TIMP-3. RESULTS: Among all MMPs studied, only staining for MMP-26 was significantly more intense in cancer cells of the post-transplant group compared with the IC group (p = 0.01), whereas MMP-9 expression was more abundant in stromal macrophages surrounding SCCs of IC patients (p = 0.02). MMP-26 expression in cancer cells (p = 0.04) and that of MMP-9 in neutrophils (p = 0.005) were more abundant in SCCs of patients using cyclosporine. CONCLUSIONS: We conclude that MMP-26 and MMP-9 may contribute to the more aggressive behavior of SCCs in OTRs.

A trend of increase in periodontal interleukin-6 expression across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.

BACKGROUND AND OBJECTIVE: Epidemiological studies have established that patients with diabetes have increased prevalence and severity of periodontal disease. However, the periodontal expression of inflammatory cytokines and matrix metalloproteinases (MMPs) in diabetic patients has not been well characterized. The objective of this study was to determine the difference in the periodontal expression of MMP-1, MMP-8, interleukin-6, tumor necrosis factor-alpha and interleukin-1beta between diabetic and nondiabetic patients. MATERIAL AND METHODS: Periodontal tissue specimens were collected from nine nondiabetic patients without periodontal disease (group 1), from 11 nondiabetic patients with periodontal disease (group 2) and from seven diabetic patients with periodontal disease (group 3). The expression of MMP-1, MMP-8, interleukin-6, tumor necrosis factor-alpha and interleukin-1beta was quantified using real-time polymerase chain reaction. RESULTS: The nonparametric Kruskal-Wallis test showed that the difference in interleukin-6 expression among the groups was statistically significant (p = 0.04). Furthermore, the generalized Kruskal-Wallis nonparametric linear-by-linear association test showed a statistically significant trend of increase in the expression of interleukin-6 from group 1 to group 2 to group 3 (p = 0.02) and a suggestion of such a trend for MMP-1 (p = 0.05). No increase in MMP-8 expression was observed in patients in group 3 compared to patients in groups 1 and 2. Although the average expression levels of MMP-1, interleukin-1beta and tumor necrosis factor-alpha were increased from group 1 to group 3, the differences were not statistically significant. CONCLUSION: A trend of increased interleukin-6 expression in periodontal tissues was observed across patients with neither diabetes nor periodontal disease, patients with periodontal disease alone, and patients with both diseases.

Experimental tooth pain elevates substance P and matrix metalloproteinase-8 levels in human gingival crevice fluid.

OBJECTIVE: Tooth pain can induce a neurogenic inflammatory reaction in gingiva in association with local elevations of matrix metalloproteinase (MMP)-8, which is considered the major tissue destructive protease in gingival crevice fluid (GCF). The pro-inflammatory neuropeptides released by sensory nerves coordinate the activities of the immuno-effector cells and may influence the secretion of MMP-8. With this background, we studied whether experimental tooth pain can trigger changes in GCF levels of the neuropeptide substance P (SP) and MMP-8. MATERIAL AND METHODS: The GCF SP levels of stimulated and non-stimulated teeth were analyzed for SP using a competitive enzyme immunoassay (EIA). The GCF MMP-8 levels were determined by quantitative immunofluorometric assay (IFMA). RESULTS: Painful stimulation of the upper central incisor caused significant elevations in GCF SP and MMP-8 levels of the stimulated tooth. At the same time, the GCF SP and MMP-8 levels of non-stimulated control teeth were unchanged. CONCLUSIONS: These data indicate that experimental tooth pain can induce local elevations of SP and MMP-8 levels in GCF simultaneously. This supports the possibility of a local neurogenic spread of inflammatory reactions from intrapulpal to surrounding periodontal tissues.