Emma Pirilä

Local and systemic responses in matrix metalloproteinase 8-deficient mice during Porphyromonas gingivalis-induced periodontitis.

ABSTRACT Periodontitis is a bacterium-induced chronic inflammation that destroys tissues that attach teeth to jaw bone. Pathologically excessive matrix metalloproteinase 8 (MMP-8) is among the key players in periodontal destruction by initiating type I collagen degradation. We studied MMP-8 in Porphyromonas gingivalis-induced periodontitis by using MMP-8-deficient (MMP8−/−) and wild-type (WT) mice. Alveolar bone loss, inflammatory mediator expression, serum immunoglobulin, and lipoprotein responses were investigated to clarify the role of MMP-8 in periodontitis and systemic inflammatory responses. P. gingivalis infection induced accelerated site-specific alveolar bone loss in both MMP8−/− and WT mice relative to uninfected mice. The most extensive bone degradation took place in the P. gingivalis-infected MMP8−/− group. Surprisingly, MMP-8 significantly attenuated (P < 0.05) P. gingivalis-induced site-specific alveolar bone loss. Increased alveolar bone loss in P. gingivalis-infected MMP8−/− and WT mice was associated with increase in gingival neutrophil elastase production. Serum lipoprotein analysis demonstrated changes in the distribution of high-density lipoprotein (HDL) and very-low-density lipoprotein (VLDL) particles; unlike the WT mice, the MMP8−/− mice underwent a shift toward a smaller HDL/VLDL particle sizes. P. gingivalis infection increased the HDL/VLDL particle size in the MMP8−/− mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total lipopolysaccharide activity and the immunoglobulin G-class antibody level in response to P. gingivalis were significantly elevated in both infected mice groups. Thus, MMP-8 appears to act in a protective manner inhibiting the development of bacterium-induced periodontal tissue destruction, possibly through the processing anti-inflammatory cytokines and chemokines. Bacterium-induced periodontitis, especially in MMP8−/− mice, is associated with systemic inflammatory and lipoprotein changes that are likely involved in early atherosclerosis.

Expression and induction of collagenases (MMP-8 and -13) in plasma cells associated with bone-destructive lesions

Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone‐destructive lesions. This study examined the ability of immunoglobulin‐producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP‐8 and ‐13) in vivo and in vitro. Phorbol‐12‐myristate‐13‐acetate, interleukin‐6, and tumour necrosis factor‐α and heparin with the tumour promoter or cytokines potently enhanced (up to nine‐fold) MMP‐8 and ‐13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi‐quantitative reverse transcriptase‐polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP‐8 and ‐13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP‐8 and MMP‐13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP‐13 was more frequently expressed than MMP‐8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP‐13 has a particularly important role in benign and malignant bone‐destructive lesions. Copyright

In vivo collagenase‐2 (MMP‐8) expression by human bronchial epithelial cells and monocytes/macrophages in bronchiectasis

The purpose of this study was to determine whether other cellular sources than neutrophils can express matrix metalloproteinase (MMP)‐8 protein and mRNA in bronchiectatic (BE) lung. The molecular forms of MMP‐8 in the BE bronchoalveolar lavage fluid (BALF) and healthy control BALF were analysed by western immunoblotting. MMP‐8 expression was demonstrated by immunohistochemistry and in situ hybridization in BE lung tissue and by immunohistochemistry in control lung tissue. In the BE BALF, different MMP‐8 species were detected: 70–80 kD MMP‐8 apparently of polymorphonuclear leukocyte (PMN) origin and also 40–60 kD MMP‐8 from non‐PMN cellular sources, such as bronchial epithelial cells, glandular cells or monocytes/macrophages. Both of these MMP‐8 species were elevated and converted to a significant extent to activated forms in BE BALF compared with healthy control BALF. The levels of high molecular weight (>80 kD) MMP‐8 complexes, evidently representing MMP‐8 trapped by endogenous MMP inhibitors and/or MMP‐8 dimers, were significantly elevated in BE BALF compared with healthy control BALF. In BE lung tissue, the MMP‐8 protein and mRNA expression was found in bronchial ciliated epithelial cells, glandular cells, neutrophils, and monocytes/macrophages infiltrating the bronchial epithelial area. Minimal MMP‐8 expression was observed in neutrophils, monocytes/macrophages, and epithelial cells in control lung tissues. In this study, new potential cellular sources have been demonstrated for MMP‐8 in the inflamed lung. MMP‐8 from multiple cellular sources, including inflamed lung epithelium, was activated to a significant extent in the BE BALF, indicating a major role for MMP‐8 in the destruction of lung and bronchial tissues. Copyright

Wound Healing in Ovariectomized Rats Effects of Chemically Modified Tetracycline (CMT-8) and Estrogen on Matrix Metalloproteinases -8, -13 and Type I Collagen Expression

Cutaneous wound healing is a complex process involving interactions of various cell types. Skin, in addition to certain other organs, is dependent on estrogen; and estrogen-deficiency is associated with impaired wound healing. Wound healing involves the action of collagenolytic matrix metalloproteinases (MMPs). We investigated the expression and localization of collagenolytic MMPs -8 and -13 by collagenase activity assay, Western immunoblot analysis, in situ hybridization and immunohistochemical staining as well as type I collagen by hydroxyproline content analysis and immunohistochemical staining in cutaneous wounds from aged Sham and ovarioectomized (OVX) rats. After wounding, OVX rats were treated with either placebo, chemically modified tetracycline-8 (CMT-8) or estrogen. We found that MMP-8 and MMP-13 mRNA were expressed in wound epithelium of all samples examined as evidenced by in situ hybridization. Type I collagen, which was abundant in all groups examined, was decreased in OVX rats, but was increased by both CMT-8 and estrogen treatments to the level of Sham group. Hydroxyproline analysis revealed similar results. Western blot data showed that all forms of MMP-8 and MMP-13 were clearly reduced in the CMT-8 treated group compared to OVX. Analysis of collagenolytic activity confirmed the decreased collagenolysis in skin wound extracts from CMT-treated rats when compared with skin wound extracts from OVX rats. Our results show for the first time that MMP-8 mRNA and protein are expressed in rat wound epithelium. We further show that CMT-8 and estrogen have a beneficial effect on skin wound healing in OVX rats by increasing the collagen content and reducing the MMP-mediated collagenolysis.

Gelatinase A (MMP-2), Collagenase-2 (MMP-8), and Laminin-5 γ2-Chain Expression in Murine Inflammatory Bowel Disease (Ulcerative Colitis)

Dextran sulfate sodium-induced inflammatory bowel disease in mice resembles human ulcerative colitis. In inflammatory bowel diseases matrix metalloproteinases contribute to tissue degradation. Laminin-5 is an anchoring filament protein in the basement membrane area that can be cleaved by matrix metalloproteinases. We investigated the expression of matrix metalloproteinases-2 and -8 and laminin-5 γ2-chain in dextran sulfate sodium-induced mice by immunohistochemistry and in situ hybridization. Matrix metalloproteinase-8 expression was evidenced in the colon surface epithelial cells and the protein was more abundant in dextran sulfate sodium-induced mice colon. Matrix metallproteinase-2 and laminin-5 γ2-chain colocalized in the colon surface epithelial cells and in the basement membrane zone as demonstrated by double immunostaining. In dextran sulfate sodium-induced colon, matrix metalloproteinase-2 immunoreactivity was detected in epithelial cells in the lower parts of the crypt and surrounding the degraded crypts. Matrix metalloproteinase-2 and -8 could participate in the local epithelial inflammatory processes and tissue destruction. The presence of laminin-5 γ2-chain indicates alternative anchoring mechanisms in the colon, a compartment devoid of hemidesmosomes.

Soluble membrane‐type 1 matrix metalloproteinase (MT1‐MMP) and gelatinase A (MMP‐2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis

The aim of this study was to investigate the involvement of the MT1‐MMP/MMP‐2 cascade in induced sputum (IS) and bronchoalveolar lavage fluid (BALF) from bronchial asthma (BA) and bronchiectasis (BE) patients and healthy controls. The molecular forms and cellular origins of MT1‐MMP and MMP‐2 were determined by Western immunoblotting, immunohistochemistry and in situ hybridization. Elevated levels of soluble activated and autocatalyzed MT1‐MMP species as well as activated forms of MMP‐2 in IS and BALF samples from BA and BE patients were evidenced. The activation degrees of soluble MT1‐MMP and MMP‐2 were significantly correlated in BA and BE IS and BALF. Only low levels of both these MMPs were observed in healthy control IS and BALF. The co‐expression of MMP‐2 with MT1‐MMP was evidenced by double immunostaining in bronchial epithelial cells, submucosal glandular cells, smooth muscle cells and monocyte/macrophages. The MT1‐MMP/MMP‐2 cascade is present and active in human inflammatory lung disease fluid and tissue samples. This cascade seemingly reflects the active destructive phases of these chronic lung diseases.

Chemically modified tetracycline (CMT-8) and estrogen promote wound healing in ovariectomized rats: Effects on matrix metalloproteinase-2, membrane type 1 matrix metalloproteinase, and laminin-5 γ2-chain

Estrogen deficiency is associated with impaired cutaneous wound healing. Remodeling of the extracellular matrix in wound healing involves the action of matrix metalloproteinases on basement membrane zone components, especially laminin‐5. We studied the effects of estrogen and a potent matrix metalloproteinase inhibitor, chemically modified non‐antimicrobial tetracycline, CMT‐8, on wound healing in ovariectomized rats. At the tissue level, laminin‐5 γ2‐chain expression was decreased and the migration‐inductive 80 kDa form of laminin‐5 γ2‐chain was absent in ovariectomized rats when compared with sham and CMT‐8‐ or estrogen‐treated ovariectomized animals as detected by Western blotting. The highest levels of gelatinolytic activity (matrix metalloproteinase‐2 and ‐9) were found in sham animals. Levels were reduced in ovariectomized rats and were lowest after treating ovariectomized rats with CMT‐8 or estrogen as analyzed by functional activity assay and zymography. The total amount of membrane type 1‐matrix metalloproteinase was unchanged in all groups. We conclude that CMT‐8 and estrogen can promote wound healing in ovariectomized rats, not only by normalizing wound bed total collagen content and structure, but also by recovering the expression and processing of key molecules in wound healing, i.e., laminin‐5 γ2‐chain. This study shows, for the first time, the role of estrogen and CMT‐8 in laminin‐5 γ2‐chain modulation in vivo.

A novel and selective membrane type-1 matrix metalloproteinase (MT1-MMP) inhibitor reduces cancer cell motility and tumor growth.

Matrix metalloproteinases (MMPs), and especially membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), play a role in cancer progression and can have a prognostic value. Various synthetic broad-spectrum MMP inhibitors have been developed but have had little success in cancer patient treatment owing to side effects. Until recently, selective targeting of specific MMPs has not been possible due to lack of specific inhibitors. Here we have developed a selective MT1-MMP peptideinhibitor GACFSIAHECGA, which did not affect the activities of many other MMPs including MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, -13, -15, -17 or -20. In a fluorescent peptide cleavage assay it displayed an IC50 value of 150 μM. The peptide effectively inhibited the migration and invasion of cancer cell lines in vitro. Furthermore, in vivo the peptide reduced the growth of tongue carcinoma xenografts and prolonged the survival of mice. Overall these results suggest that selective MT1-MMP inhibitors may have utility as anticancer agents.

Overexpression of TIMP-1 under the MMP-9 promoter interferes with wound healing in transgenic mice

We have generated transgenic mice harboring the murine matrix metalloproteinase 9 (MMP-9) promoter cloned in front of human TIMP-1 cDNA. The transgenic mice were viable and fertile and exhibited normal growth and general development. During wound healing the mice were shown to express human TIMP-1 in keratinocytes that normally express MMP-9. However, the healing of skin wounds was significantly retarded with slow migration of keratinocytes over the wound in transgenic mice. In situ zymography carried out on wound tissues revealed total blockage of gelatinolytic activity (i.e., MMP-9 and MMP-2). The results confirm studies with MMP-9 knockout mice showing that MMP-9 is not essential for general development, but they also demonstrate an important role of keratinocyte MMP-9, as well that of other keratinocyte MMPs that are inhibited by TIMP-1, in wound healing. The transgenic mice generated in this study provide a model for the role of MMPs in MMP-9-producing cells in other challenging situations such as bone fracture recovery and cancer invasion.

Wound healing in aged normal and ovariectomized rats: effects of chemically modified doxycycline (CMT-8) on MMP expression and collagen synthesis.

Skin as well as bone is an organ dependent on estrogen. Skin thickness has been correlated with bone density and hormonal parameters. 1 Recently Ashcroft et al. 2 reported a marked delay in repair of acute incisional wounds in ovariectomized young female rats. The effect was reversed by topical estrogen, suggesting that both the rate and quality of wound healing can be modulated. Tetracycline and its nonantimicrobial derivative, CMT-8, are able to inhibit bone loss in ovariectomized rats (for review see Sasaki et al. 3 ). A recent study by Ramamurthy et al. 4 has shown enhanced wound healing in diabetic rats with topically applied CMT-2. In this study we describe the effect of oral administration of CMT-8 or estrogen on the healing of skin wounds in aged female rats, 120 days post ovariectomy (OVX).