Cytokine and matrix metalloproteinase expression in fibroblasts from peri-implantitis lesions in response to viable Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: To assess inflammatory reactions of fibroblasts in the pathophysiology of peri-implantitis, we compared the pro-inflammatory and matrix-degrading responses of gingival and granulation tissue fibroblasts from periodontally healthy controls, peri-implantitis, and periodontitis lesions to an in vitro challenge with Porphyromonas gingivalis. METHODS: Fibroblasts from periodontally healthy, peri-implantitis and periodontitis donors were challenged with viable P. gingivalis. The inflammatory reactions of fibroblasts were analyzed before and after 6 h P. gingivalis challenge, and 2.5 and 18 h after removal of the challenge. Gene expression and induction of pro-inflammatory mediators, and matrix metalloproteinases (MMPs) were assessed by real-time polymerase chain reaction. Protein expression was measured by enzyme-linked immunosorbent assay. RESULTS: Non-challenged fibroblasts from peri-implantitis and periodontitis lesions expressed higher levels of interleukin (IL)-1ß, IL-8, and monocyte chemotactic protein (MCP)-1 than fibroblasts from periodontally healthy individuals. The P. gingivalis challenge induced expression of IL-1ß, IL-8, IL-6, MCP-1, and MMP-1 in periodontitis and peri-implantitis fibroblasts, but not in fibroblasts from periodontally healthy individuals. MMP-8 expression was higher in non-challenged peri-implantitis fibroblasts than in fibroblasts from periodontally healthy individuals. However, the P. gingivalis challenge downregulated MMP-8 gene expression in peri-implantitis fibroblasts. After removal of the P. gingivalis challenge, peri-implantitis fibroblasts sustained higher induction of IL-1ß, MCP-1, and MMP-1 compared to periodontitis fibroblasts. CONCLUSIONS: Fibroblasts from peri-implantitis and periodontitis lesions gave a more pronounced inflammatory response to the P. gingivalis challenge than fibroblasts from healthy donors. They may therefore be involved in the development of inflammation in peri-implantitis and periodontitis. Moreover, the sustained upregulation of inflammatory mediators and MMP-1 in peri-implantitis fibroblasts may play a role in the pathogenesis of peri-implantitis.

Defective granulation tissue formation in mice with specific ablation of integrin-linked kinase in fibroblasts - role of TGFß1 levels and RhoA activity.

Wound healing crucially relies on the mechanical activity of fibroblasts responding to TGFß1 and to forces transmitted across focal adhesions. Integrin-linked kinase (ILK) is a central adapter recruited to integrin ß1 tails in focal adhesions mediating the communication between cells and extracellular matrix. Here, we show that fibroblast-restricted inactivation of ILK in mice leads to impaired healing due to a severe reduction in the number of myofibroblasts, whereas inflammatory infiltrate and vascularization of the granulation tissue are unaffected. Primary ILK-deficient fibroblasts exhibit severely reduced levels of extracellular TGFß1, α-smooth muscle actin (αSMA) production and myofibroblast conversion, which are rescued by exogenous TGFß1. They are further characterized by elevated RhoA and low Rac1 activities, resulting in abnormal shape and reduced directional migration. Interference with RhoA-ROCK signaling largely restores morphology, migration and TGFß1 levels. We conclude that, in fibroblasts, ILK is crucial for limiting RhoA activity, thus promoting TGFß1 production, which is essential for dermal repair following injury.

Expression of matrix metalloproteinases during rat skin wound healing: evidence that membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase A.

Skin wound healing depends on cell migration and extracellular matrix remodeling. Both processes, which are necessary for reepithelization and restoration of the underlying connective tissue, are believed to involve the action of extracellular proteinases. We screened cDNA libraries and we found that six matrix metalloproteinase genes were highly expressed during rat skin wound healing. They were namely those of stromelysin 1, stromelysin 3, collagenase 3, gelatinase A (GelA), gelatinase B, and membrane type-1 matrix metalloproteinase (MT1-MMP). The expression kinetics of these MMP genes, the tissue distribution of their transcripts, the results of cotransfection experiments in COS-1 cells, and zymographic analyses performed using microdissected rat wound tissues support the possibility that during cutaneous wound healing pro-GelA and pro-gelatinase B are activated by MT1-MMP and stromelysin 1, respectively. Since MT1-MMP has been demonstrated to be a membrane-associated protein (Sato, H., T. Takino, Y. Okada, J. Cao, A. Shinagawa, E. Yamamoto, and M. Seiki. 1994. Nature (Lond.). 370: 61-65), our finding that GelA and MT1-MMP transcripts were expressed in stromal cells exhibiting a similar tissue distribution suggests that MT1-MMP activates pro-GelA at the stromal cell surface. This possibility is further supported by our observation that the processing of pro-GelA to its mature form correlated to the detection of MT1-MMP in cell membranes of rat fibroblasts expressing the MT1-MMP and GelA genes. These observations, together with the detection of high levels of the mature GelA form in the granulation tissue but not in the regenerating epidermis, suggest that MT1-MMP and GelA contribute to the restoration of connective tissue during rat skin wound healing.

Up-regulation of matrix metalloproteinase-2 and membrane-type 1-matrix metalloproteinase were coupled with that of type I procollagen in granulation tissue response after the onset of aortic dissection.

The pathophysiological significance of matrix metalloproteinases (MMPs) in aortic dissection remains poorly understood. The purpose of the present study is to clarify the significance of MMPs in aortic dissection. The activities and distributions of MMP-2, membrane-type 1-MMP (MT1-MMP), and MMP-9 were evaluated by gelatin zymography, immunohistochemistry, and in situ hybridization in 29 patients and seven autopsy cases. To assess if these MMPs are related to a tissue remodeling process, we compared the expression of these MMPs with that of type I procollagen and platelet-derived growth factor receptor beta chain (PDGF Rbeta). Patients were divided into three groups based on histological findings: acute, intermediate, and healed groups. The most remarkable changes were observed in the intermediate group, in which MMP-2 activity peaked and tissue expression of mRNAs for MMP-2 and MT1-MMP were observed in spindle-shaped cells in the neointima, organizing thrombus, and the adventitia. These expression patterns were essentially coupled with those of type I procollagen mRNA and PDGF-Rbeta protein. The association of MMP-2, MT1-MMP, type I procollagen, and PDGF-Rbeta suggests that MMP-2 and MT1-MMP could be involved not only in the degradation of aortic tissue but also in tissue remodeling, which may be associated with the healing process.

Deficiency of ascorbic acid in experimental diabetes. Relationship with collagen and polyol pathway abnormalities.

The plasma and tissue concentration of ascorbic acid (AA) is reduced in diabetes. This study was designed to investigate the mechanism and significance of this phenomenon. The low plasma AA concentration of diabetic rats can be normalized by dietary AA supplement (20-40 mg/day), a dosage approximately equal to the maximal synthetic rate of this substance in the rats. Treatment of diabetic rats with this regime prevented the decrease in activity of granulation tissue prolyl hydroxylase (PRLase), an AA-dependent enzyme required for maintaining the normal properties of collagen. The decreased plasma AA concentration and granulation tissue PRLase activity in diabetes can also be normalized by the aldose reductase inhibitor tolrestat. We conclude that in diabetic animals there is a true deficiency of AA that may be responsible for some of the changes of collagen observed in diabetes. Treatment with AA or an aldose reductase inhibitor may prevent some of the diabetic complications with underlying collagen abnormalities.

Dietary glycine inhibits angiogenesis during wound healing and tumor growth.

In this study we investigated the effects of glycine on angiogenesis during embryogenesis, wound healing and tumor growth. In chorioallantoic membrane (CAM) assay, glycine (100 microM) inhibited angiogenesis by more than 50%. We studied dietary glycine's effect on fibrin induced wound healing response in a novel (Fibrin Z-chamber) assay. Fibrin within the chamber triggers the healing cascade leading to formation of granulation tissue (GT) rich in blood vessels and stroma. GT was reduced by more than 30% (p < 0.0001) in dietary Glycine groups as compared to control. We found that microvessel density dropped significantly (15%, p < 0.0003) with dietary glycine whereas the other components of GT were unaffected. We evaluated tumor growth delay utilizing Tumor Z-Chamber (fibrin with R3230 mammary adenocarcinoma cells) since tumors take advantage of angiogenesis and matrix formation. We observed that tumor growth decreased by 15% (p < 0.03) and tumor microvessel density dropped by 20% (p < 0.03) with dietary glycine compared to controls. We found that iNOS protein levels were decreased significantly in both GT (24%-57%) and tumor tissue (19-75%). In conclusion, we found that dietary glycine is a potent anti-angiogenic agent that can reduce wound healing and tumor growth through reduction of iNOS expression.

Inactivation of substance P by granulation tissue-derived gelatinase.

An active gelatinase has been purified from the conditioned medium of granulation tissue culture formed by carrageenin injection in rats. The purified gelatinase gave a single band corresponding to a M(r) of 57 kDa on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-gelatin PAGE. The granulation tissue-derived gelatinase selectively cleaved the Gln6-Phe7 bond of substance P (SP) with a Km of 0.17 mM and a Vmax of 0.027 nmol SP7-11/min/micrograms protein, resulting in the generation of biologically inactive fragments, SP1-6 and SP7-11. Our data suggest that the gelatinase produced by granulation tissue participates in the inactivation of SP in the inflammatory site.

Expression and activity of matrix metalloproteinase-2 and -9 in experimental granulation tissue.

The restoration of functional connective tissue is a major goal of the wound healing process which is probably affected by matrix-modifying enzymes. To evaluate the spatial and temporal expression of matrix metalloproteinases (MMP) MMP-2 and MMP-9 and to study the regulation of MMP-2 in wound healing, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation for up to 3 months. MMP-2 mRNA expression was seen throughout the experiment and it was highest after 2 months. MMP-9 gene expression was low between days 8-21 and increased after 4 weeks of granulation tissue formation. Membrane-type 1 MMP (MT1-MMP) mRNA was upregulated early and tissue inhibitor 2 of MMP (TIMP-2) mRNA later during wound healing. In in situ hybridization the expression of MMP-2 mRNA was seen mostly in fibroblast-like cells and MMP-9 mRNA in macrophage-like cells. MMP-9 immunoreactivity was detected in the polymorphonuclear leukocytes and macrophage-like cells on days 3-8. MMP-9 proteolytic activity was observed only on days 3-8. The active form of the MMP-2 increased up to day 14, whereafter it remained at a constant level, whereas latent MMP-2 did not show any apparent changes during the experimental period. We conclude that MMP-2 is important during the prolonged remodelling phase, whereas the gelatinolytic activity of MMP-9 was demonstrated only in early wound healing, and the MMP-9 gene is upregulated when the granulation tissue matures.

Nitric oxide synthase isoform expression in a porcine model of granulation tissue formation.

BACKGROUND: This study was designed to determine whether the nitric oxide (NO) pathway is involved in wound granulation tissue formation. METHODS: A section of the pig abdominal wall (excluding the skin) was excised, creating an incisional hernia. The resulting defect was repaired with silicone sheeting in a manner that mimics a temporary abdominal wall closure. During the 14-day experimental period, porcine omentum adhered to the peritoneal edges of the defect and a highly vascularized granulation tissue formed on both sides of the sheeting. Granulation tissue thickness and wound fluid volume were monitored by ultrasonography and epigastric artery flow velocity was monitored by color Doppler flow analysis at days 2, 4, 7, 9, 11, and 14. Fluid was serially harvested from the wound compartment at days 2, 4, 7, 9, 11, and 14 for nitrite/ nitrate (NOx) analysis. Finally, granulation tissue was harvested at day 14 for immunohistochemical and molecular analyses. RESULTS: There was a significant increase in granulation tissue thickness and wound fluid volume during the 14-day study period. Blood flow to the wound increased significantly by day 4 and returned toward baseline by day 14. Wound fluid NOx levels significantly increased from days 7 to 11 and then decreased to near baseline values by day 14. Wound fluid arginine levels significantly decreased when compared with peritoneal fluid and plasma levels at day 14, while wound fluid ornithine levels significantly increased. Immunohistochemical analysis of granulation tissue at day 14 revealed nitric oxide synthase (NOS) 2 was present in the majority of the cells in the granulation tissue. NOS 3 was expressed in endothelial cells only, and NOS 1 expression was not observed in the granulation tissue. CONCLUSIONS: This study suggests that NO, NOS 2, and arginine may play critical roles in granulation tissue formation and wound healing. Arginase and NOS 2 may compete for available arginine as a substrate, thereby limiting later NO production in favor of sustained ornithine synthesis.

Possible participation of cyclooxygenase-2 in the recurrence of allergic inflammation in rats.

In the recurrence of allergic inflammation in a rat air pouch model, pouch fluid volume, prostaglandin E2 concentration in the pouch fluid, leukocyte infiltration into the pouch fluid, and granulation tissue weight were markedly increased by the antigen challenge. To clarify the role of cyclooxygenase-2 in the recurrence of allergic inflammation, the time-course of changes in protein levels of cyclooxygenase-1 and cyclooxygenase-2 in the granulation tissue and in the infiltrated leukocytes was examined by Western blot analysis. It was shown that cyclooxygenase-1 levels in the granulation tissue and in the infiltrated leukocytes were not changed by the antigen challenge, but cyclooxygenase-2 levels were increased. Furthermore, treatment with the selective cyclooxygenase-2 inhibitor, NS-398 ([N-2(cyclohexyloxy-4-nitrophenyl]-methanesulfonamide), suppressed the recurrence of allergic inflammation as did the non-selective cyclooxygenase-1/cyclooxygenase-2 inhibitor, indomethacin. The steroidal anti-inflammatory drug, dexamethasone, inhibited the induction of cyclooxygenase-2, and suppressed the allergic inflammation. These findings strongly suggested that cyclooxygenase-2 induced by the antigen challenge plays a role in the recurrence of inflammation induced by the allergic mechanism.

Separation and properties of rabbit buccal mucosal wound hyaluronidase.

This study establishes the existence of a mammalian buccal mucosal wound hyaluronidase (hyaluronate 4-glycohydrolase; EC 3.2.1.35) having properties distinct from those of the endogenous lysosomal hyaluronidase of normal (uninjured) buccal mucosa. A time-dependent change in hyaluronidase activity was measured, with the highest specific activity occurring on post-wound day 4 (7.7 +/- 1.3 units/mg protein), followed by consecutive decreases until activity was no longer discernible by day 21. Mucosal wound hyaluronidase closely resembled a previously described integumentary wound endoglycosidase in terms of a high pH optimum (5.0-6.0), distinct (but non-exclusive) substrate preference for hyaluronic acid, and ability to generate saturated depolymerization products by an endoglycosidic hydrolysis.

Expression of N-acetyltransferases in periodontal granulation tissue.

Smoking is a major risk of periodontal diseases. At the site of first contact, the gingiva is exposed to aromatic amines and polycyclic hydrocarbons. These are metabolized by the N-acetyltransferases (NAT), leading to local detoxification and/or activation reactions contributing to the risk of periodontal destruction in smokers. The purpose of this study was to detect the expression of N-acetyltransferase isoenzymes NAT1 and NAT2 in periodontal granulation tissue. In 24 specimens obtained from periodontitis patients or control subjects, mRNA encoding for NAT1 and NAT2 was detected by RT-PCR, and proteins were identified by immunohistochemistry. In periodontal granulation tissues, immunoreactivity for NAT1 and NAT2 was detected in infiltrating leukocytes and fibroblasts. In normal gingiva, both enzymes were found in epithelial cells, whereas NAT1 was also detected in endothelial cells. The results suggest that these enzymes may play a role in the defense against xenobiotics and the accelerated progression of periodontal disease in smokers.

Hyaluronidase activity of rabbit skin wound granulation tissue fibroblasts.

The objective of this work was to identify and compare hyaluronidase activities of normal dermal and dermal wound granulation tissue fibroblasts. Direct evidence of the fibroblast as a source of tissue hyaluronidase was obtained. Fourth passage rabbit dermal fibroblasts were harvested on culture days 4, 8, 14, 18, and 22. Hyaluronidase activity and [35S]-sulfate- or [3H]-glucosamine-labeled glycosaminoglycans (GAGs) were monitored. Hyaluronidase assays were performed on medium and cellular fractions at the designated intervals. Enzyme activity of cellular fractions for both normal dermal and 14-day post-wound granulation tissue fibroblasts increased progressively through culture day 8. Thereafter (days 14-22), an eight-fold drop in cellular activity was coupled with cell death and emergence of hyaluronidase activity in medium fractions. Marked increases in degradation of secreted matrix components were concurrent with lysis-induced release of hyaluronidase. In this culture system, hyaluronidase activity was confined exclusively to cellular fractions and was released into the medium only under non-physiological conditions conducive to cellular death and lysis. Accordingly, this work suggests that previously reported skin wound hyaluronidases may be of fibroblastic origin and that susceptible GAGs are not degraded extracellularly, but, rather, must be internalized as a prerequisite to depolymerization.

Prolyl hydroxylase activity in human normal skins and post-burn scars.

Post-burn granulation tissues showed an extraordinary high prolyl hydroxylase (EC 1.14.11.2; proline, 2-oxoglutarate dioxygenase) activity, 25 to 50 times higher than the mean value of human normal skins from the frontal thighs of 15 subjects, 385 +/- 247 (S.D.) cpm/min/g of wet weight tissue. The activity in the scars decreased sharply within 4 to 5 months, and then gradually decreased to the normal range after 2 years or so. Well-aged scars tended to show lower values than the mean value of normal skins.

Fluid secretion in arachnoid cysts as a clue to cerebrospinal fluid absorption at the arachnoid granulation.

The morphological similarity of the lining of arachnoid cysts to subdural neurothelium and the mesothelium of arachnoid granulations suggested that the latter tissues might be the origin of arachnoid cysts. Transport Na+-K+-adenosine triphosphatase was shown by enzyme ultracytochemistry to be an indication of secretory activity in the lining of arachnoid cysts and in the endothelial lining of arachnoid granulations. This secretory activity suggests the existence of a biochemical mechanism for cerebrospinal fluid absorption at these granulations separate from the mechanisms already demonstrated.

Fibroblast heterogeneity of signal transduction mechanisms to complement-C1q. Analyses of calcium mobilization, inositol phosphate accumulation, and protein kinases-C redistribution.

Fibroblasts of healthy and granulation gingiva are phenotypically heterogeneous with regard to binding C1q collagen-like (cC1qR) or C1q globular-heads (gC1qR) regions, respectively. Here, isolated fibroblast subsets, expressing either the cC1qR or the gC1qR phenotype, were stimulated with C1q, and assessed for changes in cytosolic free calcium [Ca2+]i, accumulation of inositol trisphosphate (IP3), and redistribution of Ca2+-dependent protein kinases-C (cPKCs) from cytosol to membranes. Changes in [Ca2+]i were determined using Indo-1 fluorescence in combination with adhering cell analysis and sorting (ACAS) cytometry. Accumulation of IP3 was quantified using a competitive radioreceptor binding assay. Redistribution of cPKCs was evaluated by immunoblotting with antibodies to PKCalpha/betaI-betaII/gamma. Subsets manifested different fluctuations in [Ca2+]i levels 20 seconds after C1q-stimulation in the presence of millimolar concentrations of external calcium. Whereas cC1qR fibroblasts responded with a 38% over baseline [Ca2+]i increase which was sustained for 20 to 30 minutes, gC1qR fibroblasts responded with a higher (264% over baseline) and more rapid (2 to 3 minutes) transient. Likewise, subsets exhibited different kinetics of IP3 accumulation. Whereas cC1qR fibroblasts responded with an IP3 increase of 32 +/- 3 pmol/10(4) cells over baseline after 5 seconds stimulation, gC1qR fibroblasts responded after 15 to 20 seconds with a lower increase (13 +/- 0.8 IP3 pmol/10(4) cells over baseline). Subsets differed in cPKCs redistribution which peaked in gC1qR-membranes 30 seconds after stimulation and remained sustained between 10 and 30 minutes. No cPKC redistribution was detectable in stimulated cC1qR-cells. We conclude that fibroblasts are heterogeneous in phosphoinositide-Ca2+ signaling and cPKC redistribution to C1q, and suggest that these differences may affect activities of normal and granulation gingiva.

Cathepsin activity in cholesteatoma.

Collagenolytic cathepsin, presumed to play an important role in bone destruction of cholesteatoma, was investigated in cholesteatoma epithelium, subepithelial granulation tissue, skin from the bony external auditory meatus and, temporal bone. The enzyme extracted from tissues was proven to be lysosomal cathepsin B by SDS gel electrophoresis in the use of human type I and type III collagen. alpha-N-benzoyl-DL-arginine-2-naphthylamide HCl (BANA) was supposed to be specific for cathepsin B, and so BANA-hydrolase activity was measured as collagen-degrading cathepsin. The results showed that tissues had cathepsin B with its optimal pH 6.0, and that cathepsin B activity revealed a significant increase in the subepithelial granulation tissue. A strong activity of acid phosphatase found in the subepithelial granulation tissue seems to reflect the existence of an active metabolism of substances in the granulation tissue. These findings suggest that collagen is resorbed in the subepithelial granulation tissue in the presence of cholesteatoma.

[Activity of phosphodiesterases in granulation tissue treated with electron irradiation -- experimental study on rats]. / Atividade das fosfodiesterases em tecido de granulação submetido a irradiação de elétrons--estudo experimental em ratos.

The present study evaluated the effect of low doses of electron radiation on the activity of phosphodiesterases in granulation tissue. In order to induce growth of granulation tissue, a PVC sponge disk was introduced under the dorsal skin of 84 Wistar rats. The rats were divided in two groups, control and irradiated. The enzymatic activity was evaluated according to the evolution of the granulation tissue after 5, 7, 10, 14, 17, 20 and 24 days. Irradiation was carried out 3 days after the implantation of the sponge, by means of a linear accelerator, with energy of 6 MeV, and dose of 1.0 Gy. The results of this study showed that 5'-nucleotidase and ATPase had their activity directly affected by irradiation only in the beginning of the tissue repairing process. Alkaline phosphatase did not suffer any direct effect of irradiation. It is possible that the main factor has been the damage of the cellular components responsible for the growth of granulation tissue, which determine the production of enzymes according to the necessity.

[Connective tissue function and fibril formation processes in fibrous-cavernous pulmonary tuberculosis]. / Sostoianie soedinitel'noi tkani i protsessov fibrilloobrazovaniia pri fibrozno-kavernoznom tuberkuleze legkikh.

Electronmicroscopic and histochemical examination of connective tissue in the resected lungs of patients with fibrocavernous tuberculosis was carried out. High metabolic activity and developed ultrastructural organization of fibroblasts were observed in the foci of productive tissue response. The stages of cell differentiation and the maturation of collagen fibers resulted in the multilayer structure of both the tuberculosis foci and the cavernous wall. The activation of smooth muscle cells and myofibroblasts facilitated the development of perivascular and peribronchial sclerosis. Fibroblasts were found to be in connection with lymphoid cells and macrophages that influence the intensity of fibrosis. The formation of numerous lymphoid follicles combined with the progression of fibrosis is an unfavourable prognostic symptom as it results in an excessive cirrhosis and fibrosis which inhibits the healing of destructive tuberculosis.