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.

Active hyaluronidase 2 expression in the granulation tissue formed in the healing process of equine superficial digital flexor tendonitis.

Upregulation of hyaluronidase 2 (HYAL2), one of somatic hyaluronidase (HAase), was demonstrated in granulation tissue during the healing of equine superficial digital flexor tendon injuries. The activity of HAase was assessed by hyaluronan (HA)-containing gel zymography and in situ zymography using frozen sections obtained from normal and injured tendon tissues. Elevated HAase activity was identified in the extract from the tendinopathic tissues, with lower levels of the activity in normal tendons. In situ zymography using fluorescently-labeled HA demonstrated HAase activity in the granulation tissue formed in the injured region. In addition, in situ hybridization analysis indicated that fibroblastic cells in the granulation tissue of the injured tendon actively expressed HYAL2 but not HYAL1. Quantitative RT-PCR further confirmed a higher level of amplicons corresponding to HYAL2 in tendonitis-derived samples. These results suggest that elevated HYAL2 activity in the granulation tissue could participate in controlling the healing process in equine tendonitis.

Calpain activity is essential in skin wound healing and contributes to scar formation.

Wound healing is a multistep phenomenon that relies on complex interactions between various cell types. Calpains are ubiquitously expressed proteases regulating several processes including cellular adhesion and motility as well as inflammation and angiogenesis. Calpains can be targeted by inhibitors, and their inhibition was shown to reduce organ damage in various disease models. We aimed to assess the role of calpains in skin healing and the potential benefit of calpain inhibition on scar formation. We used a pertinent model where calpain activity is inhibited only in lesional organs, namely transgenic mice overexpressing calpastatin (CPST), a specific natural calpain inhibitor. CPST mice showed a striking delay in wound healing particularly in the initial steps compared to wild types (WT). CPST wounds displayed reduced proliferation in the epidermis and delayed re-epithelization. Granulation tissue formation was impaired in CPST mice, with a reduction in CD45+ leukocyte infiltrate and in CD31+ blood vessel density. Interestingly, wounds on WT skin grafted on CPST mice (WT/CPST) showed a similar delayed healing with reduced angiogenesis and inflammation compared to wounds on WT/WT mice demonstrating the implication of calpain activity in distant extra-cutaneous cells during wound healing. CPST wounds showed a reduction in alpha-smooth muscle actin (αSMA) expressing myofibroblasts as well as αSMA RNA expression suggesting a defect in granulation tissue contraction. At later stages of skin healing, calpain inhibition proved beneficial by reducing collagen production and wound fibrosis. In vitro, human fibroblasts exposed to calpeptin, a pan-calpain inhibitor, showed reduced collagen synthesis, impaired TGFß-induced differentiation into αSMA-expressing myofibroblasts, and were less efficient in a collagen gel contraction assay. In conclusion, calpains are major players in granulation tissue formation. In view of their specific effects on fibroblasts a late inhibition of calpains should be considered for scar reduction.

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.

Phase IB study of the mTOR inhibitor ridaforolimus with capecitabine.

PURPOSE: Synergistic/additive cytotoxicity in tumor models and widespread applicability of fluoropyrimidines in solid tumors prompted the study of the combination of the mammalian target of rapamycin (mTOR) inhibitor, non-prodrug rapamycin analog ridaforolimus, with capecitabine. PATIENTS AND METHODS: Thirty-two adult patients were treated. Intravenous ridaforolimus was given once weekly for 3 weeks and capecitabine was given from days 1 to 14 every 4 weeks. Ridaforolimus was given at 25, 37.5, 50, or 75 mg with capecitabine at 1,650 mg/m(2) or 1,800 mg/m(2) divided into two daily doses. Pharmacokinetics of both drugs were determined during cycles 1 and 2. Pharmacodynamic studies in peripheral blood mononuclear cells (PBMCs) and wound tissue of the skin characterized pathways associated with the metabolism or disposition of fluoropyrimidines and mTOR and ERK signaling. RESULTS: Two recommended doses (RDs) were defined: 75 mg ridaforolimus/1,650 mg/m(2) capecitabine and 50 mg ridaforolimus/1,800 mg/m(2) capecitabine. Dose-limiting toxicities were stomatitis and skin rash. One patient achieved a partial response lasting 10 months and 10 of 29 evaluable patients had stable disease for ≥ 6 months. The only pharmacokinetic interaction was a ridaforolimus-induced increase in plasma exposure to fluorouracil. PBMC data suggested that prolonged exposure to capecitabine reduced the ridaforolimus inhibition of mTOR. Ridaforolimus influenced the metabolism of fluoropyrimidines and inhibited dihydropyrimidine dehydrogenase, behavior similar to that of rapamycin. Inhibition of the target thymidylate synthase by capecitabine was unaffected. mTOR and ERK signaling was inhibited in proliferating endothelial cells and was more pronounced at the RD with the larger amount of ridaforolimus. CONCLUSION: Good tolerability, feasibility of prolonged treatment, antitumor activity, and favorable pharmacologic profile support further investigation of this combination.

Activation of matrix metalloproteinase (MMP)-2 by membrane type 1-MMP and abnormal immunolocalization of the basement membrane components laminin and type IV collagen in canine spontaneous hemangiosarcomas.

We performed immunohistochemical investigation of the basement membrane (BM) components, namely, type IV collagen and laminin, in 83 canine hemangiosarcomas (HSAs), 22 hemangiomas, and some granulation tissues (GTs). Additionally, we analyzed the expression and activities of matrix metalloproteinase (MMP)-2, MMP-9, and membrane type 1-MMP (MT1-MMP) using the same samples by immunohistochemistry and gelatin zymography to investigate whether MMPs were associated with the BM degradation. In immunohistochemistry for the BM components, many HSAs showed discontinuous linear/negative immunoreactivity in the BM (type IV collagen: 49.4%/14.5%, laminin: 60.3%/10.8%, respectively). In contrast, almost all hemangiomas showed continuous staining in the BM (type IV collagen: 90.9%, laminin: 95.5%, respectively). Interestingly, positive cytoplasmic immunoreactivity for type IV collagen and laminin was observed in 97.6% and 91.6% HSA, respectively. Although MMP-9 immunoreactivity wasn't detected in neoplastic and active angiogenic endothelial cells (ECs), MMP-2 was detected in all ECs of GTs and in neoplastic cells of both vascular tumors. A strong immunoreactivity for MT1-MMP was observed in active angiogenic ECs in GTs and in neoplastic ECs in HSAs. However, almost all hemangiomas showed weak/negative immunoreactivity. In gelatin zymography, significantly strong activity of active MMP-2 was observed in HSAs, similar to that in active angiogenesis in GTs; however, weak/no activity of active MMP-2 was detected in hemangiomas. In canine HSA, neoplastic cells had active MMP-2, possibly activated by MT1-MMP, and discontinuous status of BM might be associated with activity of active MMP-2.

An immunolocalization study of tissue inhibitors of metalloproteinase-1 of bone graft healing on parietal bone.

This immunolocalization study was performed to investigate the temporal and spatial expression of tissue inhibitors of metalloproteinase (TIMP) 1 within endochondral and intramembranous bone grafts during the early stages of healing, in the hope of gaining a better understanding of the mechanisms of bone graft healing, which could influence the choice of bone graft used. Twenty-seven adult New Zealand White rabbits were used as the experimental model. Autogenous bone grafts taken from the cranial bone (intramembranous in origin) and the femur (endochondral in origin) were grafted into skull defects created on either side of the parietal suture. Rabbits were killed on days 1 to 9 postgrafting, and the bone graft alone was harvested for immunolocalization of TIMP-1. In endochondral bone grafts, TIMP-1 was expressed on days 1 to 3, followed by a period of absence until days 8 and 9. Intramembranous bone grafts did not express TIMP-1 until days 6 to 9. The timing and location of TIMP-1 expression coincided with osteogenesis, which indicates a role for TIMP-1 in preserving newly formed bone during the initial stages of graft healing. The differential temporal expression of TIMP-1 in endochondral and intramembranous bone grafts suggests that bone graft type plays an important role in influencing the healing process mediated by the host tissues. The earlier expression of TIMP-1 in endochondral bone grafts could be the reason for delayed vascularization of defects containing these grafts, whereas the delayed expression of TIMP-1 in intramembranous bone grafts could allow earlier vascularization of the intramembranous bone grafts.

Contraction of myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity.

During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating myofibroblasts. We examined whether regulation of myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of myofibroblasts necessary for wound closure.

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.

Energy metabolism in the granulation tissue of diabetic rats during cutaneous wound healing.

The skin cells chiefly depend on carbohydrate metabolism for their energy requirement during cutaneous wound healing. Since the glucose metabolism is greatly hampered in diabetes and this might affect wound repair process. This prompted us to investigate the intermediate steps of energy metabolism by measuring enzyme activities in the wound tissues of normal and streptozotocin-induced diabetic rats following excision-type of cutaneous injury. The activities of key regulatory enzymes namely hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6 phosphate dehydrogenase (G6PD) have been monitored in the granulation tissues of normal and diabetic rats at different time points (2, 7, 14 and 21 days) of postwounding. Interestingly, a significant alteration in all these enzyme activities was observed in diabetic rats. The activity of PFK was increased but HK, LDH and CS showed a decreased activity in the wound tissue of diabetics as compared to normal rats. However G6PD exhibited an elevated activity only at early stage of healing in diabetic rats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the wound tissue of diabetic rats may affect the energy availability for cellular activity needed for repair process and this may perhaps be one of the factor responsible for impaired healing in these subjects.

Energy metabolism during cutaneous wound healing in immunocompromised and aged rats.

Cutaneous cells primarily depend upon carbohydrate metabolism for their energy requirement during healing process. But, it may be greatly hampered during various pathological and altered physiological conditions. The present study was therefore undertaken to investigate the intermediate steps of energy metabolism by measuring enzyme activities in the granulation tissues of immunocompromised and aged rats following excision-type of cutaneous injury. The activities of key regulatory enzymes hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), citrate synthase (CS) and glucose-6 phosphate dehydrogenase (G6PD) have been monitored in the wound tissues of immunocompromised and aged rats at different time intervals (2, 7, 14 and 21 days) of postwounding. The activities of HK and CS were found significantly decreased both in immunocompromised and aged rats as compared to control subjects. However G6PD exhibited an elevated activity at early stage followed by a decreased activity at later phase of healing both in immunocompromised and aged rats. The PFK and LDH demonstrated an upward trend in immunocompromised rats but a decreasing trend in aged rats. Thus, the results suggest that significant alterations in the activities of energy metabolizing enzymes in the granulation tissues in both immunocompromised as well as in aged rats may overall affect the energy availability for cellular activity needed for repair process. Hence, this may perhaps be one of the factor responsible for impaired healing in these subjects.

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.

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.

Transient increase in telomerase activity of proliferating fibroblasts and endothelial cells in granulation tissue of the human skin.

Although granulation tissue formation is an important step for second-intention wound healing, the molecular events underlying this process are still poorly understood. To investigate the role of telomerase in the formation of granulation tissue, we measured the activity of this enzyme and determined the expression and localization of human telomerase reverse transcriptase mRNA using human skin samples. Telomerase activity in the tip of the granulation tissue where fibroblasts actively proliferate was detected at a level 5.6 +/- 1.5 times higher than that at the edge of the tissue when using a polymerase chain reaction-based telomeric repeat amplification protocol assay coupled with enzyme-linked immunosorbent assay. This, together with the findings from semiquantitative reverse transcriptase-polymerase chain reaction and in situ hybridization of human telomerase reverse transcriptase, revealed that proliferating cell nuclear antigen-positive fibroblasts and endothelial cells in the progressing granulation tissue showed de novo activation of telomerase with high human telomerase reverse transcriptase mRNA expression. This condition may be a prolongation of cellular replicative capacity taking advantage of the positive regulatory dynamics of cell growth. We conclude that the regulation of telomerase activity may play an important role in granulation tissue formation in wound healing.

[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.

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.

Induction of matrix metalloproteinases in keratinocytes by cholesteatoma debris and granulation tissue extracts.

Although it is generally accepted that destruction and remodeling of temporal bone associated with middle ear cholesteatoma is mainly caused by the action of osteoclasts, it has been shown that neutral collagenases also play a role in predigesting the osteoid layer and exposing the mineralized bone to osteoclastic activity. Here we show that gelatinase B (matrix metalloproteinase-9) is over-expressed in cholesteatoma compared to external ear canal skin (EACS). Expression of MMP-9 in cholesteatoma mainly occurs in suprabasal layers, and more rarely in basal layers of cholesteatoma epithelium, as well as in inflammatory cells of the perimatrix. We further analyzed the influence of cholesteatoma debris, cholesteatoma granulation tissue, and cholesteatoma components such as keratin, cholesterol and bacterial endotoxin on the expression of MMPs in EACS keratinocytes. We show that cholesteatoma debris and granulation tissue extract both induced the secretion of MMP-9 by EACS keratinocytes, while keratin. bacterial lipopolysaccharide (LPS) or cholesterol did not show any effect. We further performed co-incubation and immunoprecipitation experiments using neutralizing interleukin-1alpha, EGF, TGF-beta, TGF-alpha, interleukin-6 and TNF-alpha antibodies. Inhibition of MMP-9 up-regulation by debris or granulation tissue extract could be revealed with diverse cytokine antibodies. The results are discussed with regard to previously published studies.

Cyclooxygenase-2-mediated angiogenesis in carrageenin-induced granulation tissue in rats.

The possible participation of cyclooxygenase (COX)-2 in angiogenesis in granulation tissue was analyzed using an air pouch-type carrageenin-induced inflammation model in rats. Injection of carrageenin solution into an air pouch induced gradual increases in the pouch fluid volume and granulation tissue weight as well as angiogenesis in granulation tissue. NS-398 (10-100 microg) inhibited all of these parameters in a dose-dependent manner. NS-398 (100 microg), indomethacin (100 microg), and dexamethasone (10 microg) markedly reduced prostaglandin (PG) E(2) levels in the pouch fluid at day 6. NS-398 and indomethacin did not affect protein levels of COX-1 and COX-2 but dexamethasone significantly reduced the level of COX-2 in granulation tissue at day 6. Protein levels of vascular endothelial growth factor (VEGF) in granulation tissue and in the pouch fluid were higher at day 6 than at day 3, and the levels were decreased by treatment with NS-398 (10-100 microg) in a dose-dependent manner. The inhibitory effects of NS-398 (100 microg) were almost the same as those of indomethacin (100 microg). Dexamethasone (10 microg) also reduced VEGF protein levels in granulation tissue at day 6. To clarify the role of PGE(2) in VEGF production, minced granulation tissue obtained 3 days after carrageenin injection from the indomethacin-treated rats was incubated in the presence of various concentrations of PGE(2). It was shown that VEGF mRNA and protein levels in the minced granulation tissue were increased by PGE(2) in a concentration-dependent manner. These findings suggest that COX-2-derived PGE(2) plays a significant role in angiogenesis in the carrageenin-induced granulation tissue through VEGF formation.

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.