Mast cells and MMP-9 in the lamina propria during eruption of rat molars: quantitative and immunohistochemical evaluation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Differential MMP-2 and MMP-9 Activity and Collagen Distribution in Skeletal Muscle from pacu (Piaractus mesopotamicus) During Juvenile and Adult Growth Phases

Here, we evaluated collagen distribution and matrix metalloproteinases (MMPs) MMP-2 and MMP-9 activities in skeletal muscle of pacu (Piaractus mesopotamicus) during juvenile and adult growth phases. Muscle samples from juvenile and adult fishes were processed by histochemistry for collagen system fibers and for gelatin-zymography for MMP-2 and MMP-9 activities analysis. Picrosirius staining revealed a myosept, endomysium, and perimysium-like structures in both growth phases and muscle types, with increased areas of collagen fibers in adults, mainly in red muscle. Reticulin staining showed that reticular fibers in the endomysium-like structure were thinner and discontinuous in the red muscle fibers. The zymography revealed clear bands of the pro-MMP-9, active-MMP-9, intermediate-MMP-2, and active-MMP-2 forms in red and white muscle in both growth phases. MMP-2 activity was more intense in juvenile than adult muscle fibers. Comparing the red and white muscle types, MMP-2 activity was significantly higher in red muscle in adult phase only. The activity of MMP-9 forms was similar in juvenile red and white muscles and in the adult red muscle, without any activity in adult white muscle. In conclusion, our results show that, in pacu, the higher activities of MMP-2 and -9 are associated with the rapid muscle growth in juvenile age and in adult fish, these activities are related with a different red and white muscle physiology. This study may contribute to the understanding muscle growth mechanisms and may also contribute to analyse red and the white muscle parameters of firmness and softness, respectively, of the commercial product. Anat Rec, 292:387-395, 2009. (C) 2009 Wiley-Liss, Inc.

Calcaneal Tendon Regions Exhibit Different MMP-2 Activation After Vertical Jumping and Treadmill Running

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Finasteride treatment alters MMP-2 and-9 gene expression and activity in the rat ventral prostate

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Localized matrix metalloproteinase (MMP)-2 and MMP-9 activity in the rat ventral prostate during the first week of postnatal development

The initial events in prostatic morphogenesis involve cell proliferation, epithelial canalization and outgrowth toward the stroma. We have hypothesized that stromal rearrangement takes place at the sites of epithelial growth and branching and that this rearrangement involves the action of gelatinases matrix metalloproteinase (MMP)-2 and MMP-9. Thus, the purpose of the present study was to characterize structural aspects of epithelial growth during the first week of postnatal development of the rat ventral prostate and to investigate the expression, localization and activity of MMP-2 and MMP-9 during this period by histological, ultrastructural and immunocytochemical analysis, in addition to gel zymography, in situ zymography and Western blotting. An increasing complexity of prostatic architeture was observed within the first postnatal week. Concurrently, the stroma became more organized and some cells differentiated into smooth muscle cells. Reticulin fibers appeared in a basket-like arrangement around both growing tips and epithelial sprouts, associated with a fainter staining for laminin. MMP-2 and MMP-9 activities were detected. MMP-2/MMP-9 expression decreased during the first week. Developing epithelial cords showed strong and difuse gelatinolytic activity. This activity coincided with the distribution of MMP-2 as determined by immunocytochemistry. on the other hand, MMP-9 was rather concentrated at the epithelial tips. These results suggest that gelatinolytic activity (with contribution of both MMP-2 and MMP-9) in the epithelium and at the epithelium-stroma interface are at least in part responsible for the tissue remodeling that allows epithelial growth and its projection into the surrounding stroma.

Matrix metalloproteinase (MMP)-2 and MMP-9 activity and localization during ventral prostate atrophy and regrowth

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Epithelial-stromal transition of MMP-7 immunolocalization in the rat ventral prostate following bilateral orchiectorny

Epithelial cells from involuting rat ventral prostate (VP) express Matrilysin (MMP-7) mRNA. Herein, we investigated by immunohistochemistry the NIMP-7 protein location and its association with tissue changes following castration in the VP. Normal and castrated adult male Wistar rats were sacrificed at different times after surgery. VP was examined by immunocytochemistry and immunoprecipitation. Castration promoted a shrinking of prostate ducts with an extensive stromal remodeling. In the VP from normal rats, MMP-7 immunoreactivity was found in epithelial secretory granules. Three days after castration, immunostaining for MMP-7 was found in both the epithelial secretory granules and in the stroma just below the epithelium, mainly at the distal ductal tips. At seven and 21 days after castration, the immunostaining for MMP-7 was found only in the stromal space. Immunoprecipitation confirmed the specificity of the primary antibody by rescuing a pro-enzyme form (28 kDa) in the prostate extracts. The present results suggest that MMP-7 participates in the epithelial-stromal interface remodeling of the ventral prostate during the involution achieved by castration, probably in the degradation of components of the epithelial basement membrane. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

MKK3/6-p38 MAPK negatively regulates murine MMP-13 gene expression induced by IL-1 beta and TNF-alpha in immortalized periodontal ligament fibroblasts

Matrix metalloprotease-13 (MMP-13) or collagenase-3 is involved in a number of pathologic processes such as tumor metastasis and angiogenesis, osteoarthritis, rheumatoid arthritis and periodontal diseases. These conditions are associated with extensive degradation of both connective tissue and bone. This report examines gene regulation mechanisms and signal transduction pathways involved in Mmp-13 expression induced by proinflammatory cytokines in periodontal ligament (PDL) fibroblasts. Mmp-13 mRNA expression was increased 10.7 and 9.5 fold after stimulation with IL-1 beta (5 ng/mL) and TNF-alpha (10 ng/mL), respectively. However, inhibition of p38 MAPKinase with SB203580 resulted in significant (p < 0.001) induction (23.2 and 18.1 fold, respectively) of Mmp-13 mRNA as assessed by real time PCR. Negative regulation of IL-1 induced Mmp-13 expression was confirmed by inhibiting p38 MAPK gene expression with siRNA. Transient transfection of dominant negative forms of MKK3 and MKK6 also resulted in increased levels of Mmp-13 mRNA after IL-1 beta stimulation. Mmp-13 mRNA expression induced by TNF-alpha was decreased by JNK and ERK inhibition. Western blot and zymogram analysis indicated that Mmp-13 protein expression induced by the proinflammatory cytokines were also upregulated by inhibition of p38 MAPK. Reporter gene experiments using stable cell lines harboring 660-bp sequence of the murine Mmp-13 proximal promoter indicated that transcriptional mechanisms were at least partially involved in this negative regulation of Mmp-13 expression by p38 MAPK and upstream MKK3/6. These results suggest a negative transcriptional regulatory mechanism mediated by p38 MAPK and upstream MKK3/6 on Mmp-13 expression induced by proinflammatory cytokines in PDL fibroblasts. (c) 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase

Matrix metal loprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

MMP-2 and MMP-9 localization and activity in the female prostate during estrous cycle

The gerbil female prostate undergoes morphological and physiological changes resulting from hormonal fluctuations that occur during the reproductive cycle. These repetitive cycles of glandular growth and regression are followed by an extensive reconstruction and remodeling of prostate stroma throughout the reproductive life of the female gerbil. The objective of this study was to evaluate the effect that the hormonal fluctuations of the reproductive cycle have on the stromal remodeling and the expression and activity of matrix metalloproteinases MMP-2 and -9 in the adult female gerbil prostate. For this, serological, ultrastructural, immunohistochemical and biochemical methods were employed. The results showed that the major stromal alteration coincide with the peak of estradiol, which occurs in estrus, and with the peak of progesterone, occurring during diestrus II. MMP-2 and -9 presented a similar pattern of expression and activity during estrous cycle. The estrus was the phase of greater expression and activity of MMP-2 and -9. On the other hand, in DI and DII, the tissue expression and activity of MMP-2 and -9 was very weak. These results are important since they suggest the involvement of estradiol and progesterone in regulating the expression and activity of MMP-2 and -9 in adult gerbil female prostate. © 2011 Elsevier Inc.

MMP-2 and TIMP-2 in the prostates of male and female mongolian gerbils: Effects of hormonal manipulation

TIMPs in the prostates of male and female gerbils and evaluated the effects of testosterone on the expression of these enzymes. Ventral prostates from male gerbils that were either intact or had been castrated for 7 or 21 days, along with prostates from female gerbils that were either intact or had been treated with testosterone for 7 or 21 days, were submitted to histological, stereological and immunohistochemical analyses. Stereology of prostatic components showed significant alterations of tissue compartments in the ventral male prostate after castration, especially after 21 days, with a significant increase in stroma. Administration of testosterone led to disorganization in the female prostate, with a significant increase in collagen fibers and smooth muscle cells after 21 days, along with the development of epithelial lesions such as PINs. MMP-2 increased after 21 days of castration in males; however, the TIMP-2 immunoreaction for this group was weak or absent. In females, the expression of MMP-2 appeared to decrease after 7 days of treatment with testosterone, but after 21 days, both epithelium and stroma showed a stronger reaction for MMP-2 than the controls. The expression of TIMP-2 in the treated females was similar to its expression in the castrated males. We conclude that the distribution of MMPs and TIMPs in both male and female prostates is altered by androgen manipulation, but the mechanism of stromal regulation appears to be distinct between genders because both the lack of T in castrated males and the excess levels of T in treated females lead to the same effect.

Low level laser therapy increases angiogenesis in a model of ischemic skin flap in rats mediated by VEGF, HIF-1α and MMP-2

It is known that low level laser therapy is able to improve skin flap viability by increasing angiogenesis. However, the mechanism for new blood vessel formation is not completely understood. Here, we investigated the effects of 660 nm and 780 nm lasers at fluences of 30 and 40 J/cm2 on three important mediators activated during angiogenesis. Sixty male Wistar rats were used and randomly divided into five groups with twelve animals each. Groups were distributed as follows: skin flap surgery non-irradiated group as a control; skin flap surgery irradiated with 660 nm laser at a fluence of 30 or 40 J/cm2 and skin flap surgery irradiated with 780 nm laser at a fluence of 30 or 40 J/cm2. The random skin flap was performed measuring 10 × 4 cm, with a plastic sheet interposed between the flap and the donor site. Laser irradiation was performed on 24 points covering the flap and surrounding skin immediately after the surgery and for 7 consecutive days thereafter. Tissues were collected, and the number of vessels, angiogenesis markers (vascular endothelial growth factor, VEGF and hypoxia inducible factor, HIF-1α) and a tissue remodeling marker (matrix metalloproteinase, MMP-2) were analyzed. LLLT increased an angiogenesis, HIF-1α and VEGF expression and decrease MMP-2 activity. These phenomena were dependent on the fluences, and wavelengths used. In this study we showed that LLLT may improve the healing of skin flaps by enhancing the amount of new vessels formed in the tissue. Both 660 nm and 780 nm lasers were able to modulate VEGF secretion, MMP-2 activity and HIF-1α expression in a dose dependent manner. © 2013 Published by Elsevier B.V.

Zymographic patterns of MMP-2 and MMP-9 in the CSF and cerebellum of dogs with subacute distemper leukoencephalitis

Distemper leukoencephalitis is a disease caused by the canine distemper virus (CDV) infection. It is a demyelinating disease affecting mainly the white matter of the cerebellum and areas adjacent to the fourth ventricle; the enzymes of the matrix metalloproteinases (MMPs) group, especially MMP-2 and MMP-9 have a key role in the myelin basic protein fragmentation and in demyelination, as well as in leukocyte traffic into the nervous milieu. To evaluate the involvement of MMPs during subacute distemper leukoencephalitis, we measured the levels of MMP-2 and MMP-9 by zymography in the cerebrospinal fluid (CSF) and in the cerebellum of 14 dogs naturally infected with CDV and 10 uninfected dogs. The infected dogs presented high levels of pro-MMP-2 in the CSF and elevated levels of pro-MMP-2 and pro-MMP-9 in the cerebellar tissue. Active MMP-2 was detected in the CSF of some infected dogs. As active MMP-2 and MMP-9 are required for cellular migration across the blood-brain barrier and any interference between MMPs and their inhibitors may result in an amplification of demyelination, this study gives additional support to the involvement of MMPs during subacute distemper leukoencephalitis and suggests that MMP-2 and MMP-9 may take part in the brain inflammatory changes of this disease. © 2013 Elsevier B.V.

Stimulation of MMP-1 and CCL2 by NAMPT in PDL cells

Periodontitis is an inflammatory disease caused by pathogenic microorganisms and characterized by the destruction of the periodontium. Obese individuals have an increased risk of periodontitis, and elevated circulating levels of adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), may be a pathomechanistic link between both diseases. The aim of this in vitro study was to examine the regulation of periodontal ligament (PDL) cells by NAMPT and its production under inflammatory and infectious conditions. NAMPT caused a significant upregulation of 9 genes and downregulation of 3 genes, as analyzed by microarray analysis. Eight of these genes could be confirmed by real-time PCR: NAMPT induced a significant upregulation of EGR1, MMP-1, SYT7, ITPKA, CCL2, NTM, IGF2BP3, and NRP1. NAMPT also increased significantly the MMP-1 and CCL2 protein synthesis. NAMPT was significantly induced by interleukin-1β and the periodontal microorganism P. gingivalis. NAMPT may contribute to periodontitis through upregulation of MMP-1 and CCL2 in PDL cells. Increased NAMPT levels, as found in obesity, may therefore represent a mechanism whereby obesity could confer an increased risk of periodontitis. Furthermore, microbial and inflammatory signals may enhance the NAMPT synthesis in PDL cells and thereby contribute to the increased gingival and serum levels of this adipokine, as found in periodontitis. © 2013 Marjan Nokhbehsaim et al.