In vivo preservation of the hybrid layer by chlorhexidine

Host-derived proteases have been reported to degrade the collagen matrix of incompletely-resin-infiltrated dentin. This study tested the hypothesis that interfacial degradation of resin-dentin bonds may be prevented or delayed by the application of chlorhexidine (CHX), a matrix metalloproteinase inhibitor, to dentin after phosphoric acid-etching. Contralateral pairs of resin-bonded Class I restorations in non-carious third molars were kept under intra-oral function for 14 months. Preservation of resin-dentin bonds was assessed by microtensile bond strength tests and TEM examination. In vivo bond strength remained stable in the CHX-treated specimens, while bond strength decreased significantly in control teeth. Resin-infiltrated dentin in CHX-treated specimens exhibited normal structural integrity of the collagen network. Conversely, progressive disintegration of the fibrillar network was identified in control specimens. Auto-degradation of collagen matrices can occur in resin-infiltrated dentin, but may be prevented by the application of a synthetic protease inhibitor, such as chlorhexidine.

Gene expression changes associated with myocarditis and fibrosis in hearts of mice with chronic chagasic cardiomyopathy

Chronic chagasic cardiomyopathy is a leading cause of heart failure in Latin American countries. About 30% of Trypanosoma cruzi-infected individuals develop this severe symptomatic form of the disease, characterized by intense inflammatory response accompanied by fibrosis in the heart.We performed an extensive microarray analysis of hearts from a mouse model of this disease and identified significant alterations in expression of ~12% of the sampled genes. Extensive up-regulations were associated with immune-inflammatory responses (chemokines, adhesion molecules, cathepsins, and major histocompatibility complex molecules) and fibrosis (extracellular matrix components, lysyl oxidase, and tissue inhibitor of metalloproteinase 1). Our results indicate potentially relevant factors involved in the pathogenesis of the disease that may provide newtherapeutic targets in chronic Chagas disease. © 2010 by the Infectious Diseases Society of America.

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.

Effect of phosphoric acid on the degradation of human dentin matrix

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We acid-etched experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices. © 2013 International & American Associations for Dental Research.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

Triacontyl p-coumarate: An inhibitor of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A2). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37 °C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH-minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments. © 2012 Elsevier Ltd. All rights reserved.

Serum Metalloproteinases 2 and 9 as Predictors of Gait Status, Pressure Ulcer and Mortality after Hip Fracture

Introduction: The aim of this study is to evaluate the serum activity of metalloproteinases (MMPs) -2 and -9 as predictors of pressure ulcer (PU), gait status and mortality 6 months after hip fracture. Methods: Eighty-seven patients over the age of 65 admitted to the orthopedic unit from January to December 2010 with hip fracture were prospectively evaluated. Upon admission, patient demographic information, including age, gender and concomitant diseases, was recorded. Blood samples were taken for analysis of MMP -2 and -9 activity by gel zymography and for biochemical examination within the first 72 hours of the patient's admission, after clinical stabilization. The fracture pattern (neck, trochanteric or subtrochanteric), time from admission to surgery, surgery duration and length of hospital stay were also recorded. Results: Two patients were excluded due to the presence of pathological fractures (related to cancer), and three patients were excluded due to the presence of PU before admission. Eighty-two patients, with a mean age of 80.4 ± 7.3 years, were included in the analysis. Among these patients, 75.6% were female, 59.8% had PU, and 13.4% died 6 months after hip fracture. All patients underwent hip fracture repair. In a univariate analysis, there were no differences in serum MMP activity between hip fracture patients with or without PU. In addition, the multiple logistic regression analysis models, which were adjusted by age, gender, length of hospital stay and C-reactive protein, showed that the pro-MMP-9 complexed with neutrophil gelatinase-associated lipocalin form (130 kDa) was associated with gait status recovery 6 months after hip fracture. Conclusions: In conclusion, serum pro-MMP-9 is a predictor of gait status recovery 6 months after hip fracture. © 2013 Gumieiro et al.

Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Mechanism and effect of esculetin in an experimental animal model of inflammatory bowel disease

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

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.

Mechanisms Involved in the Beneficial Effects of Spironolactone after Myocardial Infarction

Introduction:Our objective was to analyze the effect of spironolactone on cardiac remodeling after experimental myocardial infarction (MI), assessed by matricellular proteins levels, cardiac collagen amount and distribution, myocardial tissue metalloproteinase inhibitor-1(TIMP-1) concentration, myocyte hypertrophy, left ventricular architecture, and in vitro and in vivo cardiac function.Methods:Wistar rats were assigned to 4 groups: control group, in which animals were submitted to simulated surgery (SHAM group; n=9); group that received spironolactone and in which animals were submitted to simulated surgery (SHAM-S group, n=9); myocardial infarction group, in which animals were submitted to coronary artery ligation (MI group, n=15); and myocardial infarction group with spironolactone supplementation (MI-S group, n=15). The rats were observed for 3 months.Results:The MI group had higher values of left cardiac chambers and mass index and lower relative wall thicknesses compared with the SHAM group. In addition, diastolic and systolic functions were worse in the MI groups. However, spironolactone did not influence any of these variables. The MI-S group had a lower myocardial hydroxyproline concentration and myocyte cross-sectional area compared with the MI group. Myocardial periostin and collagen type III were lower in the MI-S group compared with the MI-group. In addition, TIMP-1 concentration in myocardium was higher in the MI-S group compared with the MI group.Conclusions:The predominant consequence of spironolactone supplementation after MI is related to reductions in collagens, with discrete attenuation of other remodeling variables. Importantly, this effect may be modulated by periostin and TIMP-1 levels. © 2013 Minicucci et al.

The effect of dimethyl sulfoxide (DMSO) on dentin bonding and nanoleakage of etch-and-rinse adhesives

Objective The objective was to examine the effect of a solvent dimethyl sulfoxide (DMSO) on resin-dentin bond durability, as well as potential functional mechanisms behind the effect. Methods Microtensile bond strength (μTBS) was evaluated in extracted human teeth in two separate experiments. Dentin specimens were acid-etched and assigned to pre-treatment with 0.5 mM (0.004%) DMSO as additional primer for 30 s and to controls with water pre-treatment. Two-step etch-and-rinse adhesive (Scotchbond 1XT, 3M ESPE) was applied and resin composite build-ups were created. Specimens were immediately tested for μTBS or stored in artificial saliva for 6 and 12 months prior to testing. Additional immediate and 6-month specimens were examined for interfacial nanoleakage analysis under SEM. Matrix metalloproteinase (MMP) inhibition by DMSO was examined with gelatin zymography. Demineralized dentin disks were incubated in 100% DMSO to observe the optical clearing effect. Results The use of 0.5 mM DMSO had no effect on immediate bond strength or nanoleakage. In controls, μTBS decreased significantly after storage, but increased significantly in DMSO-treated group. The control group had significantly lower μTBS than DMSO-group after 6 and 12 months. DMSO also eliminated the increase in nanoleakage seen in controls. 5% and higher DMSO concentrations significantly inhibited the gelatinases. DMSO induced optical clearing effect demonstrating collagen dissociation. Significance DMSO as a solvent may be useful in improving the preservation of long-term dentin-adhesive bond strength. The effect may relate to dentinal enzyme inhibition or improved wetting of collagen by adhesives. The collagen dissociation required much higher DMSO concentrations than the 0.5 mM DMSO used for bonding. © 2013 Academy of Dental Materials.

Platelet-Rich Plasma And Chronic Wounds: Remaining Fibronectin May Influence Matrix Remodeling And Regeneration Success

Background: Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules. Aim: We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations. Methods: Recent reports were analyzed and presented as direct evidences of this hypotheses. Results: Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration. Conclusions: Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment. © 2013 International Society for Cellular Therapy.