The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal region of TIMP and the C-D B-strand connector which occupy the primed (right side of the active site) and unprimed (left side) regions of the active site. Substitutions for Thr2 of N-TIMP- 1 strongly influence MMP selectivity. In this study we found that Arg and Gly, which generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the NTIMP-1 mutant with AB loop of TIMP-2, it produced a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and MMP-9, respectively. The Gly mutant has a Ki of 2.1 nM for MMP-9 and > 40 uM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily. In collaboration with Dr. Yingnan Zhang at Genentech, we have developed a protocol for the phage display of full-length human TIMP-2 to identify high-affinity selective inhibitors of human MMP-1, a protease that plays a role in cleaving extracellular matrix (ECM) components, connective tissue remodeling during development, angiogenesis, and apoptosis. We have generated a library containing 2x1010 variants of TIMP-2 randomized at residues 2-6 (L1), at residues 34-40 (L2) and 67-70 (L3). / The L1 library yielded a positive signal for MMP-1 binding. Clones from the L1 library, designated TM1, TM8, TM13, and TM14, were isolated after 5 rounds of selection on immobilized MMP-1 and MMP-3 and found to show a greater selectivity for MMP-1 relative to MMP-3. TM8, which has Ser2 to Asp and Ser4 to Ala substitutions, showed the greatest apparent selectivity of 10-fold toward MMP-1 compared to MMP-3. The various mutations identified by phage display were introduced into recombinant Nterminal TIMP-2 and the variants characterized as inhibitors of an array of MMP catalytic domains. The TM8-based mutant showed pronounced selectivity (> 1000-fold for MMP-1 vs. MMP-3) and may be a step towards the generation of MMP-1-specific inhibitors. Molecular modeling was used to rationalize the structural basis of MMP selectivity in the mutants. / by Harinathachari Bahudhanapati. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Matrix metalloproteinases (MMPs) constitute the major class of enzymes capable of degrading all protein components of extracellular matrix (ECM) and have important roles in normal physiologic processes of maintaining tissue integrity and remodeling. However, excess MMP activities are associated with many diseases including rheumatoid arthritis and osteoarthritis, cardiomyopathy, and macular degeneration. The activity of MMPs is regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) which are avid broad-spectrum inhibitors of numerous human matrixins (MMPs and ADAMs). Uncontrolled matrix degradation occurs when the balance between TIMPs and MMPs is disrupted, resulting in serious diseases such as cancer, arthritis and chronic tissue ulcers. Thus, the engineering of TIMPs to produce highly selective and efficacious inhibitors of individual MMPs may be utilized for future treatment of diseases. Such engineering requires detailed analysis for the structural and biophysical information of MMP-TIMP interaction. Changes in the dynamics of proteins and solvent that accompany their associations with different binding partners, influence the specificity of binding through entropic effects. From the current studies it appears that the interactions of the inhibitory domains of TIMPs-1 and -2 (N-TIMPs) with MT1-MMP are driven by entropy increases that are partitioned between solvent and conformational entropy (ΔSsolv and ΔSconf), and a large conformational entropy penalty is responsible for the weak inhibition of MT1-MMP by NT1.We investigated how mutations that modify N-TIMP selectivity affect the thermodynamics of interactions with MMP1, MMP3 and MT1-MMP. The weak inhibition of MT1-MMP by N-TIMP-1 is enhanced by mutation of threonine 98, on the edge of the binding ridge, to leucine. This mutation increases the large ΔSconf cost for binding to MT1-MMP but this is offset by a greater increase in ΔSsolv. In contrast, this mutation enhances binding to MMP3 by increasing ΔSconf for the interaction. ΔSsolv and ΔSconf show mutual compensation for all interactions, with characteristic ranges for each MMP. Distinct electrostatic and dynamic features of MMPs are key factors in their selective inhibition. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Esta pesquisa teve como objetivo estudar in situ o efeito protetor de um gel à base de Cranberry aplicado sobre a dentina submetida à erosão. Este estudo duplo cego e cruzado constou de 2 fases com duração de 5 dias cada. Para tal, 10 voluntários utilizaram2 aparelhos palatinos (um em cada fase) com 4 blocos de dentina bovina divididos em 2 grupos. Na primeira fase estiveram presentes os grupos: G1 Ação da bebida ácida (Coca-cola®) sobre a dentina bovina sem nenhum tipo de tratamento prévio; G2 - Ação da bebida ácida sobre a dentina tratada com gel à base de Cranberry e na segunda fase foi testado os grupos: G3 Ação da bebida sobre a dentina tratada com gel de aplicação tópica sem nenhum princípio ativo; G4 - Ação da bebida ácida sobre a dentina tratada previamente com gel de Clorexidina. Cada aparelho foi imerso na bebida ácida, 3x/dia, durante 5 minutos por 5 dias. A porcentagem de perda de microdureza de superfície (%PDS) e a perfilometria foram as variáveis utilizadas para quantificar as alterações dadentina. A comparação dos grupos por meio da análise de variância de medidas repetidas seguido do teste de Fisher mostrou haver diferenças estatisticamente significativas entre os grupos para o desgaste (G1: 4,98 μm ± 1,36; G2: 3,29 μm ± 1,16; G3: 4,38 μm ± 1,19; G4: 3,32 μm ± 1,55) e não apresentou diferenças entre eles na %PDS (G1: 28,12 ± 5,71; G2: 24,92 ± 5,38; G3: 25,74 ± 9,15; G4: 29,83 ± 8,63).Assim, quanto ao desgaste não houve diferença estatisticamente significativa entre os grupos controle e placebo, também não houve uma diferença entre os grupos Cranberry e clorexidina. Porém, os grupos Cranberry e clorexidina apresentaram menores valores de desgaste em relação aos grupos Placebo e Controle (sem gel), sendo esta diferença estatisticamente significativa. Os resultados obtidos nesse estudo sugerem uma significativa eficácia dos géis ativos à base de Cranberry e de Clorexidina na prevenção do desgaste da dentina submetida à erosão dentária. / The aim of this study is to evaluate the in situ effect of a Cranberry gel over dentine submitted to a erosive challenge. This crossover doble-blinded study was performed in 2 phases of 5 days each. For that purpose, 10 volunteers wore 2 palatal devices (1 for each phase) with 4 dentin specimens divided into 2 groups: First Phase: G1 - Erosive challenge (Coca-cola®) over dentine without any previous treatment; G2 - Erosive challenge over dentine previously treated with Cranberry gel; and Second Phase: G3 - Erosive challenge over dentine previously treated with a gel withot any active principle; G4 - Erosive challenge over dentine previously treated with Clorexidine gel. Each device was immersed into the acid beverage, 3 times daily for 5 minutes during 5 days. The surface microhardness change percentage (%SMC) and profilometry were be used to quantify the dentin alteratons. The comparison between groups by repeated measures analysis of variance followed by Fisher\'s test showed statistically significant differences between groups for wear (G1: 4.98 ± 1.36 mM, G2: 3.29 ± 1 mM, 16, G3: 4.38 ± 1.19 mM; G4: 3.32 ± 1.55 mM) and showed no differences b in% SMH (G1: 28.12 ± 5.71; G2: 24.92 ± 5.38, G3: 25.74 ± 9.15; G4: 29,83 ± 8,63). So as to wear there was no statistically significant difference between the placebo and control groups.There was also no difference between Cranberry andchlorhexidine groups. However, Cranberry and chlorhexidine groups presented lower wear compared to placebo and control groups (without gel), and this difference was statistically significant. The results of this study suggest a significant efficacy of active gels based Cranberry and chlorhexidine in preventing wear of dentin subjected to dental erosion.

Background: Matrix metalloproteinases (MMPs) have been shown to be involved in cancers and atherosclerosis, the leading causes of present day mortality. The objectives of the cancer element of this project were to investigate single nucleotide polymorphisms (SNPs) in MMP1 and MMP8 regarding breast cancer and malignant melanoma, and a functional characterisation of the genetic variants, including the MMP1 polymorphism rs19799750, previously associated with multiple cancers. The objective of the second part of this project was to investigate whether MMP8 played a role in the development of atherosclerotic lesions and if so, the underlying mechanisms. Methods/Results: Genetic investigations found the MMP8 SNP rs11225395 to be associated with the occurrence of both breast cancer and malignant melanoma; furthermore it was also associated with reduced lymph node metastasis, reduced cancer relapse and greater survival. Functional luciferase assays showed that the minor allele of the polymorphism has higher promoter activity in breast cancer and melanoma cell lines. They also showed haplotypic effects on MMP1 promoter activity in several cancer cell lines by the 2G allele of polymorphism rs1799750 and one or more MMP1 promoter SNPS. The second part of the study found an association between a MMP8 SNP and the extent of coronary atherosclerosis; additionally a relationship among MMP8 gene variation, plasma VCAM-1 level, and atherosclerosis progression was observed in a prospective study. Murine studies showed reduced atherosclerosis in MMP8/ApoE knockout mice compared with ApoE knockout littermate controls. Biochemical studies confirmed that MMP8 can convert angiotensin I to angiotensin II. Conclusions: The data of the first part of this project support the notion that genetic polymorphisms in the MMP1 and MMP8 influence the expression of these genes and the development and progression of cancer. The results of the second part of this project indicate an important role of MMP8 in the pathogenesis of atherosclerosis.

A exposição à radiação ultravioleta promove o desenvolvimento de efeitos indesejáveis, incluindo o fotoenvelhecimento e fotocarcinogênese. Uma forma de prevenção é a utilização de antioxidantes de origem natural. O extrato de raiz de Pothomorphe umbellata (PU) apresenta conhecida atividade antioxidante e fotoprotetora, atividade esta atribuída, em parte, ao princípio ativo 4-nerolidilcatecol (4-NC). Artigos publicados sobre a atividade do extrato de raiz de PU, conhecida popularmente como \"pariparoba\" e do princípio ativo, 4-NC, mostraram que o extrato bruto de raiz é mais ativo do que o 4-NC isolado como inibidor de metaloproteinases (MMPs) e como antioxidante, sugerindo a presença de outros compostos com estas atividades, além de um possível efeito sinérgico. O objetivo deste estudo foi o de obter frações do extrato de PU isentas de 4-NC e avaliar a atividade antioxidante, fotoprotetora e inibitória de metaloproteinases 2 e 9 na pele. O extrato hidroalcoólico de raiz de PU foi submetido ao fracionamento líquido/líquido sucessivamente com hexano e n-butanol, restando um resíduo aquoso. As frações n-butanólica (NBUT) e aquosa (AQ) mostraram-se isentas de 4-NC. A avaliação da atividade antioxidante por DPPH e ORAC das 3 frações obtidas mostrou maior capacidade antioxidante para a fração hexânica (HEX), seguido das frações NBUT e AQ. O mesmo perfil foi observado quanto à presença de compostos fenólicos. Entretanto não foi detectada a presença de flavonóides nas frações. O tratamento tópico com formulação gel-creme contendo o extrato de PU, a fração NBUT e a fração NBUT+4-NC, 2h antes da exposição aguda à radiação UVB (2 MED - 204.36 mJ/cm2), mostrou-se insuficiente para repor os níveis basais do sistema antioxidante da pele depletados em aproximadamente 20% pela radiação após 12h quando comparado ao controle. O mesmo tratamento não foi capaz de reduzir a formação de dímeros de pirimidina (CPD) após 2h. A avaliação após 12h mostrou redução da marcação de células de queimadura solar (SBC) e do antígeno de proliferação nuclear (PCNA) para o grupo PU e NBUT. A marcação para p53 e p21 também foi diminuída para o grupo PU e NBUT+4NC, respectivamente. A atividade de metaloproteinases (MMPs) 2 e 9 ativas extraídas do homogenato de pele de camundongos sem pelo após 24h da exposição à radiação UVB, avaliada por zimografia foi inibida pela fração NBUT. Os resultados observados pela fração NBUT de menor atividade antioxidante, comparados à fração HEX, sugerem que a atividade inibitória de MMPs não esteja necessariamente relacionada à capacidade antioxidante. Neste estudo não foi possivel identificar o(s) composto(s) provavelmente responsável(is) pela ação inibitória de MMPs da fração NBUT. / Ultraviolet radiation exposure induces the development of undesirable effects, including photoageing and photocarcinogenesis. One way of preventing these effects is the use of natural antioxidants. The Pothomorphe umbellata (PU) root extract shows antioxidant and photoprotective activities, which have been attributed, in part, to 4-nerolidylcatechol (4-NC), the main compound from this plant species. In previous studies, the plant extract was more efficient than 4-NC as an antioxidant and in metalloproteinases (MMPs) inhibitory activities, suggesting the presence of others compounds that could be related with these properties, as well as a possible synergic effect. The purpose of this study was to obtain fractions from PU without 4-NC, and to evaluate the antioxidant, photoprotection and skin MMPs 2 and 9 inhibitory activity of this fractionation. The P.umbellata hidroalcoholic root extract was submitted to liquid/liquid extraction with hexane followed by n-buthanol, from which derived a final residue of aqueous extract. The n-buthanolic (NBUT) and aqueous (AQ) fractions did not show the presence of 4-NC. The evaluation of the antioxidant activity by DPPH and ORAC for the 3 fractions showed higher antioxidant activity for the hexanic (HEX) fraction, followed by the NBUT and AQ fractions. The same profile was observed for phenolic compound concentration. However the presence of flavonols in the fractions and PU root extract was not detected. The gel-cream topic treatment containing PU root extract, NBUT fraction and NBUT+4NC, 2h before acute UVB exposure (2 MED - 204,36 mJ/cm2), was insufficient to promote the normal conditions of basal level antioxidant system depleted approximately 20% after 12h of the exposure compared with the control. The same treatment evaluated by immunohistochemistry, was not able to reduce the CPD (cyclobutane pyrimidine dimers) 2h after the exposure. In 12h reduction of SBC (sunburn cells) and PCNA (proliferating cell nuclear antigen) in PU and NBUT mouse groups was observed. The p53 and p21 proteins were reduced in PU and NBUT+4NC groups, respectively. The activity of metalloproteinases 2 and 9 (MMPs) of mouse skin homogenates after 24h acute UVB exposure, evaluated by zymography, was inhibited by the NBUT fraction. The observed results for NBUT that showed lower antioxidant activity than HEX suggest that the inhibitory effects of MMPs activity may not be related to the antioxidant capacity. It has not been possible to identify the compound(s) that are probably responsible for inhibitory activity of MMPs from the NBUT fraction.

While the genetic control of ureteric bud (UB) morphogenesis has been extensively studied, the cellular basis of this process remains unclear. The renal organoid system is a novel technique in which embryonic kidneys are dissociated into single cells and then reaggregated, where they reassociate to form organotypic structures. This system may be very beneficial for investigating the cellular basis of ureteric bud development. Here, we first used a fluorescent UB marker, Hoxb7:myrVenus, and time-lapse microscopy to characterize the cellular and tissue-level events during self-organization and UB morphogenesis of E12.5 or E14.5 renal organoids. Briefly, we found that UB structures self-assembled by aggregation of individual cells that sent out long cell processes. The cellular aggregates grew and elongated into epithelial tubes that displayed characteristic ampullae, bifurcated, and appropriately expressed UB tip markers analogous to their in vivo counterparts. We also found that cap mesenchymal cells are attracted to newly formed epithelial structures early in renal organoid development, and were later found in cell clusters surrounding new branches. RET is a trans-membrane tyrosine kinase receptor (RTK), expressed in ureteric bud cells, whose expression is gradually restricted to the tips of the growing ureteric tree. We demonstrate that the renal organoid system can be used, as an alternative to the generation of in vivo chimeric embryos, to study Ret-dependent cell rearrangements previously shown to establish and maintain the UB tip progenitor domain. Chimeric renal organoids that juxtaposed wild-type cells with Sprouty1–/– mutant cells (higher Ret-signaling) or with Ret51/cre (lower Ret-signaling) mutant cells recapitulated the cell sorting pattern observed in similar in vivo chimeras. The cells with higher Ret-signaling preferentially sorted to, and were maintained in, the forming and growing tips of these mosaic ureteric bud structures, out-competing cells with lower Ret-signaling. We then used the mosaic organoid system to ask if fibroblast growth factor receptor 2 (Fgfr2), another RTK expressed in the ureteric bud and important for its development, also mediates individual cell rearrangements that generate and maintain the UB tips. UB cells null for Fgfr2 were largely unable to compete with wild-type cells for occupancy of the UB tips in chimeric renal organoids. Using the innovative MASTR (Mosaic Mutant Analysis with Spatial and Temporal Control of Recombination) technique in vivo, mosaic homozygous deletion of Fgfr2 in newly formed ureteric buds also revealed that mutant cells were slightly deficient in their ability to contribute to Fgfr2 heterozygous UB tips. This demonstrates a novel, cell-autonomous role of Fgfr2 in ureteric bud development. Matrix metalloproteinase 14 (MMP14) is a membrane-bound protein known to participate in a wide variety of cell functions including degradation of the extracellular matrix (ECM), cell signaling, and cell-autonomous cell migration. It is expressed in the UB and was discovered to act downstream of Ret-signaling. Although needed in the ureteric epithelium for ECM degradation and proper UB morphogenesis, its specific function in the UB has not been thoroughly investigated. In generating in vivo chimeras, we discovered that Mmp14 null cells could contribute to wild-type ureteric bud tips at E12.5 and E14.5, demonstrating that, despite its documented role in UB branching, Mmp14 does not have a cell-autonomous role in the cell rearrangements observed during UB morphogenesis.

Matrix metalloproteinases (MMPs) are required for tissue remodelling processes, including angiogenesis. MMP activity is generally proangiogenic but MMP-12 is suggested to be antiangiogenic and its precise role is still unclear. The work in this thesis describes the synthesis of an MMP-12 inhibitor and activity probe to address the hypothesis that MMP-12 inhibits angiogenesis. An inhibitor, synthesised in-house, selectively inhibited MMP-12 in in vitro recombinant enzyme assays. An activity probe, also synthesised in-house, was selective for MMP-12 in in vitro recombinant enzyme assays. The function of MMP-12 during angiogenesis was assessed using murine models of angiogenesis; the in vivo sponge implantation, and the ex vivo aortic ring assays. Angiogenesis and MMP activity were imaged in vivo in sponges in C57Bl6/J mice over 7 − 21 days (D) using commercial probes (MMPSense™ and AngioSense™). MMP-12 protein concentration and activity were higher in sponges during early angiogenesis (D 3 − 7) when gene expression of vascular endothelial growth factor (a proangiogenic marker) was also high. Gene expression for MMP-12 and platelet-derived growth factor receptor (a marker of vascular maturation) were both higher on D 21 as angiogenesis started to stabilise. The MMP-12 activity probe was unsuccessful in selectively detecting MMP-12 activity in sponge lysate mixtures from D 7 − 21. Administration of an MMP-12 inhibitor did not increase angiogenesis in the sponges in vivo. Additionally, sponges implanted in MMP-12-/- mice did not exhibit significant changes in angiogenesis or MMP activity when imaged in vivo using commercial probes (MMPSense™ and AngioSense™) on D 7. Supporting this, histological analysis of the sponges (removed on D 21) showed that deletion of MMP-12 also did not increase angiogenesis within the sponges.

Melanoma é uma neoplasia maligna com comportamento agressivo considerado o câncer mais comum da cavidade bucal de cães. Ele pode ser classificado quanto a morfologia celular em epitelióide, fusiforme e misto ou quanto ao fenótipo: melânico ou amelânico. Estudos têm sugerido que os melanomas amelânicos são mais agressivos. O objetivo deste estudo foi avaliar o comportamento dos melanomas da cavidade bucal dos cães, quantificando a expressão das Cx26 e 43, caderina-E, MMPs 2 e 9 e proliferação celular. Para tanto foram coletados 25 melanomas provenientes de cães atendidos no HOVET FMVZ-USP (16 melânicos e 9 amelânicos). Após a cirurgia os animais foram acompanhados até a morte, sendo que 5 animais foram eutanasiados (2 antes da cirurgia e 3 após o procedimento cirúrgico, devido ao sofrimento físico). Os tumores eram diagnosticados através do histopatológico e classificados de acordo com OMS (1998). Para confirmação dos melanomas amelânicos foi utilizado à técnica de imuno-histoquímica com o perfil de anticorpos pré-estabelecidos. A proliferação celular foi quantificada através do uso de PCNA, índice apoptótico e caspase-3. O comportamento tumoral foi avaliado através da técnica de imuno-histoquímica para caderina-E e MMPs 2 e 9. E a expressão das Cxs foram avaliadas através da imunofluorescência e western blot. Cães com melanma amelânico apresentaram menor sobrevida, com aumento do número de metástase, fraca marcação para caderina-E e alta intensidade para as MMPs 2 e 9, aumento de células positivas para PCNA e índice mitótico, e diminuição da caspase-3, não havendo diferença significante quanto aos tipos histotógicos. Houve diminuição de expressão das Cxs entre os amelânicos, no entanto, aumento da síntese do gene de CX 26 entre o mesmo grupo, o que se verificou através do PCR em Tempo Real. Nossos achados sugerem que os melanomas da cavidade bucal de cães apresentam um comportamento mais agressivo / Melanoma is a malignant neoplasm with aggressive behavior considered the most common cancer of the buccal cavity in dogs. It can be classified as cell morphology in epithelioid, spindle and mixed or on the phenotype, in melanotic or amelanotic. Studies have suggested that amelanotic melanomas are more aggressive. The aim of this study was to evaluate the behavior of melanoma of the buccal cavity of dogs, quantifying the expression of Connexins 26 and 43, E-cadherin, MMPs 2 and 9 and cell proliferation. For both melanomas were collected from 25 dogs attended at HOVET FMVZ (USP) (16 melanotic and 9 amelanotic melanomas). After the surgery, the animals were followed until death, so 5 animals were euthanized (2 before surgery and 3 after surgery, due to physical suffering). The tumors were diagnosed by histopathology and classified according to WHO (1998). For confirmation of amelanotic melanomas it was used the technique of immune-histochemistry with the antibody profile pre-established. Cell proliferation was quantified by using PCNA, apoptotic index and caspase-3. The tumor behavior was evaluated using the technique of immune-histochemistry for E-cadherin and MMPs 2 and 9. And the expression of Cxs was evaluated by immune-fluorescence and western blot. Dogs with amelanotic melanoma had lower survival with increasing number of metastatic, weak immunoreactivity for E-cadherin and high intensity for MMP 2 and 9, an increase of cells positive for PCNA and mitotic index, and decreased caspase-3, no significant difference in the morphologic subtypes. There was a decrease of expression of Cxs between amelanotic, however, increased synthesis of CX 26 gene among the same group, which was verified by real time PCR. Our findings suggest that melanomas of the buccal cavity of dogs exhibited more aggressive behavior.

A hipertensão arterial é uma condição clínica grave acompanhada por alterações estruturais do aparelho cardiovascular. Os antagonistas dos receptores 1-adrenérgicos são drogas usadas na terapêutica anti-hipertensiva. O nebivolol é um antagonista seletivo dos receptores 1 de terceira geração, que estimula a liberação endotelial de NO e diminui a ativação da -nicotinamida adenosina dinucleotído fosfato (NAD(P)H) oxidase. O metoprolol é um antagonista seletivo dos receptores 1de segunda geração, que não apresenta efeitos vasodilatadores. As metaloproteinases da matriz (MMPs), principalmente a MMP-2, são enzimas que participam ativamente do processo de remodelamento vascular. Elas passam de seu estado latente para seu estado ativo pela ação de proteases e espécies reativas de oxigênio (EROs). Como na hipertensão há aumento de EROs, de MMPs e remodelamento vascular, é possível que o nebivolol impeça o aumento dos níveis de MMPs vasculares e o remodelamento vascular hipertrófico associados à hipertensão, além do seu efeito de antagonismo do receptor 1. Primeiro, realizamos uma avaliação das alterações aórticas da hipertensão 2-rins,1-clipe (2R-1C), pois há poucas informações sobre essas alterações durante o desenvolvimento da hipertensão. Para isso, ratos hipertensos e controles foram estudados com 2, 4, 6 e 10 semanas após a indução da hipertensão. A pressão arterial sistólica foi monitorada semanalmente. As alterações na parede aórtica foram estudadas em hematoxilina/eosina (H&E), picrosirius e orceína. Foram avaliados também a atividade da NAD(P)H oxidase, a produção de ânion superóxido, a atividade gelatinolítica por zimografia in situ; os níveis e localização de MMP-2, MMP-14 e TIMP-2 por imunofluorescência zimografia e imunohistoquímica. No segundo protocolo, após 6 semanas de hipertensão foi iniciado o tratamento com metoprolol (Meto -20/mg/kg/dia) ou nebivolol (Nebi -10mg/kg/dia), realizado durante 4 semanas. Foram avaliados a pressão arterial sistólica, as alterações na parede aórtica por H&E, a atividade da NAD(P)H oxidase, produção de ânion superóxido, a atividade gelatinolítica, os níveis e localização de MMP-2 por imunofluorescência e zimografia em gel, e os níveis de nitrotirosina por imunohistoquímica. Observamos no protocolo temporal que a pressão foi gradualmente aumentada nos animais hipertensos quando comparado aos controles. Houve hiperplasia e hipertrofia da aorta, com aumento da deposição de colágeno e elastina. Observamos aumento nos níveis de estresse oxidativo, MMPs e atividade gelatinolítica em todas as semanas de estudo. Ao final do tratamento com metoprolol e nebivolol, observamos que a pressão diminuiu nos animais hipertensos, e que essa redução da pressão ocorreu de modo semelhante com os diferentes tratamentos. Os animais hipertensos apresentaram hipertrofia da aorta, aumento nos níveis de EROs, de MMP-2 e da atividade gelatinolítica. Essas alterações foram revertidas apenas pelo tratamento com nebivolol, mas não pelo metoprolol. Assim, o remodelamento da hipertensão 2R-1C parece estar estabelecido com duas semanas de hipertensão arterial, com elevados níveis de espécies reativas de oxigênio e MMPs, e o tratamento com nebivolol, mas não o metoprolol, atenuou o estresse oxidativo e o remodelamento vascular associado à hipertensão. / Hypertension is a serious clinical condition with structural changes in the cardiovascular system. Beta-adrenoreceptor antagonists are used in hypertension therapy. Nebivolol is a third-generation selective 1-adrenoreceptor antagonist that stimulates endothelial cell NO production and prevents vascular NAD(P)H oxidase activation. Metoprolol is a second-generation selective 1-adrenoreceptor antagonist, without vasodilatory effect. The matrix metalloproteinases (MMPs), especially MMP-2, participate actively in the vascular remodeling process, and are activated by reactive oxygen species (ROS). There are increases in ROS, of MMPs, and vascular remodeling in hypertension. Because of these, it´s possible that nebivolol prevent MMP increases and vascular remodeling associated with hypertension beyond its 1-receptorblocking properties. First, structural aortic changes in the development of two-kidney, one-clip hypertension (2K-1C) were evaluated. Sham or 2K1C hypertensive rats were studied after 2, 4, 6, and 10 weeks of hypertension. Systolic blood pressure (SBP) was monitored weekly. Morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin, orcein and picrosirius red sections. Aortic NAD(P)H activity and superoxide production was evaluated. Aortic gelatinolytic activity was determined by in situ zymography, and MMP-2, MMP-14, and tissue inhibitor of MMPs (TIMP)-2 levels were determined by gelatin zymography, immunofluorescence and immunohistochemistry. Six weeks after surgery, hypertensive and sham rats were treated with metoprolol (20 mg/kg/ day) or nebivolol (10 mg/kg/day), for four weeks, in the second-protocol. Systolic blood pressure (SBP) was monitored weekly Aortic structural were studied in hematoxylin/eosin sections. NAD(P)H oxidase activity and ROS and nitrotyrosine production were evaluated. MMPs levels and activity were determined by zymography and in situ zymography. In the temporal study, 2K-1C hypertension was associated with increased aortic collagen and elastin content in the early phase of hypertension, which was associated with vascular hypertrophy, increased vascular MMPs levels, and increased gelatinolytic activity, possibly as a result of increased vascular NAD(P)H oxidase activity and oxidative stress. In the second-protocol, similar reductions in SBP were found with both metoprolol and nebivolol treatments. However, only Nebi reversed aortic hypertrophy, the increases in aortic NAD(P)H oxidase activity, in aortic ROS levels, in nitrotyrosine staining, in aortic MMP-2 levels and in aortic MMP activity. These results indicate that vascular remodeling of renovascular hypertension is an early process associated with increases in MMPs activities, enhanced matrix deposition and oxidative stress, and that treatment with Nebi attenuate the oxidative stress and the vascular remodeling associated with 2K-1C hypertension.

Melanoma é uma neoplasia maligna com comportamento agressivo considerado o câncer mais comum da cavidade bucal de cães. Ele pode ser classificado quanto a morfologia celular em epitelióide, fusiforme e misto ou quanto ao fenótipo: melânico ou amelânico. Estudos têm sugerido que os melanomas amelânicos são mais agressivos. O objetivo deste estudo foi avaliar o comportamento dos melanomas da cavidade bucal dos cães, quantificando a expressão das Cx26 e 43, caderina-E, MMPs 2 e 9 e proliferação celular. Para tanto foram coletados 25 melanomas provenientes de cães atendidos no HOVET FMVZ-USP (16 melânicos e 9 amelânicos). Após a cirurgia os animais foram acompanhados até a morte, sendo que 5 animais foram eutanasiados (2 antes da cirurgia e 3 após o procedimento cirúrgico, devido ao sofrimento físico). Os tumores eram diagnosticados através do histopatológico e classificados de acordo com OMS (1998). Para confirmação dos melanomas amelânicos foi utilizado à técnica de imuno-histoquímica com o perfil de anticorpos pré-estabelecidos. A proliferação celular foi quantificada através do uso de PCNA, índice apoptótico e caspase-3. O comportamento tumoral foi avaliado através da técnica de imuno-histoquímica para caderina-E e MMPs 2 e 9. E a expressão das Cxs foram avaliadas através da imunofluorescência e western blot. Cães com melanma amelânico apresentaram menor sobrevida, com aumento do número de metástase, fraca marcação para caderina-E e alta intensidade para as MMPs 2 e 9, aumento de células positivas para PCNA e índice mitótico, e diminuição da caspase-3, não havendo diferença significante quanto aos tipos histotógicos. Houve diminuição de expressão das Cxs entre os amelânicos, no entanto, aumento da síntese do gene de CX 26 entre o mesmo grupo, o que se verificou através do PCR em Tempo Real. Nossos achados sugerem que os melanomas da cavidade bucal de cães apresentam um comportamento mais agressivo / Melanoma is a malignant neoplasm with aggressive behavior considered the most common cancer of the buccal cavity in dogs. It can be classified as cell morphology in epithelioid, spindle and mixed or on the phenotype, in melanotic or amelanotic. Studies have suggested that amelanotic melanomas are more aggressive. The aim of this study was to evaluate the behavior of melanoma of the buccal cavity of dogs, quantifying the expression of Connexins 26 and 43, E-cadherin, MMPs 2 and 9 and cell proliferation. For both melanomas were collected from 25 dogs attended at HOVET FMVZ (USP) (16 melanotic and 9 amelanotic melanomas). After the surgery, the animals were followed until death, so 5 animals were euthanized (2 before surgery and 3 after surgery, due to physical suffering). The tumors were diagnosed by histopathology and classified according to WHO (1998). For confirmation of amelanotic melanomas it was used the technique of immune-histochemistry with the antibody profile pre-established. Cell proliferation was quantified by using PCNA, apoptotic index and caspase-3. The tumor behavior was evaluated using the technique of immune-histochemistry for E-cadherin and MMPs 2 and 9. And the expression of Cxs was evaluated by immune-fluorescence and western blot. Dogs with amelanotic melanoma had lower survival with increasing number of metastatic, weak immunoreactivity for E-cadherin and high intensity for MMP 2 and 9, an increase of cells positive for PCNA and mitotic index, and decreased caspase-3, no significant difference in the morphologic subtypes. There was a decrease of expression of Cxs between amelanotic, however, increased synthesis of CX 26 gene among the same group, which was verified by real time PCR. Our findings suggest that melanomas of the buccal cavity of dogs exhibited more aggressive behavior.