The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

OBJECTIVE: To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. METHODS: The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. RESULTS: Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-ß regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. CONCLUSIONS: These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Endothelial Glycocalyx as a Regulator of Fibrotic Processes.

The endothelial glycocalyx, the gel layer covering the endothelium, is composed of glycosaminoglycans, proteoglycans, and adsorbed plasma proteins. This structure modulates vessels' mechanotransduction, vascular permeability, and leukocyte adhesion. Thus, it regulates several physiological and pathological events. In the present review, we described the mechanisms that disturb glycocalyx stability such as reactive oxygen species, matrix metalloproteinases, and heparanase. We then focused our attention on the role of glycocalyx degradation in the induction of profibrotic events and on the possible pharmacological strategies to preserve this delicate structure.

Sistema adesivo universal: análise da interface adesiva e degradação colagenolítica em dentina submetida à diferentes protocolos de condicionamento ácido / Universal adhesive system: analysis of adhesive interface and collagenolytic degradation of demineralized dentin submitted to different acid etching ptotocols

O objetivo desse estudo foi analisar a interface adesiva do sistema adesivo Single Bond Universal (SBU) em dentina submetida à diferentes protocolos de condicionamento ácido em 24 h e 12 meses. E a degradação colagenolítica (DC) mediada por metaloproteinases (MMPs) e Catepsina-K (CAT-K) em tempo imediato. Esse estudo foi conduzido em 3 etapas: 1) Caracterização química da dentina em FTIR; 2) DC por meio de fragmentos do Telelopeptídeo Carboxiterminal do Colágeno Tipo I (ICTP) e do Terminal C do Telopeptídeo ligado ao Colágeno Tipo I (CTX) e a resistência à tração (RT) da do colágeno; 3) Análise da interface adesiva através da resistência de união (RU), análise de fratura, microscopia eletrônica de varredura (MEV) e nanoinfiltração (NI). Para FTIR foram utilizados 6 discos de dentina, divididos em 2 grupos: 1) Ácido fosfórico 32 %15 s (AF), 2) Ácido poliacrílico 25 % 10 s (AP). Para análise da DC, 12 discos de dentina foram completamente desmineralizados e divididos em 3 grupos: 1) AF, 2) AP e 3) Água deionizada (Controle) 15 s. Após, foram incubados e armazenados por 1 semana. Seguindo-se a análise da concentração de proteína total (PT). 50 µl da solução de incubação foram utilizadas para analisar ICTP e CTX. As concentrações foram calculadas em relação à PT. Para RT, foram testados 36 palitos obtidos dos discos de colágeno. Para RU foram utilizados 48 dentes, divididos em 2 grupos, de acordo com o período de armazenamento, divididos em três subgrupos: 1) AF, 2) AP e 3) Autocondicionante SBU 20 s (SE). Os dentes foram restaurados e armazenados em água destilada 37 °C. Após, foram submetidos ao teste de microtração e análise de fraturas. Para as análises MEV e NI foram utilizados 2 espécimes de cada subgrupo. Para análise estatística utilizou-se ANOVA 1-Fator, ANOVA- 2 Fatores e teste de Tukey (α=0.05). Para FTIR, AF reduziu a quantidade de fosfato e carbonato quando comparado ao AP. Para DC, a liberação de ICTPPT para AF foi significantemente maior do que para AP (p < 0,05). Não houve diferença na liberação de CTXPT para AF e AP (p > 0,05). Para RT não houve diferença entre AP e Controle, porém, apresentaram valores maiores do que AF (p<0,05). Para RU em MPa, não houve diferença estatisticamente significativa para todos os tratamentos nos diferentes períodos de análise (p < 0,05). A análise de fraturas evidenciou a predominância de fraturas adesivas e mistas. MEV mostrou melhor qualidade da interface após 12 meses para AF e AP. Após 12 meses apenas SE não apresentou NI. Assim sendo, o autocondicionamento ainda parece ser a melhor opção para sistemas adesivos universais que possuam monômeros funcionais em sua composição.(AU)

The aim of this study was to analyze the adhesive interface of the Single Bond Universal (SBU) to dentin submitted to different acid etching protocols in 24 h and 12 months, and the collagenolytic degradation (CD) by matrix metalloproteinases (MMPs) and Cathepsin-K (CAT-K) in the immediate time. This study was divided into three stages: 1) Dentin chemical characterization by FTIR; 2) CD by release of the collagen telopeptide fragment cross-linked carboxyterminal telopeptide of type I collagen (ICTP), and C-terminal crosslinked telopeptide of type I collagen (CTX) and ultimate tensile strength (UTS); 3) Analysis of the adhesive interface by microtensile bond strength (µTBS), failure mode, scanning electron microscopy (SEM) of the AI, and nanoleakage by SEM (NL). For FTIR, six dentin disks were divided into two groups: 1) Phosphoric acid 15 s (PA), 2) Polyacrylic acid 10 s (PAA). For CD twelve dentin disks were completely demineralized, then were divided into 3 groups 1) PA, 2) PAA, and 3) deionized water (Control) for 15 s. All disks were incubated in a buffered solution (BS) for 1 week. Total protein (TP) concentrations were measured using Nanodrop™ at 280 nm. 50 µl of BS was used to analyze solubilized telopeptide fragments using ICTP and CTX . ICTP and CTX average ratios were calculated in relation to TP concentration (ICTPtp and CTXtp). For UTS, 36 dentin beams obtained from collagen disks were tested. For µTBS, forty-eight teeth were divided into two groups according to the period of storage, then subdivided into three subgroups: 1) PA, 2) PAA, and 3) Self-etch 20 s (SE). After, composite build up, the specimens were stored in distilled water at 37 °C. Two specimens of each group were used for SEM analysis of AI and NL. Data were analyzed by one-way ANOVA, two-way ANOVA and Tukey tests (p<0.05). According to the results of the FTIR etching with PA reduced the amount of phosphate and carbonate when compared to PAA. ICTPtp release of PA was significantly higher A than PAA (p > 0,05). CTXtp showed no difference between the PA and PAA (p < 0,05). For UTS there was no difference between PAA and control, but they were significantly higher (p<0.05) than PA. For µTBS in MPa, there is no statistical difference among all the etching protocols tested, as well in both storage periods of analysis (p < 0,05). The most prevalent failure mode were adhesives associated with mixed. SEM analysis highlighted a better quality of AI after 12 months for PA and PAA. However, after 12 months only SE did not show NL. Then, the self-etching protocol seems to be a better choice regarding universal adhesive systems which have functional monomers in their blend(AU)

Avaliação das metaloproteinases de matriz -2 e -9 em gatos com desmineralização óssea secundária à tirotoxicose induzida / Evaluation of matrix metalloproteinases -2 and -9 in cats under bone demineralization secondary to induced thyrotoxicosis

Observou-se significativo aumento de atividade das formas ativas das metaloproteinases -2 e -9 em gatos com tirotoxicose induzida e desmineralização óssea. As formas pró e intermediária da metaloproteinase -2 elevaram-se com 14 dias de administração hormonal, porém, posteriormente, houve uma tendência de queda. Observou-se correlação negativa entre a forma ativa das metaloproteinases de matriz -2 e -9 e a densidade mineral óssea da extremidade distal do rádio. Os resultados sugerem aumento da degradação da matriz colágena secundária com a elevação dos hormônios tiroidianos.

Significant increase of activity of active forms of matrix metalloproteinases -2 and -9 in cats under induced thyrotoxicosis and bone demineralization was observed. Pro and intermediated forms of matrix metalloproteinases -2 and -9 increased at 14 days of hormonal treatment, followed by decrease tendency. A negative correlation between active forms of matrix metalloproteinases -2 and -9 and bone mineral density of radius distal extremity was also observed. The results suggest an increase of collagen matrix degradation secondary to high levels of thyroid hormones.

Matrix metalloproteinase-induced fibrosis and malignancy in breast and lung.

Fibrosis is a pathological condition in which tissue structure is disrupted by production of excess extracellular matrix (ECM), and chronic tissue fibrosis is associated with tumor development. Myofibroblasts are the principal mediators of fibrosis, producing abundant ECM as well as inflammatory and angiogenic factors. Myofibroblasts are also abundant in tumor stroma, where they facilitate tumor growth and progression. Matrix metalloproteinases (MMPs), enzymes that degrade and remodel the ECM, are believed to play a critical role in the development of fibrotic tissue, though the mechanism by which this occurs is unclear. Expression of MMP-3 in mammary epithelial cells of transgenic mice stimulates development of fibrosis and subsequent tumor formation. We have recently determined that exposure of mammary epithelial cells to MMP-3 induces a specialized form of epithelial-mesenchymal transition in which the cells acquire myofibroblast-like characteristics and that this process is dependent upon the generation of cellular reactive oxygen species (ROS). New data from culture models in which MMPs are inducibly expressed in human lung cell lines, and transgenic mouse models in which MMPs are inducibly expressed in lung alveolar epithelial cells, suggest that similar processes likely exist in the lung.

Matrix metalloproteinases in asthma and COPD.

Asthma and chronic obstructive pulmonary disease (COPD) are both highly prevalent, chronic inflammatory lung diseases that lead to significant morbidity and mortality. Matrix metalloproteinases (MMPs) are extracellular matrix degrading enzymes that play a critical role in normal development and physiological tissue remodeling and repair. In addition, they play an important role in the regulation of the kinetics and function of inflammatory cells. There is increasing evidence that MMPs are involved in the pathogenesis of both asthma and COPD, and several MMPs are possible therapeutic targets in these common chronic airway diseases.

Alveolar macrophage deficiency in osteopetrotic mice deficient in macrophage colony-stimulating factor is spontaneously corrected with age and associated with matrix metalloproteinase expression and emphysema.

Macrophage colony-stimulating factor (M-CSF) is one of several hematologic growth factors capable of regulating the survival, proliferation, and differentiation of macrophages, but its role in modulation of the accumulation and function of alveolar macrophages (AMs) in vivo is not well defined. Osteopetrotic (Op/Op) mice have no detectable M-CSF and show variable tissue-specific reductions in macrophage numbers. It was hypothesized that AMs would be decreased in number and have altered function in Op/Op mice because of the absence of M-CSF. Lung macrophages identified by Mac-3 staining in lung sections were decreased in number in 20-day-old Op/Op mice (P <.001) but not Op/Op mice older than 4 months (P =.68) compared with findings in age-matched littermate controls. The numbers of AMs recovered by bronchoalveolar lavage (BAL) were also reduced in young but not adult Op/Op mice compared with controls. Expression of interleukin-3 (IL-3) was increased in the lungs of Op/Op mice compared with controls as determined by quantification of IL-3 cytokine levels (P =.04), bioactivity (P =.02), and messenger RNA transcript levels. AMs of Op/Op mice spontaneously released higher levels of matrix metalloproteinases (MMPs) than AMs of controls as determined by immunohistochemical staining of AMs and zymographic assessment of BAL fluid and AM lysates. Consistent with an increased release of MMP, Op/Op mice had abnormal elastin deposition and spontaneously developed emphysema in the absence of molecular or cellular evidence of lung inflammation. These data show that the AM deficiency observed in young Op/Op mice is spontaneously corrected with age and is associated with increased lung levels of IL-3, spontaneous MMP expression by AMs, and destruction of lung tissue.