A ação das metaloproteinases de matriz (MMP-2, MMP-9 e MT1-MMP) é necessária para degradação da membrana basal em carcinomas da cérvice uterina. O objetivo deste trabalho consistiu na avaliação da expressão das metaloproteinases MMP -2, -9 e MT1-MMP, do gene supressor de metástase RECK e do inibidor tecidual de MMPs (TIMP-2) em modelo de células de neoplasia da cérvice-uterina cultivadas em substratos de matriz extracelular. As linhagens celulares de carcinoma de cérvice uterina SiHa, CaSki, ambas HPV 16 positivas, e C33A, HPV negativa, foram cultivadas em gel de colágeno tipo I, Matrigel e plástico. Avaliou-se o crescimento, invasão, expressão gênica, através de ensaios de real-time PCR, e atividade de metaloproteinases, através de ensaios de zimografia. Os resultados demonstraram que estas linhagens de carcinoma cervical quando cultivadas em gel de colágeno tipo I apresentaram uma diminuição no crescimento, morfologia modificada na presença de substrato de matriz extracelular, e que nas linhagens HPV positivas há um aumento da expressão de MMP-2, MT1-MMP e TIMP-2 e da atividade de pró-MMP-2 em relação à linhagem HPV negativa. Observou-se também que, RECK apresentou maior expressão gênica em CaSki associada à atividade de pró-MMP-2. MMP-9 apresentou muito baixa expressão gênica em todas as linhagens e condições estudadas. Quando analisamos as linhagens separadamente, observamos que o Matrigel influenciou a expressão gênica de MMP-2, e que o gel de colágeno tipo I aparece como indutor da atividade de pró-MMP-2 em todas as linhagens. Em conclusão, nossos resultados mostram que a expressão de MMP-2, MT1-MMP e TIMP-2 e que a atividade de pró-MMP-2 estão aumentadas nas células HPV positivas, sugerindo que o HPV está associado com a expressão de MMPs e TIMP-2. / Matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in three human invasive cervical carcinoma cell lines. Two HPV-16- positive cell lines (SiHa and CaSki), HPV-negative cell line (C33A) were cultured either onto type-I collagen gel, Matrigel or plastic, in order to recreate their three-dimensional growth environment and evaluate the growth and invasion of the cells and expression of these genes using quantitative real-time PCR (Q-PCR). We also analyzed the gelatinolytic activity of MMP-2 and -9 by zymography. We found that the growth curves carcinoma cells are decreased and cells morphology are modified in ECM substrate. HPV-positive cell lines expressed higher levels of MMP-2, MT1-MMP and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with pro-MMP-2 activity. The Matrigel substrate influence MMP-2 expression for SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP -2, MT1-MMP and TIMP-2 and pro-MMP-2 activity in HPV-positive cells than in HPV-negative cells.

Introdução: O Câncer de próstata (CaP) é o mais comum do homem brasileiro. É importante a identificação de alterações moleculares que possam prever o seu desenvolvimento e potencial biológico. Polimorfismos de nucleotídeo único (SNP) são alterações da seqüência do DNA onde somente uma base é trocada com uma freqüência superior a 1% na população, que podem levar a modificações estruturais e funcionais na proteína, ou afetar a sua quantidade, e podem constituir marcadores de predisposição e prognóstico de neoplasias. Metaloproteinases (MMP) são proteínas da família de enzimas proteolíticas, que degradam a matriz extracelular, e SNP na sua estrutura têm sido associados ao comportamento de tumores. Objetivos: Avaliar a freqüencia de SNP nos genes das MMP1, 2, 7 e 9, em pacientes com CaP e grupo controle, relacionando com suscetibilidade para o desenvolvimento da doença e previsão de seu potencial biológico. Material e Métodos: A amostra é constituída por tecido não tumoral de 100 indivíduos com CaP, e 100 amostras controle representadas por soro de indivíduos saudáveis, sem câncer de próstata. O DNA foi obtido utilizando protocolos convencionais de extração. Para genotipagem foi utilizada técnica de identificação de base única com uso de sondas marcadas com fluoróforos (Taqman®) pela técnica de reação em cadeia da polimerase em tempo real. As freqüências alélicas foram calculadas e a comparação entre os grupos foi feita utilizando-se o teste de qui-quadrado com valor de significância de 0,05. Resultados: Nos genes das MMP1 a freqüência do genótipo homozigoto polimórfico esteve mais presente no grupo controle que no CaP (p>0,001). No gene da MMP9 o alelo polimórfico esteve mais presente em pacientes com CaP (p>0,001), e em tumores com escore de Gleason6 (p=0,003). No gene da MMP2 de acordo com estadiamento patológico o alelo polimórfico foi mais freqüente em tumores pT3 (p=0,026) e Gleason maior ou igual a 7(p=0,042). Conclusão: Nossos resultados sugerem que o polimorfismo no gene da MMP1 está associado a um caráter de proteção aos indivíduos quanto ao desenvolvimento do CaP. O polimorfismo no gene da MMP9 está associado a um aumento no risco de desenvolvimento desta neoplasia, e quando analisamos as associações com os fatores prognósticos encontramos uma correlação com tumores de melhor prognóstico. Por outro lado o polimorfismo do gene da MMP2 se associa a tumores não órgãoconfinados / Introduction: Prostate cancer (PCa) is the most frequent tumor in males in Brazil. Research has been directed for the identification of molecular markers that can predict the PCa predisposition and prognosis. Single nucleotide polymorphisms (SNPs) are genome variations, present in a frequency of 1% or more. The matrix metalloproteinases (MMPs) are a family of enzymes responsible for the degradation of extracellular matrix. SNPs have been demonstrated in the promoter region of these genes and have been associated with development and progression of some cancers. Objective: To investigate the correlation between polymorphisms of MMP1, 2, 7, 9 with susceptibility and classical prognostic parameters in PCa. Patients and methods: The sample is constituted by normal tissue of 100 patients with PCa, and 100 healthy men as controls (serum). DNA genomic was extracted from paraffin blocks and serum using conventional protocols. The DNA sequence containing the polymorphic sites was amplified by Real-Time polymerase chain reaction, using fluorescent probes (Taqman®). The allelic frequency was calculated and the comparison between the groups was made using the qui-square test with value of significance of 0.05. Results: The polymorphic homozygote genotype of the MMP1 was more frequent in the control group than in the PCa (p<0.001). The polymorphic allele of MMP9 was more frequent in the PCa group (p<0.001), and in tumors Gleason6 (p=0,003). The polymorphic allele of MMP2 was more frequent in tumors of higher stage (pT3) (p=0.026) and higher Gleason Score (7) (p=0.042). Conclusion: We have shown that MMP1 polymorphism is more frequent in the control group, than in patients with PCa, it may be associated to protection for the development of PCa. The MMP9 polymorphism was related to higher risk for development of this neoplasia, but associated with lower Gleason score. MMP2 polymorphism was associated with non organconfined disease.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study evaluated as overall purpose the influence of doxycycline used as a matrix metalloproteinase (MMPs) inhibitor on the microtensile bond strength (μTBS) of a total etching adhesive system to dentin, considering two formulations (different pHs) and two substrates (source and demineralized) of primary and permanent teeth. The study is divided into two articles, the aim of the first article was to evaluate the application s effect of acidic or neutral doxycycline on the μTBS to dentin. Twenty extracted noncarious third molars were divided into 4 groups (doxycycline acidic pH, doxycycline neutral pH, chlorhexidine and control - no treatment). A flat dentin surface was exposed, wet grinding and submitted to: acid etch, application of the substance corresponding treatment group, application of adhesive system and resin composite. The specimens were stored (24 h in distilled water) and sectioned to obtain sticks (cross-sectional area of approximately 0.9 mm2). The sticks were submitted to tensile test. The mean μTBS were submitted to a one-way ANOVA and Tukey s test (α = 0.05). No significant difference of immediate μTBS (MPa) was observed between the neutral and acidic doxycycline, furthermore the solutions of different pH values were statistically similar to control and chlorhexidine groups. For the second study 30 permanent molars and 30 second primary molars were divided into 12 experimental groups (n = 5) according to the surface treatment after etching: doxycycline, chlorhexidine and control, and the condition substrate: sound and demineralized (pH cycling). The specimens preparation, restorative procedures and tensile test were performed in the same way as in the previous study. The μTBS were subjected to a two-way ANOVA and Tukey's test (α = 0.05), considering separately primary and permanent teeth. For both teeth types the application of doxycycline did not affect the immediate μTBS, while the demineralized substrate values were statistically lower than the sound group. The doxycycline solution, even at acidic pH, does not seem to affect the immediate bond strength to dentin, while demineralized substrate needs attention by presenting μTBS immediately reduced compared to sound. / Este trabalho teve como objetivo geral, avaliar a influência de uma substância inibidora de metaloproteinases (MMPs), a doxiciclina, na resistência de união (RU) à dentina de um sistema adesivo com condicionamento prévio, considerando duas formulações (diferentes pHs) e dois substratos (hígido e desmineralizado) de dentes decíduos e permanentes. O trabalho está dividido em dois artigos, sendo o primeiro realizado com o intuito de avaliar o efeito da aplicação da doxiciclina ácida ou neutra na RU à dentina. Para isso, foram selecionados 20 terceiros molares hígidos, os quais foram divididos em 4 grupos experimentais (doxiciclina pH ácido, doxiciclina pH neutro, clorexidina e controle nenhum tratamento). Os dentes foram seccionados próximo ao terço médio da coroa para obtenção de uma superfície dentinária plana, a qual foi submetida a: condicionamento ácido, tratamento de acordo com o grupo, ou seja, aplicação da substância correspondente na superfície condicionada, aplicação do sistema adesivo e restauração com resina composta. Após armazenamento (24h em água destilada a 37°C) os espécimes foram seccionados para obtenção de palitos (área de secção transversal de aproximadamente 0,9mm2), os quais foram submetidos a ensaio de tração. Os valores médios de RU foram submetidos à análise de variância com um fator e ao teste de Tukey (α=0,05). Os valores de RU (em MPa) obtidos para os grupos tratados com as soluções de doxiciclina em diferentes pHs apresentaram valores de RU estatisticamente semelhantes entre si e aos grupos controle e clorexidina. Para o segundo estudo, foram selecionados 30 terceiros molares permanentes e 30 segundos molares decíduos hígidos, os quais foram divididos em 12 grupos experimentais (n=5) de acordo com o tratamento da superfície após condicionamento ácido: doxiciclina, clorexidina e controle, e a condição do substrato: hígido e desmineralizado (ciclagem de pH). O preparo dos espécimes, procedimentos restauradores e ensaio de tração foram realizados da mesma forma que no estudo anterior. Os valores de RU obtidos foram submetidos à análise de variância de dois fatores e ao teste de Tukey (α=0,05), considerando separadamente dentes decíduos e permanentes. Para os dois tipos de dentes, a aplicação de solução de doxiciclina não afetou a resistência de união imediata, enquanto o substrato desmineralizado apresentou valores estatisticamente inferiores ao grupo hígido. A solução de doxiciclina, mesmo em pH ácido, parece não afetar a resistência de união imediata à dentina, sendo que o substrato desmineralizado merece atenção por apresentar valores de RU imediata reduzidos comparado ao hígido.

Abstract Endometrial carcinoma is the most common gynegologic malignancy in developed countries. Due to early symptoms, including abnormal uterine bleeding, endometrial cancer is often diagnosed at an early stage and in that case usually has a good prognosis and high cure rates. However, the nature of the disease is heterogeneous. During the last decades, the improvement in survival rates among endometrial cancer patients has not been significant, suggesting that the traditional clinicopathological factors may be inadequate to identify patients with high-risk disease. Furthermore, aggressive adjuvant treatments can be costly and very toxic. Therefore, better prognostic markers associated with biological aggressiveness of endometrial carcinoma are needed to identify the patients with high-risk disease, and to be able to select the treatment more individually. Gelatinases (MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) have been found to play a role in tumor progression. In the present work, the expression and prognostic value of MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed in endometrial carcinoma. The patient material consisted of a total of 266 women diagnosed with primary endometrial carcinoma. The tissue expression of immunoreactive proteins was examined in paraffin-embedded tumor sections by immunohistochemical staining using specific antibodies, and the pretreatment serum levels of the proteins were quantitatively measured by ELISA. Tissue MMP-2 expression associated with a worsened prognosis, whereas tissue TIMP-2 overexpression was an indicator of a favorable outcome. Furthermore, we observed a combination of strong MMP-2 and weak TIMP-2 tissue expression to identify a group of women at high risk of adverse outcome in endometrial carcinoma. Patients with negative MMP-2 immunostaining had the best prognosis, regardless of TIMP-2 staining result. In serum measurements, high preoperative TIMP-1 concentration was a prognostic indicator of unfavorable outcome. These results indicate that tissue MMP-2 and TIMP-2 as well as circulating TIMP-1 may be prognostic markers in endometrial carcinoma. Of these, tissue MMP-2 seems to be the most potent prognostic marker. Studies with larger patient materials are needed to further explore the value of these enzymes in clinical practice in endometrial cancer. / Tiivistelmä Kohdunrungon syöpä on yleisin gynekologinen maligniteetti kehittyneissä maissa. Varhaisten oireiden, kuten poikkeavan verisen vuodon, vuoksi kohdunrungon syöpä havaitaan usein varhaisessa vaiheessa, jolloin sen ennuste on hyvä. Taudin käyttäytyminen voi kuitenkin olla moninaista. Viime vuosikymmenten aikana kohdunrungon syöpään sairastuneiden ennuste ei ole merkittävästi parantunut. Vaikuttaisi siltä, että perinteiset ennustetekijät eivät ole riittävän tarkkoja ennustamaan syövän taudinkulkua. Lisäksi liitännäishoidot voivat olla kalliita, ja niihin voi liittyä vakavia haittavaikutuksia. Uusien biologisten ennustetekijöiden löytäminen olisi tärkeää, jotta aggressiivista syöpätyyppiä sairastavat potilaat pystyttäisiin tunnistamaan entistä paremmin, ja hoito kyettäisiin räätälöimään yksilöllisemmin taudinkuvaa vastaavasti. Gelatinaasien (MMP-2 ja MMP-9) sekä niiden kudosinhibiittoreiden (TIMP-1 ja TIMP-2) on havaittu osallistuvan syövän etenemiseen. Tässä tutkimuksessa tarkasteltiin MMP-2:n ja MMP-9:n sekä niiden kudosinhibiittoreiden TIMP-1:n ja TIMP-2:n ilmentymistä ja ennusteellista merkitystä kohdunrungon syövässä. Aineisto käsitti yhteensä 266 primaariseen kohdunrungon syöpään sairastunutta naista. Määritysmenetelminä käytettiin sekä immunohistokemiallista värjäystä parafiiniin valettujen kudosnäytteiden osalta että ELISA-määrityksiä ennen hoitoa otettujen seeruminäytteiden osalta. Syöpäkudoksen runsas MMP-2 -proteiinin ilmentyminen liittyi epäsuotuisaan ennusteeseen, kun taas kasvainkudoksen voimakas TIMP-2 -proteiinin ilmentyminen oli hyvän ennusteen merkki. Lisäksi kasvainkudoksen voimakkaan MMP-2- ja heikon TIMP-2 -proteiinien ilmentymisen yhdistelmän havaittiin liittyvän suurempaan syövästä johtuvaan kuolleisuuteen. MMP-2 -negatiivisten potilaiden eloonjäämisennuste oli paras, TIMP-2 -värjäystuloksesta riippumatta. Seerumin korkea TIMP-1 -pitoisuus oli merkittävä huonontuneen ennusteen merkki. Tutkimuksen tulokset viittaavat siihen, että kasvainkudoksessa esiintyvät MMP-2- ja TIMP-2 -proteiinit samoin kuin seerumin TIMP-1 -pitoisuus voivat ennustaa kohdunrungon syövän kliinistä käyttäytymistä. Kasvainkudoksessa esiintyvä MMP-2 -proteiini vaikuttaisi olevan merkittävin ennusteellinen tekijä, mutta tulosten varmistamiseksi tarvitaan lisää tutkimuksia suuremmilla potilasaineistoilla.

Abstract The dentin-pulp complex comprises mineralized dentin and the vital soft tissues encased inside dentin, i.e. odontoblasts and pulp tissue. During caries progression, the dentinal minerals are dissolved and eventually the collagenous organic matrix is degraded. However, the exact mechanisms and enzymes responsible for the organic matrix breakdown remain unknown. Matrix metalloproteinases (MMPs), a family of endopeptidases capable of degrading in concert virtually all extracellular matrix components, are expressed during normal dentin-pulp complex formation and maintenance. MMP activity has also been suggested to contribute to the organic matrix degradation during dentin caries progression and to the repair and defense reactions elicited by caries in the dentin-pulp complex cells. The aim of the study was to further elucidate the role of host MMPs in dentin caries progression and the origin of MMPs in carious dentin as well as the possible changes in MMP expression in the cells of the dentin-pulp complex in response to caries. MMP inhibitors decreased the area of dentin caries lesions in vivo, suggesting the involvement of host MMPs in dentin caries pathogenesis. When the overall MMP gene expression was examined by cDNA microarray, pooled pulp samples demonstrated a high level of MMP-13 expression, but failed to show any unequivocal changes in MMP expression due to caries. MMP-13 expression is rare among normal human adult tissues. Real-time quantitative PCR of individual pulp and odontoblast samples demonstrated a rather large variation in relative MMP-13 mRNA expression between samples, especially pulp samples. Protein expression of MMP-13 was detected in pulp and odontoblasts without any major differences between the tissues of sound and carious teeth. This was also the case with the MMP-20 (enamelysin) protein, which was demonstrated in odontoblasts and the pulp tissue of fully developed human teeth. MMP-20, MMP-8, and gelatinases (especially MMP-2) were demonstrated in human dentin, and dentinal MMPs exhibited activity against native and denatured type I collagen when released. The study demonstrates the presence of MMPs in the soft and hard tissue compartments of the dentin-pulp complex. These enzymes may also contribute to dentin caries progression and response reactions to caries.

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds that is the causative agent of Primary Amoebic Meningoencephalitis (PAM). Matrix metalloproteinases (MMPs) have been described in protozoa, such as Plasmodium falciparum, Trypanosoma brucei, and Balamuthia mandrillaris, and have been linked to their increased motility and invasive capability by degrading components of the extracellular matrix (ECM). In addition, MMPs are often upregulated in tumorigenic cells and have been attributed as responsible for the metastasis of certain cancers. In the present study, in vitro experiments indicated that MMPs are linked functionally to the ECM degradation process. Gelatin zymography demonstrated protease activity in N. fowleri whole cell lysates, conditioned media, and media collected from in vitro invasion assays. Western immunoblotting confirmed the presence of the metalloproteinases MMP-2, -9, and -14. The highly virulent mouse-passaged amoebae expressed higher levels of MMPs than the weakly virulent axenically grown amoebae. The functional relevance of MMPs found in media in degradation of ECM components was confirmed through the use of MMP inhibitors. The collective in vitro results suggest that MMPs may play a critical role in the invasion of the CNS. Furthermore, the expression of select metalloproteinases may serve as amenable targets for therapeutic manipulation of expansive PAM.

Alarmins and damage associated molecular pattern (DAMP) are endogenous proteins with distinct and various intracellular roles that when released extracellularly act as startingsignals for inflammatory immune responses. The endogenous protein High mobility group box 1 (HMGB1) acts as a DAMP and has been shown to drive progression of multiple inflammatory and autoimmune diseases. During homeostasis HMGB1 is localized in the nucleus of almost any cell, where its main function is organization of the DNA and regulation of transcription. Upon cell death or immune cell activation HMGB1 can be translocated into the cytoplasm for subsequent release into the extracellular space. Extracellular HMGB1 can act as a DAMP by activating several receptors of the immune system. Recent studies focus on HMGB1 release and functional regulation due to prost-translational modifications (PTMs) on cysteine residues. However, little is known about enzymatic regulation of HMGB1. The aim of this thesis was to investigate the possibility of proteolytic processing of HMGB1 by enzymes, which play a crucial role in inflammatory diseases and their progression. We utilized an in vitro model that mimics natural conditions of the autoimmune disease arthritis. Enzymatic digestion of HMGB1 was performed in kinetics studies using the neutrophilic enzymes cathepsin G, neutrophil Elastase as well as matrix metalloproteinase-3, which is released from tissues at the site of inflammation. We defined that HMGB1 is a novel substrate of all of the tested enzymes. All enzymes induced different cleavage pattern. In conclusion, my findings open up the possibility for future studies involving the observed fragments of HMGB1 and their functional features. It also demonstrated that HMGB1 is affected by protease modifications in a disease relevant environment.

La métalloprotéinase matricielle (MMP)-8 ou collagénase-2 est capable de rompre les molécules natives, triples hélices, du collagène interstitiel, initiant ainsi le remodelage cellulaire lors du déplacement dentaire induit par une force orthodontique. C'est un bio-marqueur incontournable du remaniement tissulaire parodontal. L'augmentation de l'expression et de l'activation de MMP-8 dans le fluide gingival reflète l'activité du remodelage parodontal induit par les forces orthodontiques. En moyenne, la concentration de MMP-8 prélevée dans le fluide gingival des patients orthodontiques est 12 fois plus élevée (56 ± 50 µg/l contre 4,6 ± 4 µg/l) que chez les patients non orthodontiques. Le suivi des fluctuations de MMP-8 durant le déplacement orthodontique nécessite la mise au point d'un biocapteur. L'objectif de ce travail est d'utiliser une structure photonique à base de silicium poreux pour la conception d'un biocapteur optique de la MMP-8. Nous avons déterminé le choix du substrat de silicium poreux (PSi) le plus adapté à notre application avec une surface spécifique élevée et pores suffisamment ouverts pour l'infiltration des biomolécules qui sont des anticorps anti-MMP-8. Ensuite nous avons mis en place un procédé de fonctionnalisation chimique et biologique de la surface interne de ces échantillons. / The matrix métalloprotéinase (MMP) 8 or collagenase 2 is able to cleave native molecules, triple helixes, of the interstitial collagen, so introducing the cellular reshaping during orthodontic tooth movement. It is a major biomarker of the periodontal tissular remodeling. The increase of the expression and the activation of MMP-8 in the gingival fluid reflects the activity of the periodontal remodeling. On average, the concentration of MMP-8 taken in the gingival fluid of the orthodontic patients is 12 times as raised(brought up) (56 ± 50 µg / l against 4,6 ± 4 µg / l) that at the not orthodontic patients. Followed it by fluctuations in MMP-8 during the orthodontic movement require the development of a biosensor. The objective of this work is to use a photonique structure with porous silicon for the conception of an optical biosensor of the MMP-8. We determined the choice of the substratum of porous silicon (PSi) the most adapted to our application with a high specific surface and pores opened enough for the infiltration of the biomolecules which are antibodies anti-MMP-8. Then we set up a process of chemical and biological fonctionnalisation of the internal surface of these samples.

Integrin αvβ6 has emerged as a potential novel target for anticancer and plays a major role in promoting malignant tumor progression. Recent studies indicate that integrin αvβ6 occurs in many cancers. However, whether and how αvβ6 is regulated by genetic and epigenetic mechanisms in breast cancer remain unknown. In the present study, two different short hairpin RNAs (shRNAs) targeting the β6 gene were designed and constructed into pSUPER, respectively, which were transfected into the MCF-7 human breast adenocarcinoma cell line. The β6-shRNA stably transfected cells were successfully established, and significant lower levels of αvβ6 mRNA and protein expression were confirmed. Furthermore, inhibition of integrin αvβ6 markedly downregulated the expression of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-3 (MMP-3) and urokinase plasminogen activator (uPA) in tumor conditioned medium. Furthermore, β6-shRNA-mediated silencing of the αvβ6 gene obviously decreased the expression of ERK1/2. In particular, supression of integrin αvβ6 caused significant downregulation of the degradation of basement membrane type IV collagen secretion via modulation of the plasminogen activation cascade. Our results thus indicate that αvβ6 plays a fundamental role in promoting invasion and growth of breast adenocarcinoma cells. Taken together, this study revealed that targeting of the β6 gene by RNA interference (RNAi) could efficiently downregulate αvβ6 expression and suppress the ERK1/2-dependent extracellular matrix degradation in vitro, which is dependent upon inactivation of the mitogen-Activated protein kinase (MAPK) pathway. These findings may offer a useful therapeutic approach to block invasion and migration of breast cancer cells.

Objective: To determine if cannabinoid receptor 2 (CB2) plays a role in atherosclerosis, we investigated the effects of systemic CB2 gene deletion on hyperlipidemia-induced atherogenesis in low density lipoprotein receptor-deficient (Ldlr-/-) mice. Methods and results: Ldlr-/- and CB2/Ldlr double knockout (CB2-/-Ldlr-/-) mice were fed an atherogenic diet for 8 and 12 weeks. Morphometric analysis revealed no significant difference between the atherosclerotic lesion area in the proximal aortas of Ldlr-/- and CB2-/-Ldlr-/- mice after 8 or 12 weeks on the atherogenic diet. The macrophage and smooth muscle cell (SMC) content, as revealed by immunohistochemical staining, did not differ significantly between Ldlr-/- and CB2-/-Ldlr-/- lesions after 8 weeks. However, after 12 weeks, CB2-/-Ldlr-/- lesions displayed greater macrophage content (86.6±4.1 versus 75.2±7.5%, P<0.05) and SMC content (11.1±5.1 versus 4.2±2.4%, P<0.05) compared to controls. Lesional apoptosis, as determined by in situ TUNEL analysis, was reduced ∼50% in CB2-/-Ldlr-/- lesions after 12 weeks. CB2-/-Ldlr-/- lesions displayed significantly reduced collagen content and increased elastin fiber fragmentation after 12 weeks, which was associated with an ∼57% increase in matrix metalloproteinase 9 (MMP) levels. In vitro, CB2-/- macrophages secreted ∼1.8-fold more MMP9 activity than CB2+/+ macrophages. Conclusions: CB2 receptor deficiency affects atherogenesis in Ldlr-null mice by increasing lesional macrophage and SMC content, reducing lesional apoptosis and altering extracellular matrix components, in part, by upregulating MMP9. These results suggest that pharmacological manipulation of CB2 receptors might exert multiple and complex effects on atherogenesis and plaque stability.