The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis, abdominal aortic aneurysms, and heart valve disease in regions exposed to disturbed flow leading to low or oscillatory shear stress at the wall of the blood vessel or the surface of the valve leaflet. The novel and significant finding of this study is that mouse aortic endothelial cell exposure to pro-atherogenic oscillatory shear stress (OS) (+/- 5 dynes/cm2) increased their production of cathepsins, the family of lysosomal cysteine proteases that are potent elastases and collagenases leading to protease degradation and remodeling of the extracellular matrix structural components. Conversely, atheroprotective unidirectional laminar shear stress (LS) (15 dynes/cm2) decreased elastase and gelatinase activities of endothelial cells through a shear stress mediated reduction in cathepsins K, L, and S activity. Their endogenous inhibitor, cystatin C, was found to be inversely regulated by shear stress; LS increased its secretion by endothelial cells while OS decreased it. Binding of free cystatin C in the conditioned media to carboxymethylated papain coated agarose beads led to an increase in cathepsin activity since the available cathepsin was not inhibited. To verify these findings in human samples, immunohistochemical analysis of cystatin C and cathepsin K was performed on human coronary arteries. Cathepsin K stained strongly in the endothelial layer of vessels with degraded internal elastic lamina while cystatin C staining intensity was strongest overlying minimally diseased vessels. Additional roles for cathepsins K, L, and S were found in endothelial cell alignment in response to unidirectional laminar shear stress, endothelial cell migration, and programmed cell death. We conclude that there is an inverse regulation of cathepsins and cystatin C in endothelial cells by LS and OS and identify the cathepsin family of proteases as potential targets for therapeutic intervention of cardiovascular disease development at sites of disturbed flow.

Major advances in highly active antiretroviral therapies (ARVs) have extended the lives of people living with HIV, but there still remains an increased risk of death by cardiovascular diseases (CVD). HIV proteins and ARVs have been shown to contribute to cardiovascular dysfunction with effects on the different cell types that comprise the arterial wall. In particular, HIV-1 transactivating factor, Tat, is a cationic polypeptide that binds to endothelial cells, inducing a range of responses that have been shown to contribute to vascular dysfunction. It is well established that hemodynamics also play an important role in endothelial cell mediated atherosclerotic development where upon exposure to low or oscillatory shear stress, such as that found at branches and bifurcations, endothelial cells contribute to proteolytic vascular remodeling, by upregulating cathepsins, potent elastases and collagenases. The results of this work demonstrate that upregulation of cathepsins in vivo and in vitro is caused by a synergism between pro-atherogenic shear stress and HIV-1 proteins, elucidates pathways that are activated by HIV-1 Tat and pro-atherogenic shear stress - leading to cathepsin-mediated ECM degradation, and identifies cathepsins as novel biomarkers to monitor the adherence of patients on efavirenz- and tenofovir-containing antiretroviral regimens.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 97 pages. Includes Vita. Includes bibliographical references.

Lors de pathologies pulmonaires inflammatoires chroniques comme la mucoviscidose ou la BPCO, le déséquilibre de la balance protéases/antiprotéases aboutit à la dégradation du tissu pulmonaire et à l’inactivation des défenses antimicrobiennes. Les cathepsines à cystéine participent à l’inactivation protéolytique de peptides et protéines antimicrobiens (PAMs) pulmonaires comme le SLPI, la lactoferrine, et les β-défensines HBD-2 et -3 lors de l’emphysème ou de la mucoviscidose. Lors de cette thèse, nous avons étudié la capacité des cathepsines à cystéine B, K, L et S à hydrolyser le peptide LL-37, qui est un PAM important dans l’immunité innée pulmonaire. Seules les cathepsines K et S clivent le LL-37 et inactivent efficacement son activité antimicrobienne. A l’inverse, le LL-37 est un inhibiteur compétitif de la cathepsine L. D’autre part, l’expression pulmonaire de la cathepsine S est fortement augmentée chez les individus fumeurs atteints ou non de BPCO. La fumée de cigarette qui est une source importante de stress oxydatif induit une augmentation significative de l'expression et l'activité de la cathepsine S. Malgré un environnement oxydatif non favorable à l'activité des cathepsines, la cathepsine S parvient à hydrolyser le peptide LL-37 et pourrait ainsi augmenter le risque d’exacerbation lors de la BPCO. / During chronic inflammatory lung diseases like cystic fibrosis or COPD, proteases/antiproteases imbalance leads to pulmonary tissue degradation and compromise antimicrobial barrier. Cysteine cathepsins are involved in the proteolytic inactivation of several lung antimicrobial peptides (AMPs) such as SLPI, lactoferrin and β- defensins -2 and -3 during emphysema or cystic fibrosis. During this thesis, we studied the ability of cathepsins B, K, L and S to degrade LL-37, which is an important AMP in lung immunity. Only cathepsins K and S degrade readily LL-37 and inactivate its antimicrobial property. Conversely, LL-37 is a competitive inhibitor of cathepsin L. Beside, lung expression of human cathepsin S is significantly increased in smokers with or without COPD compared to non-smokers. Cigarette smoke that is a major source of oxidative stress significantly increases the expression and activity of cathepsin S. Despite an unfavorable oxidative environment, cathepsin S retains its proteolytic activity toward LL-37 and thus could participate to COPD exacerbation.

Adesivos dentinários aplicados diretamente sobre dentina aumentam a atividade de enzimas endógenas deste tecido que degradam colágeno, colocando em risco a integridade da camada híbrida de restaurações estéticas. Estes adesivos podem também alcançar a polpa dentária indiretamente através do fluído dos túbulos dentinários por substâncias liberadas pelos mesmos. Desta forma, a polpa dentária poderia responder a estas substâncias por meio de síntese e/ou aumento da atividade de colagenases, o que poderia colaborar na degradação da camada híbrida. Sendo assim, o objetivo desse trabalho foi avaliar o efeito das substâncias liberadas por sistemas adesivos dos tipos autocondicionante e condicione e lave sobre a atividade e a expressão gênica de metaloproteinases (MMPs) e cisteíno-catepsinas (CTs) em células-tronco da polpa dentária humana. Foram aplicados meios de cultura condicionados por adesivos do tipo autocondicionante e condicione e lave polimerizados e não polimerizados sobre culturas celulares por 24 horas. O meio de cultivo fresco foi usado como controle. Depois de 24, 48, 72 e 96 horas, as atividades gelatinolíticas de MMP-2 e de MMP-9 foram avaliadas por meio da técnica de zimografia em gel de gelatina. Nos mesmos tempos experimentais, a modulação da expressão gênica das MMPs (1, 2, 3, 7, 9, 13 e 14) e das CTs (B e K) foi analisada por meio de reação de transcriptase reversa quantitativa em tempo real (qRT-PCR). Os resultados obtidos dos dois experimentos foram avaliados por meio do teste estatístico ANOVA, complementado pelo teste de Tukey (p<0.05). Todos os grupos mostraram atividade gelatinolítica aumentada de MMP-2 e MMP-9. Até 72 horas, as atividades foram similares em todos os grupos experimentais. Diferenças significativas apareceram somente em 96 horas. De forma geral, as maiores atividades de MMPs foram observadas nas culturas celulares tratadas com o adesivo autocondicionante. Para a MMP-2, o grupo do adesivo autocondicionante polimerizado mostrou atividade intermediária, enquanto o grupo não polimerizado mostrou a maior atividade. Os dois grupos do adesivo condicione e lave polimerizado e não polimerizado mostraram atividade de MMP-9 intermediária, enquanto o grupo autocondicionante polimerizado mostrou maior atividade que o grupo controle. O qRT-PCR revelou que a maioria das MMPs e CTs analisadas tiveram a expressão gênica positivamente modulada em 24 e 48 horas. MMP-7 e MMP-9 não foram expressos em nenhum grupo experimental. Baseados nas limitações deste estudo in vitro, concluímos que substâncias liberadas por sistemas adesivos são capazes de influenciar células-tronco de polpa dentária humana levando ao aumento da atividade de MMP-2 e MMP-9 e também à modulação positiva de genes das MMPs e CTS estudadas. / Adhesive systems directly applied to dentin increase the activity of endogenous collagen degrading proteinases of the dentin, which jeopardizes the integrity of the hybrid layer of aesthetic restorations. These adhesives can also reach the dental pulp through the dentinal fluid indirectly by substances leached from them. Then, the dental pulp tissue could respond by synthetizing and/or increasing the activity of collagen proteases, which in turn could collaborate to the hybrid layer degradation. Then, the aim of this study was to evaluate the effect of substances leached from self-etch and etch-and-rinse adhesive systems on the expression and activities of matrix metalloproteinases (MMPs) and cysteine cathepsins (CT-B and CT-K) in human dental pulp stem cells. Culture media conditioned by polymerized or non-polymerized self-etch and etch-and-rinse adhesive systems were applied to the cultures for 24 hours. Fresh medium was used as control. After 24, 48, 72 and 96 hours, the gelatinolytic activities of MMP-2 and MMP-9 were assessed by zymography technique. At the same experimental time gene expression of MMPs (1, 2, 3, 7, 9, 13 e 14) and CTs (B e K) were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR). Data was compared by ANOVA complemented by the Tukey´s test (p<0.05). All experimental groups showed increased gelatinolytic activity for MMP-2 and MMP-9. Until 72 hours, the activities were similar regardless the group. Significant differences appeared only after 96 hours. Overall, the highest activities of MMPs were observed in the cultures treated with the self-etch adhesive. For MMP-2, the group of polymerized self-etch adhesive showed intermediary activity, while the group of non-polymerized adhesive showed the highest activity. Both polymerized and non-polymerized etch-and-rinse adhesive groups showed intermediary MMP-9 activity, while the group of polymerized self-etch adhesive showed higher activity than control. The qRT-PCR revealed that most of MMPs and CTs analyzed presented the gene expression positively modulated at 24 and 48 hours. MMP-7 and MMP-9 were not expressed in any experimental group.Based on the limitations of this in vitro study, it was concluded that substances leached from adhesive systems are able to influence human dental pulp stem cells leading to the increase of the activity of MMP-2 and MMP-9 along with positive modulation of MMPs and CTS studied genes.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-27T14:18:58Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Michelle Nauara Gomes do Nascimento - 2014.pdf: 1938197 bytes, checksum: a67bc5040425a846cdd4a7a73cefa5cd (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-28T12:06:41Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Michelle Nauara Gomes do Nascimento - 2014.pdf: 1938197 bytes, checksum: a67bc5040425a846cdd4a7a73cefa5cd (MD5) / Made available in DSpace on 2015-01-28T12:06:41Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - Michelle Nauara Gomes do Nascimento - 2014.pdf: 1938197 bytes, checksum: a67bc5040425a846cdd4a7a73cefa5cd (MD5) Previous issue date: 2014-07-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This work describes the chemical study of E. suberosum, aiming to elucidate structures of secondary metabolites in the leaves, stem and roots. Cathepsins assays were used to evaluate in vitro activity of extracts, fractions and isolated compounds, using fluorogenic substrate ZFR-MCA. These enzymes, also known as lysossomal cysteine peptidases are involved in many physiological processes, and are associated with many pathological conditions. Cathepsins K, L and V are involved in the development of diseases such as osteoporosis, skin cancer and atherosclerosis, respectively. Based on pathological processes in which cathepsins are involved, the search for specific inhibitors can be a new approach for the treatment these diseases. The study of ethanolic extract from leaves led to the identification of four flavonoids belonging to the flavonol class: quercetin and its derivatives 3-O-monoglycosides hyperin and isoquercitrin, and 3-O-diglycosides, ombuin-3-rutinoside, all previously reported for this species. The mixture of flavanols, catechin and epicatechin were isolated from ethanolic extract from roots, besides the fatty ester of sitosterol. The mixture of steroids campesterol, stigmasterol and β-sitosterol were identified from the stem extract. This study also suggests the presence of tropane alkaloids, tropacocaine and nortropacocaine, in the ethanolic extract from leaves of E. suberosum, reported for the first time in this species. In the evaluation for cathepsins K, L and V, the extracts (concentration of 50 mg/mL), showed significant inhibition of all enzymes with percentage inhibition values above 60%. The fractions showed significant activity against cathepsin V and L when evaluated at concentrations of 5 and 50 mg/mL. The quercetin flavonol showed IC50 value of 2.2 ± 0.2 μM against cathepsin V and low affinity for cathepsins K and L (100 μM). This is an important result since literature reports for flavonoids as inhibitors of cathepsin V are quite limited. / O presente trabalho descreve o estudo químico de E. suberosum, no intuito de elucidar estruturas de metabólitos secundários presentes nas folhas, caule e raiz, além de avaliar, por meio de teste in vitro, a atividade dos extratos, frações e, quando possível das substâncias isoladas, em busca de inibidores de catepsinas utilizando como ferramenta o substrato fluorogênico ZFR-MCA. Estas enzimas, também conhecidas como cisteíno peptidases lisossomais, estão envolvidas em diversos processos fisiológicos, além de estarem associadas a muitas condições patológicas, estando as catepsinas K, L e V, envolvidas no desenvolvimento de doenças como osteoporose, câncer de pele e aterosclerose, respectivamente. Baseado nos processos patológicos em que estas catepsinas estão envolvidas, a busca de inibidores específicos pode ser uma nova abordagem para o tratamento destas doenças. O estudo do extrato etanólico das folhas levou a identificação de quatro flavonoides pertencentes à classe dos flavonois, sendo estes a quercetina e seus derivados 3-Omonoglicosídeos hiperina e isoquercitrina e 3-O-diglicosídeo, ombuina-3- rutinosídeo, todos já relatados para esta espécie. Do extrato etanólico da raiz, foi isolada a mistura dos flavanois catequina e epicatequina, além do éster graxo do β-sitosterol. No caule foram identificados a mistura dos esteroides campesterol, estigmasterol e β-sitosterol. Este estudo também sugere a presença dos alcaloides tropanos, tropacocaína e nortropacocaína no extrato etanólico das folhas, sendo estes relatados pela primeira vez na espécie. Nos ensaios frente às catepsinas K, L e V, todos os extratos apresentaram inibição superior a 60% quando avaliados na concentração de 50 μg/mL. Já as frações oriundas da partição líquido-líquido, apresentaram inibição mais expressiva frente às catepsinas V e L, quando avaliadas nas concentrações de 5 e 50 μg/mL. O flavonol quercetina apresentou um valor de IC50 de 2,2 ± 0,2 μM para a catepsina V e baixa afinidade para as catepsinas K e L quando avaliado na concentração de 100 μM. Este é um resultado importante, uma vez que os relatos na literatura para flavonoides como inibidores de catepsinas são bastante restritos.

In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention. / In the last decade, a tremendous gain in knowledge concerning the molecular events of apoptosis signaling and execution has been achieved. The aim of this thesis was to clarify the role of lysosomal membrane permeabilization and lysosomal proteases, cathepsins, in signaling for apoptosis. We identified cathepsin D as an important factor in staurosporine-induced human fibroblast cell death. After release to the cytosol, cathepsin D promoted mitochondrial release of cytochrome c by proteolytic activation of Bid. Cathepsin D-mediated cleavage of Bid generated two fragments with the apparent molecular mass of 15 and 19 kDa. By sequence analysis, three cathepsin D-specific cleavage sites, Phe24, Trp48, and Phe183, were identified. Moreover, we investigated the mechanism by which cathepsins escape the lysosomal compartment, and found that Bax is translocated from the cytosol to lysosomes upon staurosporine treatment. In agreement with these data, recombinant Bax triggered release of cathepsins from isolated rat liver lysosomes. Conceivably, the Bcl-2 family of proteins may govern release of pro-apoptotic factors from both lysosomes and mitochondria. The importance of lysosomal cathepsins in apoptosis signaling was studied also in oral squamous cell carcinoma cells following exposure to the redox-cycling drug naphthazarin or agonistic anti-Fas antibodies. In this experimental system, cathepsins were released to the cytosol, however, inhibition of neither cathepsin D, nor cysteine cathepsin activity suppressed cell death. Interestingly, cysteine cathepsins still appeared to be involved in activation of the caspase cascade. Cathepsins are often overexpressed and secreted by cancer cells, and it has been reported that extracellular cathepsins promote tumor growth and metastasis. Here, we propose that cathepsin B secreted from cancer cells may suppress cancer cell death by shedding of the Fas death receptor. Defects in the regulation of apoptosis contribute to a wide variety of diseases, such as cancer, neurodegeneration and autoimmunity. Increased knowledge of the molecular details of apoptosis could lead to novel, more effective, treatments for these illnesses. This thesis emphasizes the importance of the lysosomal death pathway, which is a promising target for future therapeutic intervention.