Type 1 diabetes (T1D) is an autoimmune disease whereby the insulin-producing β-cells of the pancreas are destroyed by the immune system, possibly related to an inappropriate immune reaction to dietary antigens and/or microbes in the gut. We previously observed a deficit in gut-resident CD163+ M2 anti-inflammatory macrophages in BioBreeding diabetes-prone (BBdp) rats. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of the CD163 pathway and through the breakdown of toxic heme releases potent antioxidants. We hypothesized that the treatment of animals with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, would inhibit development of T1D through modulation of the CD163/HO-1 pathway and increase M2 macrophages. HO-1 expression was significantly increased in the pancreas and gut. T1D incidence was inhibited in CoPP-treated rats and these animals showed an unexpected increase in cells expressing CD68 (an M1 pro-inflammatory macrophage marker) in the pancreas and gut. CoPP induced the expression of cathelicidin anti-microbial peptide (CAMP) in the jejunum, which co-localized with CD163+ (M2) macrophages. KLF4, an M2 macrophage-specific transcription factor, was significantly upregulated in the pancreas and jejunum of CoPP-treated animals and co-localized with CD68 and HO-1 in the pancreas. We conclude that HO-1 induction prevented T1D through modulation of the gut immune system and potential recruitment of a unique population of anti-inflammatory M2 macrophages in the gut and pancreas

Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases. Maintenance of vascular reactivity and endothelial viability is essential to preserving oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. These results suggest that hypoxic induction of MMP-2 activity potentiates contraction in systemic conduit and resistance arteries through proteolytic activation of big ET-1. The second part of the thesis investigated oxygen regulation of HO-2 protein and whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but not HO-1, protein level was maintained during hypoxia in human endothelial cells through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression increased the production of reactive oxygen species, decreased mitochondrial membrane potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but not during normoxia. These data indicate that HO-2 is translationally regulated and important in maintaining endothelial viability and function during hypoxia. In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in preserving vascular function during prolonged systemic hypoxia. These enzymatic pathways may, therefore, represent novel therapeutic targets that may be exploited to ameliorate the effects of hypoxia in patients with cardiopulmonary disease.

Stroke is the third largest cause of death, and the leading cause of disability worldwide. Treatments are sought to reduce mortality, and increase survival time following an ischaemic stroke. Hypoxia-ischaemia (HI) is the combination of cerebral ischaemia and global hypoxia that can lead to neuronal damage, particularly perinatally. The complex neurodegenerative cascade following ischaemic stroke and HI activates many stress pathways, including heme oxygenase (HO). HO metabolises free heme to release iron, carbon monoxide, and biliverdin, which is subsequently metabolised to bilirubin. This thesis aims to elucidate the role HO plays following HI, and assess any neuroprotective mechanisms using HO modulators. The 26 day old rat model of HI was used to induce the neurodegenerative cascade. All animals were sacrificed 3 days post-insult. Immunohistochemistry and Western blotting demonstrated that HO-1 was increased in the ipsilateral hemisphere of both HI (by 1.7 � 0.1 fold: p = 0.016, n = 4) and middle cerebral artery occlusion (MCAO) brains (by 1.6 � 0.1 fold: p = 0.037, n = 4), compared to controls. HO-2 was constitutively expressed throughout the control brain, but HI upregulated HO-2 expression (by 1.7 � 0.2 fold: p = 0.027, n = 4) ipsilaterally, whereas MCAO did not alter HO-2 expression. Administration of the HO inhibitor tin protoporphyrin (SnPP; 30[mu]mol/kg intraperitoneally) daily, beginning 1 day prior to HI until sacrifice, reduced infarct volume to 50% � 10 of saline-treated animals (p = 0.039, n = 6-8). The HO inducer ferriprotoporphyrin (FePP; 30[mu]mol/kg) had no effect on infarct volume. HO activity and protein expression were not significantly altered following treatment with SnPP. Therefore, the neuroprotective actions of SnPP may be through alternative mechanisms. SnPP treatment increased HI + saline-induced total nitric oxide synthase (NOS) activity by 1.5 � 0.06 fold (p < 0.001, n = 6-8). Conversely, SnPP inhibited both inducible NOS (50% � 7 of HI + saline; p = 0.045, n = 7-8) and cyclooxygenase (COX) activity (32% � 6 of HI + saline; p = 0.049, n = 4-8). SnPP treatment also increased mitochondrial complex I activity by 1.6 � 0.25 fold (p = 0.04, n = 4-8) and complex V activity by 1.7 � 0.26 fold (p = 0.046, n = 4-8) in the ipsilateral hemisphere. It appears that SnPP is acting on inflammatory and mitochondrial enzymes to produce neuroprotection. In vitro analysis of cultured RAW264.7 macrophages exposed to lipopolysaccharide (LPS; 10[mu]g/mL) treated with SnPP (dose range: 10⁻�⁰M - 10⁻⁵M) did not alter nitrite levels or cell viability. However, high dose SnPP (10⁻⁵M) in the absence of LPS increased nitrite levels from control cells by 2.7 � 0.7 fold (p = 0.043, n = 6), complementing the in vivo total NOS data. Other mechanisms such as NMDA receptor activation were not affected by 100[mu]M SnPP or 100[mu]M SnCl₂ in patch clamped cortical pyramidal neurons. Overall, the role that HO plays following HI remains unclear, but this thesis provides definitive evidence that SnPP (an established HO inhibitor) provides neuroprotection. This neuroprotection may be due to its effects on inducible pathways such as NOS and COX. Therefore, further experimentation is required to fully elucidate the role that HO plays following cerebral ischaemia, and additional in vivo evidence will be necessary to establish HO inhibitors as a putative candidate for cerebral ischaemia neuroprotection.

Stroke is the third largest cause of death, and the leading cause of disability worldwide. Treatments are sought to reduce mortality, and increase survival time following an ischaemic stroke. Hypoxia-ischaemia (HI) is the combination of cerebral ischaemia and global hypoxia that can lead to neuronal damage, particularly perinatally. The complex neurodegenerative cascade following ischaemic stroke and HI activates many stress pathways, including heme oxygenase (HO). HO metabolises free heme to release iron, carbon monoxide, and biliverdin, which is subsequently metabolised to bilirubin. This thesis aims to elucidate the role HO plays following HI, and assess any neuroprotective mechanisms using HO modulators. The 26 day old rat model of HI was used to induce the neurodegenerative cascade. All animals were sacrificed 3 days post-insult. Immunohistochemistry and Western blotting demonstrated that HO-1 was increased in the ipsilateral hemisphere of both HI (by 1.7 � 0.1 fold: p = 0.016, n = 4) and middle cerebral artery occlusion (MCAO) brains (by 1.6 � 0.1 fold: p = 0.037, n = 4), compared to controls. HO-2 was constitutively expressed throughout the control brain, but HI upregulated HO-2 expression (by 1.7 � 0.2 fold: p = 0.027, n = 4) ipsilaterally, whereas MCAO did not alter HO-2 expression. Administration of the HO inhibitor tin protoporphyrin (SnPP; 30[mu]mol/kg intraperitoneally) daily, beginning 1 day prior to HI until sacrifice, reduced infarct volume to 50% � 10 of saline-treated animals (p = 0.039, n = 6-8). The HO inducer ferriprotoporphyrin (FePP; 30[mu]mol/kg) had no effect on infarct volume. HO activity and protein expression were not significantly altered following treatment with SnPP. Therefore, the neuroprotective actions of SnPP may be through alternative mechanisms. SnPP treatment increased HI + saline-induced total nitric oxide synthase (NOS) activity by 1.5 � 0.06 fold (p < 0.001, n = 6-8). Conversely, SnPP inhibited both inducible NOS (50% � 7 of HI + saline; p = 0.045, n = 7-8) and cyclooxygenase (COX) activity (32% � 6 of HI + saline; p = 0.049, n = 4-8). SnPP treatment also increased mitochondrial complex I activity by 1.6 � 0.25 fold (p = 0.04, n = 4-8) and complex V activity by 1.7 � 0.26 fold (p = 0.046, n = 4-8) in the ipsilateral hemisphere. It appears that SnPP is acting on inflammatory and mitochondrial enzymes to produce neuroprotection. In vitro analysis of cultured RAW264.7 macrophages exposed to lipopolysaccharide (LPS; 10[mu]g/mL) treated with SnPP (dose range: 10⁻�⁰M - 10⁻⁵M) did not alter nitrite levels or cell viability. However, high dose SnPP (10⁻⁵M) in the absence of LPS increased nitrite levels from control cells by 2.7 � 0.7 fold (p = 0.043, n = 6), complementing the in vivo total NOS data. Other mechanisms such as NMDA receptor activation were not affected by 100[mu]M SnPP or 100[mu]M SnCl₂ in patch clamped cortical pyramidal neurons. Overall, the role that HO plays following HI remains unclear, but this thesis provides definitive evidence that SnPP (an established HO inhibitor) provides neuroprotection. This neuroprotection may be due to its effects on inducible pathways such as NOS and COX. Therefore, further experimentation is required to fully elucidate the role that HO plays following cerebral ischaemia, and additional in vivo evidence will be necessary to establish HO inhibitors as a putative candidate for cerebral ischaemia neuroprotection.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2007" Includes bibliographical references.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Lipoxinas (LXs) são metabólitos do ácido araquidônico com reconhecidas atividades antiinflamatórias e pró-resolução. Apesar do grande número de trabalhos publicados descrevendo o papel das LXs e seus análogos em leucócitos e outros tipos celulares envolvidos em doenças inflamatórias, pouco é sabido a respeito dos mecanismos de ação que desencadeiam estas respostas. Neste trabalho investigamos o papel do 15-epi-16-(para-flúor)-fenoxi-lipoxina A4 (ATL-1), um análogo sintético da 15-epi-lipoxina A4, sobre diversos processos de ativação de monócitos. Caracterizamos, pela primeira vez, o receptor da lipoxina A4 (ALX) na linhagem monocítica U937, através da avaliação de sua expressão gênica e protéica e de sua funcionalidade analisando a ativação de ERK-2, o que torna esta célula uma ferramenta apta para estudo dos mecanismos de ação das LXs e seus análogos sobre os monócitos. Além disso, demonstramos que o ATL-1 aumenta a expressão da enzima heme oxigenase (HO) -1 em células U937 via ativação da p38 MAP quinase (MAPK) e diminui a secreção da Monocyte chemoattractant protein-1 (MCP-1), uma quimiocina envolvida com o recrutamento de monócitos para o foco inflamatório, em células U937 estimuladas com LPS. A inibição da secreção de MCP-1 foi revertida pela utilização do SB203580, sugerindo que este efeito é dependente da ativação da via p38 MAPK. O presente estudo elucida alguns dos mecanismos envolvidos na ativação de monócitos pelas lipoxinas que podem levar a novas abordagens para o controle de diversas doenças nas quais o componente inflamatório é importante / Lipoxins (LXs) are arachidonic acid metabolites with well recognized anti-inflammatory and pro-resolution activities. Despite the large number of studies describing the role of LXs and their analogs in leukocytes and other cell types involved in inflammatory diseases, little is known about the mechanisms of action that trigger these responses. This work investigated the role of 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1), a synthetic analog of 15-epi-lipoxin A4 on various processes of monocyte activation. We characterized, for the first time, the lipoxin A4 receptor (ALX) in the monocytic lineage U937, through the assessment of its gene expression and protein and its functionality through the activation of ERK-2, which makes this cell line a suitable tool to study the mechanisms of action of LXs and their analogs on the monocytes. Furthermore, we demonstrated that ATL-1 increases the expression of the enzyme heme oxygenase (HO)-1 in the U937 cells via activation of p38 MAP kinase (MAPK) and decreases the secretion of Monocyte chemoattractant protein-1 (MCP-1), a chemokine involved in the recruitment of monocytes to the inflammatory focus in LPS-stimulated U937 cells. MCP-1 secretion inhibition by ATL-1 was reverted by SB203580 indicating that this effect is dependent on the activation of p38 MAPK pathway. This study clarifies some of the mechanisms involved in the activation of monocytes by lipoxins which may lead to new approaches for the control of different pathologies where the inflammatory component is relevant

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O Trypanosoma cruzi é o agente etiológico da doença de Chagas, transmitida através de insetos vetores triatomíneos durante a alimentação no hospedeiro vertebrado. Os triatomíneos ingerem numa única alimentação cerca de 10 mM de heme ligado à hemoglobina. O heme é uma importante molécula no metabolismo dos organismos. Um mecanismo intracelular importante no controle de sua homeostase é a degradação enzimática pela Heme Oxigenase (HO) formando biliverdina (Bv), monóxido de carbono e ferro. Como esta enzima não está presente no genoma de T. cruzi, esse trabalho tem por objetivo identificar uma atividade funcional de HO neste parasito, uma vez que dados do nosso laboratório mostram a presença de biliverdina nas incubações dessas células com heme. No presente trabalho testamos o efeito do SnPPIX (inibidor da HO-1), CoPPIX (indutor da HO-1) e Bv sobre a proliferação da forma epimastigota do parasito. A adição de SnPPIX diminuiu a proliferação do parasito na tanto na ausência quanto na presença de heme. Quando a Bv foi adicionada à cultura esse efeito foi revertido; a Bv aumenta a proliferação celular na presença de heme. Por outro lado, a adição de CoPPIX não interferiu na proliferação. Posteriormente, mostramos através da técnica de immunoblotting, utilizando anticorpo monoclonal contra a HO-1, um aumento da expressão de uma proteína em resposta ao heme. Diferentemente das HO-1 já descritas que possuem massa molecular de 32 kDa, a única banda reconhecida pelo anticorpo apresenta 45 kDa. Analisamos também a expressão da HO-1 na presença de CoPPIX, SnPPIX e biliverdina, e somente o CoPPIX foi capaz de modular os níveis de expressão da HO-1. A análise estrutural através da técnica de imunocitoquímica mostrou uma maior expressão da enzima na presença de heme, e que a HO-1 de T. cruzi pode ter mais de uma localização, apresentando marcação citoplasmática e glicossomal. A fim de investigar a sequência da HO-1 de T. cruzi, o DNA genômico foi extraído para amplificação por PCR do gene da HO-1 utilizando oligonucleotídeos desenhados no genoma de T. cruzi. Os dois pares de oligonucleotídeos utilizados nao foram capazes de amplificar uma sequência equivalente a uma HO. Em seguida, utilizamos a técnica de imunoprecipitação, seguida de immunoblotting, com anticorpo anti-HO-1, com objetivo de concentrar a proteína alvo, e observamos um aumento significativo do imunocomplexo nas células tratadas com heme 300 mM, cerca de 2 vezes em relação ao controle. Dando seguimento à tentativa de identificação da HO-1 de T. cruzi, utilizamos a técnica de espectrometria de massa a partir de eletroforese unidimensional, que mostrou uma grande alteração do perfil protéico na presença de heme, mas futuros experimentos são necessários, como eletroforese 2D, para a identificação da proteína alvo / Trypanosoma cruzi, the ethiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate host. These hematophagous insects ingest blood about 6 to 12 times its original weight, reaching in a single meal about 10 mM heme bound to hemoglobin. Heme (iron protoporphyrin IX) is an important molecule in metabolism of all living organisms. One important intracellular mechanism to control heme homeostasis is its enzymatic degradation by heme oxygenase (HO). HO catalyzes the degradation of heme to biliverdin (Bv), carbon monoxide and iron. HO is absent in T. cruzi genome, thus we have been investigating the presence of a functional HO in this parasite, since our previous results showed a presence of biliverdin in heme-treated epimastigotes. In the present work, we evaluated the effect of SnPPIX, a HO-1 inhibitor, CoPPIX, a HO inducer, and Bv upon T. cruzi epimastigotes proliferation. The addition of SnPPIX decreased the parasite proliferation in the absence or in the presence of heme. When Bv was added to the culture this effect was reversed; Bv increases the parasite proliferation in the presence of heme. On the other hand, CoPPIX did not interfered on proliferation. Furthermore, we showed through immunoblotting, using an anti-HO-1 monoclonal antibody, an increase in the protein expression in heme-treated epimastigotes. Differently of described HO-1 that has a mass molecular of a 32 kDa, we showed a 45 kDa protein, the only band recognize by the HO-1 antibody. HO-1 expression analysis in the presence of CoPPIX, SnPPIX and biliverdin, showed that only CoPPIX was able to modulate its expression level. Ultrastructural immunocytochemistry analysis suggests a higher expression of the enzyme in heme-treated epimastigotes, and that T. cruzi HO-1 might have a dual distribution, since the anti-HO-1 antibody labeled both cytosol and glycosomes. In order to investigate the T. cruzi HO-1 gene sequence, we isolated genomic DNA from T. cruzi for PCR amplification using primers designed as from the parasite genome. Unfortunately, the two pairs of the designed oligonucleotides tested were unable to amplify a sequence equivalent to a HO-1. In order to isolate the target protein, immunoprecipitation was performed and subsequently immunoblotted with anti-HO-1 antibody. The immunocomplex level was two-fold higher in heme-treated cells. Following the attempt to identify a HO-1 in T. cruzi, we used mass spectrometry from unidimensional electrophoresis. We showed a significant protein profile modification in heme-treated epimastigotes, but further experiments will be necessary, such as mass spectrometry from bidimensional electrophoresis, for identification of the target protein

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T14:23:59Z No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-04T14:24:19Z (GMT) No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) / Made available in DSpace on 2016-02-04T14:24:19Z (GMT). No. of bitstreams: 1 Nivea Farias Luz Ativação...2015.pdf: 10567971 bytes, checksum: 1479b4bbd75d3db2ffcf895208002d81 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A Leishmaniose visceral (LV) apresenta ampla distribuição geográfica e é fatal caso não seja tratada. As manifestações hematológicas são constantes na LV e em casos não tratados os pacientes evoluem à óbito por sangramento maciço ou anemia grave. Neste cenário, mecanismos ligados à morte celular, hemólise, metabolismo do heme e atividade da enzima heme oxigenase podem estar envolvidos na imunopatogênese da LV. A heme oxigenase (HO) tem importantes propriedades regulatórias e está envolvida em processos fisiológicos e patofisiológicos como citoproteção e inflamação. Nesse projeto testamos a hipótese de que a ativação da enzima heme oxigenase-1 (HO-1) favorece a infecção por Leishmania infantum chagasi, principal agente etiológico da LV humana no Brasil e de que mecanismos de morte celular inflamatória induzida por heme estão associados com a resistência ao parasita. Nossas observações nesse trabalho indicam que a enzima HO-1 é induzida em macrófagos durante a infecção por L. chagasi e que a indução farmacológica da HO-1, pela CoPP aumenta a carga parasitária de macrófagos infectados por L. chagasi e reduz a produção de mediadores próinflamatórios. Além disso, a HO-1 favorece um ambiente anti-inflamatório onde prevalece a presença de IL-10 sobre a de TNF. Macrófagos derivados de medula óssea de camundongos deficientes no gene HO-1 têm menor carga parasitária, quando infectados por L. chagasi em comparação aos macrófagos de camundongos selvagens. Além disso, pacientes com LV apresentam maiores níveis de heme-oxigenase 1 e de heme no soro. Nossas observações indicam que heme é capaz de induzir necroptose em macrófagos humanos, e de que moléculas da via da necroptose estão associadas com a resistência na infecção por Leishmania. A molécula RIPK1 controla a replicação de Leishmania por um mecanismo independente da produção de IL-1β, enquanto que a molécula PGAM5 depende de IL-1βpara controlar o crescimento do parasita. Por fim, encontramos que essas proteínas participam do controle da replicação por Leishmania em um modelo experimental de Leishmaniose cutânea. Esses achados indicam um potencial deletério para a HO-1 na infecção por L. chagasi, e um papel protetor da necroptose na infecção porLeishmania. / Visceral leishmaniasis (VL) is a widespread disease and is fatal if left untreated. Hematological manifestations are common in VL and untreated patients evolve to death from massive bleeding and severe anemia. In this scenario, mechanisms related to cell death pathways, hemolysis, heme metabolism and enzymatic activity of heme oxygenase may be involved in the immunopathogenesis of the disease. Heme oxygenase (HO) has important regulatory properties and is involved in patho-physiological processes such as cytoprotection and inflammation. This project tested the hypothesis that heme oxygenase- 1 (HO-1) activation favors Leishmania infantum chagasi infection, the main etiologic agent of human VL in Brazil, we also tested whether heme induced inflammatory cell death pathways are involved in resistance to Leishmania infection. Our observations indicate that HO-1 is induced in macrophages infected with L. infantum chagasi and pharmacological induction for HO-1 by CoPP increases parasite load of infected macrophages and reduces production on inflammatory mediators. In addition, HO-1 contributes to the anti inflammatory pathway that favors L. chagasi replication through a higher IL-10/TNF-α ratio in macrophages. We also observed that bone marrow derived macrophages knockout to HO-1 gene have a significant lower parasite load when infected by L. infantum chagasi than their wild type counterparts. Beyond this, we found that patients with VL presented higher systemic concentrations of HO- 1 and heme than healthy individuals. We found that heme is able to induce programmed necrosis “necroptosis” in human cells and that molecular players from necroptosis pathway contribute to resistance to Leishmania infection. RIPK1 controls Leishmania replication through a mechanism independent of IL-1β production, while PGAM5 requires IL-1β to control Leishmania replication. Finally, we found that RIPK1 and PGAM5 play an important role in controlling Leishmania replication in a cultaneous leishmaniasis experimental model. Our findings argue that HO-1 has a critical role in L. chagasi replication and necroptosis pathway is involved in resistance against Leishmania infection.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As doenças cardiovasculares representam a principal causa de morte nos países ocidentais. Dentre essas doenças, a aterosclerose é que mais se destaca, sendo caracterizada pelo acúmulo de células musculares lisas vasculares (CMLV). O efeito patológico das CMLV em resposta a diferentes estímulos pode acarretar em disfunções nestas células. É notável que a aterosclerose ocorra principalmente em vasos sinuosos onde ocorre um forte turbilhonamento do fluxo sanguíneo, que pode acarretar em hemólise e, consequentemente, acúmulo de heme livre. Além disso, no processo de aterogênese as moléculas de adesão, principalmente integrinas, são de crucial importância durante a resposta de CMLV. Nesse trabalho nosso objetivo inicial foi avaliar o efeito do heme livre nas funções de CMLV, bem como os mecanismos moleculares por trás desses efeitos. Em uma segunda parte, investigamos o envolvimento da integrina &#945;1ß1 no efeito da Angiotensina II (Ang II) em CMLV. Nós observamos que o heme livre é capaz de induzir a proliferação e migração de CMLV via espécies reativas de oxigênio (ERO) provenientes da NADPHoxidase (NADPHox). Adicionalmente vimos que o heme ativa vias de sinalização redox-sensíveis relacionadas à proliferação celular, como MAPKinases e o fator de transcrição NF&#954;B. Também observamos que há uma ligação entre a NADPHox e o sistema heme oxigenase (HO), uma vez que o heme induz a expressão de HO-1 e o pré-tratamento das CMLV com inibidores de HO levam ao aumento tanto o efeito proliferação quanto a indução de ERO promovidas pelo heme. Além disso, vimos que o efeito contra-regulatório promovido pela HO ocorre devido as metabolites do heme: biliverdina, bilirrubina e monóxido de carbono. Por último, quando bloqueamos tanto a NADPHox quanto o sistema HO o heme não teve efeito algum na proliferação de CMLV. Em um segundo estudo, observamos que o efeito da Ang II sobre a migração de CMLV foi inibido quando as células foram pré-tratadas com o ligante da integrina &#945;1ß1, a desintegrina Obtustatina. A seguir observamos que o efeito da Ang II na ativação de FAK e na colocalização actina-ILK é dependente da integrina &#945;1ß1, que possivelmente ativa PKC&#945;, uma vez que vimos que a produção de ERO induzida por Ang II foi inibida pela Obtustatina. Vimos que a indução da expressão de ILK por Ang II em CMLV é dependente da integrina &#945;1ß1 e também observamos que a Obtustatina inibibiu o desacoplamento de ILK da FAK, uma vez que a Obtustatina bloqueou a fosforilação de FAK induzida por Ang II (processo crucial para o desacoplamento da ILK). Nós também observamos que a Ang II induz, via integrina &#945;1ß1, a fosforilação de AKT e a diminuição da expressão de p21, provavelmente via ILK. Corroborando estes dados, nós mostramos que o pré-tratamento com Obtustatina induziu um estacionamento na fase G0 e diminuição da proliferação de CMLV tratadas com Ang II. Portanto, mostramos nesse trabalho que o heme livre induz a ativação de CML via NADPHox, que é elegantemente contra-regulado pelo sistema HO. Além disso, sugerimos que a integrina &#945;1ß1 pode ser um importante alvo molecular para o desenvolvimento de intervenções mais efetivas para a aterosclerose. / Cardiovascular diseases represent the major mortality reason in western countries. Among these diseases, atherosclerosis is the most prominent one, which is characterized by vascular smooth muscle cell (VSMC) accumulation. The pathological effect of VSMC in response to different stimuli is able to induce VSMC dysfunctions. Notably, this cardiovascular disease occurs mainly in sinuous vessels with turbulent blood flow, which may lead to hemolysis and consequent free heme accumulation. Furthermore, in atherogenesis the adhesion molecule, mainly integrins, were of crucial importance during the VSMC response. In this work our aim was to elucidate the effect of free heme in VSMC, as well the molecular mechanisms underlying this process. In a second part, we investigated the role of &#945;1ß1 integrin in Angiotensin II (Ang II) effect on VSMC. We observed that free heme is able to induce VSMC proliferation in a Reactive Oxygen Species (ROS) derived from NADPHoxidase (NADPHox) dependent manner. Additionally, heme activates proliferation-relationed redox-sensitive signaling routes, such as MAPKinases and the transcription factor NF&#954;B. It was also observed a critical crosstalk between NADPHox and heme oxygenase (HO) system, once heme induces HO-1 expression and VSMC pretreatment with HO inhibitors increased heme proliferative effect and ROS production. Accordingly, we observed that the counter-regulatory effect promoted by HO occurs due heme metabolites: biliverdin, bilirubin and carbon monoxide. Finally, when both NADPHox and HO system were blocked, heme had no effect on VSMC proliferation. In a second part, we observed that the chemotactic effect of Ang II on VSMC was abolished when the cells were pretreated with the &#945;1ß1 integrin ligand, the disintegrin Obtustatin. Then, we observed that the Ang II effect on FAK activation and actin-ILK colocalization is integrin &#945;1ß1 dependent, which possibly activates PKC&#945;, once we observed that the ROS production induced by Ang II was inhibited by Obtustatin. We demonstrated by western blotting that ILK induction by Ang II is dependent of &#945;1ß1 integrin and we also observed that Obtustatin inhibited the uncoupling of ILK to FAK, once Obtustatin blocked the FAK phosphorylation induced by Ang II (crucial process to ILK uncoupling). We also observed that Ang II induced, via &#945;1ß1 integrin, AKT phosphorylation, and p21 expression reducement, probably via ILK. Corroborating these data we demonstrated that the pretreatment with Obt induced G1 phase arrest and diminishment of VSMC proliferation treated with AngII. Thus we showed that free heme induces VSMC activation via NADPHox, which is elegantly counter-regulated by HO-1. Furthermore, we suggest that &#945;1ß1 integrin may be an important target molecule to the development of more effective therapeutic interventions in atherosclerosis.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Lipoxinas (LXs) são metabólitos do ácido araquidônico com reconhecidas atividades antiinflamatórias e pró-resolução. Apesar do grande número de trabalhos publicados descrevendo o papel das LXs e seus análogos em leucócitos e outros tipos celulares envolvidos em doenças inflamatórias, pouco é sabido a respeito dos mecanismos de ação que desencadeiam estas respostas. Neste trabalho investigamos o papel do 15-epi-16-(para-flúor)-fenoxi-lipoxina A4 (ATL-1), um análogo sintético da 15-epi-lipoxina A4, sobre diversos processos de ativação de monócitos. Caracterizamos, pela primeira vez, o receptor da lipoxina A4 (ALX) na linhagem monocítica U937, através da avaliação de sua expressão gênica e protéica e de sua funcionalidade analisando a ativação de ERK-2, o que torna esta célula uma ferramenta apta para estudo dos mecanismos de ação das LXs e seus análogos sobre os monócitos. Além disso, demonstramos que o ATL-1 aumenta a expressão da enzima heme oxigenase (HO) -1 em células U937 via ativação da p38 MAP quinase (MAPK) e diminui a secreção da Monocyte chemoattractant protein-1 (MCP-1), uma quimiocina envolvida com o recrutamento de monócitos para o foco inflamatório, em células U937 estimuladas com LPS. A inibição da secreção de MCP-1 foi revertida pela utilização do SB203580, sugerindo que este efeito é dependente da ativação da via p38 MAPK. O presente estudo elucida alguns dos mecanismos envolvidos na ativação de monócitos pelas lipoxinas que podem levar a novas abordagens para o controle de diversas doenças nas quais o componente inflamatório é importante / Lipoxins (LXs) are arachidonic acid metabolites with well recognized anti-inflammatory and pro-resolution activities. Despite the large number of studies describing the role of LXs and their analogs in leukocytes and other cell types involved in inflammatory diseases, little is known about the mechanisms of action that trigger these responses. This work investigated the role of 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 (ATL-1), a synthetic analog of 15-epi-lipoxin A4 on various processes of monocyte activation. We characterized, for the first time, the lipoxin A4 receptor (ALX) in the monocytic lineage U937, through the assessment of its gene expression and protein and its functionality through the activation of ERK-2, which makes this cell line a suitable tool to study the mechanisms of action of LXs and their analogs on the monocytes. Furthermore, we demonstrated that ATL-1 increases the expression of the enzyme heme oxygenase (HO)-1 in the U937 cells via activation of p38 MAP kinase (MAPK) and decreases the secretion of Monocyte chemoattractant protein-1 (MCP-1), a chemokine involved in the recruitment of monocytes to the inflammatory focus in LPS-stimulated U937 cells. MCP-1 secretion inhibition by ATL-1 was reverted by SB203580 indicating that this effect is dependent on the activation of p38 MAPK pathway. This study clarifies some of the mechanisms involved in the activation of monocytes by lipoxins which may lead to new approaches for the control of different pathologies where the inflammatory component is relevant