Aims There are few proven therapies that can protect against the inevitable ischaemia reperfusion injury (IRI) that occurs during renal transplantation. IRI increases the likelihood of delayed graft function (DGF), which negatively impacts on the long-term survival of a transplanted kidney. One enzyme of interest, heme oxygenase-1 (HO-1), degrades heme and protects against the oxidative stress that occurs secondary to IRI. Clinical renal recipients with higher HO-1 levels have improved graft function post transplant. Heme arginate (HA), a form of hemin, which has been used to treat porphyria for over 30 years, has repeatedly been shown to induce HO-1 in in vivo and in vitro macrophages. It is one of the few HO-1 inducers approved for clinical use and healthy volunteer studies confirmed that HA could also safely induce HO-1 in humans. Prior to the formal start of the MD, the University of Edinburgh successfully applied to NHS Blood and Transplant for funding to investigate whether giving HA to recipients of deceased donor renal grafts prior to transplant could upregulate HO-1 and whether this had any effect on the function and health of the grafts. This MD aims to explain the background behind the proposed study, the process of study approval, planning and trial logistics and protocol. This thesis then describes the methods of sample analysis, the results and future directions for the HOT (Heme Oxygenase-1 in renal Transplantation) study. Methods The HOT study planning and approval process took eight months and the first participant was randomised in January 2012. The study was sponsored by ACCORD, a joint company from University of Edinburgh and NHS Lothian, and recruited patients from the Edinburgh Royal Infirmary Transplant Unit. The protocol was followed to ensure that 40 recipients were randomised blind to either active (two doses 3mg kg-1 HA: pre-operatively, day 2) or placebo (NaCl: same schedule). To ensure that the primary outcome was fulfilled, recipient blood was taken daily for peripheral blood mononuclear cells (PBMC) extraction. After further blinding steps, the PBMCs were analysed for HO-1 protein and mRNA. The secondary outcome measures involved collecting urine for analysis of urinary biomarkers (KIM-1 and NGAL), taking renal graft biopsies pre-op and day 5 for renal HO-1 analysis and collecting renal function data. DGF was calculated daily. To ensure that all adverse event data was captured, the recipients were closely reviewed for 7 days and their renal function was monitored for 90 days. Results The final participant was recruited in May 2013 within the predicted timescale and to budget. This participant completed follow-up in August 2013. Of the 40 participants, three received the infusion but did not receive a transplant and therefore could not give primary outcome data. The remaining 37 did and this was analysed. Adverse events were equivalent between groups and there were no adverse reactions to HA. HA upregulated PBMC HO-1 protein at 24 hours compared to placebo: HA 11.1ng/ml (1.0- 37.0) vs. placebo 0.14ng/ml (-0.7- 0.3)(p= < 0.0001). PBMC HO-1 mRNA was also increased: HA 2.73 fold (1.8- 3.2) vs. placebo 1.41 fold (1.2- 2.2) (p=0.02). HA increased HO-1 protein immunopositivity in day 5 renal tissue compared with placebo: HA 0.21 (-24- 0.7) vs. placebo -0.03 (-76- 0.15) (p=0.02) and the percentage of HO-1 positive renal macrophages also increased: HA 50.8 cells per HPF (40.0- 59.8) vs. placebo 22.3 (0- 34.8) (p=0.012). Renal HO-1 mRNA was also increased in HA group: 2.02 (0.20- 4.03) fold increase compared to 1.68 (0.75- 10.39) fold in the placebo group but it was not significant (p= 0.451). Urinary biomarkers were reduced after HA but not significantly so. Histological injury and DGF rates were similar between the groups. Conclusion HA is safe and effective in renal transplant recipients as reported in this phase II, randomised, placebo controlled, blinded, single-centre study. The primary outcome was achieved and demonstrated for the first time that HA induces HO-1 in peripheral and renal macrophages in kidney transplant recipients. There was also evidence that HA increased HO-1 expression in renal tissue. There was no evidence that HA improved renal function or reduced injury as seen in animal models but it is recognised that the sample size was small and the study was not powered to these endpoints. Larger studies are planned to determine the impact of HO-1 upregulation on clinical outcomes and evaluate the benefit to patients at risk of IRI. The plans for HOT2 are expanded in this thesis.

Heme is the major iron source for the deadly human pathogen, Group A Streptococcus (GAS). During infection, GAS lyses host cells releasing hemoglobin and other hemoproteins. This dissertation aims to elucidate the general mechanism by which GAS obtains and utilizes heme as an iron source from the host hemoproteins. GAS encodes a heme relay system consisting of Shr, Shp and the SiaABC transporter. We specifically determine the role of Shr in the heme uptake process, by conducting a detailed functional characterization of its constituent domains. We also undertake to solve the long-standing mystery surrounding the catabolism of heme in streptococci. The studies presented herein established Shr as a prototype of a new family of NEAT-containing hemoproteins receptors. They demonstrate its importance in heme acquisition by GAS and provide a molecular model for heme scavenging and transfer by the protein. We show that Shr modulates heme uptake depending on heme availability by a mechanism where NEAT1 facilitates fast heme scavenging and delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface. Finally, we identified and characterized for the first time, a heme oxygenase (HO) in the Streptococcus genus which was named HupZ. Sequence comparison between HupZ and several HOs from different structural families indicates that this enzyme is unrelated to any of the previously characterized HOs. However, orthologs of the protein are found in other important pathogens. The structure and the catalytic mechanism of HupZ suggest that it is the representative of a new family of flavoenzymes capable of degrading heme using their reduced flavin cofactor as a source of electrons. Overall, this work contributes significant knowledge to the topic of heme utilization by pathogens and importantly, provides new direct evidence that associates flavins with heme metabolism in bacteria. Thus it sets a new direction in the field and lays the ground for future fundamental and applied discoveries.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains vii, 104 p. : ill. Includes abstract. Includes bibliographical references.

Some pathogenic bacteria derive significant amounts of iron heme from their hosts. In this study we investigated SiaA, a heme binding protein from Streptococcus pyogenes. The wildtype methionine79 putative axial ligand was mutated to alanine. SiaA M79A was expressed in E. coli in three production runs, lysed by sonication or French press, and purified by fast protein liquid chromatography (FPLC). Nickel affinity FPLC was found to give much purer SiaA when 30 mM imidazole was added to the binding buffer. The protocol using extensive sonication resulted in SiaA weighing 30464 Da. The protocol using French press resulted in SiaA weighting 33358 Da. Despite the difference in masses, the two forms of SiaA interacted with heme similarly.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references (p. 99-116).

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

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-02-07T19:06:15Z No. of bitstreams: 1 Graziele Quintela de Caravalho... O Papel do Neme na infecção....pdf: 1591650 bytes, checksum: 3f661f0e0b742c04c4ad3e8907c1cf24 (MD5) / Made available in DSpace on 2014-02-07T19:06:15Z (GMT). No. of bitstreams: 1 Graziele Quintela de Caravalho... O Papel do Neme na infecção....pdf: 1591650 bytes, checksum: 3f661f0e0b742c04c4ad3e8907c1cf24 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Laboratório de Engenharia Tecidual e Imunofarmacologia. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / A Leishmaniose é uma doença que atinge milhões de pessoas em todo o mundo, sendo endêmica em muitas áreas. A doença pode apresentar diferentes manifestações clínicas, tais como a Leishmaniose Visceral (LV), que é a forma mais grave e letal, caso não seja tratada. As manifestações hematológicas são comumente associadas à LV, onde os mecanismos relacionados à hemólise e a presença do heme livre, podem interferir no comportamento de neutrófilos. Já foi descrito que o heme livre é uma molécula pró-inflamatória, com a capacidade de induzir migração e ativação dos neutrófilos. No entanto, o efeito do heme sobre neutrófilos humanos durante a infecção por Leishmania chagasi, ainda não foi explorado. A nossa hipótese é que o heme induz ativação de neutrófilos humanos favorecendo o processo inflamatório na infecção por L. chagasi. Nossos resultados mostraram que o heme induz a ativação e apoptose em neutrófilos humanos infectados com L. chagasi, seguido pela sobrevivência do parasita. Moléculas presentes na composição do heme, como a protoporfirina IX (PPIX) e o Fe+2, não alteram o status de ativação dos neutrófilos, mas mantêm o aumento do crescimento parasitário como observado em presença do heme. Além disso, o heme e o Fe+2 aumentam a produção do TGF-β e a atividade da SOD. A inibição farmacológica da enzima SOD com dietilditiocarbamato (DETC) reduz a taxa de proliferação da L. chagasi em neutrófilos infectados. Em conjunto, esses dados indicam que o heme e o Fe+2 podem contribuir como fonte nutricional e controlar o ambiente inflamatório, com a indução de TGF-β e SOD, permitindo a sobrevivência da L. chagasi em neutrófilos humanos. Esse estudo poderá abrir novas perspectivas para o entendimento dos mecanismos imunopatogênicos envolvendo neutrófilos e suas implicações no processo inflamatório da LV. / The Leishmaniasis is a disease that affects millions of people worldwide, being endemic in many areas. The disease may present different clinical manifestations, such as Visceral Leishmaniasis (VL), which is the most severe form and fatal if left untreated. The hematologic manifestations are commonly associated with VL, where the mechanisms related to hemolysis and the presence of free heme may modify the behavior of neutrophils. It has been reported that the free heme is a pro-inflammatory molecule with the ability to induce migration and activation of neutrophils. However, the effect of the heme on human neutrophils during infection by Leishmania chagasi yet hasn't been explored. Our hypothesis is that the heme induces activation of human neutrophils favoring the inflammatory process on the infection by L. chagasi. The results showed that the heme induces activation and apoptosis in human neutrophils infected with L. chagasi, followed by parasite survival. Molecules present in the composition of heme, as protoporfirin IX (PPIX) and Fe+2, do not alter the activation status of neutrophils, but maintain the increased parasite growth as viewed in the presence of heme. Furthermore, the heme and Fe+2 increase the production of TGF-β and SOD activity. The pharmacological inhibition of SOD with diethyldithiocarbamate (DETC) reduces the rate of L. chagasi proliferation in infected neutrophils. Together, these data indicate that the heme and Fe+2 may contribute as a nutritional source and control the inflammatory environment with the induction of TGF-β and SOD, allowing the survival of the L. chagasi in human neutrophils. This study may open new perspectives for the understanding of the immunopathogenic mechanisms involving neutrophils and its implications in the inflammatory process of the VL.

O Trypanosoma cruzi, agente etiológico da doença de Chagas, possui um ciclo de vida complexo, deve lidar com diversas condições do ambiente e depende dos hospedeiros para suprir suas necessidades nutricionais. Uma delas é a necessidade de captar a molécula de heme (Fe-protoporfirina IX) que será utilizada como fator de crescimento. Os mecanismos envolvendo o metabolismo de heme são cruciais para a sobrevivência do T. cruzi pois o parasito não possui várias enzimas de biossíntese dessa porfirina e o heme livre pode apresentar citotoxicidade para célula. Na tentativa de perseguir o destino final do heme no parasito, nós estudamos essa via inexplorada no T. cruzi. Nessa tese, nós demonstramos que epimastigotas cultivados com heme, produziram os compostos, &#945;-meso hidroxiheme, verdoheme e biliverdina (identificados por HPLC acoplado á espectrofotômetria). Além disso, nós observamos através de análise dos extratos de epimastigotas no espectrômetro de massas (LQT Orbitrap), espécies iônicas de m/z 583,4 e m/z 619,3. A fragmentação subsequente desses íons originaram espécies filhas típicas das moléculas de biliverdina e verdoheme, respectivamente. Nós observamos também, espécies iônicas de m/z 1397,4 e m/z 1135,4. A fragmentação dessas espécies produziram íons, sendo um deles com a mesma massa molecular de heme (m/z 616,3). Essa espécie iônica por sua vez, gerou fragmentos iônicos idênticos a uma molécula de heme, confirmando que esses intermediários são produtos da modificação da porfirina. Baseado nesses resultados, nós propomos um modelo onde o catabolismo de heme em T. cruzi, envolveria a conjugação da bis(glutationil)spermina, um derivado da tripanotiona presente em tripanossomatídeos, à porfirina (m/z 1137,4), seguido da remoção de dois resíduos de ácidos glutâmicos (m/z 1135,4). Embora o significado bioquímico e fisiológico da adição desse resíduo tiol na molécula de heme ainda é pouco compreendido, alguns trabalhos demonstram a abilidade desses compostos em ligar na porfirina, sem contar também, que esse heme conjugado poderia resultar em uma forma efetiva de prevenção de danos à membrana e a célula ocasionados pelo acúmulo de heme livre. Em conjunto, esses resultados fornecem novas abordagens do metabolismo de heme em T. cruzi, revelando possíveis alvos de intervenção quimioterápica futuros. Nossa proposta está direcionada para uma via ativa de catabolismo de heme que inclui a adição de grupos tiol (derivado da tripanotiona) à heme e a clivagem do anel porfirínico originando a molécula de biliverdina. / Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and they must cope with diverse environmental conditions and depends on hosts for its nutritional needs. One of the nutritional characteristic is that they need a heme compound (Fe-protoporphyrin IX) as a growth factor. The mechanisms involved in these processes are crucial for their survival mainly because of trypanosomatids lack of the complete heme biosynthetic pathway and the cytotoxic activity of free heme. Following the fate of this porphyrin in the parasite we studied this missing pathway in T. cruzi. Here, we show that epimastigotes cultivated with heme yielded the compounds, &#945;-meso hydroxyheme, verdoheme and biliverdin (as determined by HPLC with diode array detector). Furthermore, we observed ion species of m/z 583.4 and m/z 619.3 from epimastigotes extracts detected by direct infusion on LQT Orbitrap platform. A tipical biliverdin and verdoheme doughter-ion species were generated by m/z 583.4 and m/z 619.3 fragmentations, respectively. We also observed an ion species at m/z 1397.4 and m/z 1135. The subsequent fragmentation of this species produced a daughter-ions whose one with the same molecular mass as heme (m/z 616.4). This species, in turn, generated daughter species identical to an authentic heme, confirming that these intermediates were modified heme products. Based on these findings, we propose that heme catabolism in T. cruzi involves a additions of Bis(glutathionyl)spermine, a low molecular mass thiols occurring in trypanosomatids, to heme (m/z 1397.4), followed by removal of the glutamic residues (m/z 1135). Although the biochemical and physiological significance of the addition of thiol residues to heme molecule is underexplored, some works, already demonstrated their ability to bind heme and also this modified heme may resulting in the effective prevention of membrane damage and cytotoxicity by the heme accumulation. Taken together, these results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets. We propose an active heme catabolism pathway that includes a trypanotione derivate additions and cleavage of the heme porphyring ring to biliverdin.

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.