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Universidade Federal do Rio grande do Sul
Faculdade de Medicina - UFRGS
Pós - Graduação em Cardiologia e Ciências Cardiovasculares
Tese de Doutorado
Estudos do perfil de estresse oxidativo e mediadores de
remodelamento ventricular em modelo experimental de infarto agudo
do miocárdio e o papel do uso precoce de terapia celular
Angela Maria Vicente Tavares
Orientação: Profa. Dra. Nadine Clausell
Co-Orientação: Prof. Dr. Luis Eduardo P. Rohde
Porto Alegre – Dezembro de 2008.
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2
SUMÁRIO
EPÍGRAFE 03
AGRADECIMENTOS 04
RESUMO 07
INTRODUÇÃO 08
REFERÊNCIAS BIBLIOGRÁFICAS 22
JUSTIFICATIVAS 33
HIPOTESES 34
OBJETIVO GERAL 35
Objetivos específicos (artigos 01 e 02) 35
ARTIGO 01 36
ARTIGO 02 51
CONCLUSÕES 70
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3
“ Não ande pelo caminho traçado;
Ele conduz somente até onde os outros já foram.”
A. Einstein
...“Não DIGAS nada!
Não, nem a verdade!
Há tanta suavidade
Em nada se dizer
E tudo se entender –
Tudo metade
De sentir e de ver...
Não digas nada!
Deixa esquecer.
Talvez que amanhã
Em outra paisagem
Digas que foi vã
Toda esta viagem
Até onde quis
Ser quem me agrada...
Mas ali fui feliz...
Não digas nada.”
Fernando Pessoa
4
AGRADECIMENTOS
“A única coisa que não nos pode ser tomado é o conhecimento, mas a tarefa de
obtê-lo não deve ter objetivo de passagem; ela deve servir a um propósito humano e
este, causar felicidade a quem esteja buscando, pois é para isso que se vive. A busca
de grandes ideais é um universo de pequenos gestos, portanto, retirar o melhor
aprendizado da pior das situações, não é uma tarefa fácil, mas nos deixa mais fortes
para a próxima etapa”. Dissertação de Mestrado - A. Tavares, 2003.
Tenho um único mérito em todo este trabalho, uma inexplicável capacidade de
agregar e manter próximas pessoas competentes, comprometidas com os seus ideais
de pesquisa e, o mais importante: que acreditaram nos meus objetivos e confiaram na
minha miserável condição de começar um processo novo.
Tenho a exata noção de quanto este trabalho foi incapaz de demonstrar o enorme
esforço de todos para que tivéssemos a oportunidade de contribuir com a ciência de
forma inequívoca. Por isso, peço desculpas; fomos capazes de gerar tantos dados
importantes, mas a incoerência das circunstâncias, às vezes, faz nosso tempo limitado
e diminuto perante tudo o que gostaríamos e poderíamos mostrar.
Por fim, eu tive o privilégio de conviver, porque eu escolhi conviver, com pessoas
muito melhores do que eu, seres humanos admiráveis, pesquisadores apaixonados,
profissionais impressionantes e amigos muito especiais.
O Hospital de Clínicas foi minha casa por mais de quatro anos, não tenho palavras
para agradecer a oportunidade de ter estado aqui e o orgulho que sinto disso.
Uma pesquisadora por excelência, um ser humano, uma pessoa fantástica e uma
médica preocupada em levar o melhor aos seus pacientes; que saibam todos: esta foi e
sempre será minha orientadora, Dra. Nadine Clausell; ser orientada por ela foi, sem
dúvida, uma experiência única. Sob sua orientação se aprende ou se aprende... E se
5
aprende! Talvez esta seja a melhor tradução para “vai dar certo!” O que posso dizer é
que simplesmente, mais que um privilégio, foi uma honra!
Agradeço ao Dr. Luis Eduardo Rohde, meu Co-Orientador por acreditar, muito mais
do que eu, que realmente eu pudesse manusear uma ferramenta tão importante como
é o Eco. Seu apoio para que eu pudesse utilizar o equipamento me deu a oportunidade
de ajudar outros pesquisadores e participar de outros projetos, me tornando mais útil
para todos.
Agradeço a toda a equipe que a compõe a Unidade de Experimentação Animal,
pois foram incansáveis para facilitar o nosso trabalho com os animais, em todos os
sentidos.
Um agradecimento especial a Profa. Roseli Moleck(†), veterinária chefe que nos
deu todo o suporte para que pudéssemos iniciar o trabalho e nos acolheu com tanto
carinho e empenho, sempre comprometida com tudo e com todos.
Agradeço as colegas do Laboratório de Pesquisa Cardiovascular pelo apoio e os
momentos de alegria;
Aos meus colegas do LaFiEx, agradeço a oportunidade de poder participar das
suas pesquisas e principalmente pelo voto de confiança.
Agradeço aos colegas da Terapia Gênica, onde me sinto em casa e tive apoio para
realizar minha pesquisa, em especial ao Guilherme, sempre pronto a colaborar.
A Dra. Ursula Matte, um daqueles seres humanos admiráveis, pesquisadora
apaixonada, profissional impressionante e amiga muito especial. Sentirei falta das
descobertas inusitadas. As cartilagens que nos perdoem...
Aos meus bolsistas (Andréia Taffarel, Gabriela Nicolaidis e Rafael Dall’Alba) um
agradecimento especial, pois foram muito mais do que alunos de iniciação científica;
foram uma amostra de pesquisadores de futuro, profissionais competentes e pessoas
6
que vou lembrar sempre com carinho. A qualidade do trabalho passou pelas mãos
deles e também por isso foi um trabalho de qualidade.
Dra. Adriane Belló-Klein também faz parte daqueles seres humanos admiráveis
pesquisadores apaixonados, muito melhores do que eu, em quem, não só me espelhei
como modelo de pesquisadora, mas que fez toda a diferença para que este doutorado
pudesse ser concretizado. Parece mais o meu anjo da guarda, pois me acompanha
desde o mestrado, sempre intervindo para que as coisas nunca dêem erradas para
mim. Faz parte da minha família de amigos. Espero, um dia, poder retribuir.
Agradeço ao Laboratório de Fisiologia Cardiovascular como um todo, à Tânia,
sempre pronta a ajudar com muita dedicação, aos colegas de Pós-graduação,
extremamente prestativos e em especial ao Alex Sander, um dos melhores
pesquisadores de bancada que já tive o privilégio de conhecer e conviver, um dos
meus melhores amigos.
Agradeço a Dra. Maria Flávia que abriu seu laboratório para que eu pudesse
terminar meus experimentos de Western blot.
Por fim, a minha família! Afinal, família são aquelas pessoas que a gente escolhe...
As pessoas que me acolhem e que eu me dedico, um agradecimento especial: Maria,
sempre uma inspiração de humanidade e competência para tratar com as pessoas;
Guega, Jaci e Denise, pessoas que me lembram sempre que mesmo longe, eu faço
parte de alguma coisa; Lucia, mesmo à distância nunca esquece de me manter por
perto e Daniel Merel e Charles Knoblish... Lembram-me sempre, que aproveitar a vida
é impressionantemente fácil, quando se sabe!
Vó Olívia, Vô Padão… Estes são meus avós perfeitos, sempre que penso neles
tenho certeza que o dia de amanhã será melhor… E fico em paz!
7
Resumo
A insuficiência cardíaca (IC) é uma síndrome clínica complexa com diversas etiologias e elevada
prevalência. No infarto agudo do miocárdio, a oclusão aguda da artéria coronária resulta em alterações
complexas da arquitetura ventricular. Estas alterações começam a se estabelecer imediatamente após a
oclusão arterial, progredindo por várias semanas após o dano isquêmico inicial. O processo inflamatório e a
liberação de citocinas fazem parte da resposta a injuria sofrida pelo tecido e desempenha um papel
importante no período pós-infarto. Dentre os múltiplos fatores que contribuem para a progressão do
remodelamento ventricular, a participação das espécies ativas de oxigênio (EAOs) neste processo é de
extrema relevância. Diante deste quadro, novas estratégias terapêuticas têm sido propostas e a terapia
celular surge como uma alternativa viável promissora objetivando, em última análise, o reparo cardíaco.
A hipótese inicial de que as células-tronco adultas poderiam ter efeito regenerativo do tecido
miocárdio tem sido revisada e hoje, é cada vez mais aceito e reconhecido que as células-tronco adultas tem
pouca ou nenhuma capacidade regenerativa e que os seus efeitos benéficos envolvam ações parácrinas
sobre o tecido hospedeiro.
Neste estudo avaliamos o perfil oxidativo e sua correlação com o processo inflamatório e função
cardíaca, precocemente, 48 horas pós-infarto agudo do miocárdio (IAM). Analisamos também estes
mesmos parâmetros no tecido cardíaco de animais infartados que receberam tratamento com terapia
celular.
Nosso estudo sugere que o comprometimento da função ventricular precocemente pós-IAM parece
se associar com um desequilíbrio do estado redox e este por sua vez interage com os processos iniciais do
fenótipo de remodelamento ventricular.
A terapia celular, com células derivadas da medula óssea num modelo experimental de 48 horas
pós-IAM, foi associada com redução da hipertrofia ventricular e menor secreção de citocinas inflamatórias
sugerindo ações parácrinas das células, nesta janela temporal.
8
INTRODUÇÃO
A insuficiência cardíaca (IC) é uma síndrome clínica complexa com diversas
etiologias e elevada prevalência. Embora grandes avanços tenham ocorrido, consiste em
uma preocupação crescente dos diferentes sistemas de saúde pública pelo seu elevado
impacto econômico associado, particularmente, os custos das internações hospitalares.
Este, e o seu prognóstico clínico sombrio, apesar de tratamentos otimizados, tem sido a
tônica na busca de novas terapias.
Aspectos Epidemiológicos
As doenças cardiovasculares tornaram-se o principal problema de saúde em todo o
mundo, ultrapassando o câncer e infecções como a principal causa de morte de muitos
países em desenvolvimento
1;2
. Embora a mortalidade por doença arterial coronariana
tenha diminuído devido a avanços nos tratamentos de aterosclerose, hipercolesterolemia,
hipertensão e diabetes
3
, as doenças cardiovasculares ainda representam 1 em cada 2,7
mortes nos Estados Unidos, traduzindo-se em aproximadamente 2,5 milhões de mortes
todos os anos
4
. Além disso, a prevalência dos fatores de risco para doenças
cardiovasculares, como hipertensão, diabetes tipo II e obesidade, tem aumentado nos
últimos anos
5-7
.
Num estudo realizado em mais de 300 hospitais, nos Estados Unidos, foram
rastreadas 2,5 milhões de internações, onde 496.534 pacientes (19,7%) tiveram IC, como
desfecho primário ou secundário com uma média de permanência hospitalar de 8,7±28,6
dias e mortalidade intra-hospitalar de 7,1%. Neste estudo, as admissões de pacientes
com IC como desfecho primário ou secundário foram associadas com pior prognóstico
8
.
No Brasil, de um total de 1.006.375 de mortes por qualquer causa, 6,4% foram por
IAM, somente no ano de 2005. O número de internações hospitalares por IC foi de
9
293.473, cerca de 2,6% de todas as internações hospitalares por qualquer causa,
ocorridas somente no ano de 2007
9
.
Remodelamento Ventricular Pós-Infarto Agudo do Miocárdio
Os estudos que descreveram pioneiramente o remodelamento ventricular (RV) na
progressão da IC são aqueles utilizando como modelo o desenvolvimento de infarto
agudo em ratos no inicio da década de 80 por Pfeffer e colaboradores
10
. Estes estudos
demonstraram que a oclusão aguda da artéria coronária, particularmente quando ocorrem
lesões transmurais de grandes proporções, resulta em alterações complexas da
arquitetura ventricular. Estas alterações começam a se estabelecer imediatamente após a
oclusão arterial, progredindo por várias semanas após o dano isquêmico inicial
11
.
Uma série de respostas compensatórias ocorre no ventrículo esquerdo após o IAM,
com o objetivo primordial de preservar o débito cardíaco. A distensão aguda do tecido
miocárdico viável e a ação do mecanismo de Frank-Starling, bem como o aumento da
atividade cronotrópica e inotrópica secundária a estimulação simpática, buscam manter a
função de bomba do ventrículo esquerdo, apesar da perda abrupta do tecido contrátil
12
. A
expansão da zona infartada ocorre poucas horas após o dano ao miocárdio, resultando
em estresse e dilatação da parede ventricular
13
. A dilatação ventricular, embora
represente um mecanismo eficiente de compensação, restabelecendo o volume sistólico,
também tem sido consistentemente associada com uma diminuição de sobrevida
14
.
Embora grandes avanços tenham sido alcançados no entendimento dos
mecanismos hemodinâmicos, histológicos e moleculares envolvidos nas alterações, que
ocorrem no tecido cardíaco após um dano isquêmico, muitos pacientes que sofrem
eventos agudos, mesmo tratados de forma otimizada, desenvolvem dilatação ventricular
progressiva e podem evoluir para quadros clínicos de IC
15
.
10
Muito além de adaptações hemodinâmicas secundárias, eventos celulares e
moleculares precoces pós-infarto, como a ativação de citocinas inflamatórias, estresse
oxidativo, hipertrofia de miócitos, apoptose, necrose, fibrose e ativação proteolítica
parecem exercer papeis centrais na progressão do RV
16-19
. Além de implicações
mecanísticas, estes eventos podem ser alvo de terapias mais objetivas e eficazes no
combate a progressão da doença
20
.
Papel da Modulação Inflamatória
O processo inflamatório e a liberação de citocinas fazem parte da resposta à injuria
sofrida pelo tecido e desempenham um papel importante no período pós-infarto. A
conseqüência dos efeitos das citocinas inflamatórias podem ser favoráveis, levando à
cicatrização e restauração da função cardíaca, ou desfavoráveis levando, agudamente, ao
um processo de dilatação e posteriormente, aos desfechos que culminam em IC
21
.
A modulação imunoinflamatória tem papel fundamental relacionada a fisiopatogenia
da IC e sua correlação direta com o estresse oxidativo
22
. A indução da expressão gênica
pelo fator nuclear kappa-beta (NFk-B), envolvendo sinalização redox é um dos
mecanismos moleculares que envolvem as sinalizações redox-sensíveis. O NFk-B pode
ser ativado por agentes oxidantes e inibido por agentes antioxidantes
23
. A proteína NFk-B
se transloca para o núcleo, onde se liga ao gene alvo, ativando a transcrição de genes,
cujos produtos atuam como mediadores pró-inflamatórios
24
.
Citocinas pró-inflamatórias como interleucina 1 beta (IL-1β), fator de necrose
tumoral alfa (TNF-α) e interleucina 6 (IL-6) não são constitutivamente expressas no tecido
cardíaco normal
25
. O aumento da produção destas citocinas representa uma resposta
intrínseca ao estresse mecânico provocado pela injúria. Das primeiras horas até um dia
pós-infarto estas citocinas tem sua expressão de mRNA aumentada na área infartada,
assim como, em áreas não infartadas do miocárdio
26;27
.
11
Estas citocinas inflamatórias são mediadores biológicos que têm sido encontradas
em concentrações séricas elevadas em pacientes com IC
28
. Em um estudo avaliando
marcadores inflamatórios no momento do infarto comparados após um seguimento de oito
anos mostrou que aqueles pacientes sobreviventes tinham os menores níveis séricos de
IL-6 no momento do infarto
29
.
Papel do Estresse Oxidativo
Dentre os múltiplos fatores que contribuem para a progressão do RV, vários
estudos apontam para a participação de radicais livres e em especial as espécies ativas
de oxigênio (EAOs) neste processo
30;31
. Por exemplo, as EAOs tem sido referenciadas
como fortemente implicadas na gênese da hipertrofia cardíaca
32
. A hipertrofia cardíaca
pode ser tanto compensatória como adaptativa ou um indício de mal-adaptação, como um
precursor da IC. Muitos dos fatores extracelulares que são capazes de induzir a hipertrofia
dos cardiomiócitos e os fatores que medeiam esta podem ser ativados direta ou
indiretamente pelas EAOs
33
.
Estudos experimentais mostram diminuição da atividade antioxidante e aumento do
estresse oxidativo, resultando no aumento da produção de radicais livres
34
. Neste
contexto, a contratilidade miocárdica pode estar deprimida em decorrência da redução de
Ca
++
proveniente do retículo sarcoplasmático e da atividade da Ca
++
ATPase no tecido
cardíaco
35
. Outros estudos relacionam alterações morfológicas, como hipertrofia e
apoptose dos cardiomiócitos, e disfunção da contratilidade miocárdica com o aumento da
expressão das isoformas da NOS (óxido nítrico sintase): NOS endotelial (eNOS) e NOS
induzida (iNOS)
36
.
Devido a estas alterações metabólicas, é possível inferir que as conseqüentes
modificações nos processos de oxi-redução que ocorrem na célula, e conseqüentemente
no “status” de estresse oxidativo ao qual este órgão está submetido, são também
responsáveis diretos pelo processo de RV e pelos fenótipos apoptóticos
37;38
.
12
Durante a fase de inflamação aguda pós-IAM, neutrófilos e monócitos produzem
mieloperoxidases (MPO) que, apesar de constituírem uma das primeiras linhas de defesa
do sistema imunitário inato, induzem a formação de EAOs e subseqüentemente
contribuem para o processo de remodelamento do miocárdio
39
. Clinicamente, altos níveis
plasmáticos de MPO predizem aumento de mortalidade de pacientes que sofreram IAM
40
.
Em um estudo utilizando MPO marcada com quelato de gadolínio e análise por
ressonância magnética, foi demonstrado que o uso de atorvastatina em camundongos
infartados diminuía de forma significante a expressão dessa enzima já em 48 horas pós-
infarto. O grupo controle também demonstrou que o maior pico de MPO se deu nas
mesmas 48 horas demonstrando que as alterações bioquímicas no RV associadas a
estresse oxidativo ocorrem muito precocemente após o infarto
41
.
Evidencias de estudos clínicos na IC, apontam para a importância dos processos
de oxi-redução. Através de estudos envolvendo lipoperoxidação (LPO) e atividade
antioxidante (GPx e vitamina C) de pacientes com diferentes graus de IC congestiva, foi
encontrado um progressivo aumento de danos induzidos por radicais livres e diminuição
das reservas antioxidantes diretamente proporcionais ao grau da IC
42
.
A busca de intervenções que reduzam o dano oxidativo justifica algumas iniciativas
clinico-experimentais. Por exemplo, em pacientes portadores de cardiopatia isquêmica ou
com cardiomiopatia dilatada, a atividade de NADPH-oxidase está elevada e foi associada
ao aumento da atividade GTPase rac-1. O uso de estatinas nestes pacientes diminuiu a
atividade da rac-1 no miocárdio, possivelmente demonstrando o efeito destas drogas
sobre a atividade das EAOs
43
. O tratamento com alopurinol, um inibidor da XO (xantina-
oxidase), enzima que induz a formação de EAOs, melhora a contratilidade do miocárdio e
restaura a reatividade vasomotora
44;45
. Por fim, o probucol, droga com função redutora de
colesterol e importantes propriedades antioxidantes, foi capaz de diminuir a expressão
gênica de IL-6 e o estresse oxidativo, 24 horas após o infarto
46
.
13
Terapia celular
As terapias biológicas estão cada vez mais presentes trazendo expectativas de
renovação na área de saúde com perspectivas terapêuticas melhores nas diversas áreas
do conhecimento.
As células-tronco (stem cells) se diferenciam das outras células do organismo por
apresentarem três características: a) são indiferenciadas e não especializadas; b) tem um
potencial de replicação sem a necessidade de diferenciar-se, de tal forma que um
pequeno número de células pode gerar uma maior população de células semelhantes; c)
também são capazes de diferenciar-se em células especializadas de um tecido
específico
47-49
.
Tipos de células candidatas a uso em terapia celular
Cardiomiócito Fetal; Fenótipo de cardiomiócito; Imunossupressão;
debate ético; curta
sobrevida e limitada
disponibilidade;
Mioblásto Sem Imunogenicidade, autó- Arritmias, sem “gap”
Esquelético logo; fadiga e isquemia
resistente e disponível;
C. Progenitoras sem rejeição; autóloga; Necessita Expansão;
Endoteliais;
C. Tronco Pluripotente, altamente Imunossupressão;
Embrionárias expansível; debate ético; dispo
nibilidade, tumor?
C. Tronco Sem rejeição, autólogo, Propriedade funcional
Mesenquimais Pluripotente, criopreservável; e não esclarecida;
adultas dificuldade para
isolar em cultura;
Fonte Vantagem Desvantagem
Cardiomiócito Fetal; Fenótipo de cardiomiócito; Imunossupressão;
debate ético; curta
sobrevida e limitada
disponibilidade;
Mioblásto Sem Imunogenicidade, autó- Arritmias, sem “gap”
Esquelético logo; fadiga e isquemia
resistente e disponível;
C. Progenitoras sem rejeição; autóloga; Necessita Expansão;
Endoteliais;
C. Tronco Pluripotente, altamente Imunossupressão;
Embrionárias expansível; debate ético; dispo
nibilidade, tumor?
C. Tronco Sem rejeição, autólogo, Propriedade funcional
Mesenquimais Pluripotente, criopreservável; e não esclarecida;
adultas dificuldade para
isolar em cultura;
Fonte Vantagem Desvantagem
Cardiomiócito Fetal; Fenótipo de cardiomiócito; Imunossupressão;
debate ético; curta
sobrevida e limitada
disponibilidade;
Mioblásto Sem Imunogenicidade, autó- Arritmias, sem “gap”
Esquelético logo; fadiga e isquemia
resistente e disponível;
C. Progenitoras sem rejeição; autóloga; Necessita Expansão;
Endoteliais;
C. Tronco Pluripotente, altamente Imunossupressão;
Embrionárias expansível; debate ético; dispo
nibilidade, tumor?
C. Tronco Sem rejeição, autólogo, Propriedade funcional
Mesenquimais Pluripotente, criopreservável; e não esclarecida;
adultas dificuldade para
isolar em cultura;
Fonte Vantagem Desvantagem
Tabela – Tipos de células utilizadas em pesquisas pré-clínicas e/ou clínicas – vantagens e desvantagens
observadas no uso destas células in vivo
50
;
Um exemplo de regeneração de tecido são as células que compõem o músculo
esquelético, onde células satélites (mioblastos esqueléticos) armazenadas entre as
lâminas basais e as fibras musculares têm papel importante de regeneração, pois são
14
células quiescentes à espera de um estímulo de resposta a uma injúria que as
possibilitará se diferenciarem em miócitos maduros, com o objetivo de reparar a área
lesada
51
.
De acordo com a sua origem, as células podem ser classificadas como: células-
tronco embrionárias e adultas ou somáticas.
Células-tronco Embrionárias
Células-tronco embrionárias são isoladas e retiradas de blastocistos embrionários
capazes de proliferar e se diferenciar em células das três linhagens germinativas
diferentes em células de qualquer tecido. Também são capazes de se proliferarem
indefinidamente, mantendo sua capacidade de diferenciação, resultando, assim, em uma
fonte potencialmente ilimitada
52;53
.
Células-tronco embrionárias são consideradas as mais versáteis, podendo dar
origem a diversos tipos celulares incluindo cardiomiócitos
54
. Em estudos experimentais,
realizados em animais, foi observada uma grande capacidade proliferativa das células-
tronco embrionárias. Uma vez implantadas diretamente no miocárdio foram responsáveis
pela diminuição da área de infarto com melhora da função cardíaca e acoplamento
elétrico observado pela detecção da expressão da proteína conexina 43
55
.
Apesar dos benefícios mostrados com o uso destas células, não é possível
descartar a possibilidade de formação de teratomas e tumores (Figura 1)
56
.
15
Desenvolvimento das Células-Tronco Embrionárias
Figura 1 - Células-tronco embrionárias. Durante o desenvolvimento, as células-tronco embrionárias (CES)
dão origem a ectoderme, mesoderme e endoderme. CES são obtidas a partir da massa celular interna do
blastocisto e in vitro forma organismos embrióides (EBS) que contêm células indiferenciadas (amarelo),
ectodérmicas (verde), mesodérmicas (azul), e endodérmicas (vermelho). A implantação da EBS, in vivo,
pode reparar órgãos danificados e gerar tumores
57
.
Células-tronco adultas ou somáticas
As células-tronco adultas comportam, pelo menos, três diferentes grupos: 1)
células-tronco derivadas da medula óssea; 2) células-tronco circulantes ou células
progenitoras que também, pelo menos em parte, são derivadas a partir da medula óssea
e 3) células-tronco residentes do tecido. A medula óssea contém uma complexa
variedade de células progenitoras, incluindo as células-tronco hematopoiéticas (HSCs), as
células-tronco mesenquimais (CTMs) ou células estromais, e as células progenitoras
adultas (MAPCs), um subgrupo das células CTMs
58
.
Outra população de células progenitoras a mostrar um potencial terapêutico foram
encontradas no sangue circulante, são as chamadas células progenitoras endoteliais
16
(EPCs), definidas pela sua capacidade de formar novos vasos sangüíneos pós-
isquemia
59
.
As células-tronco de medula óssea são, basicamente, responsáveis por manter a
homeostase dos tecidos, substituindo células que foram perdidas na maturação, no
envelhecimento ou por qualquer tipo de injúria que o tecido tenha sofrido. A partir de um
processo fisiológico, elas são capazes de migrar até o tecido alvo, através do sangue
periférico, bem como, retornar novamente para a medula
60
.
Estas células, cujo potencial parece ilimitado, têm se mostrado como candidatas
ideais, já bastante utilizadas no tratamento de doenças hematológicas.
Terapia Celular e Cardiopatias
Um dos motivos pelo qual a terapia celular tem sido amplamente estudada
objetivando reparo cardíaco, é o fato de haver um conceito de uma relativa incapacidade
das células-tronco residentes na medula óssea em migrar para o miocárdio lesado ou
quando no miocárdio, se auto-regenerar.
61;62
Entretanto, a definição do coração como um
órgão de diferenciação terminal, foi contestada pela observação de células cardíacas
precoces em um estado mitótico. De fato, Beltrami e colaboradores analisaram a
proliferação de cardiomiócitos em pacientes que foram a óbito entre 4 e 12 dias pós-
infarto e observaram células em mitose na zona adjacente ao infarto, bem como, em
zonas distantes da área de lesão
63
.
Atualmente, diversos tipos de regeneração de celular estão sendo investigados,
bem como vários tipos de células que possam demonstrar alguma capacidade de
proliferar e se diferenciar em cardiomiócitos funcionais
64;65
.
Em 2004, pesquisadores procuraram reproduzir os experimentos de Orlic e
colaboradores de transdiferenciação de células de medula óssea para o reparo do tecido
cardíaco lesado
66
. Os resultados não foram concordantes com os experimentos
17
anteriores, pois as células implantadas não expressaram marcadores específicos do
tecido cardíaco. Além disso, estas células adotaram seu tradicional destino
hematopoiético
67;68
.
Experimentalmente, em um estudo utilizando uma sub-população de células Lin
-
ckit
+
, isoladas a partir de corações de ratas adultas, in vitro e também in vivo, exibiram
propriedades de células-tronco cardíacas. As células apresentaram capacidade de auto-
renovação, sendo clonáveis e multipotentes e que puderam se diferenciar não somente
em cardiomiócitos, mas também em células endoteliais e músculo liso. Embora estas
células não apresentassem contração espontânea em cultivo, ao serem implantadas no
miocárdio infartado melhoraram a função cardíaca mostrando que o miocárdio possui
células-tronco residentes com capacidade regenerativa quando estimuladas
69
Os mioblastos esqueléticos, ou células satélites são células progenitoras que
normalmente permanecem em um estado quiescente, são capazes de serem cultivadas in
vitro, e se diferenciar conservando propriedades do músculo esquelético quando
transplantadas em regiões infartadas
70
.
Num estudo clínico realizado em cinco pacientes com cardiomiopatia isquêmica e
listados para transplante cardíaco, que receberam o transplante de mioblastos
esqueléticos, houve diferenciação das células musculares esqueléticas em fibras
musculares maduras e um pequeno aumento na formação de vasos em três pacientes
71
.
O transplante celular utilizando mioblastos no coração, também conhecido como
cardioplastia celular tem sido estudado como uma alternativa terapêutica para
cardiopatias isquêmicas e não isquêmicas
72
. Como terapia para o tratamento do infarto, a
cardioplastia celular tem como objetivo repovoar a cicatriz necrótica e as zonas
adjacentes ao infarto com células potencialmente contráteis, capazes de substituir os
cardiomiócitos mortos, restaurando a função das áreas acinéticas
73
.
18
Por fim, há na literatura intenso interesse em avaliar desfechos clínicos com o uso
de transplante autólogo de células-tronco de medula óssea. Alguns são exemplos
clássicos de algumas estratégias utilizadas
74;75
. O TOPCARE-AMI (Transplantation of
progenitor cells and regeneration enhancement in acute myocardial infarction), um estudo
de fase I incluiu 54 pacientes com IAM tratados com stent intracoronário que tiveram um
seguimento de quatro meses enquanto trinta e sete foram avaliados após um seguimento
de doze meses. Os pacientes receberam infusão intracoronária de células de medula
óssea vinte e quatro horas após o IAM. Os grupos foram divididos por tempo de
seguimento. Houve um aumento na fração de ejeção de 50±10%, antes do procedimento,
para 58,3±10% (p<0.001). Uma limitação deste estudo foi a falta de um grupo controle.
Foi demonstrado com isso, apenas a viabilidade e a segurança deste tipo de
procedimento
76;77
.
Num estudo observacional realizado em dez pacientes com histórico de IAM e
fração de ejeção <35%, foram administrados mioblástos esqueléticos por via
intramiocárdica. Após um seguimento de dez meses foi observado um aumento da fração
de ejeção de 23% para 32% (p<0.03) e uma melhora do encurtamento funcional. Apesar
destes resultados, a classe funcional passou de 2,7, antes do transplante, para 1,6, após
o tratamento. Além disso, quatro pacientes apresentaram taquicardia ventricular
sustentada e foi necessário o implante de um cardio-desfibrilador
78
.
Em outro estudo, de fase II, foi utilizado um estimulador de mobilização de células-
tronco, a partir de sangue periférico (Granulocyne-colony-stimulating factor) G-CSF,
infundido por infusão intracoronária. Foram arrolados 27 pacientes com IAM que
receberam stent coronário. Após seis meses de seguimento, o grupo de pacientes, que
recebeu terapia celular, obteve melhora da fração de ejeção de 48,7±8,3%, antes do
tratamento, para 55,1±7,4% (p<0.005), após os seis meses de seguimento. Um resultado
19
inesperado deste estudo foi a alta taxa de reestenose nos pacientes que receberam o
tratamento com terapia celular
79
.
O estudo BOOST (Bone Marrow Transfer to Enhance ST-Elevation Infarct
Regeneration) foi conduzido com objetivo de avaliar o efeito da transferência de autóloga
intracoronária de células da medula óssea sobre a fração de ejeção. Foi um estudo
controlado, randomizado que envolveu 60 pacientes que haviam sofrido infarto agudo
com elevação ST sustentada submetidos à reperfusão coronária percutânea. A fração de
ejeção global avaliada por ressonância magnética foi significativamente aumentada nos
pacientes tratados com transplante de medula óssea
de 50±10% antes do tratamento para 56.7±12.5% versus controles, 51.3±9.3% para
52.0±12.4%, avaliados após seis meses de seguimento (P=0.0026). Apesar dos
resultados revelarem melhora na função sistólica em seis meses, este aumento da fração
de ejeção global não foi sustentado até 18 meses de seguimento destes pacientes. O
estudo mostrou uma melhora na função cardíaca, com pouca viabilidade, a longo prazo,
não se traduzindo em permanente reparação do miocárdio
80
.
O uso de terapia celular merece ainda algumas considerações críticas da sua
aplicabilidade e segurança. Uma das limitações do uso das células-tronco de medula
óssea é a possibilidade de que estas possam diferenciar-se em outros tipos celulares,
como os fibroblastos, ao serem implantadas um uma zona onde esteja se desenvolvendo
uma cicatriz fibrótica
81
, contribuindo para o risco de eventuais arritmias ventriculares.
Neste sentido, a utilização mais precoce destas células parece ser uma alternativa mais
atraente nos tratamentos com terapia celular
56
.
Embora estas informações sejam pouco encorajadoras, abre-se a possibilidade de
que estas células tenham um papel muito mais complexo, de ação parácrina, junto ao
tecido cardíaco lesado e, outras abordagens devam ser avaliadas como respostas
importantes no entendimento dos efeitos da terapia celular
82
.
20
Segundo Menasché, a hipótese inicial de que as células-tronco adultas poderiam
ter efeito regenerativo do tecido miocárdio levou a um habitual enfoque na fração de
ejeção como o principal parâmetro a ser analisado. Hoje, é cada vez mais aceito e
reconhecido que as células-tronco adultas, em contraste com as células embrionárias tem
pouca ou nenhuma capacidade regenerativa e que os seus efeitos benéficos envolvam
ações parácrinas sobre o tecido hospedeiro
82
.
Reparo Cardíaco por Efeitos Parácrinos
Figura 2 - O transplante de células progenitoras pode ter um impacto favorável no desempenho contrátil e
perfusão tecidual, promovendo a vascularização e formação de miócitos. Dependendo do tipo, local e meio
de administração das células-tronco, sua contribuição relativa na incorporação (transdiferenciação e/ou
fusão) versus efeitos parácrinos podem variar. O número de células progenitoras e sua capacidade
funcional são diretamente influenciadas pela idade, sexo, fatores de risco cardiovasculares e doenças
subjacentes
83
.
Em suma, parece claro que houve uma migração talvez um pouco precoce para
estudos clínicos com terapia celular em cardiopatias, ainda sem que houvesse um mais
21
completo e abrangente entendimento dos diversos mecanismos envolvidos nos seus
efeitos benéficos e além de explorar potenciais efeitos indesejáveis associados, que
poderiam surgir ao longo do tempo.
Os estudos experimentais que compõem este documento procuram dissecar com
maior detalhamento aspectos de estresse oxidativo, processo inflamatório e morte celular
muito precocemente pós-IAM e estudar o efeito do uso de terapia celular nestes
processos e sua repercussão em parâmetros ecocardiográficos.
22
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33
JUSTIFICATIVAS
I - Dentre os fatores que podem interferir com o processo de hipertrofia ventricular
pós-infarto e conseqüente disfunção cardíaca, as interações entre o processo inflamatório
e de estresse oxidativo, progredindo para um quadro de IC, poderiam ser melhor
exploradas em janelas temporais mais precoces após o insulto isquêmico.
II - O uso de células-tronco adjuvante a tratamentos já otimizados é, certamente,
uma abordagem terapêutica a ser considerada, porém, sua melhor utilização depende de
um maior conhecimento dos mecanismos de interação dos processos que permeiam seu
desempenho no tecido miocárdico, seja por proliferação, fusão, transdiferenciação ou
efeitos parácrinos.
34
HIPOTESES
I - A janela temporal de 48 horas pós-infarto permitiria avaliar o papel do estresse
oxidativo na gênese do remodelamento ventricular, sendo o H
2
O
2
um modulador dos
processos que alteram a função cardíaca precocemente.
II - Neste contexto, o tratamento com células-tronco de medula óssea seria capaz
de modular a função cardíaca, podendo retardar o início do remodelamento ventricular,
através dos seus efeitos parácrinos sobre a hipertrofia ventricular e o processo
inflamatório.
35
OBJETIVO GERAL
Avaliar o perfil do estresse oxidativo e sua associação com a função ventricular em 48
horas após o insulto isquêmico, num modelo experimental de IAM, analisando sua
correlação com os efeitos parácrinos do tratamento com terapia celular com células da
medula óssea.
Objetivos específicos
Artigo 01:
Avaliar precocemente (48 horas) pós-IAM o perfil de estresse oxidativo e sua associação
com parâmetros ecocardiográficos de função ventricular em ratos Wistar.
Artigo 02:
Analisar os efeitos do uso de terapia celular utilizando células da medula óssea em
modelo de IAM, 48 horas após a lesão, avaliando parâmetros ecocardiográficos de função
ventricular, perfil oxidativo, inflamatório e apoptótico no miocárdio.
36
Artigo 1
Early oxidative stress profile and
left ventricular function evaluation by
echocardiography in rats at 48h post-experimental infarction
Avaliação Precoce do Perfil de Estresse Oxidativo e Função Ventricular por
Ecocardiografia em Ratos 48 horas Pós-Infarto Experimental
37
Early oxidative stress profile and
left ventricular function evaluation by
echocardiography in rats at 48h post-experimental infarction
Cardiovascular Laboratory – Research Centre,
Hospital de Clínicas de Porto Alegre
Porto Alegre - RS, BRAZIL
Abstract
Background: Events occurring subsequent to acute myocardial infarction (AMI) are partially determinants of
the cardiac damage extent later on. The role of redox balance in the post-ischemic cardiac tissue may be
critical in this process.
Objectives: To assess cardiac function and its correlation with redox balance in cardiac tissue 48 hours post-
experimental AMI.
Methods: Male Wistar rats, 8-week-old (n=6/group), weighing 229±24g, were randomized in two groups:
Sham-operated (S) and AMI. AMI was produced in rats via ligation of the left coronary artery. Cardiac function
parameters were evaluated by echocardiography 48h later. Oxidative profile was studied by measuring
antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)
and peroxiredoxine 6 (Prx-6). Oxidative damage was quantified by protein oxidation (carbonyls), lipid
peroxidation (chemiluminescence - CL), reduced (GSH) and oxidized (GSSG) glutathione ratio and hydrogen
peroxide (H
2
O
2
) concentration by spectrophotometer.
Results: Ejection fraction (EF) was lower in the infarct group: AMI (51±5%) vs. S (77±6%) (p=0.0001).
Hydrogen peroxide values (H
2
O
2
) (nmol/mg protein) was diminished 48 hours post-AMI: AMI (0.022 ± 0.005)
vs. S (0.032 ± 0.008) (p=0.024). We found a correlation between reduced/oxidized glutathione ratio
(GSH/GSSG) and EF (r=0.79; p=0.009) at 48 hours post-MI. CL and carbonyls were not different between
groups.
Conclusion: These data suggest that the loss of myocardial function and impaired redox balance may be
associated with the activation of mechanisms that trigger the process of ventricular remodeling in heart failure.
Moreover, we speculate that countervailing survival mechanisms act against H
2
O
2
in order to maintain cardiac
function in this early temporal window following AMI.
Key words: heart failure, redox balance, hydrogen peroxide.
Introduction
Subsequent to acute myocardial
infarction (AMI), the heart undergoes a
remodeling process that includes hypertrophy of
surviving myocytes and hyperplasia of non-
myocytes [1;2].
During left ventricular (LV) remodeling,
the expression of several growth factors and
cytokines is activated [3;4]. ROS (reactive
oxygen species) are involved in modulating the
activity of specific transcription factors, such as
NF-kB [5;6], which are responsible for the
transcription of inflammatory cytokines. Besides
modulating inflammatory processes, ROS have
been shown to activate matrix
metalloproteinases (MMPs) in cardiac
fibroblasts [7]. In a model of MMP-2 knockout
mice, a significant improvement in post-AMI
survival was observed [8]. Thus, because
MMPs can be activated by ROS, a proposed
mechanism for LV remodeling is the activation
of MMPs secondary to increased ROS
production following injury [9].
An adequate oxygen supply is
necessary to sustain cell viability and oxygen
Tavares, A.M.V. / Article 1
38
metabolism is also central to the generation of
ROS, which participate as benevolent
molecules in many cellular functions, such as
cell signaling process, but can also induce
irreversible cellular damage [10].
Reactive oxygen species can oxidize
membrane phospholipids, proteins, and DNA
and are implicated in a wide range of
pathological conditions. Toxic effects of ROS
can be abrogated by enzymatic scavengers, as
well as by non-enzymatic antioxidants [11]. For
instance, H
2
O
2
is neutralized by the enzymes
glutathione peroxidase (GPx) and catalase
(CAT).
Glutathione peroxidase, a selenium-
containing enzyme that catalyzes removal of
H
2
O
2
through oxidation of reduced glutathione
(GSH), and is recycled from oxidized
glutathione (GSSG) by glutathione reductase
(GRed) [12]. Similar to GPx, thioredoxin (Trx)
complements the GSH system by protecting
against ROS toxicity. GSH and Trx both
function in the reduction of peroxides through
the action of multiple GSH peroxidases and Trx
peroxidases (peroxiredoxins), respectively [13].
The peroxiredoxin (Prx) superfamily,
expressed in all the vital organs, including the
heart [14], possesses H
2
O
2
scavenging
activities and also removes hydroperoxides
[15]. Thus, the regulation of redox status is
essential for maintaining cellular homeostasis,
and can be compromised by injurious events
such as cardiac ischemia. Increased oxidative
stress results in a phenotype characterized by
hypertrophy and apoptosis in isolated cardiac
myocytes. Although these responses initially
serve as an adaptative process, ultimately are
often accompanied by depressed contractile
function [16].
The importance of redox status in the
development of heart failure subsequent to MI
is evident from the association that both
increased myocardial oxidative stress, as well
as an antioxidant deficit are correlated with
cardiac dysfunction at different stages of
failure [17]. Although many studies have
explored the mechanisms of ventricular
remodeling, relatively little is known regarding
the role of redox balance in cardiac cells post-
ischemia during the very early stages of heart
failure.
In this study, we evaluated multiple
parameters of cardiac function via
echocardiography and its correlation with
oxidative stress and antioxidant reserve in
cardiac tissue examined 48 hours post-
experimental MI.
Material and Methods
Animals and Groups
We studied male Wistar rats at the
age of 8 weeks (n=6/group), weighing
229±24g. Animals were maintained in
compliance with the “Principles of Laboratory
Animal Care” formulated by the National
Institutes of Health (publication No. 96-23,
revised, 1996).
The study was approved by the ethics
and research committee of Hospital de
Clínicas de Porto Alegre. Animals were
randomized in two groups: (a) sham-operated
(S), with fictitious myocardial infarction
surgery, and (b) acute myocardial infarction
(AMI), with surgical procedure to induce
myocardial infarction. All animals were
maintained at temperatures ranging from
20
o
C to 25
o
C and in light/dark cycles of 12
hours and fed ad libitum with rat chow.
Myocardial Infarction Surgery
Myocardial infarction was induced
according to a previously described procedure
[18] and adapted in our laboratory. Briefly,
animals were placed in dorsal decubitus
position and anesthetized with xylazine
(0.67mg/Kg i.p.) and ketamine (0.33 mg/Kg
i.p.). Following orotracheal intubation, animals
were submitted to mechanical ventilation
(Harvard ventilator, Model 683). Thoracotomy
Tavares, A.M.V. / Article 1
39
was performed without exteriorization of the
heart. The left anterior descending coronary
artery was occluded with a 6-0 mononylon.
The thoracic cavity was closed with a 5-0
mononylon thread.
Echocardiogram
Animals underwent echocardiography
prior to surgical interventions (baseline) and
forty-eight hours following the surgical
procedures. The EnVisor Philips system
(Andover, MA, USA) was used, with a 12-
13MHz transducer, at 2cm depth and
fundamental and harmonic imaging.
Left ventricular dimensions – The end-
diastolic and end-systolic transverse areas
(cm
2
) were obtained by tracing the
endocardial border at three levels: basal (at
the tip of the mitral valve leaflets), middle (at
the papillary muscle level) and apical (distal
from the papillary muscle but before the final
curve cavity) [19]. The end-diastolic and end-
systolic diameters (cm) were also measured
in the three planes using the M-Mode. The
final value for each animal was taken as the
mean of all three planes.
Myocardial infarction size – On each
echocardiographic transverse plane, the arc
corresponding to the infarcted segments
(systolic movement akinesis and/or
hypokinesis - AHR) and the total endocardial
perimeter (EP) were measured at end-
diastole. Infarction size (IS) was estimated
[20] as % IS by using the following formula:
IS= (AHR/EP) x 100.
Left Ventricular systolic function - Fractional
area change (FAC) was calculated as follows:
(FAC = diastolic area – systolic area / diastolic
area). LV EF was calculated as: (End-diastolic
volume – End-systolic volume / End-diastolic
volume) x 100; end-diastolic end-systolic
cavity volumes were calculated using
Simpson´s rule [21]. Ejected volume (EV) was
determined by equation: EV = (πR)
2
x VTI,
where VTI is velocity time integral of the trace
of Doppler flow profile, R is radius cross
section of aorta artery and π value is 3.1415.
Cardiac output (CO) was obtained by the
equation: CO = EV x Heart Rate (HR). LV
fractional shortening (LVFS) was obtained by
the equation: LVFS = Dd – Sd/Dd x 100
(diastolic diameter – Dd; systolic diameter –
Sd);
Left Ventricular diastolic function – Left
ventricular diastolic function was assessed by
E/A ratio, which was determined by dividing
the peak velocity of the E wave by peak
velocity of the A wave of the mitral diastolic
flow profile.
Myocardial performance index (MPI) - MPI
was obtained via the trace of Doppler flow
profile as expressed by the following
equation: MPI = (MFT - ET)/ET (MF - mitral
flow time; ET -ejection time of aortic artery)
[22].
Left Ventricular Mass – Mass (in grams) was
calculated according to the equation
established by the American Society of
Echocardiography M(g)=1.04[(Dd + AWDT +
PWDT) – Dd
3
]
3
(anterior wall diastolic
thickness – AWDT; posterior wall diastolic
thickness – PWDT) [23;24].
Final values for all calculations for
each animal were based on three transverse
planes. Data were recorded in CD for later
review and off-line analysis. Subsequent to
echocardiography assessment, the animals
were killed by cervical dislocation.
Tissue preparation
After animals were killed, hearts were
rapidly excised, weighed, and homogenized
(1.15% w/v KCl and phenyl methyl sulphonyl
fluoride PMSF 20 mmol/L) in Ultra-Turrax.
The resulting suspension was centrifuged at
600 g for 10 min at 0 - 4°C to remove nuclei
and cell debris [25] and supernatant was used
for oxidative stress measurements.
Immediately following killing, left ventricle
samples were removed and frozen at -80°C
Tavares, A.M.V. / Article 1
40
for the evaluation of glutathione content and
protein expression.
Determination of Lipid peroxidation
Tert-butyl-hydroperoxide-initiated
chemiluminescence –
Chemiluminescence (CL) was measured in a
liquid scintillation counter in the out-of-
coincidence mode (LKB Rack Beta Liquid
Scintillation Spectrometer 1215, LKB –
Produkter AB, Sweden). Homogenates were
placed in low-potassium vials at a protein
concentration of 0.5-1.0 mg/mL in a reaction
medium consisting of 120 mmol/L KCl and
30mmol/L phosphate buffer (pH=7.4).
Measurements were started by the addition of
3 mmol/L tert-butyl hydroperoxide and data
expressed as counts per second per milligram
of protein (cps/mg protein) [26].
Carbonyl Assay
Tissue samples were incubated with
2,4 dinitrophenylhydrazine (DNPH 10 mol/L)
in 2.5 mol/L HCl solution for 1 h at room
temperature, in the dark. Samples were
vortexed every 15 min. Subsequently, in tube
samples, 20% TCA (w/v) solution was added,
left in ice for 10 min and centrifuged for 5 min
at 1000 g to collect protein precipitates.
Another wash was performed with 10% TCA
(w/v). The pellet was washed three times with
ethanolethyl acetate (1:1) (v/v). The final
precipitates were dissolved in 6 mol/L
guanidine hydrochloride solution, incubated
for 10 min at 37°C, and read at 360 nm [27].
Determination of antioxidant enzyme activities
Catalase (CAT) – CAT activity was
determined by following the decrease in
hydrogen peroxide (H
2
O
2
) absorbance at 240
nm. CAT activity was expressed as
nanomoles of H
2
O
2
reduced per minute per
milligram of protein [28].
Superoxide dismutase (SOD) – SOD activity,
expressed as units per milligram of protein,
was based on the inhibition of superoxide
radical reaction with pyrogallol [29].
Glutathione peroxidase (GPx) – GPx activity
expressed as nanomoles of peroxide/
hydroperoxide reduced per minute per
milligram of protein, was based on the
consumption of NADPH a 480 nm [30].
Determination of oxidized and reduced
glutathione concentration
To determine oxidized (GSSG) and
reduced (GSH) glutathione concentration,
tissue was deproteinized with 2 mol/L
perchloric acid, centrifuged for 10 min at 1000
g and supernatant was neutralized with 2
mol/L potassium hydroxide. The reaction
medium contained 100 mmol/L phosphate
buffer (pH 7.2), 2 mmol/L NADPH, 0.2 U/mL
glutathione reductase and 70 µmol/L 5,5’
dithiobis (2-nitrobenzoic acid). To determine
reduced glutathione, the supernatant was
neutralized with 2 mol/L potassium hydroxide,
reacted with 70 µmol/L 5,5’ dithiobis (2-nitro
benzoic acid), and read at 420 nm [31].
Determination of hydrogen peroxide (H
2
O
2
)
The assay was based on horseradish
peroxidase (HRPO)-mediated oxidation of
phenol red by H
2
O
2.
Ventricle slices were
incubated for 30 min. at 37ºC in 10 mmol/L
phosphate buffer (NaCl 140 mmol/L and
dextrose 5 mmol/L). Supernatants were
transferred to tubes with 0.28 mmol/L phenol
red and 8.5 U/mL HRPO. After a 5 min
incubation, 1 mol/L NaOH was added and
was read at 610 nm. The results were
expressed in nanomoles H
2
O
2
/ g tissue [32].
Determination of nitric oxide (NO) metabolites
(Nitrates)
Nitrates were determined as total
nitrates (initial nitrite plus nitrite reduced from
nitrate) after its reduction using nitrate
reductase, from Aspergillus species in the
Tavares, A.M.V. / Article 1
41
presence of NADPH. A standard curve was
established with a set of serial dilutions (10
-8
–
10
-3
mol/L) of sodium nitrite. Results were
expressed as mmol/mg protein [33].
Determination of protein concentration
Protein was measured by the method
of Lowry [34], using bovine serum albumin as
standard.
Western Blot Analysis
Tissue homogenization, electrophore-
sis, and protein transference were performed
as described elsewhere [35;36]. Membranes
were processed for immunodetection using
rabbit anti-CAT polyclonal antibody, sheep
anti-Cu/Zn SOD polyclonal antibody, and
rabbit anti-Prx-6 as primary antibodies (Santa
Cruz Biotechnology, Santa Cruz, CA).
The bound primary antibodies were
detected using rabbit anti-sheep or goat anti-
rabbit horseradish peroxidase-conjugate
secondary antibodies and membranes were
exposed for chemiluminescence.
Autoradiographs were quantitatively analyzed
with an image densitometer (Image Master
DS CI, Amersham Biosciences Europe, IT).
Molecular weights of the bands were
determined by referring to a standard
molecular weight marker (RPN 800 rainbow
full range Bio-Rad, CA, USA). Results from
each membrane were normalized by the
Ponceau red method [37].
Statistical analysis
Data are presented as mean ± SD and
were compared by Student’s t-test. Simple
linear regression analysis (Pearson
correlation coefficient) was used to test
associations between continuous variables.
Tests with P<0.05 were considered
statistically significant. Data were analyzed
using the Sigma Plot 11.0 program.
Results
Heart rate, infarction size and mortality
The mean HR after sedation during
echocardiography was not significantly
different between groups (243±12 bpm for the
S group and 222±22 bpm for the AMI group).
Area of infarction (% of circumference) was
46.91±8.55% in the AMI group. Mortality
occurring during or immediately after surgical
procedure was around 10%.
Cardiac function
Systolic function - SD, EF, FAC, and LVFS
were lower in rats with AMI than in the control
animals (p<0.0001) [Table 1] [Figure 1]. Both
ET and EV were (p=0.0184 and p=0.0498,
respectively) decreased in AMI group,
although CO was not significantly different
between groups [Table 1].
Diastolic function - Velocity of the E wave in
the curve of diastolic mitral flow was not found
to be different between groups, but the
velocity of the A wave was lower in AMI group
(p=0.0038). E/A ratio was increased in the
AMI group (p=0.0048) [Figure 2]. In addition,
a negative correlation between E/A ratio and
GSH/GSSG ratio (r = -0.71; p = 0.0321) was
found [Figure 4A]. The mitral diastolic flow
deceleration time of E wave as well as mitral
time flow was also not different between
groups, [Table 1].
Myocardial performance index - MPI
increased in the AMI group (0.24 ± 0.06 vs.
0.59 ± 0.14) (p = 0.0009) [Table 1]. A negative
correlation was also found between MPI and
EF (r = -0.64; p = 0.036).
Left Ventricular mass -The AMI group showed
loss of LV mass compared to control (p =
0.0146) [Table 1].
Tavares, A.M.V. / Article 1
42
Table 1 – Values of functional variables obtained by echocardiographic analysis
Variable S AMI p Value
SD (cm) 0.27 ± 0.08 0.50 ± 0.03 0.0001
DD (cm) 0.64 ± 0.07 0.72 ± 0.05 0.0599
EF (%) 77 ± 7 51 ± 5 0.0001
FAC (cm) 0.71 ± 0.06 0.47 ±0.06 0.0001
CO (mL/min) 87 ± 17 72 ± 28 0.3262
LVFS (%) 59 ± 10 30 ± 4 0.0001
E (m/sec) 1.24 ± 0.11 1.43 ± 0.24 0.1419
A (m/sec) 0.70 ± 0.13 0.45 ± 0.09 0.0038
E/A 1.78 ± 0.23 3.24 ± 0.84 0.0048
MDDT (msec) 46.2 ± 9.26 45.8 ± 6.59 0.9405
TF (msec) 125.2 ± 26.3 124.3 ±15.7 0.9474
ET (msec) 108.6 ± 9.6 90.8 ±10.7 0.0184
EV (mL) 0.366±0.05 0.310 ± 0.1 0.0498
MPI 0.24 ± 0.06 0.59 ± 0.14 0.0009
LV Mass (g) 0.36 ± 0.06 0.28 ± 0.02 0.0146
Groups
Abbreviations: - S, Sham; SD, Systolic Diameter; DD, Diastolic Diameter; EF, Ejection Fraction; FAC, Fractional Area
Change; CO, Cardiac Output; LVFS, Left Ventricular Fractional Shortening; E, E wave; A, A wave; E/A, E/A ratio; MDDT,
Mitral Diastolic Flow Deceleration Time; TF, Time Flow; ET, Ejection Time; EV, Ejected Volume; MPI, Myocardial
Performance Index. Data expressed as mean ± SD; p<0.05 vs. S group (Student’s t-Test).
Tavares, A.M.V. / Article 1
43
Figure 1 - Illustrative example of an M mode echocardiogram image of the left ventricle (LV) of the same animal before
(A) and after (B) myocardial infarction (AMI group). Note the increase of the left ventricular cavity and loss of LV free
wall contractility.
Figure 2 - Illustrative example of mitral inflow velocity profile determined by pulsed wave Doppler before (C) and after (D)
acute myocardial infarction (AMI group). Note decreased A wave after acute myocardial infarction (D).
Oxidation products
Protein oxidation (carbonyls) and lipid
peroxidation (CL) were not different in the AMI
group compared to controls [Table 2].
Antioxidant enzyme activities and expression
Myocardial SOD and CAT activities
were not different in the AMI group compared
with controls. Similarly, the protein expression
of SOD and CAT in the cardiac tissue was not
different between the two groups [Table 2;
Figure 3]. GPx activity was depressed by
about 44% in the AMI group in relation to
controls (p=0.0025). Conversely, Prx-6 protein
expression was increased in the AMI group
compared to control S (p<0.0003). A
significant positive correlation between GPx
and EF (r = 0.71; p = 0.0162) was also
observed.
GSH/GSSG - reduced to oxidized glutathione
ratio
GSH/GSSG was assessed as being
significantly (p=0.0226) depressed by about
44% in the AMI group as compared to
controls [Table 2]. A positive correlation
existed between GSH/GSSG and EF (r =
0.79; p = 0.0097) [Figure 4C].
Hydrogen peroxide (H
2
O
2
)
Myocardial hydrogen peroxide
concentration was lower (p=0.0244) in the
AMI group as compared to controls [Table 2].
Nitric oxide (NO) metabolites (Nitrates)
NO metabolite levels in the myocardial
tissue were not altered in the AMI group as
compared to controls [Table2].
E
A
C
E
A
D
E
A
C
E
A
E
A
C
E
A
D
E
A
E
A
D
Tavares, A.M.V. / Article 1
44
Table 2
– Oxidative stress and antioxidant enzymes parameters
Variable S AMI p Value
CL (cps/mg prot) 5526 ± 1228 6219 ± 969 0.3250
Carbonyl (nmol/mg prot) 2.23 ± 0.54 1.7 ± 0.41 0.0826
SOD activity (U/mg prot) 5.13 ± 2 5.22 ± 1.4 0.7561
CAT activity (µmol/mg prot) 40.57 ± 8.6 34.06 ± 9.01 0.2375
GPx (nmol/mg prot) 46.88 ± 9.0 26.32 ± 7.53 0.0025
GSH/GSSG ratio 14.61 ± 3.36 8.21 ± 3.81 0.0226
H
2
O
2
(nmol/mg prot)
0.032 ± 0.008 0.022 ± 0.005 0.0244
Nitrates (mmol/mg protein) 0.0448 ± 0.003 0.0429 ± 0.007 0.5738
Groups
Abbreviations – S, Sham; CL, chemiluminescence;, superoxide dismutase (SOD), catalase (CAT), and glutathione
peroxidase (GPx), reduced (GSH) and oxidized (GSSG) glutathione ratio, hydrogen peroxide (H
2
O
2
) and NO metabolites
(Nitrates); Data expressed as mean ± SD; p<0.05 vs. S group (Student’s t-Test).
0
60
120
180
240
300
360
SOD CAT Prx-6
Densitity Normalized by
Ponceau Red
S
AMI
*
0
60
120
180
240
300
360
SOD CAT Prx-6
Densitity Normalized by
Ponceau Red
S
AMI
*
Figure 3 – Graph of Western blot analysis in cardiac homogenates using Cu/Zn SOD antibody, CAT antibody and Prx-6
antibody. p<0.05 as statistically significant. Note illustrative bands of blots on top part of graphs.
Abbreviations: S, Sham; AMI, acute myocardial infarction; SOD, superoxide dismutase; CAT, catalase; Prx-6,
peroxiredoxin-6.
Tavares, A.M.V. / Article 1
45
Figure 4 – A) Graph showing correlation between the E/A ratio and GSH/GSSG ratio.
Figure 4 – B) Graph showing correlation between ejection fraction and glutathione
peroxidase activity.
Figure 4 - C) Graph showing correlation between ejection fraction and GSH/GSSG ratio.
Discussion
In the present study, we found an
early (48 hours post-MI) depression of cardiac
function as reduced ejection fraction,
fractional shortening and LV mass loss were
evident; however no significant alteration in
cardiac output was observed. Also, from the
analyses of redox status at this time point, we
found that the steady-state myocardial H
2
O
2
concentration was diminished, the activity of
the antioxidant GPx was lower, and the
r = - 0.71
p = 0.032
0
4
8
12
16
20
0 1 2 3 4
E/A Ratio
GSH/GSSG Ratio
r = 0.71
p = 0.016
0
20
40
60
80
100
0 20 40 60 80 100
Ejection Fraction (%)
Glutathione Peroxidase
(nmol/mg prot)
r = 0.79
p = 0.009
0
4
8
12
16
20
0 20 40 60 80 100
Ejection Fraction (%)
GSH/GSSG Ratio
Tavares, A.M.V. / Article 1
46
oxidative stress index, GSH/GSSG ratio, was
decreased.
This apparent balance of various
redox state contributors present in a
decompensated cardiac scenario was also
evident in functional readouts, where cardiac
flow assessment, E/A ratio and ET were
depressed in the AMI group, while TF and
MDDT did not change. Moreover, MPI was
found to be markedly increased and was
significantly negatively correlated with EF.
The clear reduction of LVFS characterizes an
impairment of ventricular contractility
associated with an expressive LV chamber
enlargement, as can be seen by the
enhanced systolic diameter.
Although diastolic diameter was not
changed, an evident loss of systolic function
was witnessed. Additionally, it was observed
that although the LV rapid filling was normal,
the slow filling was depressed and the EV
was minor, showing that diastolic function was
also depressed in the AMI group at this time
point. A strong correlation between diastolic
dysfunction (higher E/A ratio) and oxidative
stress was also demonstrated (Figure 4A).
It has previously been shown that
ROS generated in the immediate post-AMI
period can also function to modulate cardiac
inotropism, inducing cardiac hypertrophy and
apoptosis. These events reduce calcium
myofilament sensitivity and cardiac
contractility, contributing to the development
of heart failure [6].
While we did not find increased
myocardial oxidative damage, as observed by
protein oxidation and lipid peroxidation data,
we did observe diminished antioxidant
defenses through decreased GPx activity at
48 hours post-MI. GPx is an important
enzyme that catalyzes the reduction of H
2
O
2
and hydroperoxides, preventing the formation
of other more toxic radicals, such as
.
OH [38].
Furthermore, we found a positive
correlation between GPx and EF, suggesting
a strong association between cardiac function
and the activity of this enzyme. These data
corroborate previous findings which
demonstrated that GPx overexpression
inhibits the development of LV remodeling
and failure after AMI, which could contribute
to the improved survival [38], attenuation of
diastolic dysfunction, myocyte hypertrophy,
and interstitial fibrosis in diabetes [39].
We also analyzed myocardial CAT
activity and protein expression, but found no
significant differences between groups.
Studies suggest that CAT activity and
expression have minor relevance to GPx in
cardiac tissue [11]. We also did not note
important differences in myocardial SOD
activity or protein expression between groups,
leading us to believe that O
2
.-
concentration is
not significantly altered in our model.
Tissue production of O
2
.-
and,
consequently, low levels of H
2
O
2
, are
maintained by the basal activity of endothelial
NADPH oxidases and by mitochondrial
electron chain leakage, and is necessary for
endothelial growth and proliferation [40].
Under pathological conditions such as AMI,
agonist-induced activation of NADPH oxidase
and the subsequent activation of xanthine
oxidase, as well as the uncoupling of eNOS,
result in the production of large quantities of
H
2
O
2
, leading to cardiac dysfunction. For
instance, when H
2
O
2
diffuses to adjacent
muscle tissue, it can induce hypertrophy [41].
Interestingly, myocardial concentration
of H
2
O
2
was diminished at 48 hours post-AMI
in our study. We believe that this finding
suggests countervailing survival mechanisms
acting against H
2
O
2
in order to maintain
cardiac function in this temporal window
following MI. Among these mechanisms are
novel antioxidant proteins designated as
peroxiredoxins (Prxs). Among six known
mammalian Prxs, Prx-1,-2, -3, and -4 require
the small redox protein thioredoxin (Trx) as an
electron donor to remove H
2
O
2
. Prx-3 is
Tavares, A.M.V. / Article 1
47
especially efficient as an antioxidant, perhaps
because it can utilize lipid peroxides as well
as H
2
O
2
as substrates. This may also help to
explain the minimization of lipid damage
evident in our present CL data.
Moreover, Prx-3 overexpression was
also associated with heart protection,
reducing LV remodeling and heart failure
progression in mice following AMI [42].
Importantly, Prx-3 functions not only to
remove H
2
O
2
formed after SOD-catalyzed
dismutation reactions, but also detoxifies
peroxynitrite [43].
This may also help to explain why we
did not observe differences in nitrate level
between groups. A key role of Prx-6 in
ischemic injury has also been suggested. In a
study of Prx-6
-/-
mice subjected to 30 min of
global ischemia followed by 120 min of
reperfusion at normothermia, Prx6
-/-
mice
exhibited reduced post-ischemic ventricular
recovery, increased infarct size, and
increased cardiomyocyte apoptosis as
compared with wild-type controls [44].
In our findings, we observed that Prx-6
was significantly increased in the AMI group
compared with the control group,
demonstrating that in an ischemic
environment, during this time point, Prx-6 can
also act as a reperfusion-independent H
2
O
2
scavenger.
In the present study, we also observed
a marked decrease in the GSH/GSSG ratio,
possibly due to a depletion of GSH in cardiac
tissue. Moreover, this redox imbalance was
positively correlated with EF. In addition, the
observed decrease in GPx activity may also
result from decreased GSH concentration at
later time points [45].
In summary, our data suggest that this
period (48 hours post-AMI) represents a point
at which compensated cardiac function begins
to shift toward the progression to heart failure.
Our results show a strong correlation between
oxidative stress and cardiac function and that
H
2
O
2
may play an important role in this
potential transitional phase.
Conclusions
It is widely accepted that events
transpiring in the period immediately following
cardiac injuries, such as those imparted by
AMI, have important implications for ultimate
functional capacity and potential for
progression to heart failure.
Our evaluation of cardiac function and
redox status at the relatively early period of 48
hours post-MI suggest that it may represent a
point of critical transition from a compensated
to decompensated state. In particular, our
data show a depressed GPx and GSH/GSSG
ratio, indicating redox imbalance, which may
pave the way for activation of inflammatory
and apoptotic responses, which represent key
next steps in the process of ventricular
remodeling.
Our observations of differential H
2
O
2
concentration lead us to believe that the
above-mentioned transition may be
dependent on, or otherwise influenced by
H
2
O
2
levels. In this study, the low H
2
O
2
concentrations noted may act as a ‘sensor’ for
survival pathway activation within this
timeframe following AMI. This ‘sensor’ could
be regulated by Prx-6 in this time point.
To conclude, this temporal window
seems to be an important period in which we
may better exploit the role and contribution of
oxidative stress as a trigger for signaling in
the processes leading to heart failure.
Acknowledgements
We would like to thank Ms. Tania R. G.
Fernandes for technical assistance and to Andreia
C. Taffarel (Veterinary Medicine), Gabriela
Nicolaidis (Medicine) and Rafael Dall’Alba
(Biology), undergraduate students of UFRGS, for
their generous contribution. This work was
supported by HCPA-FIPE and CNPq grants.
Tavares, A.M.V. / Article 1
48
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Tavares, A.M.V. / Article 2
52
Artigo 2
Paracrine Effects of Early Bone Marrow Cells Treatment in Experimental
Myocardial Infarction in Rats: Tissue Evaluation of Inflammatory Process, Redox
Status and Echocardiographic Parameters
Efeitos Parácrinos do Tratamento Precoce com Células-Tronco de Medula
Óssea no Infarto do Miocárdio Experimental em Ratos: Avaliação Tecidual do
Processo Inflamatório, Estado Redox e Parâmetros Ecocardiográficos
Tavares, A.M.V. / Article 2
53
Paracrine Effects of Early Bone Marrow Cells Treatment in Experimental Myocardial
Infarction in Rats: Tissue Evaluation of Inflammatory Process, Redox Status and
Echocardiographic Parameters
Cardiovascular Laboratory – Research Centre,
Hospital de Clínicas de Porto Alegre
Porto Alegre - RS, BRAZIL
Abstract
Background: Association among redox unbalance, inflammation and apoptosis are associated with cardiac
dysfunction post-acute myocardial infarction (AMI). Transplant of bone marrow cells (BMC) can exert beneficial
effects through paracrine actions on the host tissue.
Objective: To assess cardiac function and its correlation with redox balance, inflammatory process and
apoptosis in cardiac tissue 48 hours post-experimental AMI treated with cellular therapy.
Methods: Male 8-week-old Wistar rats were randomized into four groups: Sham-operated (S) with fictitious
myocardial infarction surgery; AMI; S + treatment (ST) and AMI + treatment (AMIT). Induction of AMI was
accomplished through ligation of the left anterior descending coronary artery (LAD), with open-chest under
mechanic ventilation. Determination of ejection fraction (EF) and akinetic or hypocinectic regions (AHR) were
evaluated by echocardiography. Tumor necrosis factor (TNF)-α, Interleukin(IL)-6 and Caspase-3 were measured
by Western Blot, and the reduced and oxidized glutathione ratio (GSH/GSSG) were measured by
spectrophotometer
.
Results: Infarcted area (%) was not different between groups AMI (52.8±5.7) vs. AMIT (54.2±4.3). EF (%) was
lower in the infarcted groups: AMI (51±5%) vs. S (74±7%) (p=0.001) and AMIT (56±10%) vs. ST groups (73±3%)
(p=0.001). Oxidative stress assessed by GSH/GSSG ratio was increased in infarcted groups, AMI (8.21±3.8) vs.
S (14.61±3.4) (p<0.05), AMIT (2.1±0.7) vs. ST (4.7±1.5) (p<0.05) and with treatment the oxidative stress was
high, AMIT (2.1±0.7) vs. AMI (8.21±3.8) (p<0.005), as well as capase-3 expression. However, it was observed
that BMC treatment was able to minimize ventricular hypertrophy (mg/g) in AMIT (2.86±0.2) vs. AMI group
(3.40±0.6) (p<0.001) and minimize TNF-α and IL-6 expression in infarcted treated group. We found a positive
correlation between ventricular hypertrophy and cytokines’ expression of TNF-α (r=0.732; p=0.001), and IL-6
(r=0.720; p=0.001).
Conclusions: Our data suggest that BMC treatment was able of attenuate ventricular hypertrophy and reduced
the expression of pro-inflammatory cytokines through its paracrine effects, at least in this time point.
Key words: oxidative stress, inflammation, cardiac hypertrophy, stem cell therapy, experimental infarction
Introduction
Considerable progress has been
achieved in the search for new therapies for the
management of acute myocardial infarction
(AMI) and consequent heart failure (HF) due to
ventricular remodeling; nonetheless, HF
continues to be a major world-wide problem
1
.
Cell therapy has been proposed a
stimulating approach to promote cardiac repair
post-AMI, although clinical data have brought
Tavares, A.M.V. / Article 2
54
somewhat disappoint long term results
2
. Clearly
in depth experimental studies are required to
better understand multiple and distinct
mechanisms involved in both ventricular
remodeling and how cellular therapy may
function in this scenario. In fact, several
mechanisms have been proposed to explain the
effects of cell therapy. These include cell
transdifferentiation, cell fusion and/or paracrine
effects.
Recent evidence suggests that
transplanted cells can exert a beneficial
influence through paracrine effects, secreting
cytokines and growth factors that stimulate
proliferation an differentiation of host tissue
3
.
Several studies have shown that the
mechanical wall stress associated with
myocardial infarction leads to the prompt
production in the myocardium of pro-
inflammatory cytokines such as tumor necrosis
factor (TNF)-α and Interleukin (IL)-6. These
cytokines are rapidly released in the central
ischemic zone during infarction but are usually
maximal in the ischemic zone border
4;5
.
Similarly, it has been proposed an association
between increased myocardial oxidative stress
and an antioxidant deficit correlating with cardiac
dysfunction at different stages of failure
6
.
Excessive reactive oxygen species
(ROS) may derive from intracellular sources
such as mitochondrial dysfunction or may be
secreted by infiltrating cells during the
inflammatory response to the ischemic insult, as
reviewed by Ungvári and colleagues
7
.
Isolated adult rat cardiomyocytes
exposed to different TNF- α concentrations
showed that this exposure caused a significant
decrease in both protein and mRNA for
manganese (Mn) superoxide dismutase (SOD)
and catalase (CAT), as well as, glutathione
peroxidase protein (GPx), increased intracellular
ROS and lipid peroxidation
8
.
Recently, oxidative stress was shown to
trigger cardiomyocyte apoptosis in myocardial
infarction ischemia/reperfusion injury,
atherosclerosis, and heart failure
9-11
.
Bone marrow cells (BMC) administered
after coronary ligation in mice showed that, four
days after myocardial infarction, BMC were
found within injured myocardium. However,
cardiomyocytes were observed in low number
within per-infarct area in BMC group, which
suggests that other mechanisms than
differentiation and fusion may be responsible for
the beneficial effects of cell therapy
12
.
Ventricular remodeling is characterized
by changes in left ventricular (LV) geometry,
mass, volume, and function, which include
hypertrophy and cellular apoptosis of
cardiomyocytes, in response to myocardial injury
or alteration in load
13
.
Although the role of oxidative stress and
inflammatory process are considered part of the
cardiac injury process leading to remodeling, the
effects of early treatment with BMC in the redox
status and its consequences on inflammatory
pathways and cell cycle remain unclear.
In this study, we aimed to evaluate the
profile of oxidative stress, inflammatory process
and apoptosis in myocardial tissue, submitted to
BMC treatment, 48 hours post- infarction.
Material and Methods
Animals and Groups
We studied male Wistar rats 8-week-old.
Animals were kept at Experimental Animal Unit
of Research Centre of Hospital de Clínicas de
Porto Alegre. All animals were treated in
accordance with the Guidelines for the Care and
Use of Laboratory Animals prepared by the
National Academy of Sciences and published by
the National Institutes of Health (NIH publication
no. 85-23, Revised 1996). The study was
Tavares, A.M.V. / Article 2
55
approved by the Research ethics committee of
Hospital de Clínicas de Porto Alegre. Animals
were randomized in four groups: Sham-operated
(S) with fictitious myocardial infarction surgery;
Acute Myocardial Infarction (AMI); S + treatment
(ST) and MI + treatment (AMIT). All animals
were kept in cages in room with temperature
ranging from 20
o
C to 25
o
C and light/dark cycles
of 12 hours, and fed ad libitum with rat chow.
Myocardial Infarction Surgery
Myocardial infarction was induced
according to a procedure previously described in
the literature and adapted in our laboratory
14
.
Briefly, animals were placed in dorsal decubitus
position and anesthetized with xilazine
(0.67mg/Kg) and ketamine (0.33 mg/Kg)
administered intraperitoneally.
Then, following orotracheal intubation,
animals were submitted to mechanical ventilation
with Harvard ventilator, Model 683 (frequency:
60m/min, tidal volume: 1.5 mL). Next, a surgical
incision was performed in the skin along the left
sternal margin, and divulsion of pectoralis and
transverses muscle was performed.
Thoracotomy was performed at the 2
nd
intercostal space and thorax was opened,
without exteriorization of the heart. The left
anterior descending coronary artery (LAD) was
identified and occluded with a 6-0 mononylon
suture between the left atrial appendage margin
and the pulmonary artery. Next, the thoracic
cavity was closed with a 5-0 mononylon thread,
muscles were repositioned and skin sutured. All
animals received analgesic after surgical
procedure (dipirone 0.1mL-i.m).
Isolation and staining of BMC
Bone marrow cells from male donors
were obtained as follows: after killing animals in
CO
2
chamber, the tibia and femur were flushed
with cell culture media (DMEM, Invitrogen, USA)
and mononuclear bone marrow cell fraction was
isolated by centrifugation using FICOLL (GE-
Healthcare, USA) gradient
15
. The cells were then
stained with DAPI (4-6-diamine-2-phenylindole
dihydrocloride, Roche Mannheim, Germany)
16
,
2.7 mg/mL. Cells were counted using Neubauer
chamber and Trypan Blue exclusion test to verify
cell viability, and adjusted to 2x10
6
cells/mL final
concentration.
Cell Transplantation
Five injections of 10µl (cells
concentration) were administered after LAD
occlusion or fictitious myocardial infarction
surgery in five different sites around ischemic
size of LV free wall (ST and AMIT groups).
Flow Cytometry Analysis
Approximately 2 x 10
6
mononuclear BMC
were prepared. They were placed in sterile tubes
and washed twice by centrifugation at 2000 rpm
for 5 minutes at 4°C. BMC were then
ressuspended in 200 µL of PBS and incubated
for 20 minutes at 4°C with FITC-anti-CD45
(CALTAG lab. CA,USA) and PE-anti-CD34
(Santa Cruz Biotechnology, Santa Cruz, CA) for
characterization of hematopoietic stem cells
through the ISHAGE protocol
17
. Anti-KDR
(Abcam, Inc, CA, USA) and PE-anti-CD34
(Santa Cruz Biotechnology, Santa Cruz, CA)
were used to quantify endothelial precursors
cells
18
. We also characterized the mesenchymal
stem cell population in our sample performing
triple staining for FITC-anti-CD44 (Abcam Inc,
CA, USA), PE-anti-CD71 (AbD Serotec -
Munich, DEU) and PECy5-anti- CD29
(BioLegend, San Diego, CA)
19
.
All assays were conducted using
antibodies' concentrations as recommended by
manufactures. Phycoerythrin-PE and FITC
mouse anti-rat IgG1, IgG2a and IgM were used
as isotype controls. After antibody incubation,
Tavares, A.M.V. / Article 2
56
lysis solution (1 mL) was added for 15 minutes to
hemolize erythrocytes; then 1 mL of PBS was
added for another 15 minutes to stop the
hemolytic treatment. Cells were collected and
washed with PBS by centrifugation and 500 µL
of PBS was added to the cell suspension.
Analysis was carried out with the BD FACS-
Calibur flow cytometry system with a one-laser
system that is capable to detect three
fluorochromes excited by the 488 nm laser in a
multiparameter manner. The samples were read
in the cell quest and PAINT-A-GATE software.
Echocardiogram
Before surgical interventions (baseline
evaluation) and forty-eight hours after surgical
procedures, animals underwent
echocardiography. Animals were placed in left
lateral decubitus position (45
o
) to obtain cardiac
images. EnVisor HD System, Philips Medical
(Andover, MA, USA) was used, with a 12-13MHz
transducer, at 2cm depth and fundamental and
harmonic imaging. Images were captured by a
trained operator with experience in gruel animal
echocardiography.
Left ventricular dimensions – The end-diastolic
and end-systolic transverse areas (cm
2
) were
obtained by tracing the endocardial border at
three levels: basal (at the tip of the mitral valve
leaflets), middle (at the papillary muscle level)
and apical (distal from the papillary muscle but
before final curve cavity)
20
. End-diastolic and
end-systolic diameters (cm) were measured
using the M-Mode, also in the three planes. Final
value for each animal was obtained by the
average of these all three planes.
Myocardial infarction size – On each
echocardiographic transverse plane (basal,
middle and apical) the arc corresponding to the
segments with infarction (regions or segments of
the myocardium showing one the following
changes in myocardial kinetics: systolic
movement akinesis and/or hypokinesis - AHR)
and to the total endocardial perimeter (EP) were
measured at end-diastole. Infarction size (IS)
was estimated as %IS = (AHR/EP) x 100
21
.
LV systolic function - LV ejection fraction (EF)
was calculated as: (End-diastolic volume – End-
systolic volume / End-diastolic volume) x 100;
end-diastolic and end-systolic cavity volumes
were calculated using Simpson´s rule
22
. Left
ventricular fractional shortening (LVFS) was
obtained by equation: LVFS = Dd – Sd/Dd x 100
(diastolic diameter – Dd; systolic diameter – Sd);
LV Mass – Mass (in grams) was calculated
according to the equation established by the
American Society of Echocardiography
M(g)=1.04 [(Dd + AWDT + PWDT) – Dd
3
]
3
(anterior wall diastolic thickness – AWDT;
posterior wall diastolic thickness – PWDT)
23;24
.
Final value volumes to all calculations for
each animal were based on three transverse
planes. Two-dimensional images and tracings of
the M-Mode were recorded in CD for later review
and off-line analysis.
Organs weight data
After undergoing echocardiography,
animals were killed by anesthesia with xylazine
(0.67 mg/Kg) and ketamine (0.33 mg/Kg),
followed by cervical dislocation and underwent
thoracotomy for heart and lung removal, as well
as open peritoneum was opened for liver
removal. In order to obtain the wet/dry weight
ratio of the lungs and liver, these organs were
removed and freed from adhering tissue. In each
case, the tissue was weighed and placed in the
oven (100°C) for 72 h. The hearts were weighted
(only ventricles) to analyze ventricular
hypertrophy. Hypertrophy was calculated by left
and right ventricles/body weight.
Tavares, A.M.V. / Article 2
57
Hemodynamic and tissue weight determinations
After undergoing echocardiography, the
animals were placed to allow pressure
measurements (MP100 - Biopac Systems, Inc –
CO) through a catheter inserted into the right
carotid artery and then advanced into the left
ventricle. Left ventricular end-diastolic (LVEDP)
and left ventricular peak systolic pressures
(LVSP) were recorded for an offline analysis.
After these assessments, the rats were killed
and the heart and other organs removed for
further studies.
Cardiac tissue preparation for analysis of
oxidative processes
After completing cardiac hemodynamic
measurements, rats were killed and hearts were
rapidly excised, weighted, and immediately
frozen in liquid nitrogen and stored at −80 °C for
the evaluation of glutathione content and protein
expression or homogenized in 1.15% w/v KCl
and phenyl methyl sulphonyl fluoride PMSF 20
mmol/L in Ultra-Turrax.
The resulting suspension was centrifuged
at 600 g for 10 min at 0 - 4°C to remove the
nuclei and cell debris
25
and supernatants were
used for oxidative stress measurements.
Immediately following killing, and prior to
homogenization, cardiac tissue samples were
removed and frozen at -80°C for the evaluation
of glutathione content and protein expression.
Determination of lipid peroxidation
Tert-butyl-hydroperoxide-initiated
chemiluminescence
Chemiluminescence (CL) was measured in a
liquid scintillation counter in the out-of-
coincidence mode (LKB Rack Beta Liquid
Scintillation Spectrometer 1215, LKB – Produkter
AB, Sweden). Homogenates were placed in low-
potassium vials at a protein concentration of 0.5-
1.0 mg/mL in a reaction medium consisting of
120 mmol/L KCl, 30 mmol/L phosphate buffer
(pH=7.4). Measurements were started by the
addition of 3 mmol/L tert-butyl hydroperoxide and
data expressed as counts per second per
milligram of protein (cps/mg protein)
26
.
Determination of antioxidant enzyme activities
Catalase (CAT) – activity was determined by
following the decrease in hydrogen peroxide
(H
2
O
2
) absorbance at 240nm. It was expressed
as nanomol of H
2
O
2
reduced per minute per
milligram of protein
27
.
Superoxide dismutase (SOD) activity, expressed
as units per milligram of protein, was based on
the inhibition of superoxide radical reaction with
pyrogallol
28
.
Glutathione peroxidase (GPx) – activity
expressed as nanomols of peroxide/
hydroperoxide reduced per minute per milligram
of protein, was based on the consume of
NADPH at 480 nm
29
.
Determination of oxidized and reduced
glutathione concentration
To determine oxidized and reduced
glutathione concentration, tissue was
deproteinized with 2 mol/L perchloric acid,
centrifuged for 10 min at 1000 g and supernatant
was neutralized with 2 mol/L potassium
hydroxide. The reaction medium contained 100
mmol/L phosphate buffer (pH 7.2), 2 mmol/L
nicotinamide dinucleotide phosphate acid, 0.2
U/mL glutathione reductase, 70 µmol/L 5,5’
dithiobis (2-nitrobenzoic acid). To determine
reduced glutathione, the supernatant was
neutralized with 2 mol/L potassium hydroxide, to
react with 70 µmol/L 5,5’ dithiobis (2-nitro
benzoic acid) and read at 420 nm
30
.
Determination of hydrogen peroxide (H
2
O
2
)
The assay was based in horseradish
peroxidase (HRPO)-mediated oxidation of
phenol red by hydrogen peroxide, leading to the
Tavares, A.M.V. / Article 2
58
formation of a compound that absorbs at 610
nm. Ventricle slices were incubated for 30 min.
at 37ºC in phosphate buffer 10 mmol/L (NaCl
140 mmol/L and dextrose 5 mmol/L). The
supernatants were transferred to tubes with
phenol red 0.28 mmol/L and 8.5 U/mL HRPO.
After 5 min incubation, NaOH 1 mol/L was added
and it was read at 610 nm. The results were
expressed in µmoles H
2
O
2
/ g tissue
31
.
Determination of protein concentration
Protein was measured by the method of
Lowry
32
, using bovine serum albumin as
standard.
Western Blot (WB) analysis
Tissue homogenization, electrophoresis,
and protein transference were performed as
described elsewhere
33;34
. The membranes were
processed for immunodetection using rabbit anti-
CAT polyclonal antibody, sheep anti-Cu/Zn SOD
polyclonal antibody, goat anti-Prx-6 polyclonal
antibody primary as (Santa Cruz Biotechnology,
Santa Cruz, CA) and rabbit anti-IL-6 polyclonal
antibody, mouse anti-TNF-α monoclonal
antibody, rabbit anti-Caspase-3 monoclonal
antibody primary as (Abcam Inc, CA, USA). The
bound primary antibodies were detected using
rabbit anti-sheep, rat anti-mouse or goat anti-
rabbit horseradish peroxidase-conjugate
secondary antibodies and membranes were
revealed for chemiluminescence. The generated
auto-radiographs were quantitatively analyzed
with an image densitometer (Image master VDS
CI, Amersham Biosciences Europe, IT).
Molecular weights of the bands were determined
by reference to a standard molecular weight
marker (RPN 800 rainbow full range Bio-Rad,
CA, USA). Results from each membrane were
normalized through Ponceau red method
35
.
Histological analyses
From each animal, the cardiac tissue was
cut according echocardiographic transverse
planes (basal, middle and apical). Tissue was
embedded in 10% formalin neutral buffer (pH
7.4), during 48 hours, then embedded in paraffin
and cut into semi thin sections 3 to 4 µm thick for
microscopy and histological analysis using
hematoxylin and eosin staining.
Statistical analysis
Data were expressed as mean ± SD of n
independent experiments. To compare multiple
groups, we used two-way ANOVA test (Student-
Newman-Keuls Method). The correlation
between two variables was analyzed by
Pearson’s correlation. Tests with P<0.05 were
considered statistically significant. Data were
analyzed with the SigmaPlot 11.0 program.
Results
Heart rate, infarction size and mortality
The mean HR after sedation during basal
echocardiographic evaluation was not
significantly different among groups (data not
shown). In data observed in the final
echocardiogram performed 48 hours post-
infarction there was a rise in HR in BMC-treated
animals as compared with the non-treated ones;
where AMIT HR was increased (p<0.01)
compared with AMI group. Infarction area (% of
circumference) was not different between AMI
and AMIT groups [Table; Figure 1]. Mortality
occurred during or immediately after the surgical
procedure was around 10% in all groups.
Tavares, A.M.V. / Article 2
59
Representative myocardium images – Left ventricular free wall
Figure 1 - Representative images of myocardium stained with hematoxylin and eosin (HE) illustrating myocardial transverse
diameter showing inflammatory infiltration of infarcted region compared to their respective control groups: (A) Myocardium
sample S group (Sham), (B) Myocardium sample AMI group. (C) Non-infarcted myocardium sample with treatment, ST
(Sham-treated) group and (D) Infarcted myocardium sample with treatment, AMIT group;
Cardiac dimensions and systolic function
In basal echocardiographic evaluation
there was no statistical difference between
treated and non-treated groups in all the
parameters evaluated (data not shown). In final
echocardiographic evaluation assessed 48
hours post-infarction, EF, SD and LVFS were
lower in the groups AMI and AMIT, in relation to
their respective controls (S and ST,
respectively) (p<0.001). At this time point, with
the use of BMC was not able to change the
profile of these variables in infarcted groups,
after 48 hours. Diastolic dimensions and LV
mass were not different among groups [Table].
Hemodynamic data
Animals were assessed for LVEDP and
LVSP values. AMI and AMIT rats, compared
with their controls showed increase in the
LVEDP (p<0.001), but without change of LVSP
among groups. However, BMC treatment has
not attenuated the rise in LVEDP in the AMIT
compared with AMI group [Table].
Organs weight data
The ratio of wet to dry weight in lung
and liver of animals were not different in treated
and non-treated groups, indicating that there
was no lung or liver congestion at this time
point. The body weight gain in both treated and
non-treated groups was slightly lower than in
their respective control groups, however not
statistically significant [Table]. Ventricular
hypertrophy was observed in infarcted animals
compared with the respective controls, AMI vs.
S (p<0.001) and AMIT vs. ST (p=0.039) groups.
In addition, we observed that the BMC
treatment was able to minimize ventricular
hypertrophy (AMIT vs. AMI groups) (p<0.001)
[Table].
Tavares, A.M.V. / Article 2
60
Table – Parameters of cardiac function, hemodynamic profile, oxidative profile and organs weight for
each group, with and without BMC treatment.
Parameters S AMI ST AMIT
n 11 10 12 14
243 ± 12 230 ± 22 267 ± 29
270 ± 24
†
AHR,%
...
52.8 ± 5.7
...
54.2 ± 4.3
SD, cm 0.339 ± 0.1
0.519 ± 0.04
ψψ
0.373 ± 0.07 0.519 ± 0.07
**
DD, cm 0.704 ± 0.1 0.746 ± 0.06 0.744 ± 0.06 0.758 ± 0.07
EF, % 74 ± 7
51 ± 5
ψψ
73 ± 3 56 ± 10
**
LVFS, % 53 ± 9
30 ± 4
ψψ
50 ± 8 31 ± 8
**
0.316 ± 0.08 0.265 ± 0.06 0.282 ± 0.10 0.250 ± 0.08
n 5 4 10 12
6.7 ± 2.3
15 ± 4.7
ψψ
6.3 ± 1.4 13 ± 2.8
**
127.8 ± 16.1 122.1 ± 30.9 114.9 ± 15.0 111.1 ± 15.2
n 11 10 10 14
4.75 ± 0.8 4.96 ± 0.3 4.99 ± 0.7 4.85 ± 0.3
3.38 ± 0.2 3.47 ± 0.2 3.54 ± 0.5 3.31 ± 0.3
295.2 ± 67.8 265.7 ± 46.4 325.8 ± 47.6 319.9 ± 37.7
2.72 ± 0.3
3.40 ± 0.6
ψψ
2.55 ± 0.3
2.86 ± 0.2
&
††
n 5 5 5 8
5.13 ± 2.0 5.22 ± 1.4
9.34 ± 0.7
7.2 ± 0.8
†
40.57 ± 8.6 34.06 ± 9.0 31.98 ± 9.6 33.03 ± 9.5
46.88 ± 9.0
26.32 ± 7.5
ψ
36.9 ± 10.7 30 ± 6.5
n 5 7 6 8
5526 ± 1228 6219 ± 969 4169 ± 1035 7392 ± 2223
*
32 ± 0.008 22 ± 0.005
116 ± 0.029
131 ± 0.023
††
14.61 ± 3.4
8.21 ± 3.8
≠
4.7 ± 1.5
2.1 ± 0.7
§
CL, cps/mg protein
H
2
O
2
(µmol/mg prot)
GSH/GSSG, ratio
SOD, U/mg protein
CAT, µmol/mg protein
GPx, nmol/mg protein
Oxidative Stress
Liver, ratio (wet/dry weight) mg/g
Body (weight), g
Ventricular weight/body weight, mg/g
Antioxidant Enzymes
LVEDP, mmHg
LVSP, mmHg
Organs weight data
Lung, ratio (wet/dry weight) mg/g
Final Echocardiographic Data
Heart rate, bpm
LV Mass, g
Hemodynamic data
Characteristic of Animal Groups Non-treated Treated
Abbreviations: Akinetic and/or Hypokinetic region, AHR; SD, Systolic Diameter; DD, Diastolic Diameter; EF, Ejection Fraction; Left
Ventricular Fractional Shortening, LVFS; Left Ventricular End-Diastolic, LVEDP; and Left Ventricular Systolic Pressures, LVSP;
Ventricular weight, Vwt/body weight; Superoxide Dismutase, SOD; Catalase, CAT and Glutathione Peroxidase, GPx activity;
Hydrogen Peroxide, H
2
O
2
concentration; Chemiluminescence, CL; reduced (GSH) and oxidized glutathione ratio, GSH/GSSG; Data
expressed as mean ± SD; p<0.05 was considered statistically significant (Two-way ANOVA test). p<0.001
ψψ
, p<0.01
ψ
, p<0.05
≠
to S
vs. AMI; p<0.001
**
, p<0.01
*
, p<0.05
&
to ST vs. MIT; p<0.001
ҰҰ
, p<0.01
Ұ
to S vs. ST and p<0.001
††
, p<0.01
†
, p<0.005
§
to AMI vs. AMIT;
Tavares, A.M.V. / Article 2
61
Flow Cytometry Analysis
To characterize the precursor cells
injected, we quantified endothelial precursor
cells (KDR
+
/CD34
+
), mesenchymal stem cells
(CD44
+
/CD71
+
/CD29
+
) and hematopoietic stem
cells (using ISHAGE protocol) in four animals
used as cell donors. Endothelial precursor cells
corresponded to 1.39% ±0.87 of the total cells
injected, while mesenchymal and hematopoietic
fractions corresponded to 0.48% ± 0.09 and
0.48% ± 0.37, respectively.
Antioxidant enzyme activities and expression
Myocardial SOD and CAT activities
were not different between AMI compared with
S group. On the other hand, treatment with
BMC was able to increase SOD activity in both
groups (p<0.001). Moreover, infarcted animals
that received BMC compared with those that
did not received (AMIT vs. AMI), had increased
activity of this enzyme (p<0.01), showing the
protective role of BMC against O
2
.-
[Table].
Similar results were found in protein
expression of SOD and CAT in cardiac tissue of
untreated animals (S and AMI); without
difference between groups. However, BMC-
treated animals had minor protein expression of
SOD (p<0.05) compared to non-treated group
[Table; Figure 2A].
GPx activity was depressed in the AMI
group in relation to S group (p<0.01). Despite
the absence of differences between groups AMI
and AMIT, the treatment with BMC was able to
maintain the redox balance activity for this
antioxidant enzyme in AMIT compared to ST
group [Table].
In contrast, CAT protein expression in
BMC-treated groups was higher (p<0.002)
compared to non-treated groups. In addition,
between infarcted groups, cellular therapy was
able to increase CAT protein expression
(p<0.001). Therefore, the highest CAT protein
expression was in AMIT group (p<0.001),
showing that although not changing the activity
[Table], the treatment was able to increase the
CAT protein expression in the presence of an
ischemic damage [Figure 2B]. Prx-6 protein
expression, responsible by H
2
O
2
scavenging,
was increased in the AMI group compared to
control S (p<0.01) 48 hours post-infarction.
Similar result was also found between AMIT
compared to control ST group (p<0.01).
Nonetheless, the treatment with BMC was not
capable of modifying the profile of this protein
[Figure 2C].
Oxidation products
Infarction did not cause lipid damage in
myocardial tissue at this time point. On the
other hand, lipid damage was observed in
treated groups, since lipid peroxidation (CL)
was increased in AMIT group compared to its
control (ST) (p<0.01), although without
difference between infarcted groups (AMI and
AMIT) [Table].
Hydrogen peroxide (H
2
O
2
)
H
2
O
2
myocardial concentration was not
different between AMI and control S groups,
while treatment with BMC was capable of
modifying their pattern, greatly increasing H
2
O
2
concentration in both groups (p<0.001). ST and
AMIT groups were not different compared to
each other, but the concentration of H
2
O
2
was
increased in AMIT compared with AMI group
(p<0.001), as well as, in ST compared with S
group (p<0.001), showing that treated groups
had increased production of H
2
O
2
[Table]. In
addition, a positive correlation between H
2
O
2
Tavares, A.M.V. / Article 2
62
and caspase-3 (r=0.803; p=0.0001), was also
observed.
GSH/GSSG - reduced to oxidized glutathione
ratio
GSH/GSSG ratio was assessed, being
depressed in the AMI group compared to
control S (p<0.003), showing an increase of
oxidative stress in this group. We also found a
decrease in AMIT as compared with AMI group
(p<0.005), and between ST and S groups
(p<0.001). The GSH/GSSG ratio was also
different between ST and AMIT groups,
showing that BMC was not capable of
maintaining redox balance.
In treated groups, there was an increase
in oxidative stress showed by major
consumption of glutathione [Table]. In addition,
it was found a positive correlation between
GSH/GSSG ratio and LVFS (r=0.639; p=0.004),
showing its interaction with cardiac function.
Figure 2 – Graphs illustrating analysis of antioxidant enzymes in cardiac homogenates
Figure 2 – A) Graph of Western blot analysis in cardiac homogenates using Cu/Zn SOD antibody;
Abbreviations: S, sham; AMI, acute myocardial infarction; ST, sham treated; AMIT, AMI treated; SOD, superoxide
dismutase; Note illustrative bands of blots on top part of graphs. p<0.05 was considered as statistically significant;
0
60
120
180
240
300
360
S AMI ST AMIT
SOD
Density Normalized
by Ponceau Red
P<0.05
0
60
120
180
240
300
360
S AMI ST AMIT
SOD
Density Normalized
by Ponceau Red
P<0.05
0
60
120
180
240
300
360
S AMI ST AMIT
SOD
Density Normalized
by Ponceau Red
P<0.05P<0.05
Tavares, A.M.V. / Article 2
63
F
i
g
Figure 2 – B) Graph of Western blot analysis in cardiac homogenates using CAT antibody;
Abbreviations: S, sham; AMI, acute myocardial infarction; ST, sham treated; AMIT, AMI treated; CAT, catalase;
Note illustrative bands of blots on top part of graphs. p<0.05 was considered as statistically significant;
–
F
F
F
i
g
u
r
e
Figure 2 – C) Graph of Western blot analysis in cardiac homogenates using Prx-6 antibody;
Abbreviations: S, sham; AMI, acute myocardial infarction; ST, sham treated; AMIT, AMI treated; Prx-6, peroxiredoxin-6;
Note illustrative bands of blots on top part of graphs. p<0.05 was considered as statistically significant;
P<0.001
P<0.001
0
60
120
180
240
300
360
S AMI ST AMIT
CAT
Density Normalized
by Ponceau Red
P<0.001
P<0.001
P<0.001
P<0.001
0
60
120
180
240
300
360
S AMI ST AMIT
CAT
Density Normalized
by Ponceau Red
P<0.001
P<0.001
P<0.001
P<0.001
0
60
120
180
240
300
360
S AMI ST AMIT
CAT
Density Normalized
by Ponceau Red
P<0.001
P<0.001
0
60
120
180
240
300
360
S AMI ST AMIT
Prx-6
Density Normalized
by Ponceau Red
P<0.01
P<0.05
P<0.001
0
60
120
180
240
300
360
S AMI ST AMIT
Prx-6
Density Normalized
by Ponceau Red
P<0.01
P<0.05
P<0.001
P<0.01
P<0.05
P<0.001
Tavares, A.M.V. / Article 2
64
Inflammation and apoptotic markers assessed
TNF-α protein expression was not
different between non-treated (AMI vs. S)
groups. On the other hand, treatment with BMC
was able to reduce this marker expression in
infarcted group AMIT, when compared to
control AMI (p<0.0001), although the TNF-α
expression has been increased in AMIT
compared to ST group (p<0.05) [Figure 3A].
Additionally, a moderate correlation between
ventricular hypertrophy and TNF-α protein
expression was found (r=0.732; p=0.001).
IL-6 expression was increased in AMI
compared to S group (p<0.001), as well as, in
the AMIT compared to ST group (p<0.001).
Nevertheless, the treatment with BMC was
capable to reduce this cytokine’s expression
(p<0.0001). In the infarcted groups, IL-6
expression was minor in AMIT as compared to
AMI group (p<0.001) [Figure 3B]. In addition, a
moderate correlation between ventricular
hypertrophy and IL-6 protein expression
(r=0.720; p=0.001) was also observed.
Caspase-3 protein expression was not
different between non-treated (AMI vs. S)
groups. On the other hand, caspase-3 in AMIT
was higher compared to ST group (p<0.01). We
observed an increase of caspase-3 in treated
groups (ST and AMIT), (p=0.0002), and
between non-treated and treated (S vs. ST)
groups (p<0.01) and (AMI vs. AMIT) groups
(p<0.001), showing that BMC treatment was
capable to stimulate apoptotic process [Figure
3C].
Figure 3 – Graphs illustrating analysis of inflammatory and apoptotic markers in cardiac
homogenates
Figure 3 – A) Graph of Western blot analysis in cardiac homogenates using TNF-α antibody;
Abbreviations: S, sham; AMI, acute myocardial infarction; ST, sham treated; AMIT, AMI treated; TNF-α, tumor necrosis
factor- α. Note illustrative bands of blots on top part of graphs. p<0.05 was considered as statistically significant;
0
60
120
180
240
300
360
S AMI ST AMIT
TNF-α
Density Normalized
by Ponceau Red
P<0.001
P<0.01
P<0.05
0
60
120
180
240
300
360
S AMI ST AMIT
TNF-α
Density Normalized
by Ponceau Red
P<0.001
P<0.01
P<0.05
0
60
120
180
240
300
360
S AMI ST AMIT
TNF-α
Density Normalized
by Ponceau Red
P<0.001
P<0.01
P<0.05
P<0.001
P<0.01
P<0.05
TESE - Dezembro / 2008
63
Figure 3 – B) Graph of Western blot analysis in cardiac homogenates using IL-6 antibody;
Abbreviations: S, sham; AMI, acute myocardial infarction; ST, sham treated; AMIT, AMI treated; IL-6, interleukin-6. Note
illustrative bands of blots on top part of graphs. p<0.05 was considered as statistically significant;
Figure 3 – C) Graph of Western blot analysis in cardiac homogenates using caspase-3 antibody;
Abbreviations: S, sham; AMI, acute myocardial infarction; ST, sham treated; AMIT, AMI treated. Note illustrative bands of
blots on top part of graphs. p<0.05 was considered as statistically significant;
0
30
60
90
120
150
180
S AMI ST AMIT
IL-6
Density Normalized
by Ponceau Red
P<0.001
P<0.001
P<0.001
P<0.001
0
30
60
90
120
150
180
S AMI ST AMIT
IL-6
Density Normalized
by Ponceau Red
P<0.001
P<0.001
P<0.001
P<0.001
0
30
60
90
120
150
180
S AMI ST AMIT
Caspase-3
Density Normalized by
Ponceau Red
P<0.01
P<0.01
P<0.001
0
30
60
90
120
150
180
S AMI ST AMIT
Caspase-3
Density Normalized by
Ponceau Red
P<0.01
P<0.01
P<0.001
P<0.01
P<0.01
P<0.001
P<0.01
P<0.01
P<0.001
TESE - Dezembro / 2008
64
Discussion
Early after AMI, cytokines produced
locally participate in the recruitment of
inflammatory cells, helping the natural process
of healing the myocardium and, in this
pathologic condition, high concentrations of
TNF-α can induce LV dysfunction and cardiac
dilation
36
. In the present study, we were able to
show the paracrine effect of BMC treatment in
myocardial infarction, assessed 48 hours post-
cardiac insult. We observed a decrease in
proteic expression of TNF-α and IL-6 in treated
groups compared to non-treated, showing that
the treatment with BMC was able to interfere in
the inflammatory process.
In a study using isolated myocytes and
nonmyocytes from non-infarcted myocardium,
investigators demonstrated that after
myocardial infarction the expression of IL-1β,
IL-6, and TNF-α mRNAs is clearly induced in
the nonmyocyte fraction, suggesting possible
autocrine and paracrine effect of cardiac
nonmyocytes. In a rat model of AMI, membrane
bound and soluble TNF-α proteins and mRNA
expression significantly increased after 1 and 4
weeks of post-AMI, which correlated positively
with LV end diastolic pressure
37
. This early
increase in cytokine levels may reflect the
inflammatory response induced by ischemia,
and the subsequent rise may reflect healing
after myocyte necrosis or a response to heart
failure
38
. The expression of these cytokines can
occur simultaneously with early cardiac
dysfunction and ventricular compliance
changes, suggesting a relation of between
these phenomena in a model of ischemia and
reperfusion
39
.
In our study, we saw that there was a
decrease in cardiac hypertrophy in BMC-treated
group which was directly related to both TNF-α
and IL-6 reduction in its expression. Our
findings are in accordance with Franz and
colleagues that, recently highlighted
amelioration of ventricular remodeling by the
use of unfractionated BMC injection compared
to other BMC populations and suggested that
this effect may be mediated by paracrine
secretions of cytokines of transplanted cells,
although they only have observed improvement
of cardiac hypertrophy, without significant
difference in other functional parameters
40
.
The exposure of cells to different
concentrations of H
2
O
2
, increases the endogen
levels of cellular TNF-α
41
. In neonatal rat
cardiac myocytes, it was found an increase in
H
2
O
2
resulting in hypertrophy and apoptosis
42
.
In contrast to the literature, in our findings, high
concentrations of H
2
O
2
were not capable of to
stimulate TNF-α and/or IL-6 expression in
treated groups. In fact, we found an increase of
H
2
O
2
and consequently increased oxidative
stress by GSH/GSSG ratio in treated groups
but this was not accompanied by ventricular
hypertrophy, at this time point.
A previous study has shown that
oxidative stress is one of the mechanisms
directly responsible for cardiac dysfunction
43
.
After AMI, oxidative stress develops in both
infarcted and non-infarcted myocardium
44
.
Moreover, the loss of cardiac function observed
by reduced EF, SD and LVFS, and increased of
LVEDP in both groups of infarcted animals, can
be directly correlated to redox unbalance, as
described in other studies
45;46
.
In a study in patients with HF, it was
observed an increase in inflammatory
cytokines, TNF-α and IL-6, as well as an
increase in the production of hydroxyl radical,
post-myocardial infarction. The elevation of
both molecules suggests that the interaction
between ROS and inflammatory process should
TESE - Dezembro / 2008
65
be, probably, related to the progression of LV
dysfunction after MI
47
. Our present data indicate
that despite of increased oxidative stress, the
reduced cytokine expression, use of BMC,
probably prevented interaction between ROS
and inflammatory process at this time point.
According to Mann and colleagues it is
thought that myocardial response to
environmental stress is made up of at least two
interdependent homeostatic mechanisms: one
that allows this tissue to delimit cell injury
through upregulation of cytoprotective factors
and a second that facilitates tissue repair when
and if these cytoprotective responses are
insufficient to prevent cell death. However, the
mechanisms that are responsible for
orchestrating these different stress responses
within the myocardium are not known
48
.
Another study using circulating
progenitor cells (EPCs), isolated from
peripheral blood showed that protein
expression of MnSOD, CAT, and GPx was
significantly higher in these cells, compared
with other cell types. In addition, EPCs due to
their higher expression of genes encoding for
antioxidative proteins, have low baseline ROS
levels and a reduced sensitivity toward ROS-
induced cell death
49
.
In our study we assessed expression
and activities of antioxidant enzymes, and
cellular treatment was capable of increasing
expression of SOD and CAT. These effects
may be attributed to an increased ROS
production, caused by ischemia and
consequent O
2
.-
production
50
.
Previous observations from our group
on the profile of H
2
O
2
48 hours, post-AMI led us
to believe that the transition between
compensated and decompensated cardiac
function may be influenced by H
2
O
2
levels,
acting as a signaling molecule activating
survival pathways (unpublished data). In our
present study the high H
2
O
2
concentrations in
treated groups led to an increase of oxidative
stress which correlated with increased caspase-
3 expression. Apoptotic myocyte cell death
precedes cell necrosis, which is a major
determinant of infarct size
51
. Although cardiac
tissue exhibits apoptotic characteristics at this
time point, this process may be considered as a
survival pathway to the adjacent cells not
affected (or barely affected) by ischemia.
Our findings are in agreement with
speculations made by Von Harsdorf and
colleagues that the induction of apoptosis in
cardiomyocytes exposed to ROS in ischemic
zones may represent an evolutionarily-
conserved protective mechanism that these
cells dispose in order to avoid necrosis of other
cells and thus risking cardiac function and
integrity so early after AMI
52
.
In conclusion, taken together results
from the present study revealed that treatment
with BMC had a key role in the attenuation of
cardiac hypertrophy following infarction, as well
as a marked reduction in pro-inflammatory
cytokines. It can also be suggested that the
apoptotic mechanism observed may lead to
decreased necrotic events in remanescent
myocardium which are more deleterious to the
cardiac function, considering that cells in
apoptosis cannot signal. In our view, 48 hours
post-AMI seems to be an attractive time point to
evaluate both paracrine effects exerted by the
transplanted cells in the heart tissue as well as
the influences imposed upon the cells by the
surrounding ischemic environment. Further
studies at later time-points are warranted to
fully describe potential beneficial effects of BMC
therapy in the post-AMI scenario.
TESE - Dezembro / 2008
66
Acknowledgements
The authors are grateful to Andreia C. Taffarel
(Veterinary Medicine), Gabriela Nicolaidis (Medicine)
and Rafael Dall’Alba (Biology), undergraduate
students of UFRGS, for their generous contribution.
This work was supported by HCPA-FIPE and CNPq
grants.
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TESE - Dezembro / 2008
70
CONCLUSÕES DA TESE
I - O comprometimento da função ventricular precocemente pós-IAM parece se associar com
um desequilíbrio do estado redox e este, por sua vez interage com os processos iniciais do
fenótipo de remodelamento ventricular.
II - A terapia celular, com células derivadas da medula óssea num modelo experimental de 48
horas pós-IAM foi associada com redução da hipertrofia ventricular e menor secreção de
citocinas inflamatórias sugerindo ações parácrinas das células, nesta janela temporal.
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