FUNDAMENTO: O Imatinib é um inibidor do receptor tirosina-quinase que foi confirmada como exercendo um efeito inibidor sobre a atividade do receptor do PDGF, fator de crescimento plaquetário (PDGFRα e PDGFRβ). OBJETIVO: Investigar o efeito protetor do Imatinib na fibrose miocárdica em acetato de deoxicorticosterona (DOCA)/ratos com hipertensão induzida por sal. MÉTODOS: Sessenta ratos Sprague-Dawley machos, uninefrectomizados foram distribuídos em três grupos: ratos controles (grupo CON): grupo deoxicorticosterona (grupo DOCA); grupo deoxicorticosterona e Imatinib (grupo DOCA IMA). A Pressão Arterial Sistólica (PAS) foi medida quinzenalmente. Foi estudada a porção apical do ventrículo esquerdo. Foram empregados: coloração vermelho sirius, coloração de hematoxilina-eosina, imuno-histoquímica e ensaio de western blot. RESULTADOS: A PAS nos grupos DOCA e IMA+DOCA foi maior que no grupo CON nos dias 14 e 28. Os animais do grupo DOCA apresentaram fibrose intersticial e perivascular grave no dia 28, e as expressões de PI, PIII, tenascina-C e fibronectina foram significativamente maiores que nos grupos DOCA+IMA e CON. Quando comparados com o grupo CON, os grupos DOCA e DOCA+IMA apresentaram resposta inflamatória de tecido miocárdico e infiltração de monócitos/macrófagos de diferentes graus. As expressões proteicas do PDGF-A, PDGF-C e PDGFRα foram significativamente maiores nos grupos DOCA e DOCA+IMA que no grupo CON, mas a expressão proteica do p-PDGFRα no grupo DOCA+IMA foi menor que no DOCA. CONCLUSÃO: O Imatinib pode exercer efeitos inibitórios sobre a fibrose miocárdica em ratos com hipertensão induzida por DOCA/sal, os quais podem ser atribuídos à inibição da atividade do PDGFR-α.
Imatinib atenua a fibrose miocárdica em associação com a inibição da atividade do PDGFR
Department of Cardiology, Anhui Provincial Hospital, Hefei – China
A fibrose miocárdica é um problema clínico comum, pode ser encontrado em várias doenças cardíacas e está frequentemente associada com o processo inflamatório. No coração, o acúmulo excessivo de componentes da matriz extracelular (MEC), especialmente os tipos I, III, e colágeno tipo V, é responsável pela fibrose miocárdica. Mudanças na estrutura tecidual e disfunção cardíaca após a fibrose miocárdica contribuem para a insuficiência cardíaca, arritmias, morte súbita cardíaca e outros eventos cardiovasculares graves. Os mecanismos moleculares da fibrose miocárdica são complexos, sobres os quais ainda há controvérsias. Estudos anteriores sugeriram que a fibrose miocárdica está estreitamente relacionada com o aumento do mineralocorticoide endógeno. No entanto, os papéis de algumas vias de sinalização molecular na fibrose miocárdica, induzida por mineralocorticoide, permanecem obscuros. Várias citocinas e fatores de crescimento têm demonstrado desempenhar papéis cruciais no desenvolvimento da fibrose miocárdica. Esses fatores incluem o fator β de crescimento transformante (TGFβ)1, interleucina-1 (IL-1)2, fator de necrose tumoral α (TNFα)3, e fator de crescimento plaquetário (PDGF)4. O PDGF é um membro da família de fatores de crescimento, e se encontrou que promove a divisão e proliferação de fibroblastos e células musculares lisas. Há evidências mostrando que altas expressões proteicas dos PDGF podem levar a fibrose miocárdica, o que sugere que vias de sinalização PDGF-PDGFR podem servir como importantes mecanismos moleculares da fibrose miocárdica5.
O Imatinib é um inibidor do receptor tirosina-quinase, especialmente utilizado no atual tratamento clínico de tumores, e foi confirmado como exercendo um efeito inibidor sobre a atividade do receptor de PDGF (PDGFRα e PDGFRβ). As vias sinalização PDGF-PDGFR são reguladas pelo PDGFR5, e assim, o Imatinib pode modular a resposta biológica celular induzida pelas vias de sinalização PDGF-PDGFR. Neste estudo, os efeitos de Imatinib na inflamação do miocárdio e resposta fibrogênica, infiltração de monócitos/macrófagos, e as expressões proteicas de PDGF-A, PDGF-C, PDGFRα, p-PDGFRα, e o procolágeno (PI), procolágeno III (PIII), tenascina-C e fibronectina foram investigados em ratos com hipertensão induzida por sal/acetato de deoxicorticosterona (DOCA), com o objetivo de estudar se o Imatinib pode efetivamente atenuar a fibrose miocárdica, e explorar a relação entre a via de sinalização PDGFRα e a fibrose miocárdica.
Sessenta ratos Sprague Dawley machos (SD), pesando entre 200 ~ 250 g, foram adquiridos do Centro Experimental Animal, filiado à Universidade Médica de Anhui, e alojados em ambiente livre de patógenos específicos. Antes de experiência, os animais foram anestesiados por injeção intraperitoneal de 10% de hidrato de cloral (400 mg kg-1) e foi realizada nefrectomia direita. Uma semana após a cirurgia, os ratos foram alimentados com água contendo 1% de NaCl e 0,2% de KCl, e divididos aleatoriamente em três grupos (n = 20 por grupo): 1) grupo controle (grupo CON): água destilada foi administrada diariamente por via intragástrica, e injeções subcutâneas de óleo de gergelim foram administradas uma vez a cada quatro dias; 2) grupo deoxicorticosterona (grupo DOCA): deoxicorticosterona (60 mg.kg-1.4d-1) foi administrada por via subcutânea, e água destilada foi administrada diariamente por via intragástrica; 3) grupo deoxicorticosterona mais Imatinib (grupo DOCA+IMA): deoxicorticosterona (60 mg.kg-1.4d-1) foi administrada subcutaneamente uma vez a cada quatro dias, e simultaneamente o Imatinib (60 mg.kg-1.d-1) foi administrado intragastricamente, diariamente. O volume de água destilada usada no grupo de controle foi idêntico ao volume de água destilada contendo as drogas nos restantes dois grupos. Além disso, o volume de óleo de gergelim utilizado no grupo de controle foi equivalente ao volume de óleo de gergelim contendo os agentes nos outros dois grupos. O tratamento com agentes durou quatro semanas.
Foram utilizados no presente estudo: deoxicorticosterona, óleo de gergelim (Sigma, EUA), Imatinib (Novartis, Suíça), corante vermelho sirius (Beijing Hede Biocompany), hematoxilina, kits de coloração imuno-SP (Beijing Goldenbridge Biotechnology CO., Ltd), anticorpo ED-1 (Beijing Biosynthesis Biotechnology CO., Ltd), PDGF-A, PDGF-C, PDGFRα, p-PDGFRα, PI, PIII, fibronectina, tenascina-C e anticorpos primários e secundários para β-actina (Santa Cruz Inc) e kits ECL (Pierce Inc).
A Pressão Arterial Sistólica (PAS) foi medida em 1d, 14d e 28d, usando o método do manguito cauda. No dia 14 e dia 28 após a intervenção farmacológica, os animais (n = 10) foram anestesiados intraperitonealmente com 10% de hidrato de cloral (400 mg kg-1) e depois mortos. Foram obtidos os corações e removidos os átrios bilaterais, ventrículos direitos, grandes vasos e tecidos conectivos, a parte apical do ventrículo esquerdo foi coletada para exames complementares. As amostras coletadas no dia 14 foram fixadas em paraformaldeído 4%, embebidas em parafina, seguidas de corte e imunocoloração. As amostras obtidas no dia 28 foram divididas em duas: uma foi fixada em paraformaldeído 4% para coloração HE e coloração com colágeno vermelho sirius, e a outra foi armazenado a -80v para ensaio de western blot.
Dois patologistas que foram cegos para o estudo avaliaram as seções. Após os dias 14 e 28, a porção apical do ventrículo esquerdo foi submetida a coloração H&E para exame de inflamação cardíaca. A infiltração de macrófagos do miocárdio foi observada por imuno-histoquímica, 14 dias após a intervenção com agentes. O ED-1 é um tipo de antígeno específico sobre a superfície de monócitos/macrófagos. A imuno-histoquímica foi realizada para detectar a expressão do ED-1 no coração, de acordo com as instruções do fabricante. Macrófagos com membrana marrom foram considerados positivos para ED-1. Dez campos na região positiva foram selecionados aleatoriamente com uma ampliação de 400 ×. O software Image-Proplus 6.0 foi utilizado para contar o número de células positivas, seguido pelo cálculo do número de macrófagos por mm2 de campo visual. Em seguida, a média foi obtida para cada secção.
A fração de volume do colágeno miocárdico (FVC) e área de colágeno de vasos periféricos miocárdicos (PVCA) foram medidas por pícrico coloração vermelho sirius-ácido pícrico no dia 28. O método de cálculo foi o indicado a seguir. Foi usado o sistema fotográfico Nikon, e o software Image-Proplus 6.0 foi utilizado para a análise. A FVC foi expressa como a relação entre a área de colágeno do miocárdio e a área total de campo visual. Oito campos (400×) foram selecionados aleatoriamente e as correspondentes FVC foram medidas em cada seção, e a média foi considerada como a FVC final de cada seção. A PVCA foi expressa como a relação entre a área de colágeno periférica em torno da artéria pequena e a área do lúmen arterial. Quatro pequenas artérias (400×) foram escolhidas aleatoriamente para a detecção em cada seção, e a média foi considerada como a PVCA final para a seção correspondente.
As expressões proteicas de PDGF-A, PDGF-C, PDGFRα, p-PDGFRα, PI, PIII, fibronectina e tenascina-C no coração foram detectadas pelo ensaio de western blot no dia 28 após a intervenção com agentes. A proteína total foi extraída usando uma solução tampão de lise celular (Principais componentes: Tris 20 mM (pH 7,5), NaCl 150 mM, Triton X-100 1%, deoxicolato 1%, SDS 0,1%, PMSF 1 mM, leupeptina 2 mg/mL, EDTA 1 mM, pirofosfato de sódio, β-glicerofosfato e Na3VO4), o sobrenadante contendo as proteínas foi recolhido por centrifugação, e a concentração de proteína foi detectada com o método BCA. Todas as amostras (uma amostra representa uma) foram diluídas na mesma concentração. Três amostras foram escolhidas aleatoriamente de cada grupo e comparadas com os dos restantes dois grupos. Em seguida, 50
O software SPSS versão 13.0 foi utilizado para a análise estatística. Os dados foram apresentados como média ± erro padrão (EPM). As médias entre vários grupos foram comparadas com a análise de variância de uma via (ANOVA). As relações foram comparadas com o teste de qui quadrado. Um valor de p < 0,05 foi considerado estatisticamente significativo.
Não houve diferença significativa na PAS entre os grupos antes da experiência. No entanto, a PAS nos grupos DOCA e DOCA+IMA foram significativamente mais elevadas que no grupo CON nos dias 14 e 28. Não foi observada significância estatística na PAS entre os grupos DOCA e DOCA+IMA, em relação aos níveis (
A coloração H&E revelou infiltração evidente de células inflamatórias e fibrose leve no coração no dia 14, no grupo DOCA. Uma grande quantidade de células inflamatórias se infiltrou no coração no grupo DOCA+IMA, enquanto a fibrose estava ausente. Vinte e oito dias após a intervenção com agente, as células do miocárdio estavam regularmente organizadas, e infiltração de células inflamatórias e fibrose grave foram observadas no grupo DOCA. No grupo DOCA+IMA, células miocárdicas estavam regularmente organizadas. Embora não houvesse células inflamatórias infiltrando o coração, não foram encontrados tecidos fibróticos. As células do miocárdio no grupo controle foram normais durante todo o experimento, e não se encontrou infiltração de células inflamatórias nem tecido fibrótico (
O número de células positivas ED-1 reflete a extensão da infiltração de monócitos/macrófagos. A imuno-histoquímica para ED-1 mostrou que o número de células positivas ED-1 nos grupos DOCA e DOCA+IMA era significativamente maior que no grupo CON (grupo DOCA: 6,3 ± 0,4/mm2; grupo DOCA + IMA: 6,0 ± 0,3/mm2; grupo CON: 1,1 ± 0,2/mm2 [
Aos 28 dias da intervenção, a coloração vermelho sirius mostrou que o colágeno do miocárdio vermelho-claro, e os restantes tecidos cardíacos se apresentavam amarelos. A coloração vermelho sirius revelou que fibrose miocárdica no grupo DOCA foi a mais grave entre todos os grupos, caracterizada pelo maior nível de colágeno intersticial. Além disso, a FVC e PVCA no grupo DOCA foram também significativamente maiores quando comparados com os do grupo CON. A FVC e PVCA no grupo DOCA+IMA foram também mais elevadas que no grupo CON, embora acentuadamente menor que no grupo DOCA (
Quando comparado com o grupo CON, as expressões proteicas de PDGF-A, PDGF-C e PDGFRα no grupo DOCA e DOCA+IMA grupo foram notavelmente aumentadas. A expressão proteica de p-PDGFRα no grupo DOCA foi significativamente elevada, mas no DOCA+IMA foi marcadamente diminuída, em comparação com o grupo CON. O ensaio de western blot (
PI e PIII, os índices precisos e diretos da fibrose miocárdica, são os principais componentes de proteína do colágeno. Expressões proteicas aumentadas de fibronectina e tenascina-C também foram encontradas na fibrose miocárdica grave. No presente estudo, as expressões proteicas de PI, PIII, fibronectina e tenascina-C no grupo DOCA foram maiores que nos grupo CON e DOCA+IMA. O ensaio de western blot (
Neste estudo, nossos achados mostraram que: 1) Os ratos com hipertensão induzida por sal-DOCA apresentaram inflamação aparente nas células do miocárdio. Houve mais infiltração de macrófagos no miocárdio, acompanhada por sobre-expressões de PDGF-A, PDGF-C e PDGFRα. Esses achados sugerem que a ocorrência de fibrose miocárdica está intimamente associada com a ativação aumentada da via de sinalização de PDGF; 2) Embora não tenha havido diferença na PAS antes da experiência, a FVC e PVCA no grupo DOCA+IMA foram drasticamente reduzidas quando comparadas com o grupo DOCA, indicando que o Imatinib é capaz de aliviar a fibrose miocárdica independentemente da diminuição da PAS; 3) A expressão proteica de p-PDGFRα diminuiu significativamente no grupo DOCA+IMA quando comparado com o grupo DOCA. Como inibidor do receptor de tirosina-quinase, o Imatinib pode suprimir a atividade de PDGFRα. O efeito inibidor sobre a fibrose miocárdica é possivelmente atribuída à proliferação comprometida de fibroblastos com a inibição da via de sinalização de PDGF; 4) Fibronectina e tenascina-C são considerados índices do grau de fibrose. Os nossos resultados mostraram que as expressões proteicas de fibronectina e tenascina-C foram proporcionalmente relacionadas com o grau de fibrose muscular cardíaca.
Estudos anteriores sugeriram que a fibrose miocárdica induzida por mineralocorticoide foi acompanhada por uma fase inicial de inflamação aguda no miocárdio, sugerindo que a inflamação é um evento inicial na fibrose miocárdica. Além disso, a inflamação crônica em fases média e tardia agrava o processo fibrótico6-8, o que foi confirmado no presente estudo. Nos dias 14 e 28, após a intervenção, a coloração HE revelou a infiltração de células inflamatórias no coração do grupo DOCA. O aumento da infiltração de macrófagos mononucleares durante a inflamação é um passo característico e fundamental na fibrose tecidual9,10. Além disso, cito-hormônios múltiplos e fatores de crescimento secretados por células mononucleares e macrófagos são críticos para a fibrose miocárdica. A remoção de Receptores de Mineralocorticoide (MR) sobre as células mononucleares e macrófagos por técnica de
Os PDGF atuam como fatores de crescimento essenciais, são capazes de promover a divisão e proliferação de fibroblastos e células musculares lisas vasculares, e desempenham um papel quimiotáctico. Achados recentes indicam que os membros da família PDGF participam na fibrose de múltiplos órgãos e tecidos. A inflamação intersticial evidente e a secreção aumentada de PDGF por células inflamatórias (células mononucleares e macrófagos) têm sido observadas na fibrose do pulmão, coração, fígado e rim15-17, acompanhadas pela expressão elevada de PDGFR nos fibroblastos18-21. Algumas investigações sugeriram que a fibrose miocárdica foi associada com a ativação excessiva de vias de sinalização de PDGF e proliferação de fibroblastos elevada22-24. Os membros da família PDGF incluem PDGF-A, PDGF-B, PDGF-C e PDGF-D. PDGF-A e PDGF-C são considerados intimamente relacionados com a fibrose miocárdica25-28. PDGF-AA é a isoforma dimérica de PDGF-A, que tem sido demonstrado ser um mitogênio para os fibroblastos do miocárdio e está implicado na patogênese da fibrose miocárdica29,30. PDGF-C, um ligando recém-descoberto para PDGFRα31,32, também desempenha um papel importante no desenvolvimento da fibrose miocárdica. Os PDGF funcionam ligando-se aos receptores da proteína tirosina quinase transmembrana (PDGFRα e PDGFRβ). As vias de sinalização de PDGF foram divididas em vias de sinalização PDGFRα e PDGFRβ, com base no tipo de PDGFR33. Provou-se que a fibrose miocárdica está relacionada especialmente à via de sinalização PDGFRα5,34. Além disso, a via de sinalização PDGF é especialmente regulada por PDGFR. A inibição do PDGFR pode bloquear completamente as respostas celulares e biológicas mediadas por esta via5,35. Os resultados no presente estudo confirmam os achados antes mencionados. O ensaio de western blot mostrou que as expressões proteicas de PDGF-A, PDGF-C e PDGFR- α nos grupos DOCA e DOCA+IMA foram dramaticamente aumentadas, quando comparadas com o grupo CON, e a expressão proteica de p-PDGFRα foi positivamente associada com a fibrose miocárdica.
Como os PDGF e seus receptores são mediadores importantes para a proliferação, sobrevivência e migração de fibroblastos, os inibidores de PDGFR podem tornar-se agentes promissores para o tratamento da fibrose miocárdica. O Imatinib é um inibidor do receptor tirosina-quinase. Estudos anteriores sugeriram que inibindo-se a transformação de PDGFR em PDGFRα ativado era possível bloquear a via de sinalização de PDGF, prevenindo com o tempo a ocorrência de fibrose miocárdica36-38. Neste estudo, a fibrose do miocárdio e dos tecidos dos vasos sanguíneos circundantes, no grupo DOCA+IMA, foi acentuadamente mitigada, e as expressões proteicas de PI e PIII também diminuíram significativamente se comparado com o grupo DOCA. A fibronectina e tenascina-C são também componentes principais de tecidos fibróticos39,40. Encontrou-se que a tenascina-C está envolvida na adesão entre as células do miocárdio e os tecidos fibróticos, e na regulação do recrutamento de fibroblastos após a lesão do miocárdio. Assim, a tenascina-C tem sido considerada como um novo índice de fibrose miocárdica e remodelação ventricular. Além disso, a tenascina-C pode também ser usada para avaliar a função do ventrículo esquerdo. As expressões proteicas de fibronectina e tenascina-C no grupo DOCA+IMA foram significativamente diminuídas, quando comparadas com o grupo DOCA. Esses achados confirmam que o Imatinib é capaz de inibir a fibrose miocárdica em ratos com hipertensão induzida por mineralocorticoides.
Nossos achados demonstraram que a via de sinalização de PDGF participa na fibrose miocárdica induzida por mineralocorticoide. Além disso, os PDGF desempenham um papel como fatores substitutos de macrófagos. Assim, especula-se que o mineralocorticoide induz a inflamação do miocárdio, ativa MR sobre os macrófagos, e recruta os macrófagos no miocárdio, conduzindo à produção de uma grande quantidade de PDGF. Então, os PDGF ligam-se e ativam o PDGFRα sobre os fibroblastos do miocárdio, o qual promove a infiltração e proliferação de fibroblastos em locais de inflamação e facilita a síntese e secreção de colágeno excessivo, resultando, no final das contas, em fibrose miocárdica22,36. O Imatinib exerce um efeito antifibrose miocárdica, através do bloqueio da via de sinalização de PDGF. Assim, os animais do grupo DOCA+IMA apresentaram inflamação do miocárdio evidente, e expressões elevadas de PDGF-A, PDGF-C, e PDGF- α. Contudo, a fibrose miocárdica grave estava ausente.
O presente estudo confirma que a via de sinalização de PDGF participa na fibrose miocárdica induzida por mineralocorticoide. O Imatinib é capaz de suprimir eficazmente a fibrose miocárdica, inibindo a atividade do PDGFR α e, finalmente, bloqueando a via de sinalização de PDGF.
Agradecemos aos assistentes técnicos do laboratório central da Universidade Médica de Anhui por sua ajuda na imunohistoquímica e ensaio western blot.
Declaro não haver conflito de interesses pertinentes.
O presente estudo não teve fontes de financiamento externas.
Não há vinculação deste estudo a programas de pós-graduação.
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24. Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev. 2004;15(4):237-54.
25. Liao CH, Akazawa H, Tamagawa M, Ito K, Yasuda N, Kudo Y, et al. Cardiac mast cells cause atrial fibrillation through PDGF-A-mediated fibrosis in pressure-overloaded mouse hearts. J Clin Invest. 2010;120(1):242-53.
26. Yang D, Qiu D. Linkage between elevated PDGF-C expression and myocardial fibrogenesis in coxsackievirus B3-induced chronic myocarditis. Eur Heart J. 2005;26(7):642-3.
27. Grün K, Markova B, Böhmer FD, Berndt A, Kosmehl H, Leipner C. Elevated expression of PDGF-C in coxsackievirus B3-induced chronic myocarditis. Eur Heart J. 2005;26(7):728-39.
28. Pontén A, Folestad EB, Pietras K, Eriksson U. Platelet-derived growth factor D induces cardiac fibrosis and proliferation of vascular smooth muscle cells in heart-specific transgenic mice. Circ Res. 2005;97(10):1036-45.
29. Simm A, Nestler M, Hoppe V. Mitogenic effect of PDGF-AA on cardiac flbroblasts. Basic Res Cardiol. 1998;93(Suppl 3):40-3.
30. Simm A, Nestler M, Hoppe V. PDGF-AA, a potent mitogen for cardiac flbroblasts from adult rats. J Mol Cell Cardiol. 1997;29(1):357-68.
31. Li X, Ponten A, Aase K, Karlsson L, Abramsson A, Uutela M, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol. 2000;2(5):302-9.
32. Heldin CH, Eriksson U, Ostman A. New members of the platelet-derived growth factor family of mitogens. Arch Biochem Biophys. 2002;398(2):284-90.
33. Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 2008;22(10):1276-312.
34. Zymek P, Bujak M, Chatila K, Cieslak A, Thakker G, Entman ML, Frangogiannis NG. The role of platelet-derived growth factor signaling in healing myocardial infarcts. J Am Coll Cardiol. 2006;48(11):2315-23.
35. Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev. 2004;15(4):255-73.
36. Chaudhary NI, Roth GJ, Hilberg F, Müller-Quernheim J, Prasse A, Zissel G, et al. Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis. Eur Respir J. 2007;29(5):976-85.
37. Vittal R, Zhang H, Han MK, Moore BB, Horowitz JC, Thannickal VJ. Effects of the Protein Kinase Inhibitor, Imatinib mesylate, on epithelial/mesenchymal phenotypes:Implications for treatment of fibrotic diseases. J Pharmacol Exp Ther. 2007;321(1):35-44.
38. Savikko J, Taskinen E, Von Willebrand E. Chronic allograft nephropathy is prevented by inhibition of platelet-derived growth factor receptor: tyrosine kinase inhibitors as a potential therapy. Transplantation. 2003;75(8):1147-53.
39. Nishioka T, Suzuki M, Onishi K, Takakura N, Inada H, Yoshida T, et al. Eplerenone Attenuates Myocardial Fibrosis in the Angiotensin II-Induced Hypertensive Mouse: Involvement of Tenascin-C Induced by Aldosterone-Mediated Inflammation. J Cardiovasc Pharmacol. 2007; 49(5): 261-8.
40. Eismann U, Sommer M, Kosmehl H, Appenroth D, Fleck C, Stein G. Fibronectin splice variants-prognostic markers for the stage of renal interstitial flbrosis in the rat. Nephron. 2002;92(2):379-88.
Correspondência: Li-kun Ma Department of Cardiology Anhui Provincial Hospital Lujiang Lu, 17 CEP 230001, Hefei – China E-mail:
Artigo recebido em 29/11/11
revisado em 01/12/11
aceito em 22/03/12
BACKGROUND: Imatinib is a tyrosine kinase receptor inhibitor that has been confirmed to exert inhibitory effect on the platelet derived growth factor PDGF receptor (PDGFRα and PDGFRβ) activity. OBJECTIVE: To investigate the protective effect of imatinib on the myocardial fibrosis in deoxycorticosterone-acetate (DOCA)/salt induced hypertensive rats. METHODS: Sixty male uninephrectomized Sprague-Dawley rats were assigned to three groups: control rats (CON group); deoxycorticosterone group (DOCA group); deoxycorticosterone and imatinib group (DOCA+IMA group). Systolic blood pressure (SBP) was measured biweekly. The apical portion of the left ventricle was studied. Sirius-Red staining, Hematoxylin-Eosin staining, immunohistochemistry and Western blot assay were employed. RESULTS: SBP in the DOCA group and DOCA+IMA group was higher than that in the CON group on day 14 and 28. Animals in the DOCA group showed severe interstitial and perivascular fibrosis on day 28, and the expressions of PI, PIII, tenascin-C and fibronectin were significantly higher than those in the DOCA+IMA group and CON group. When compared with the CON group, myocardial tissue inflammatory response and monocyte/macrophage infiltration of different degrees were observed in the DOCA group and DOCA+IMA group. Protein expressions of PDGF-A, PDGF-C and PDGFRα were signiflcantly higher in the DOCA and DOCA+IMA groups than those in the CON group, but the p-PDGFRα protein expression in the DOCA+IMA group was lower than that in the DOCA group. CONCLUSION: Imatinib can exert inhibitory effects on myocardial fibrosis in DOCA/salt induced hypertensive rats, which may be attributed to the inhibition of PDGFR-α activity.
Department of Cardiology, Anhui Provincial Hospital, Hefei – China
Myocardial fibrosis is a common clinical problem, can be found in several heart diseases and is often associated with inflammatory process. In the heart, excessive accumulation of extracellular matrix (ECM) components, especially the type I, III, and V collagens, is responsible for myocardial fibrosis. Changes in tissue structure and cardiac dysfunction following myocardial fibrosis contribute to heart failure, arrhythmia, sudden cardiac death and other serious cardiovascular events. The molecular mechanisms of myocardial fibrosis are complex, on which there are still controversies. Previous studies suggested that myocardial fibrosis is closely related to the increase of endogenous mineralocorticoid. However, the roles of some molecular signaling pathways in mineralocorticoid induced myocardial fibrosis remain unclear. Various cytokines and growth factors have been demonstrated to play crucial roles in the development of myocardial fibrosis. These factors include transforming growth factor ß (TGFß)1, interleukin-1 (IL-1)2, tumor necrosis factor α (TNFα)3, and platelet-derived growth factor (PDGF)4. PDGF is a member of growth factor family and has been found to promote the division and proliferation of fibroblasts and smooth muscle cells. There is evidence showing that high protein expressions of PDGFs may lead to myocardial fibrosis, which suggests that PDGF-PDGFR signaling pathways may serve as important molecular mechanisms of myocardial fibrosis5.
Imatinib is a tyrosine kinase receptor inhibitor that is mainly used in the treatment of tumor in clinic currently and has been confirmed to exert inhibitory effect on the PDGF receptor (PDGFRα and PDGFRβ) activity. PDGF-PDGFR signaling pathways are regulated by PDGFR5 , and thus imatinib can modulate the cell biological response induced by the PDGF-PDGFR signaling pathways. In this study, the effects of imatinib on the myocardial inflammation and fibrogenic response, monocyte/macrophage infiltration, and the protein expressions of PDGF-A, PDGF-C, PDGFRα, p-PDGFRα, procollagen I (PI), procollagen III (PIII), tenascin-C and fibronectin were investigated in deoxycorticosterone-acetate (DOCA)/salt-induced hypertensive rats, aiming to examine whether imatinib can effectively attenuate myocardial fibrosis and explore the relationship between PDGFRα signaling pathway and myocardial fibrosis.
Sixty male Sprague Dawley (SD) rats weighing 200 ~ 250 g were purchased from the Animal Experimental Center affiliated to Anhui Medical University and housed in specific pathogen free environment. Before experiment, animals were anesthetized by intraperitoneal injection of 10% chloral hydrate (400 mg·kg-1) and right nephrectomy was performed. One week after surgery, rats were fed with water containing 1% NaCL and 0.2% KCL, and randomly divided in three groups (n=20 per group): 1) control group (CON group): distilled water was intragastrically administered daily, and subcutaneous injection of sesame oil was given once every 4 days; 2) deoxycorticosterone group (DOCA group): deoxycorticosterone (60 mg.kg-1.4d-1) was subcutaneously given, and distilled water intragastrically administered daily; 3) deoxycorticosterone plus imatinib group (DOCA + IMA group): deoxycorticosterone (60 mg.kg-1.4d-1) was subcutaneously given once every 4 days, and imatinib (60 mg.kg-1.d-1) intragastrically administered daily simultaneously. The volume of distilled water used in the control group was identical to the volume of distilled water containing drugs in the remaining 2 groups. In addition, the volume of sesame oil used in the control group was equivalent to the volume of sesame oil containing drugs in the other 2 groups. Treatment with drugs lasted for 4 weeks.
Deoxycorticosterone, sesame oil (Sigma, U.S.), imatinib (Novartis, Switzerland), Sirius red dye (Beijing Hede Biocompany), hematoxylin, SP immunostaining kits (Beijing Goldenbridge Biotechnology CO., Ltd), ED-1 antibody (Beijing Biosynthesis Biotechnology CO., Ltd), PDGF-A, PDGF-C, PDGFRα, p-PDGFRα, PI, PIII, Fibronectin, Tenascin-C and β-actin primary antibodies and secondary antibodies (Santa Cruz Inc) and ECL kits (Pierce Inc) were used in the present study.
The systolic blood pressure (SBP) was measured on 1d, 14d and 28d by using the tail-cuff method. On the 14th day and 28th day after drug intervention, animals (n=10) were intraperitoneally anesthetized with 10% chloral hydrate (400 mg·kg-1) and then killed. The hearts were obtained, the bilateral atria, right ventricle, great vessels and connective tissues were removed and the apical portion of left ventricle was collected for further examinations. The samples collected on the 14th day were fixed in 4% paraformaldehyde, embedded in paraffin followed by sectioning and immunostaining. The samples obtained on the 28th day were divided into 2: one was fixed in 4% paraformaldehyde for HE staining and Sirius red collagen staining, and the other was stored at - 80 ºC for western blot assay.
Two pathologists who were blinded to the study evaluated the sections. Upon the 14th and 28th days, the apical portion of the left ventricles was subjected to H&E staining for the examination of cardiac inflammation. The infiltration of myocardial macrophages was observed by immunohistochemistry at 14 days after drug intervention. ED-1 is a kind of specific antigen on the surface of monocyte/macrophage. Immunohistochemistry was performed to detect the ED-1 expression in the heart according to manufacturer's instructions. Macrophages with brown membrane were regarded to be positive for ED-1. Ten fields in the positive region were randomly selected at a magnification of ×400. Image-Proplus 6.0 software was employed to count the number of positive cells followed by calculation of the number of macrophage per mm2 visual field. Then, average was obtained for each section.
Myocardial collagen volume faction (CVF) and myocardial peripheral vessel collagen area (PVCA) were measured by picric acid-Sirius red staining on the 28th day. The calculation method was stated below: Nikon photographic system was used, Image-Proplus 6.0 software was employed for analysis. CVF was expressed as the ratio of myocardial collagen area to whole area of visual field. Eight fields (×400) were selected randomly and the corresponding CVFs were measured in each section, and the average was considered as the final CVF for each section. PVCA was expressed as the ratio of peripheral collagen area around the small artery-to-artery lumen area. Four small arteries (×400) were chosen randomly for detection in each section, and the average was considered as the final PVCA for the corresponding section.
The protein expressions of PDGF-A, PDGF-C, PDGFRα, p-PDGFRα, PI, PIII, fibronectin and tenascin-C in the heart were detected by western blot assay on the 28th day after drug intervention. The total protein was extracted using cell lysis buffer(Main components: 20mM Tris (pH7.5), 150mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1mM PMSF, 2mg/mL leupeptin, 1 mM EDTA, sodium pyrophosphate, β-glycerophosphate and Na3VO4), the supernatant containing proteins was collected by centrifugation, and the protein concentration was detected with BCA method. All the samples (One sample stand for one) were diluted into the same concentration. Three samples were randomly selected from each group and compared with those from the remaining two groups. Then, 50 µg of proteins were subjected to polyacrylamide gel electrophoresis, transferred onto PVDF membrane, and blocked in skim milk overnight. Then, the membrane was incubated with primary antibody for 8~12 h and with secondary antibody for 2 h. Visualization was done with ECL kit. The bands were scanned with Bio-Rad imaging system, and Quantity-one software was employed for the detection of optical density. β-actin served as an internal reference. The optical density of target protein was normalized by that of β-actin representing the relative amount of target protein.
SPSS version 13.0 software program was employed for statistical analysis. Data were presented as mean ± standard error (SEM). The means among multiple groups were compared with one-way analysis of variance (ANOVA). The ratios were compared with chi square test. A value of P<0.05 was considered statistically significant.
There was no significant difference in SBP among groups prior to experiment. However, SBP in the DOCA and DOCA+IMA groups were significantly higher than that in the CON group on the 14th and 28th days. No statistical significance in the SBP was noted between DOCA group and DOCA+IMA group regarding levels (
H&E staining revealed evident infiltration of inflammatory cells and mild fibrosis in the heart on the 14th day in the DOCA group. A large amount of inflammatory cells infiltrated the heart in the DOCA+IMA group while fibrosis was absent. Twenty-eight days after drug intervention, myocardial cells were regularly arranged, and infiltration of inflammatory cells and severe fibrosis were noted in the DOCA group. In the DOCA+IMA group, myocardial cells were regularly arranged. Although there were inflammatory cells infiltrating the heart, fibrotic tissues were not found. The myocardial cells in the control group were normal throughout the experiment, and neither infiltrating inflammatory cells nor fibrotic tissues were found (
The number of ED-1 positive cells reflects the extent of monocyte/macrophage infiltration. Immunohistochemistry for ED-1 showed that the number of ED-1 positive cells in the DOCA group and DOCA+IMA group was significantly higher than that in the CON group (DOCA group: 6.3±0.4/mm2; DOCA+IMA group: 6.0±0.3/mm2; CON group: 1.1±0.2/mm2 [
At 28 days after intervention, Sirius red staining showed that the myocardial collagen became light red, and the remaining cardiac tissues presented yellow. Sirius red staining revealed that the myocardial fibrosis in the DOCA group was the most severe among all groups, characterized by the highest level of interstitial collagen. Additionally, the CVF and PVCA in the DOCA group were also significantly increased when compared with those in the CON group. The CVF and PVCA in the DOCA_IMA group were also higher than those in the CON group, whereas markedly lower than those in the DOCA group (
When compared with the CON group, the protein expressions of PDGF-A, PDGF-C and PDGFRα in the DOCA group and DOCA+IMA group were remarkably increased. The p-PDGFRα protein expression in the DOCA group was significantly elevated, but that in the DOCA+IMA group was markedly decreased as compared to the Con group. Western blot assay (
PI and PIII, the accurate and direct indexes of myocardial fibrosis, are the main components of collagen protein. The increased protein expressions of fibronectin and fenascin-C have also been found in severe fibrotic myocardium. In the present study, the protein expressions of PI, PIII, fibronectin and tenascin-C in the DOCA group were higher than those in the CON group and DOCA+IMA group. Western blot assay (
In this study, our findings showed: 1) The DOCA-salt induced hypertensive rats presented apparent inflammation in the myocardial cells. More macrophages infiltrated in the myocardium, accompanied by over-expressions of PDGF-A, PDGF-C and PDGFRα. These findings suggest that the occurrence of myocardial fibrosis is closely associated with increased activation of PDGF signaling pathway. 2) When there was no difference in SBP before experiment, and the CVF and PVCA in the DOCA+IMA group were dramatically decreased when compared with those in the DOCA group, indicating that imatinib is able to relieve myocardial fibrosis independent of lowering SBP. 3) The p-PDGFRα protein expression was significantly declined in the DOCA+IMA group when compared with the DOCA group. As an inhibitor of tyrosine kinase receptor, imatinib can suppress the activity of PDGFRα. The inhibitory effect on myocardial fibrosis is possibly attributed to the compromised proliferation of fibroblasts following inhibition of PDGF signaling pathway. (4) Fibronectin and tenascin-C are regarded as the indexes of the degree of fibrosis. Our results showed the protein expressions of fibronectin and tenascin-C were proportionally related to the degree of cardiac muscular fibrosis.
Previous studies suggested that mineralocorticoid-induced myocardial fibrosis was accompanied by early-stage acute inflammation in the myocardium, suggesting that inflammation is an initial event in the myocardial fibrosis. In addition, chronic inflammation at the mid- and late-stage aggravates the fibrosis process6-8, which had been confirmed in the present study. On the 14th and 28th days after intervention, HE staining revealed the infiltration of inflammatory cells in the heart of the DOCA group. The increased infiltration of mononuclear macrophages during inflammation is a major characteristic and key step in tissue fibrosis9,10. Moreover, multiple cytohormones and growth factors secreted by mononuclear cells and macrophages are critical for the myocardial fibrosis. Removal of mineralocorticoid receptors (MR) on the mononuclear cells and macrophages by knock-out technique or inflammatory effusion and mononuclear macrophage infiltration in drug-dependent manner can significantly alleviate the DOCA-induced myocardial fibrosis8,11,12. Besides, mineralocorticoid is able to regulate the differentiation and maturation of mononuclear cells and macrophages by activating MR on the mononuclear cells and macrophages. Thus, MR inhibitors, such as antisterone, are able to exert anti-inflammation and anti-myocardial fibrosis effects13,14. In this study, the DOCA-treated rats presented obvious myocardial inflammation and increased amount of infiltrated macrophages when compared with the control group. Therefore, it can be reasonably assumed an explicit relationship between mineralocorticoid-induced myocardial fibrosis and subsequent inflammation in the myocardium. The aggravated infiltration of interstitial macrophages is of significance towards the occurrence and development of myocardial fibrosis.
PDGFs act as vital growth factors, are able to promote the division and proliferation of fibroblasts and vascular smooth muscle cells and play a chemotactic role. Recent findings indicate that PDGF family members participate in the fibrosis of multiple organs and tissues. Obvious interstitial inflammation and increased PDGFs secretion by inflammatory cells (mononuclear cells and macrophages) have been observed in the fibrosis of the lung, heart, liver and kidney15-17, accompanied by elevated PDGFR expression on the fibroblasts18-21. Some investigations suggested that myocardial fibrosis was associated with excessive activation of PDGF signaling pathways and elevated proliferation of fibroblasts22-24. PDGF family members includes PDGF-A, PDGF-B, PDGF-C and PDGF-D. PDGF-A and PDGF-C are considered to be closely related to myocardial fibrosis25-28. PDGF-AA is the dimeric isoform of PDGF-A which has been demonstrated as a mitogen for myocardial fibroblasts and is implicated in the pathogenesis of myocardial fibrosis29,30. PDGF-C, a newly discovered ligand for PDGFRα31, 32, also plays an important role in the development of myocardial fibrosis. PDGFs function by binding to the receptors of transmembrane protein-tyrosine kinases (PDGFRα and PDGFRβ). PDGF signaling pathways have been divided into PDGFRα and PDGFRβ signaling pathways based on the type of PDGFR33. It has been proved that myocardial fibrosis is mainly related to the PDGFRα signaling pathway5,34. Besides, PDGF signaling pathway is mainly regulated by PDGFR. The inhibition of PDGFR can completely block the cellular and biological responses mediated by this pathway5,35. Results in the present study confirmed the findings mentioned above. Western blot assay revealed that the protein expressions of PDGF-A, PDGF-C and PDGFR-α in the DOCA group and DOCA + IMA group were dramatically increased when compared with those in the CON group, and p-PDGFRα protein expression was positively associated with the myocardial fibrosis.
Because PDGFs and their receptors are major mediators for the proliferation, survival and migration of fibroblasts, PDGFR inhibitors may become promising drugs for the treatment of myocardial fibrosis. Imatinib is an inhibitor of tyrosine kinase receptor. Previous studies suggested that inhibiting the transformation from PDGFRs into activated PDGFRα was able to block the PDGF signaling pathway, eventually preventing the occurrence of myocardial fibrosis36-38. In this study, the fibrosis of myocardium and tissues surrounding blood vessels in the DOCA + IMA group were markedly alleviated, and the protein expressions of PI and PIII significantly decreased when compared with the DOCA group. Fibronectin and tenascin-C are also major components of fibrotic tissues39,40. Tenascin-C has been found to be involved in the adhesion between myocardial cells and fibrotic tissues, and to regulate the fibroblast recruitment after myocardial injury. Thus, tenascin-C has been regarded as a novel index of myocardial fibrosis and ventricle remodeling. Additionally, tenascin-C can also be used to evaluate the function of left ventricle. The protein expressions of fibronectin and tenascin-C in the DOCA + IMA group were significantly decreased when compared with the DOCA group. These findings confirmed that imatinib is able to inhibit myocardial fibrosis in mineralocorticoid induced hypertensive rats.
Our findings demonstrated that PDGF signaling pathway participates in the mineralocorticoid induced myocardial fibrosis. Additionally, PDGFs play a role as succeeding factors of macrophages. Thus, we speculate that mineralocorticoid induces the myocardial inflammation, activates MR on the macrophages, and recruits the macrophages into the myocardium leading to the production of a large amount of PDGFs. Then, PDGFs bind to and activate PDGFRα on the myocardial fibroblasts, which promotes the infiltration and proliferation of fibroblasts into inflammatory sites and facilitates the synthesis and secretion of excessive collagens, eventually resulting in myocardial fibrosis22,36. Imatinib exert anti-myocardial fibrosis effect through blocking the PDGF signaling pathway. Thus, animals in the DOCA + IMA group presented obvious myocardial inflammation, and elevated expressions of PDGF-A, PDGF-C, and PDGF-α. However, severe myocardial fibrosis was absent.
This study confirms that PDGF signaling pathway participates in the mineralocorticoid induced myocardial fibrosis. Imatinib is able to effectively suppress the myocardial fibrosis by inhibiting the PDGFRα activity and eventually blocking the PDGF signaling pathway.
We appreciate the technical assistants in the central lab of Anhui Medical University for their help in the immunohistochemistry and western-blot assay.
No potential conflict of interest relevant to this article was reported.
There were no external funding sources for this study.
This study is not associated with any post-graduation program.
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15. Ostendorf T, Kunter U, Grone HJ, Bahlmann F, Kawachi H, Shimizu F, et al. Speciflc antagonism of PDGF prevents renal scarring in experimental glomerulonephritis. J Am Soc Nephrol. 2001;12(5):909-18.
16. Eitner F, Ostendorf T, Kretzler M, Cohen CD, Eriksson U, Grone HJ, et al. ERCB-Consortium. PDGF-C expression in the developing and normal adult human kidney and in glomerular diseases. J Am Soc Nephrol. 2003;14(5):1145-53.
17. Leeb SN, Vogl D, Falk W, Scholmerich J, Rogler G, Gelbmann CM. Regulation of migration of human colonic myoflbroblasts. Growth Factors. 2002;20(2):81-91.
18. Bonner JC, Lindroos PM, Rice AB, Moomaw CR, Morgan DL. Induction of PDGF receptor-alpha in rat myoflbroblasts during pulmonary flbrogenesis in vivo. Am J Physiol. 1998;274 (1 Pt 1):L72-80.
19. Lindroos PM, Wang YZ, Rice AB, Bonner JC. Regulation of PDGFR-alpha in rat pulmonary myoflbroblasts by staurosporine. Am J Physiol Lung Cell Mol Physiol. 2001;280(2):L354-62.
20. Pinzani M, Marra F. Cytokine receptors and signaling in hepatic stellate cells. Semin Liver Dis. 2001;21(3):397-416.
21. Bonner JC, Goodell AL, Coin PG, Brody AR. Chrysotile asbestos upregulates gene expression and production of alpha-receptors for platelet-derived growth factor (PDGF-AA) on rat lung flbroblasts. J Clin Invest. 1993;92(1):425-30.
22. Leipner C, Grün K, Müller A, Buchdunger E, Borsi L, Kosmehl H, et a1. Imatinib mesylate attenuates fibrosis in COXsackievirusb3-induced chronic myocarditis. Cardiovasc Res. 2008;79(1):118-26.
23. Leask A. Potential therapeutic targets for cardiac fibrosis: TGF beta, angiotensin, endothelin, CCN2, and PDGF, partners in fibroblast activation. Circ Res. 2010;106(11):1675-80.
24. Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev. 2004;15(4):237-54.
25. Liao CH, Akazawa H, Tamagawa M, Ito K, Yasuda N, Kudo Y, et al. Cardiac mast cells cause atrial fibrillation through PDGF-A-mediated fibrosis in pressure-overloaded mouse hearts. J Clin Invest. 2010;120(1):242-53.
26. Yang D, Qiu D. Linkage between elevated PDGF-C expression and myocardial fibrogenesis in coxsackievirus B3-induced chronic myocarditis. Eur Heart J. 2005;26(7):642-3.
27. Grün K, Markova B, Böhmer FD, Berndt A, Kosmehl H, Leipner C. Elevated expression of PDGF-C in coxsackievirus B3-induced chronic myocarditis. Eur Heart J. 2005;26(7):728-39.
28. Pontén A, Folestad EB, Pietras K, Eriksson U. Platelet-derived growth factor D induces cardiac fibrosis and proliferation of vascular smooth muscle cells in heart-specific transgenic mice. Circ Res. 2005;97(10):1036-45.
29. Simm A, Nestler M, Hoppe V. Mitogenic effect of PDGF-AA on cardiac flbroblasts. Basic Res Cardiol. 1998;93(Suppl 3):40-3.
30. Simm A, Nestler M, Hoppe V. PDGF-AA, a potent mitogen for cardiac flbroblasts from adult rats. J Mol Cell Cardiol. 1997;29(1):357-68.
31. Li X, Ponten A, Aase K, Karlsson L, Abramsson A, Uutela M, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor. Nat Cell Biol. 2000;2(5):302-9.
32. Heldin CH, Eriksson U, Ostman A. New members of the platelet-derived growth factor family of mitogens. Arch Biochem Biophys. 2002;398(2):284-90.
33. Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 2008;22(10):1276-312.
34. Zymek P, Bujak M, Chatila K, Cieslak A, Thakker G, Entman ML, Frangogiannis NG. The role of platelet-derived growth factor signaling in healing myocardial infarcts. J Am Coll Cardiol. 2006;48(11):2315-23.
35. Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev. 2004;15(4):255-73.
36. Chaudhary NI, Roth GJ, Hilberg F, Müller-Quernheim J, Prasse A, Zissel G, et al. Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis. Eur Respir J. 2007;29(5):976-85.
37. Vittal R, Zhang H, Han MK, Moore BB, Horowitz JC, Thannickal VJ. Effects of the Protein Kinase Inhibitor, Imatinib mesylate, on epithelial/mesenchymal phenotypes:Implications for treatment of fibrotic diseases. J Pharmacol Exp Ther. 2007;321(1):35-44.
38. Savikko J, Taskinen E, Von Willebrand E. Chronic allograft nephropathy is prevented by inhibition of platelet-derived growth factor receptor: tyrosine kinase inhibitors as a potential therapy. Transplantation. 2003;75(8):1147-53.
39. Nishioka T, Suzuki M, Onishi K, Takakura N, Inada H, Yoshida T, et al. Eplerenone Attenuates Myocardial Fibrosis in the Angiotensin II-Induced Hypertensive Mouse: Involvement of Tenascin-C Induced by Aldosterone-Mediated Inflammation. J Cardiovasc Pharmacol. 2007; 49(5): 261-8.
40. Eismann U, Sommer M, Kosmehl H, Appenroth D, Fleck C, Stein G. Fibronectin splice variants-prognostic markers for the stage of renal interstitial flbrosis in the rat. Nephron. 2002;92(2):379-88.
Mailing Address: Li-kun Ma Department of Cardiology Anhui Provincial Hospital Lujiang Lu, 17 Postal Code 230001, Hefei – China E-mail:
Manuscript received November 29, 2011
manuscript revised December 1, 2011
accepted March 22, 2012