Single-target RNA interference for the blockade of multiple interacting proinflammatory and profibrotic pathways in cardiac fibroblasts.
Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals.
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Juliane
J
Tank
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Diana
D
Lindner
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Xiaomin
X
Wang
Institute for Biometry and Clinical Epidemiology, CBF, Germany.
Andrea
A
Stroux
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Leona
L
Gilke
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Martina
M
Gast
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Christin
C
Zietsch
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Carsten
C
Skurk
Institute for Medical Immunology, CCM, Charité - Universitätsmedizin Berlin, Berlin, Germany; Berlin Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
Carmen
C
Scheibenbogen
Dept. of Molecular Pathology, Institute for Pathology, University Hospital, Tübingen, Germany.
Karin
K
Klingel
Institute for Clinical Diagnostics and Therapy, Berlin, Germany.
Dirk
D
Lassner
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Uwe
U
Kühl
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Heinz-Peter
HP
Schultheiss
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany.
Dirk
D
Westermann
Dept. of Cardiology and Pneumology, CharitéCentrum11 (Cardiovascular Medicine), CBF, Germany; Berlin Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany. Electronic address: Hwolfgang.poller@charite.de.
Wolfgang
W
Poller
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D054410:Chemokine CCL7 / Q000235:genetics / Q000378:metabolism; D054412:Chemokine CCL8 / Q000235:genetics / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism* / Q000473:pathology; D005355:Fibrosis / Q000517:prevention & control; D005786:Gene Expression Regulation; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D007249:Inflammation / Q000517:prevention & control; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D058990:Molecular Targeted Therapy; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
C526317:CCN2 protein, human; C526318:CCN2 protein, mouse; C515451:Ccl7 protein, mouse; C515454:Ccl8 protein, mouse; D054410:Chemokine CCL7; D054412:Chemokine CCL8; D034741:RNA, Small Interfering; D055513:Connective Tissue Growth Factor; D020778:Matrix Metalloproteinase 2; C522362:Mmp2 protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse
AdV; CAR; CCN1; CCN2; CCN3; CCN4; CCN5; CCN6; CVB3; Ccl2; Ccl7; Ccl8; Coxsackievirus B3; Coxsackievirus-Adenovirus-Receptor; Cx43; EMB; FCS; Fibrosis; Heart disease; IL10; Immuno-therapy; Inflammation; LVEDD; LVEF; MCP-2; MCP-3; MI; MMP2; MOI; OPN; PCFB; RNA interference; RNAi; Remodeling; WNT-inducible signaling pathway protein-1 (WISP1); WNT-inducible signaling pathway protein-2 (WISP2); WNT-inducible signaling pathway protein-3 (WISP3); adenoviral vector; chemokine C-C motif ligand 2=MCP-1; connective tissue growth factor (CTGF); connexin 43; cysteine-rich angiogenic inducer 61 (Cyr61); endomyocardial biopsy; fetal calf serum; interleukin 10; left ventricular ejection fraction; left ventricular end-diastolic diameter; matrix metalloproteinase 2; miR; microRNA; multiplicity of infection; myocardial infarction; neuroblastoma overexpressed (Nov); osteopontin; pfu; plaque forming units; primary cardiac fibroblast; qRT-PCR; quantitative reverse transcription PCR; shRNA; short hairpin RNA