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SP0164 Inflammatory-Driven Myocardial Fibrosis – the Role of Inflammatory Progenitors
  1. G. Kania
  1. Bio-Technopark Zürich Schlieren, Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zürich, Schlieren, Switzerland


Background Heart-specific inflammation-myocarditis is a common cause of pathological tissue remodelling and heart failure with the phenotype of inflammatory dilated cardiomyopathy (iDCM). During the last years there was a shift in systemic sclerosis (SSc)-related causes of death, indicating iDCM as a major cause of death in these patients. Despite the high unmet clinical need, so far little is known about the etiology of iDCM and the mechanisms leading to heart dysfunction in SSc patients.

Objective The mouse model of experimental autoimmune myocarditis (EAM) mimics human iDCM. We used this model to study mechanistic aspects of the progression from acute cardiac inflammation into iDCM. Further, we aimed to determine the role of bone marrow-derived inflammatory progenitors in myocardial fibrogenesis and pathological remodelling in iDCM and SSc-iDCM.

Methods Alpha-myosin heavy chain peptide/complete Freund's adjuvant immunization was used to induce EAM in wild-type and Nitric oxide synthase 2-deficient (Nos2 –/–) BALB/c mice. Chimeric mice, reconstituted with bone marrow from enhanced green fluorescence protein (EGFP)+ mice were used to track the fate of inflammatory progenitor cells. Inflammatory CD133+ progenitors were isolated from inflamed hearts, cultured in vitro and injected intracardially at different stages of EAM. In vitro, inflammatory CD133+ progenitors were differentiated into myofibroblasts with Transforming Growth Factor (TGF)-b, and into macrophages with Macrophage-Colony Stimulating Factor (M-CSF). Cells were also treated with Wnt proteins or their inhibitors, or co-cultured with STF reporter cells expressing luciferase under the control of TCF/LEF transcriptional reporter element. CD45+/CD14+ monocytes were isolated from the blood of SSc patients and healthy subjects.

Results Myocarditis peaked 21 days after immunization and numbers of pathological cardiac myofibroblasts progressively increased during follow-up. In chimeric mice, >60% of cardiac myofibroblasts were EGFP+ 46 days after immunization, indicating their bone marrow origin. At day 21, cardiac infiltrates contained about 30% of inflammatory CD133+ progenitors and only a small subset expressed the macrophage-specific antigen F4/80. CD133+, but not CD133 cells, isolated from acutely inflamed hearts represented the cellular source of cardiac myofibroblasts at late stages of EAM. Mechanistically, in vitro myofibroblast differentiation of inflammatory CD133+ progenitors depended on TGF-b-mediated phosphorylation of Smad proteins and activation of Wnt signalling. TGF-β initiated myofibroblast differentiation of inflammatory CD133+ progenitors and cardiac gp38+ fibroblasts via the canonical SMAD2/3-dependent pathway. In both myofibroblast cell sources, TGF-β activated both the canonical Wnt pathway (Wnt-1, Wnt-10b) that resulted in nuclear translocation of b-catenin, as well as the non-canonical pathway (Wnt-5a, Wnt-11) leading to an activation of c-Jun N-terminal kinases. Wnt secretion was crucial for myofibroblasts differentiation, and together with TGF-β controlled Wnt(s) transcription. Moreover, canonical and non-canonical Wnt signalling was activated in the mouse myocardium during EAM, and in the myocardium of patients with acute myocarditis. Blocking of Wnt activity with chemical and natural Wnt inhibitors (Sfrp-2, ICG-001, FH-535) lead to a reduction of TGF-β-mediated myofibroblast differentiation of both cell types in vitro, and prevented the formation of inflammatory-driven cardiac fibrosis in vivo. Finally, TGF-β induced myofibroblast differentiation of human blood-derived CD14+ monocytes via activation of canonical and non-canonical Wnt pathways.

Similarly, anti-TGF-b antibody treatment prevented myocardial fibrosis in immunized mice, and inhibited myofibroblast differentiation of inflammatory CD133+ cells via suppression of Wnt(s) transcription and secretion. CD133+/F4/80hi cells showed an impaired myofibrogenic potential compared to CD133+/F4/80 cells. M-CSF treatment of wild-type but not Nos2 –/– mice with EAM markedly increased CD133+/F4/80hi cells in the myocardium, and CD133+ progenitors isolated from M-CSF-treated wild-type mice failed to differentiate into myofibroblasts.

Conclusions We showed that an induction of macrophage lineage differentiation by M-CSF abrogates the TGF-b-mediated myofibrogenic differentiation potential of heart-infiltrating inflammatory CD133+ progenitors in a NOS2 dependent manner. Moreover, our data revealed novel mechanistic aspects of TGF-β in the Wnt-dependent myofibroblast differentiation pathway. A better understanding of the nature of inflammatory progenitors and the mechanisms triggering cardiac fatal fibrosis in iDCM and SSc might be helpful in developing novel and effective therapies targeting a broad spectrum of pathologic and inflammatory-mediated processes.

Disclosure of Interest None declared

DOI 10.1136/annrheumdis-2014-eular.6117

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