Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.

A Novel Role of Endothelin-1 in Linking Ro60-ssRNA Immune Complexes to Cardiac Fibrosis in Congenital Heart Block.

Alvarez-Carbonell4,  David, Zavadil5,  Jiri, Abellar1,  Rosanna, Barrat2,  Franck, Clancy3,  Robert, Buyon5,  Jill

Columbia University Medical Center
Dynavax Technologies
New York University School of Mediciine
New York University School of Medicine, New York, NY
New York University School of Medicine

Passively acquired anti-Ro associated congenital heart block (CHB) likely results from pathologic crosstalk between inflammatory and fibrosing pathways eventuating in scarring. The target, Ro60, complexed with uridine rich ssRNA induces TLR7-dependent macrophage production of supernatants capable of trans-differentiating human fetal cardiac fibroblasts. This study addressed the molecular components responsible for the TLR-dependent profibrosing effects. In seeking the link between an inflammatory response of macrophages and the profibrotic effect on fibroblasts, a microarray analysis comparing the mRNA expression profile of macrophages stimulated with hY3 (ssRNA associated with Ro60) or immune complexes consisting of Ro60-hY3-IgG fraction containing anti-Ro60 antibodies (IC) in the presence or absence of IRS661 (antagonist of TLR7) was performed. Gene expression of the vasoconstrictor endothelin-1 (ET-1), recently shown to promote dermal fibrosis, was significantly up-regulated by ~ 4 fold and confirmed by RT-PCR. Incubation of macrophages with either hY3 or IC increased secretion of ET-1 from 2.64 pg/mL (baseline) to 9.67 pg/mL (p <.0001) and 7.52 pg/mL (p =.0003) respectively. Pre-treatment with IRS661 decreased hY3-induced secretion to 3.83 pg/mL and IC to 3.98 pg/mL. The direct effect of ET-1 (100 nM) on these fibroblasts (shown to express both ETa and ETb receptors) resulted in a profibrosing phenotype as demonstrated by a) TGFbeta secretion (13 pg/mL vs. 772 pg/mL, p =.03), b) increased collagen release (18 ng/mL baseline vs. 772 ng/mL, p =.05, and c) expression of a-smooth muscle actin (a-smac). ET-1-induced TGFbeta secretion was significantly decreased (p =.03) by each of the following: BQ-123, an ETa antagonist (119 pg/mL), BQ-788, an ETb antagonist (145 pg/mL), and anti-ET-1 antibody (211 pg/mL), but not isotype control antibody (691 pg/mL). Similarly, ET-1-induced collagen secretion was significantly decreased (p =.05) by BQ-123 (147 ng/mL collagen), BQ-788 (247 ng/mL), anti-ET-1 antibody (253 ng/mL), and anti-TGFbeta antibody (239 ng/ml), but not isotype control (815 ng/mL). Predictably, pretreatment of fibroblasts with either BQ-123, BQ-788 or incubation of supernatants with anti-ET-1 antibody, but not isotype control, significantly inhibited the profibrosing readouts (increased TGFbeta, collagen and a-smac) induced by supernatants from macrophages stimulated with either hY3 or IC. Additionally, fibroblasts transfected with either ETa or ETb siRNA, but not scrambled siRNA, also significantly inhibited the profibrosing readouts induced stimulated macrophages. Immuno-histochemistry of a heart from a fetus dying with CHB revealed ET-1/2/3 antibody staining but not isotype IgG control in the septal region in areas showing calcification, fibrosis, and mononuclear cell infiltrates (not seen in an age-matched fetal heart). In conclusion, these data suggest that macrophage secretion of ET-1 may be one mechanism linking TLR inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB.

To cite this abstract, please use the following information:
Alvarez-Carbonell, David, Zavadil, Jiri, Abellar, Rosanna, Barrat, Franck, Clancy, Robert, Buyon, Jill; A Novel Role of Endothelin-1 in Linking Ro60-ssRNA Immune Complexes to Cardiac Fibrosis in Congenital Heart Block. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :2231
DOI: 10.1002/art.29995

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