Presentation: Role of Hypoxia and cAMP in the Transdifferentiation of Human Fetal Cardiac Fibroblasts in Amplifying the Scarring Effect of Anti-Ro Antibodies in Congenital Heart Block (2007)

143 Role of Hypoxia and cAMP in the Transdifferentiation of Human Fetal Cardiac Fibroblasts in Amplifying the Scarring Effect of Anti-Ro Antibodies in Congenital Heart Block

Identification of congenital heart block (CHB) predicts with near certainty that the maternal serum reacts with SSA/Ro; however, the 2% incidence in first offspring of anti-Ro+ mothers, 20% recurrence, and discordance in identical twins suggest that an environmental factor amplifies the effect of antibody. Accordingly, this study explored the hypothesis that hypoxia potentiates a profibrosing phenotype of the fetal fibroblast which may be more exaggerated in cardiac (affected organ) compared to pulmonary (unaffected organ) fibroblasts. Hypoxic insult was supported by prominent intracellular fibroblast expression of hypoxia-inducible factor-1α (HIF-1α) in fibrotic regions of conduction tissue from 2 fetuses dying with CHB. Downstream HIF-1α signaling was suggested by intra and extracellular expression of plasminogen activator inhibitor -1 ( PAI-1), findings absent in a normal 23-wk heart. Sustained hypoxia was evidenced by significantly elevated erythropoietin in cord blood from 31 CHB- versus 36 unaffected anti-Ro-exposed neonates (mean = 1290 vs 29, p = .008). Having demonstrated in vivo surrogates of hypoxic insult, in vitro experiments addressed functional consequences with regard to amplification of fibrosis. Hypoxic effects were evaluated in fibroblasts isolated from hearts and lungs of electively terminated human abortuses ( 3 sets from same donor, 11 separate cardiac fibroblast donors). Incubation of cardiac fibroblasts with 10 ng/ml TFGβ or exposure to hypoxia (<0.1% O2), resulted in scarring myofibroblasts as evidenced by increased expression of smooth muscle actin (SMAc; immunoblot, immunofluorescence). In contrast, hypoxia did not induce SMAc in the lung fibroblasts. Gene expression profiling (Affymetrix arrays, QPCR) was exploited to identify additional genes which might provide clues to the molecular basis for this amplification of the fibroblast-contribution to CHB. Hypoxia-exposed cardiac fibroblasts expressed 4-fold increases in adrenomedullin compared to normoxia, focusing attention on cAMP as a modulator of fibrosis. Under normoxia, incubation with the adenylate cyclase inhibitor, MDL12 30 μM (lowers cAMP) increased expression of fibrosis-related proteins (mTOR, SMAc, PAI-1, collagen-1) while forskolin 10 μM (raises cAMP) markedly supressed TGFβ-elicited fibrosing endpoints in the cardiac but not lung fibroblasts (immunofluorescence,immunoblot). Furthermore, forskolin inhibited cardiac fibroblast SMAc expression induced by supernatants secreted from macrophages inbubated with anti-Ro opsonized apoptotic cardiocytes. These data suggest differential scarring potential of fibroblasts as a function of origin and provide evidence that hypoxia may amplify the injurious effects of anti-Ro. Exploiting susceptibility of the cardiac fibroblast to cAMP modulation offers a new research direction in CHB.

 M. O'Mahoney, None; P. Izmirly, SLE Foundation Inc, New York, Extended Fellowship, 2; P. Zheng, None; L. Gardner, None; J. Zavadil, None; R.M. Clancy, Sjogren's Syndrome Foundation Grant, 2; J.P. Buyon, NIH Grant AR-42455 and N01-AR-4-2271, March of Dimes Birth Defects Grant, 2.