Background/Purpose:

One mechanism by which anti-Ro antibodies are linked to the pathogenesis of (cardiac-NL) neonatal lupus is the increased urokinase plasminogen activator (uPA)/urokinase-type plasminogen activator receptor (uPAR)-dependent plasminogen activation and subsequent triggering of TGFbeta signaling. Immunologic staining of affected hearts reveals cell specific expression of uPA on endothelial cells and infiltrating macrophages  in areas  of inflammation and fibrosis. Since plasminogen/plasmin-dependent TGFbeta signaling has a documented role in endothelial cell survival, this study evaluated the association of anti-Ro and an injury pathway in the fate of the cardiac vasculature. 

Methods:

Adhesion, tube formation, TGFbeta activation assays were used to assess the effect of antibodies on endothelial cell function. Immunohistology of cardiac-NL autopsies were conducted to evaluate diseased vasculature. 

Results:

Human IgGs were used from a healthy control (nl-IgG) and a mother whose serum contained reactivity to all the components of the SSA/Ro-SSB/La complex and whose child had cardiac-NL (CHB-IgG).  Adherence was enumerated (colorimetric dye) after vascular endothelial cells (CD3 cell line) were incubated with CHB-IgG (0.03 mg/ml, 2 hr) or nl-IgG, with or without plasmin inhibitor aprotinin (10 μg/ml) or TGFbeta inhibitor SB5543 (10μM). CHB-IgG coincubation significantly reduced EC adhesion on ECM (collagen-coated surfaces) compared with nl-IgG (0.38±0.1 vs 0.89±0.09 respectively (adhesion units, scale 0-3), p=0.02, n=3).  The CHB-IgG decreased adhesion was reversed in the presence of either aprotinin or SB5543. Further evidence to implicate a plasmin/TGFbeta axis was provided by the observation that ECs treated with CHB-IgG, but not nl-IgG, increased luciferase activation of a TGFbeta reporter cell line (TMLC) (432±20 vs 138±12 RLU respectively,p = 0.003;n = 3), an effect abrogated by cotreatment with aprotinin or SB5543. Vessel formation was evaluated by visual inspection of networks after ECs were plated on matrigel (8 hr). Exposure of ECs to CHB-IgG but not nl-IgG markedly attenuated blood vessel formation in vitro which again was reversed by cotreatments with aprotinin and SB5543 as observed in the adhesion studies (n=4).  As further proof of concept, during migration of ECs on collagen-coated surfaces, confocal microscopy revealed colocalization of uPAR with anti-Ro60  (but not anti-Ro52 or anti-RNP) at the tips of migrating cells. Evaluation in vivo, showed that in a fetal CHB heart, protein expression of CD31 (a surface marker of endothelium lining blood vessels) was at a substantially lower level in septal regions with involved inflammation when compared with a healthy zone of atrial tissue. 

Conclusion:

These results suggest that anti-SSA/Ro interference during the remodeling events occurring in angiogenesis results in increased uPAR-dependent uPA activity with generation of active TGFbeta.  This then leads to the attenuation of adhesion and new vessel development and ultimately a loss of vasculature in the septal region of affected hearts.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-ssaro-mediated-injury-to-the-endothelium-via-urokinase-plasminogen-activator-receptortgfbeta-activation-implications-in-the-pathogenesis-of-congenital-heart-block/