Binding of anti-SSA antibodies to apoptotic fetal cardiocytes stimulates urokinase plasminogen activator (uPA)/uPA receptor-dependent activation of TGF-β and potentiates fibrosis

Abstract

In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-β activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-β, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-β compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-β Ab or TGF-β inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-β dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-β activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-β activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar.

Figure 1. CHB-IgG treated apoptotic cardiocytes activate latent TGF beta

(A) TMLC-based assessment of the luciferase activity generated by incubation of apoptotic cardiocytes in the presence or absence of latent TGF beta (1 ng/ml). Y axis represents the relative luciferase units (RLU) and x axis each experimental condition of apoptotic cardiocytes (either untreated or treated with nl-IgG, CHB-IgG, CHB-IgG+ SB431542 or CHB-IgG+aprotinin respectively) in the presence of 1 ng/ml of latent TGF beta. (B) TMLC-based assessment of the luciferase activity generated by incubation of nl-IgG, Ro60-IgG, mouse isotype or anti-HLA-treated apoptotic cardiocytes in the presence or absence of latent TGF beta (1 ng/ml). (C) TMLC-based assessment of the luciferase activity generated by incubation of nl-IgG or CHB-IgG treated healthy cardiocytes in the presence of latent TGF beta (1 ng/ml). Sample number of independent experiments is indicated and error bars represent ±SEM of the mean. p values are given above the experimental conditions and considered significant when less that 0.05.

Figure 2. Cocultures of healthy cardiocytes with CHB-IgG bound apoptotic cardiocytes activates TGF beta

(A) TMLC-based assessment of the luciferase activity generated by cocultures of healthy cardiocytes with apoptotic cardiocytes or apoptotic cardiocytes alone. Y axis represent relative luciferase units (RLU). (B) TMLC-based assessment of the luciferase activity generated by cocultures of healthy cardiocytes with apo-nl-IgG or apo-CHB-IgG cardiocytes or antibody treated cardiocytes alone. (C) TMLC-based assessments of the luciferase activity generated by cocultures of healthy cardiocytes with apoptotic cardiocytes treated with either nl-IgG or different IgG fractions CHB-IgG sera (CHB1, CHB2, CHB3). (D) Inhibition of TGF beta luciferase activation of TMLC cells exposed to supernatants of cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes in the presence of either pan anti-TGF beta antibody or small molecule TGF beta inhibitor (SB431542). Sample number of independent experiments is indicated and error bars represent ±SEM of the mean. p values are given above the experimental conditions and considered significant when less that 0.05.

Figure 3. Anti-Ro dependent uPA/uPAR plasminogen activation is responsible for the generation of active TGFbeta

(A) A chromogenic enzymatic activity assay was used to evaluate plasminogen activation of supernatants generated from cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes or apo-CHB-IgG cardiocytes subsequently treated with antibodies against uPA, uPAR or incubated in the presence of aprotinin (10 μg/ml). (B) Inhibition of TGF beta luciferase activation of TMLC cells exposed to supernatants of cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes or apo-CHB-IgG cardiocytes subsequently treated with antibodies against uPA, uPAR or incubated in the presence of aprotinin (10 μg/ml g/ml). (C) Contribution of plasminogen to the CHB-IgG mediated TGF beta luciferase activation. Supernatants of cocultures of healthy cardiocytes with apo-nl-IgG or apo-CHB-IgG cardiocytes conducted in the presence or absence of plasminogen containing media were added to the TMLC cells and after 24 hr incubation luciferase activity was determined. (D) Contribution of cell contact in CHB-IgG mediated TGF beta luciferase activation. Healthy cardiocytes were cocultured with apo-nl-IgG or apo-CHB-IgG cardiocytes either together or separated with cell culture plate inserts to inhibit direct cell contact. After overnight incubation supernatants were collected and added to TMLC cells. Y axis represents relative luciferase units (RLU). Sample number of independent experiments is indicated and error bars represent ±SEM of the mean. p values are given above the experimental conditions and considered significant when less that 0.05.

Figure 4. Total TGF beta levels are decreased but uPA levels are increased in cocultures of apoptotic of CHB-IgG bound cardiocytes with healthy

(A) 100 μl of supernatants (healthy cardiocytes, cocultures of healthy cardiocytes with apo-nl-IgG or apo-CHB-IgG cardiocytes, apo-CHB-IgG alone and apo-nl-IgG cardiocytes alone) were acid treated with the addition of 1 N HCL in order to measure total levels (active plus latent) of TGF beta. Total TGF beta was then determined by using an enzyme-linked immunosorbent assay system ELISA (R&D Systems). (B) Total uPA levels were measured in supernatants (100 μl) of either healthy cardiocytes, cocultures of healthy cardiocytes with apo-nl-IgG cardiocytes or cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes by enzyme-linked immunosorbent assay (American Diagnostica). Error bars represent ±SEM and p values and experimental sample numbers are indicated.

Figure 5. Supernatants from cocultures of healthy cardiocytes and CHB-IgG bound apoptotic cardiocytes trigger activation of TGF beta pathway as depicted by increased phospho-Smad2 phosphorylation

Fibroblasts were prepared as monolayers and seeded on collagen-treated culture slides. Following overnight serum starvation cells were treated with supernatants from healthy cardiocyte cultures, supernatants from cocultures of healthy cardiocytes with isotype control treated apoptotic cardiocytes, cocultures of healthy cardiocytes with anti-uPAR treated apoptotic cardiocytes, cocultures of healthy cardiocytes with apo-nl-IgG cardiocytes, cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes, cocultures of healthy cardiocytes with initially apo-nl-IgG and then anti-uPAR cardiocytes, cocultures of healthy cardiocytes with initially apo-CHB-IgG and then anti-uPAR cardiocytes cocultures of healthy cardiocytes with mouse anti-uPAR treated apoptotic cardiocytes, cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes in the presence of SB431542, or treated with recombinant TGFbeta (100 pg/ml). Cell lysates were collected and subjected to electrophoresis followed by immunoblotting for anti- phospho-Smad2. Equal loading was confirmed with anti-tubulin.

Figure 6. Supernatants from cocultures of healthy cardiocytes and CHB-IgG bound apoptotic cardiocytes promote transdifferentiation of human fetal cardiac fibroblasts as depicted by increased smooth muscle actin (SMAc) staining

Fibroblasts were prepared as monolayers and seeded on collagen-treated culture slides. Following overnight serum starvation cells were treated with recombinant TGF beta (100 pg/ml)(A) or exposed to supernatants from healthy cardiocyte cultures (B), supernatants from cocultures of healthy cardiocytes with isotype control treated apoptotic cardiocytes (C), cocultures of healthy cardiocytes with anti-uPAR treated apoptotic cardiocytes (D), cocultures of healthy cardiocytes with apo-nl-IgG cardiocytes (E), cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes (F), cocultures of healthy cardiocytes with initially apo-nl-IgG and then anti-uPAR cardiocytes (G), cocultures of healthy cardiocytes with initially apo-CHB-IgG and then anti-uPAR cardiocytes cocultures of healthy cardiocytes with mouse anti-uPAR treated apoptotic cardiocytes (H), cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes in the presence of SB431542 (I). Fibroblasts were then fixed, stained and analyzed by fluorescence microscopy (original magnification X40). Results are representative of three experiments.

Figure 7. Supernatants from cocultures of healthy cardiocytes and CHB-IgG bound apoptotic cardiocytes promote fibroblast transdifferentiation as demostrated by increased smooth muscle actin (SMAc) expression

Fibroblasts were prepared as monolayers and seeded on collagen-treated culture slides. Following overnight serum starvation cells were treated with supernatants from healthy cardiocyte cultures, supernatants from cocultures of healthy cardiocytes with isotype control treated apoptotic cardiocytes, cocultures of healthy cardiocytes with anti-uPAR treated apoptotic cardiocytes, cocultures of healthy cardiocytes with apo-nl-IgG cardiocytes, cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes, cocultures of healthy cardiocytes with initially nl-IgG and then anti-uPAR treated apoptotic cardiocytes, cocultures of healthy cardiocytes with initially apo-CHB-IgG and then anti-uPAR cardiocytes cocultures of healthy cardiocytes with mouse anti-uPAR treated apoptotic cardiocytes, cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes in the presence of SB431542, or treated with recombinant TGF beta (100 pg/ml). Cell lysates were collected and subjected to electrophoresis followed by immunoblotting for anti-SMAc. Equal loading was confirmed with anti-tubulin.

Figure 8. Supernatants from cocultures of healthy cardiocytes and CHB-IgG bound apoptotic cardiocytes promote transdifferentiation of human fetal cardiac fibroblasts as depicted with increased collagen staining (Col1A) staining

Fibroblasts were prepared as monolayers and seeded on collagen-treated culture slides. Following overnight serum starvation cells were treated with recombinant TGF beta (100 pg/ml)(A) or exposed to supernatants from healthy cardiocyte cultures (B), supernatants from cocultures of healthy cardiocytes with isotype control treated apoptotic cardiocytes (C), cocultures of healthy cardiocytes with anti-uPAR treated apoptotic cardiocytes (D), cocultures of healthy cardiocytes with apo-nl-IgG cardiocytes (E), cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes (F), cocultures of healthy cardiocytes with initially apo-nl-IgG and then anti-uPAR cardiocytes (G), cocultures of healthy cardiocytes with initially apo-CHB-IgG and then anti-uPAR cardiocytes cocultures of healthy cardiocytes with mouse anti-uPAR treated apoptotic cardiocytes (H), cocultures of healthy cardiocytes with apo-CHB-IgG cardiocytes in the presence of SB431542 (I). Fibroblasts were then fixed, stained and analyzed by fluorescence microscopy (original magnification X40). Results are representative of three experiments.