Autoantibodies Targeting AT1- and ETA-Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor

Abstract

Scleroderma renal crisis (SRC) is an acute life-threatening manifestation of systemic sclerosis (SSc) caused by obliterative vasculopathy and thrombotic microangiopathy. Evidence suggests a pathogenic role of immunoglobulin G (IgG) targeting G-protein coupled receptors (GPCR). We therefore dissected SRC-associated vascular obliteration and investigated the specific effects of patient-derived IgG directed against angiotensin II type 1 (AT1R) and endothelin-1 type A receptors (ETAR) on downstream signaling events and endothelial cell proliferation. SRC-IgG triggered endothelial cell proliferation via activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent activation of the E26 transformation-specific-1 transcription factor (Ets-1). Either AT1R or ETAR receptor inhibitors/shRNA abrogated endothelial proliferation, confirming receptor activation and Ets-1 signaling involvement. Binding of Ets-1 to the tissue factor (TF) promoter exclusively induced TF. In addition, TF inhibition prevented endothelial cell proliferation. Thus, our data revealed a thus far unknown link between SRC-IgG-induced intracellular signaling, endothelial cell proliferation and active coagulation in the context of obliterative vasculopathy and SRC. Patients’ autoantibodies and their molecular effectors represent new therapeutic targets to address severe vascular complications in SSc.

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation

“…Under our experimental in vitro conditions, the proliferating effect of Ang II on HMEC-1s was very limited and more pronounced for AT 1 R-Abs ( Figure 1 B). Although it is possible that the previously observed differences between the two stimuli resulted from the effects of AT 2 R with native HMEC-1s (which showed no proliferation with Ang II [ 25 ]), AT 2 R anti-proliferative effects (as described in vascular smooth muscle cells (VSMCs) [ 74 ]) are here very unlikely due to AGTR1 mRNA being 100,000-fold more expressed than AGTR2 mRNA ( Supplementary Materials Figure S1 ). Furthermore, we recently showed that AT 2 R inhibitors fail to block SSc antibodies-induced endothelial cell proliferation [ 25 ].…”

“…Although it is possible that the previously observed differences between the two stimuli resulted from the effects of AT 2 R with native HMEC-1s (which showed no proliferation with Ang II [ 25 ]), AT 2 R anti-proliferative effects (as described in vascular smooth muscle cells (VSMCs) [ 74 ]) are here very unlikely due to AGTR1 mRNA being 100,000-fold more expressed than AGTR2 mRNA ( Supplementary Materials Figure S1 ). Furthermore, we recently showed that AT 2 R inhibitors fail to block SSc antibodies-induced endothelial cell proliferation [ 25 ]. In any case, further complementary studies are still required to determine the exact difference between Ang II- and antibodies-induced endothelial cell proliferation.…”

“…Diagnosis was confirmed by renal biopsy that assessed obliterative vasculopathy of arteries and arterioles in all cases. Moreover, all SRC individuals were tested for the presence of AT 1 R antibodies, as previously described (CellTrend GmbH, Luckenwalde, Germany) [ 25 , 26 , 61 ]. Patients were considered positive if their antibody level reached at least 17 U/mL [ 26 ].…”

“…The local Medical Ethics Committee approved the study protocol (EA1/013/705). All experiments were conducted with immunoglobulin G (IgG) extracted from patient plasma samples (AT 1 R-Abs), as previously described [ 14 , 25 , 26 ]. Based on the results obtained with the MMY yeast model in which AT 1 R was the sole GPCR expressed (see the description of the experimental model below), where AT 1 R-Abs dose-dependently activated AT 1 Rs ( Figure 5 B), we decided to perform the experiments with the smallest dose of 1 mg/mL activating AT 1 R.…”

“…Binding of AT 1 R-Abs promotes downstream signaling through the activation of AT 1 R [ 14 , 25 , 26 ]. While Ang II receptor binding has been explored in detail [ 27 , 28 , 29 , 30 , 31 ], and a receptor–ligand complex structure (PDB ID: 6oS0 [ 32 ]) is known, the detailed molecular mechanisms of the antibody–receptor interaction and associated effects are still unknown, hampering a comprehensive understanding of AT 1 R-Abs-related pathogenesis at the molecular level.…”