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

Abstract

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis.

Complement component C3 and C5b‐9 deposition on hypoxia reperfused endothelial cells by non‐HLA antibodies against RhoGDI2: A player involved in graft failure?

“…A limitation of our study is that only one MoAb has been tested. It may be that the antibody will be more effective at higher antibody concentrations, as in some papers even concentrations up to 1 mg/ml are used, 22 but we question whether such antibody concentrations are clinically relevant as we found that anti‐RhoGDI2 antibody concentrations in patients are around 10 μg/ml. Patient sera containing high anti‐RhoGDI2 antibody titers added to starved endothelial cells seem to activate complement, but the usage of patient sera exhibit some drawbacks such as effects of serum components other than anti‐RhoGDI2 antibodies on complement activation.…”

Expanded Hemodialysis ameliorates uremia-induced impairment of vasculoprotective KLF2 and concomitant proinflammatory priming of endothelial cells through an ERK/AP1/cFOS-dependent mechanism

“…1) Human microvascular endothelial cells (HMECs; catalogue no. CRL-3243; purchased from ATCC ® , Manassas, VA, USA) were cultured in MCDB131 basic media for HMECs (Invitrogen and Gibco, Waltham, MA, USA) supplemented with 5% FCS, 10 mM L-glutamine, 10 ng/ml recombinant human EGF, 10nM hydrocortisone, and 1% penicillin/streptomycin (100 U/ml and 100 ug/ml, respectively) ( 31 ).…”

“…No. : 30-2003 from ATCC ® ) supplemented with 10% FCS, 2mM GlutaMax (Gibco), 1mM sodium pyruvate, 10mM HEPES, and 100 U/ml penicillin/100 ug/ml streptomycin ( 31 ).…”

Autoantibodies from patients with kidney allograft vasculopathy stimulate a proinflammatory switch in endothelial cells and monocytes mediated via GPCR-directed PAR1-TNF-α signaling

“…Protein-DNA complexes were analyzed by electrophoresis in 6% non-denaturing polyacrylamide gels and visualized with LightShift Chemiluminescent EMSA Kit (Thermo). The activity of NFkB and NFAT signaling was analyzed with EMSA and luciferase assays, respectively, as described previously ( 50 , 51 ).…”

“…(A, B) Effect of KTx-IgG on TNF-α protein release from HMECs (A) Upon a 24-hour incubation with 1.0 mg/ml Con-IgG or KTx-IgG from patients with or without vasculopathy or baseline control (pg/ml, n=3 each) and (B) Upon a 24-hour stimulation with 1 mg/ml KTx-IgG from patients with vasculopathy (n=5 each) in the presence or absence of the following stimuli: a) mitogen phorbol-myristate-acetate (PMA, concentration 50 ng/ml), b) thrombin (0.1 U/ml), and c) PAR-1 inhibitor (BMS200261, 1 µM); and (C, D) Analysis of NFkB and NFAT activation in response to KTx-IgG from patients with vasculopathy compared to Con-IgG (each 1 mg/ml for 24 hours). The activity of NFkB and NFAT signaling was analyzed with EMSA and luciferase assays, respectively, as described previously ( 50 , 51 ). The data were analyzed with repeated ANOVA and expressed as Mean +/- SD with *P<0.05, **P<0.01, and ***P<0.001.…”