Uremic Solute-Aryl Hydrocarbon Receptor-Tissue Factor Axis Associates with Thrombosis after Vascular Injury in Humans.

Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.

The Aryl Hydrocarbon Receptor is a Critical Regulator of Tissue Factor Stability and an Antithrombotic Target in Uremia.

Patients with CKD suffer high rates of thrombosis, particularly after endovascular interventions, yet few options are available to improve management and reduce thrombotic risk. We recently demonstrated that indoxyl sulfate (IS) is a potent CKD-specific prothrombotic metabolite that induces tissue factor (TF) in vascular smooth muscle cells (vSMCs), although the precise mechanism and treatment implications remain unclear. Because IS is an agonist of the aryl hydrocarbon receptor (AHR), we first examined the relationship between IS levels and AHR-inducing activity in sera of patients with ESRD. IS levels correlated significantly with both vSMC AHR activity and TF activity. Mechanistically, we demonstrated that IS activates the AHR pathway in primary human aortic vSMCs, and further, that AHR interacts directly with and stabilizes functional TF. Antagonists directly targeting AHR enhanced TF ubiquitination and degradation and suppressed thrombosis in a postinterventional model of CKD and endovascular injury. Furthermore, AHR antagonists inhibited TF in a manner dependent on circulating IS levels. In conclusion, we demonstrated that IS regulates TF stability through AHR signaling and uncovered AHR as an antithrombotic target and AHR antagonists as a novel class of antithrombotics. Together, IS and AHR have potential as uremia-specific biomarkers and targets that may be leveraged as a promising theranostic platform to better manage the elevated thrombosis rates in patients with CKD.

The c-Cbl ubiquitin ligase regulates nuclear ß-catenin and angiogenesis by its tyrosine phosphorylation mediated through the Wnt signaling pathway.

Wnt signaling plays important roles in both the tumor-induced angiogenesis and tumorigenesis through the transcriptionally active nuclear ß-catenin. Recently, c-Cbl was identified as a unique E3 ubiquitin ligase targeting the active nuclear ß-catenin. However, little is known about the molecular mechanisms by which c-Cbl regulates ubiquitination and degradation of active ß-catenin. Here, we demonstrate that Wnt activation promotes the phosphorylation of c-Cbl at tyrosine 731(Tyr-731), which increases c-Cbl dimerization and binding to ß-catenin. Tyr-731 phosphorylation and dimerization mediate c-Cbl nuclear translocation and lead to the degradation of nuclearly active ß-catenin in the Wnt-on phase. c-Cbl activation also inhibits expression of the pro-angiogenic Wnt targets, IL-8 and VEGF. Phospho-Tyr-731-inactive mutant c-Cbl (Y731F) enhances and phosphomimetic mutant c-Cbl (Y731E) suppresses angiogenesis in zebrafish. Taken together, we have identified a novel mechanism for the regulation of active nuclear ß-catenin by c-Cbl and its critical role in angiogenesis. This mechanism can be further explored to modulate both the pathological angiogenesis and the tumorigenesis.

c-Cbl, a ubiquitin E3 ligase that targets active ß-catenin: a novel layer of Wnt signaling regulation.

Regulation of transcriptionally active nuclear ß-catenin during the Wnt-on phase is crucial to ensure controlled induction of Wnt target genes. Several ubiquitin E3 ligases are known to regulate cytosolic ß-catenin during the Wnt-off phase, but little is known about the fate of active nuclear ß-catenin in the Wnt-on phase. We now describe ubiquitination of active ß-catenin in the Wnt-on phase by a RING finger ubiquitin E3 ligase, Casitas B-lineage lymphoma (c-Cbl) in endothelial cells. c-Cbl binds preferentially to nuclearly active ß-catenin in the Wnt-on phase via the armadillo repeat region. Wild-type c-Cbl suppresses and E3 ligase-deficient c-Cbl-70Z increases Wnt signaling. Wnt induces nuclear translocation of c-Cbl where it ubiquitinates nuclear ß-catenin. Deletion of the c-Cbl UBA domain abrogates its dimerization, binding to ß-catenin, Wnt-induced c-Cbl nuclear translocation, and ubiquitination of nuclear ß-catenin. c-Cbl activity inhibits pro-angiogenic Wnt targets IL-8 and VEGF levels and angiogenesis in a ß-catenin-dependent manner. This study defines for the first time c-Cbl as a ubiquitin E3 ligase that targets nuclearly active ß-catenin in the Wnt-on phase and uncovers a novel layer of regulation of Wnt signaling.

Uremic serum and solutes increase post-vascular interventional thrombotic risk through altered stability of smooth muscle cell tissue factor.

BACKGROUND: Stent thrombosis (ST), a postinterventional complication with a mortality rate of 50%, has an incidence that rises precipitously in patients at risk. Chronic renal failure and end-stage renal disease have emerged as particularly strong ST risk factors, yet the mechanism remains elusive. Tissue factor (TF) is a crucial mediator of injury-related thrombosis and has been implicated for ST. We posit that uremia modulates TF in the local vessel wall to induce postinterventional thrombosis in patients with end-stage renal disease. METHODS AND RESULTS: As a model of the de-endothelialized, postinterventional state, we exposed primary human vascular smooth muscle cells (vSMCs) pretreated with uremic serum (obtained from ESRD patients on hemodialysis) to coronary-like blood flow. vSMC TF expression, activity, stability, and posttranslational modification were examined after vSMCs were treated with uremic serum or solutes. We found significantly greater clot formation after uremic serum exposure, which was substantially reduced with the prior treatment with anti-TF neutralizing antibody. Uremic sera induced 2- to 3-fold higher TF expression and activity in vSMCs independent of diabetes mellitus. Relevant concentrations of isolated uremic solutes such as indole-3-acetic acid (3.5 µg/mL), indoxyl sulfate (25 µg/mL), and uric acid (80 µg/mL) recapitulated these effects in cell culture and the flow loop model. We show further that TF undergoes ubiquitination at baseline and that uremic serum, indole-3-acetic acid, and indoxyl sulfate significantly prolong TF half-life by inhibiting its ubiquitination. CONCLUSIONS: The uremic milieu is profoundly thrombogenic and upregulates vSMC TF levels by increasing TF stability and decreasing its ubiquitination. Together, these data demonstrate for the first time that the posttranslational regulation of TF in uremia may have a causative role in the increased ST risk observed in uremic patients. These data suggest that interventions that reduce vSMC TF may help to prevent ST and that uremic solutes should be considered as novel risk factors for ST in patients with chronic renal failure.