Drug Repurposing Screen Identifies Foxo1-Dependent Angiopoietin-2 Regulation in Sepsis*

Abstract

Objective The recent withdrawal of a targeted sepsis therapy has diminished pharmaceutical enthusiasm for developing novel drugs for the treatment of sepsis. Angiopoietin-2 is an endothelial-derived protein that potentiates vascular inflammation and leak-age and may be involved in sepsis pathogenesis. We screened approved compounds for putative inhibitors of angiopoietin-2 production and investigated underlying molecular mechanisms. Design Laboratory and animal research plus prospective placebo- controlled randomized controlled trial (NCT00529139) and retrospective analysis (NCT00676897). Setting Research laboratories of Hannover Medical School and Harvard Medical School. Patients Septic patients/C57Bl/6 mice and human endothelial cells. Interventions Food and Drug Administration–approved library screening. Measurements and Main Results In a cell-based screen of more than 650 Food and Drug Administration–approved compounds, we identified multiple members of the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor drug class (referred to as statins) that suppressed angiopoietin-2. Simvastatin inhibited 3-hydroxy-3-methyl-glutaryl-CoA reductase, which in turn activated PI3K-kinase. Downstream of this signaling, PI3K-dependent phosphorylation of the transcription factor Foxo1 at key amino acids inhibited its ability to shuttle to the nucleus and bind cis-elements in the angiopoietin-2 promoter. In septic mice, transient inhibition of angiopoietin-2 expression by liposomal siRNA in vivo improved absolute survival by 50%. Simvastatin had a similar effect, but the combination of angiopoietin-2 siRNA and simvastatin showed no additive benefit. To verify the link between statins and angiopoietin-2 in humans, we performed a pilot matched case-control study and a small randomized placebo-controlled trial demonstrating beneficial effects on angiopoietin-2. Conclusions 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors may operate through a novel Foxo1-angiopoietin-2 mechanism to suppress de novo production of angiopoietin-2 and thereby ameliorate manifestations of sepsis. Given angiopoietin-2’s dual role as a biomarker and candidate disease mediator, early serum angiopoietin-2 measurement may serve as a stratification tool for future trials of drugs targeting vascular leakage.

Tie2 protects the vasculature against thrombus formation in systemic inflammation

“…Levels of phosphorylated (active) Tie2 were significantly decreased in endotoxemic mice at the early 3-hour time point compared with saline controls (Figure 3, G and H). The temporal profile of the Angpt-1/ Angpt-2 ratio in circulation (Supplemental Figure 2A) suggested that an early decline in Angpt-1 ( Figure 3I) contributed to reduced Tie2 signaling, which in turn, derepressed Angpt-2 ( Figure 3J), consistent with prior studies (19,(41)(42)(43)(44). To assess whether the loss of vascular quiescence indicated by reduced Tie2 signaling was more broadly manifest, we analyzed a panel of endothelial activation markers in plasma collected at 3 hours after LPS; PAI-1, von Willebrand factor (VWF), and soluble forms of endothelial adhesion molecules (sE-selectin and sVCAM-1) were all elevated at the early 3-hour time point (Supplemental Figure 2, B-E).…”

Tie1 controls angiopoietin function in vascular remodeling and inflammation

“…We confirmed these findings and additionally found reduced Tie1 mRNA in mice 3 to 12 hours after administration of endotoxin (LPS), a potent microbial mediator of the pathogenesis of Gram-negative bacterial sepsis and septic shock. We found that LPS induced the release of preformed ANG2 from Weibel-Palade bodies in endothelial cells of tracheal vessels within 30 minutes and promoted FOXO1 translocation to nucleus within 6 hours, which provides a positive feedback loop for Ang2 gene transcription (75). These rapid events are likely to contribute to the subsequent increase in Ang2 mRNA expression in the lungs and ANG2 protein in serum as well as to the reduction of Tie2 phosphorylation.…”

FoxO1 regulates asymmetric dimethylarginine via downregulation of dimethylaminohydrolase 1 in human endothelial cells and subjects with atherosclerosis

“…One alternative approach is tuning FoxO1 via upstream regulators. Two recent observations showed that both Metformin and Statins control FoxO1 nuclear entry in endothelial cells [38,39]. In this context, it will be necessary to test the validity of ADMA as a potential biomarker for FoxO1 trial in preclinical and clinical studies where FoxO1 nuclear shuttling may be modified by these agents.…”