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 leakage 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.

Conflict of interest statement

The remaining authors have disclosed that they do not have any potential conflicts of interest.

Figures

Figure 1

Food and Drug Administration (FDA) library screening identifies simvastatin (SIM) as a potent inhibitor of Angpt-2 production. A, Human umbilical vein endothelial cells (HUVECs) grown in a 96-well format were treated with an FDA-drug library for 24 hr, and secreted Angpt-2 protein was measured by enzyme-linked immunosorbent assay (ELISA). Results were analyzed as the fold-change relative to the median value and ordered from strongest inducers (left, blue bars) to strongest inhibitors (right, red bars). The yellow window spans ±1-fold change. B, Simvastatin (SIM, 10 µM) was applied to HUVECs for 24 hr at indicated doses and secreted Angpt-2 protein was measured by ELISA. C, Simvastatin (10 µM) was applied to HUVECs for indicated durations, after which Angpt-2 protein levels in the conditioned media were measured by ELISA *p < 0.05, **p < 0.01. D, HUVECs were treated with SIM (10 µM) or an excess of mevalonate (MEV, 200 µM), and secreted Angpt-2 protein was measured by ELISA. *p < 0.05, **p < 0.01, and ***p < 0.001. E, HUVECs were stained for Angpt-2 (green) and a marker of Weibel Palade bodies (ie, von Willebrand Factor, red) 24 hr after vehicle or SIM (10 µM) treatment.

Figure 2

Simvastatin reduces angiopoietin-2 (Angpt-2) transcription and binding of the transcription factor Foxo1. A, Angpt-2 mRNA concentrations were measured via real-time polymerase chain reaction (RT-PCR) 24 hr after applying simvastatin (SIM) at indicated concentrations to human umbilical vein endothelial cells (HUVECs). B, SIM (10 µM) was applied for 24 hr to HUVECs pretreated with control or Krüppel-like factor-2 (KLF2) siRNA and Angpt-2 mRNA was measured by RT-PCR. C, Putative Foxo1 binding sites were identified at –2,840 and –1,660 in the three kilobases 5′ to the translational start site of human ANGPT2 by TFsearch and cross-checked by aligning the consensus Foxo1 binding sequence (in bold). D, Nuclear extracts of HUVECs were incubated with biotin labeled DNA probes specific for the –2,840 and –1,660 sites of ANGPT2 in the presence (+) or absence (−) of six-fold excess unlabeled probes. Arrows indicate the specific band eliminated by competition. E, Nuclear extracts of HUVECs either treated with vehicle (−) or SIM (10 µM, +) for 24 hr were prepared and incubated with biotin-labeled DNA probes specific for the –2,840 and –1,660 sites of ANGPT2. Arrows indicate the band of interest and arrowhead indicates a non-specific band. F, HUVECs treated with SIM (10 µM) for 24 hr were gently lysed and chromatin immunoprecipitation was performed with anti-Foxo1. Results of RT-PCR to quantify ANGPT2 promoter concentration are shown for the –2,840 and –1,660 sites. *p < 0.05, ***p < 0.001.

Figure 3

Simvastatin prevents nuclear Foxo1 translocation by phosphorylation. A, Endothelial cells (ECs) treated with simvastatin (SIM, 10 µM) were stained for Foxo1 (red) and nuclei (4',6-diamidino-2-phenylindole, blue). White arrows indicate the edge of nuclear staining. B, Planimetric quantification of staining results by surveying 10 high-powered fields (×40) per slide. *p < 0.05 and **p < 0.01. C, HUVEC lysates 24 hr after SIM treatment (10 µM) were immunoblotted with anti-pSer256-Foxo1 (pFoxo-1), anti-Foxo1 (tFoxo-1), and anti-GAPDH as a loading control. D, Densitometric quantification of the above results. E, Immunoblotting as described above for lysates of ECs infected with a virus encoding wild-type Foxo1 (AdFoxo1) for 24 hr and treated with SIM (10 µM) for 24 hr or vehicle. F, Quantification of the above results. G, Immunoblotting as described above for lysates of ECs infected with a virus encoding triple-phosphorylation mutant Foxo1 (TM Foxo1) before 24-hr treatment with SIM (10 µM) or vehicle. *p < 0.05.

Figure 4

Foxo1 phosphorylation is critical for the suppression of Angpt-2 by simvastatin. A, Real-time polymerase chain reaction for Angpt-2 24 hr after treating virally transduced human umbilical vein endothelial cells with simvastatin (SIM, 10 µM) or vehicle. “βGal” is a control virus expressing β-galactosidase; “AdFoxo1” is a virus encoding wild-type Foxo1; and “Ad-TM-Foxo1” is a virus encoding the triple phosphorylation constitutively active mutant Foxo1. B, ELISA for secreted Angpt-2 protein for the above conditions. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 5

Simvastatin (SIM) suppresses Angpt-2 during sepsis and improves sepsis survival. A, Adult male C57BL/6 mice treated with SIM (0.2 µg/g body weight [BW] IP) or vehicle (Veh) 24 hr before the injection with gram-negative endotoxin (lipopolysaccharides [LPS] from Escherichia coli IP) or saline control. Angpt-2 mRNA was measured 16 hr after LPS by real-time polymerase chain reaction (RT-PCR) in lung homogenates. (n = 4–9 mice per group, **p < 0.01). B, Angpt-2 mRNA was measured by quantitative RT-PCR from murine lungs 48 hr after treatment with an Angpt-2 or control siRNA specifically targeting the pulmonary endothelium (n = 9 mice per group, **p < 0.01). C, Adult male C57BL/6 mice (n = 10 mice per each of the four study arms) were treated with SIM (200 µg/kg BW IP) versus vehicle solution and control siRNA versus Angpt-2 siRNA as described in the Materials and Methods section. Survival after cecal ligation and puncture (CLP) was followed. **p < 0.01 by log-rank test for all conditions versus animals treated with control siRNA and vehicle. Survival following CLP was statistically indistinguishable among the remaining groups.

Figure 6

Chronic or acute statin use is associated with lower circulating Angpt-2 among critically ill patients. A, Circulating Angpt-2 concentration at the time of enrollment in a case-control study of critically ill subjects comparing chronic statin users versus statin-naïve individuals. B, Change in circulating Angpt-2 over the first 48–72 hr after randomization in a placebo controlled clinical trial of statin-naïve septic individuals receiving 40 mg simvastatin orally once daily or placebo pill (*p < 0.05 and **p < 0.01).

Figure 7

Proposed mechanism for statin-mediated suppression of ongoing Angpt-2 production during sepsis. In endothelium of quiescent blood vessels, Tie2 is tonically expressed and activated. Through downstream activation of Akt, Foxo1 is phosphorylated and Angpt-2 transcription is suppressed. During the initial phase of sepsis, pre-formed Angpt-2 stored in Weibel Palade bodies (WPBs) is rapidly exocytosed in response to early acute-phase cytokines. Excess Angpt-2 impairs Tie2 signaling, which in turn, reduces Foxo1 phosphorylation and enables it to drive the transcription of the ANGPT2 gene. Simvastatin favors the phosphorylation of Foxo1, thus attenuating the production of Angpt-2.