Molecular Regulation of Acute Tie2 Suppression in Sepsis

Abstract

Objectives: Tie2 is a tyrosine kinase receptor expressed by endothelial cells that maintains vascular barrier function. We recently reported that diverse critical illnesses acutely decrease Tie2 expression and that experimental Tie2 reduction suffices to recapitulate cardinal features of the septic vasculature. Here we investigated molecular mechanisms driving Tie2 suppression in settings of critical illness.

Design: Laboratory and animal research, postmortem kidney biopsies from acute kidney injury patients and serum from septic shock patients.

Setting: Research laboratories and ICU of Hannover Medical School, Harvard Medical School, and University of Groningen.

Patients: Deceased septic acute kidney injury patients (n = 16) and controls (n = 12) and septic shock patients (n = 57) and controls (n = 22).

Interventions: Molecular biology assays (Western blot, quantitative polymerase chain reaction) + in vitro models of flow and transendothelial electrical resistance experiments in human umbilical vein endothelial cells; murine cecal ligation and puncture and lipopolysaccharide administration.

Measurements and main results: We observed rapid reduction of both Tie2 messenger RNA and protein in mice following cecal ligation and puncture. In cultured endothelial cells exposed to tumor necrosis factor-α, suppression of Tie2 protein was more severe than Tie2 messenger RNA, suggesting distinct regulatory mechanisms. Evidence of protein-level regulation was found in tumor necrosis factor-α-treated endothelial cells, septic mice, and septic humans, all three of which displayed elevation of the soluble N-terminal fragment of Tie2. The matrix metalloprotease 14 was both necessary and sufficient for N-terminal Tie2 shedding. Since clinical settings of Tie2 suppression are often characterized by shock, we next investigated the effects of laminar flow on Tie2 expression. Compared with absence of flow, laminar flow induced both Tie2 messenger RNA and the expression of GATA binding protein 3. Conversely, septic lungs exhibited reduced GATA binding protein 3, and knockdown of GATA binding protein 3 in flow-exposed endothelial cells reduced Tie2 messenger RNA. Postmortem tissue from septic patients showed a trend toward reduced GATA binding protein 3 expression that was associated with Tie2 messenger RNA levels (p < 0.005).

Conclusions: Tie2 suppression is a pivotal event in sepsis that may be regulated both by matrix metalloprotease 14-driven Tie2 protein cleavage and GATA binding protein 3-driven flow regulation of Tie2 transcript.

Figure 1. Posttranslational modification by Tie2 ectodomain cleavage in cells, mice and men

(A) sTie2 ELISA (detects N-terminal Tie2 ectodomain) of human umbilical vein endothelial cells (HUVEC) supernatants stimulated with 50 ng/mL TNFα or vehicle after 24 hrs (n=8, ***p<0.001) and serum from C57/Bl6 mice after sepsis induction via cecal ligation and puncture (CLP). Mice were sacrificed after 16 hrs. SHAM surgery served as control (n=7-9, **p<0.01). sTie2 ELISA of serum from septic human patients (n=57) and healthy controls (n=22) (healthy control: 12.29 ± 0.71 ng/ml vs. septic humans 18.77 ± 0.48 ng/ml, ****p < 0.0001). (B) Densitometric quantification of immunoblots for matrix metalloproteases (MMP) 14 and GAPDH from C57/Bl6 murine lungs harvested 16 h after cecal ligation & puncture (CLP) or sham surgery (n=9-11, ***p<0.001); qPCR for MMP14 mRNA from the same conditions (n=11-13, ****p<0.0001).

Figure 2. MMP14 expression is both required and sufficient for Tie2 shedding in vitro and affects Tie2 downstream signaling

(A) Human umbilical vein endothelial cells (HUVECs) transfected with control or MMP14 RNA(interference)-i were stimulated with 50 ng/mL TNFα for 24 hrs and sTie2 was quantified in supernatants by ELISA (n=6, **p<0.01). (B) sTie2 ELISA of supernatants from HUVECs stimulated with vehicle, 10 μmol/L GM6001 (a global MMP blocker as positive control) or recombinant human (rh) MMP14 at 3 different doses (low: 40 μg/mL, med: 100 μg/mL and high: 300 μg/mL) (n=6, **p<0.01, ***<0.001). (C) Tie2 downstream signaling in control or MMP14 siRNA transfected HUVECs has been evaluated by immunoblot for pAKT, AKT and GAPDH after stimulation with 500 ng/mL Angiopoietin-1 (Angpt-1) for 10 minutes. Bar graphs show densitometric quantification of the above results for pAKT over AKT (n=7, **p<0.01, ***p<0.001). Exemplary immunoblot can be found in the Supplemental Digital Content – Figure 11)

Figure 3. Tie2, GATA3 and KLF2 are highly responsive to flow but only GATA3 is required for Tie2 transcription

(A) Tie2 mRNA, GATA3 mRNA and KLF2 mRNA was assessed by qPCR in human umbilical vein endothelial cells (HUVECs) under flow (20 dyne/cm²) and no flow conditions for 48h (n=6-8, **p<0.01, ***p<0.001). (B) Tie2 mRNA level assessed by qPCR in HUVECs transfected with control or either GATA3 siRNA (black bars n=4, **p<0.01) compared to HUVECs transfected with control or KLF2 siRNA (white bars, n=4, p=ns). (C) Transendothelial electrical resistance (TER) was measured using an electrical cellsubstrate impedance sensing system (ECIS) in HUVECs pretreated with control or GATA3 siRNA for 48hrs and then stimulated with 500 ng/mL Angiopoietin-1 (Angpt-1) or control (PBS) (n=4 per condition) TER was recorded over the course of 5h.

Figure 4. GATA3 is analogously downregulated in experimental sepsis and involved in the flow-response of Tie2

(A) Black bars indicate GATA3 mRNA (assessed by qPCR) from mice that underwent sham or CLP surgery and were sacrificed 16 h later (n=10-12, ****p<0.0001). White bars indicate GATA3 mRNA from isolated murine pulmonary endothelial cells. C57Bl/6 mice received 17.5 mg/kg bodyweight LPS or vehicle control for 12 hrs. Endothelial cells were isolated using CD146⁺ magnetic beads (n=5-6, p=0.05). (B) Tie2 mRNA levels in HUVECs transfected with control or GATA3 RNAi for 48h. HUVECs were set under flow (20 dyne/cm²) or no flow conditions for additional 48h followed by qPCR analysis. Bar graphs show the delta (∆) Tie2 mRNA increase upon 20 dyne/cm² flow in control and GATA3 RNAi transfected cells (n=4, *p<0.05).

Figure 5. Regulation of GATA3 in human septic samples

(A) GATA3 mRNA expression in immediate post-mortem kidney biopsies from patients who died of sepsis (n=16) and healthy controls (n=12) (p=0.096) (B) Spearman correlation of Tie2 and GATA3 mRNA level from the same human biopsies (r=0.519, p<0.005). White dots indicate healthy control biopsies compared to the grey dots (septic shock AKI patients).