Anti-vascular endothelial growth factor antibody attenuates inflammation and decreases mortality in an experimental model of severe sepsis

Abstract

Introduction: Severe sepsis is associated with an unacceptably high rate of mortality. Recent studies revealed elevated levels of vascular endothelial growth factor (VEGF), a potent angiogenic and vascular permeability factor, in patients with sepsis. There was also an association between VEGF levels and sepsis severity. Here we investigate the effects of an anti-VEGF antibody (Bevacizumab, Bev) in an experimental model of sepsis.

Methods: Human umbilical vein endothelial cells (HUVECs), murine cecal ligation and puncture (CLP), and endotoxemia models of sepsis were used. HUVECs were treated with lipopolysaccharide (LPS) and/or Bev, harvested and cytokine mRNA levels determined using a semi-quantitative reverse transcription-polymerase chain reaction assay. The levels of inflammatory cytokine were also determined in HUVECs supernatants. In addition, the effects of Bev on mortality in the CLP and endotoxemia models of sepsis were evaluated.

Results: Treatment with Bev and LPS significantly decreased the expression and the level of inflammatory cytokines in HUVECs relative to LPS alone. In CLP and endotoxemia models, survival benefits were evident in mice given 0.1 mg/kg of Bev relative to the CLP or LPS alone (P<0.001 and P=0.028, respectively), and in 6 h post-treated mice relative to the CLP alone for the effect of different time of Bev (P=0.033). In addition, Bev treatment inhibited LPS-induced vascular leak in the lung, spleen and kidney in the murine endotoxemia model (P<0.05).

Conclusions: Anti-VEGF antibody may be a promising therapeutic agent due to its beneficial effects on the survival of sepsis by decreasing inflammatory responses and endothelial permeability.

Figure 1

The expression of VEGF mRNA in HUVECs. Human umbilical vein endothelial cells (HUVECs) were stimulated with 1 μg/ml lipopolysaccharide (LPS) for up to 5 h. Vascular endothelial growth factor (VEGF) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were semi-quantified by RT-PCR. Error bars represent SD. *P <0.01 compared to non-treated HUVECs (n = 4).

Figure 2

Expression of IL-6, MCP-1 and RANTES in HUVECs after treatment with LPS and/or bevacizumab. A, cDNA of cytokines and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). B, semi-quantitative IL-6 levels. C, semi-quantitative monocyte chemotactic protein-1 (MCP-1) levels. D, semi-quantitative RANTES (regulated on activation, normal T-cell expressed and secreted) levels. Error bars represent SD. *P <0.01 when compared to lipopolysaccharide (LPS)-only treated human umbilical vein endothelial cells (HUVECs) (n = 4).

Figure 3

Survival in murine sepsis models and the effects of differing doses of bevacizumab on mortality. A, Kaplan-Meier survival analysis following cecal ligation and puncture (CLP) comparing bevacizumab (Bev)-treated animals administered 0.1 mg/kg (n = 8) or 1.0 mg/kg (n = 8) i.p. 1 h before CLP to controls with CLP (n = 13). The group administered 0.1 mg/kg had a significantly greater survival than the CLP controls (P <0.001). B, Kaplan-Meier survival analysis following lipopolysaccharide (LPS) injection comparing Bev-treated animals administered 0.1 mg/kg (n = 10) or 1.0 mg/kg (n = 10) i.p. 1 h before LPS treatment to mice administered LPS-only (n = 8). Administration of 0.1 mg/kg led to significantly greater survival relative to the LPS-only group (P = 0.028).

Figure 4

Effects of differing bevacizumab treatment times on mortality in the murine models of sepsis. A, Delayed administration of Bev is protective in cecal ligation and puncture (CLP). Kaplan-Meier survival analysis following CLP in mice comparing the efficacy of pre- and post-surgical bevacizumab (Bev) treatment at various time intervals relative to the CLP control. Bev administration significantly enhanced survival relative to the CLP controls (P = 0.006 in the pre-treated group, and P = 0.033 in the 6 h post-treated group), except for mice in which Bev administration was delayed for 12 h (12 h delayed-treatment group, P = 0.062). B, Lethality from endotoxemia was diminished with delayed Bev administration, but not statistically significantly so.

Figure 5

Effect of bevacizumab treatment on vascular permeability in a mouse model of endotoxemia. Quantitation of Evans-blue extravasation (optical density (OD) at 620 nm). Error bars represent SD. *P <0.05 when compared to the LPS group (P = 0.011 for lung, P = 0.046 for spleen, and P <0.001 for kidney).