Vascular endothelial growth factor blockade reduces plasma cytokines in a murine model of polymicrobial sepsis

Anna Nolan, Michael D Weiden, Gavin Thurston, Jeffrey A Gold, Anna Nolan, Michael D Weiden, Gavin Thurston, Jeffrey A Gold

Abstract

Numerous cytokines, including vascular endothelial growth factor (VEGF), are implicated in the pathogenesis of sepsis. While overexpression of VEGF produces pulmonary capillary leak, the role of VEGF in sepsis is less clear. We investigated VEGF in sepsis, utilizing a VEGF trap (VEGF(T)). Polymicrobial sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP) and resulted in significantly increased plasma VEGF levels (234 vs. 46 pg/mL; p = 0.03). Inhibition of VEGF had no effect on mortality or lung leak but did attenuate plasma IL-6 (120 vs. 236 ng/mL; p = 0.02) and IL-10 (16 vs. 41 ng/mL; p = 0.03). These alterations in inflammatory cytokines were associated with increased levels of the dominant negative inhibitory C/EBPbeta. In vitro, VEGF stimulated IL-6, IL-10 and reduced the inhibitory isoform of C/EBPbeta in cultured macrophages. Together these data suggest VEGF can regulate inflammatory cytokine production in murine polymicrobial sepsis, via regulation of C/EBPbeta.

Figures

Fig. 1
Fig. 1
CLP-induced murine polymicrobial sepsis significantly increases plasma levels of VEGF. C57BL/6 female mice underwent CLP or remained as unoperated controls (four animals each). Plasma was collected after 18 h and VEGF levels were measured by ELISA. VEGF levels were increased fivefold when compared to unoperated controls (234 pg/mL ± 100 vs. 46 pg/mL ± 16; p < 0.05. Results expressed as the mean ± standard error of the mean (SEM).
Fig. 2
Fig. 2
Survival not improved with VEGFT treatment in murine sepsis. C57BL/6 female mice underwent CLP 6 h after receiving either VEGFT 500 μg/mouse (▴, N = 15) or vehicle control (■, N = 12) IP and in equal volumes. Mice were observed at regular time intervals for a total of 72 h. CLP induced a 100% mortality with a median survival of 37 h. Treatment with VEGFT did not significantly alter mortality with a median survival of 28 h. Kaplan–Meier survival analysis was performed.
Fig. 3
Fig. 3
Plasma levels of IL-6, IL-10 significantly decreased after VEGFT treatment in murine sepsis. Plasma levels of IL-6 in the VEGFT-treated mice (N = 11) was decreased compared to vehicle-treated (N = 8) controls, (119.5 ng/mL vs. 236.2 ng/mL*) after CLP. In contrast VEGFT had no affect on BAL IL-6 (850 pg/mL vs. 1520 pg/mL). Similar results were seen for IL-10 (16 ng/ml vs. 41 ng/mL*) in plasma and in BALF (93 pg/mL vs. 311 pg/mL). A similar trend was observed with IL-12, although this also did not reach statistical significance. Data represented as mean ± SEM with asterisk indicating a significant p-value.
Fig. 4
Fig. 4
Dominant negative (short) form of C/EBPβ has increased expression when VEGF is inhibited. (A) Representative examples of a Western blot for C/EBPβ protein expression of liver homogenates/whole cell extract of vehicle control (lane 1) and VEGFT-treated (lane 2) samples. (B) Ratio of Stimulatory/Inhibitory (S/I) form of C/EBPβ for vehicle control (N = 4) vs. VEGFT-treated mice (N = 9); *significant p-value.
Fig. 5
Fig. 5
IL-6 and IL-10 expression in macrophages after VEGF stimulation. (A) Levels of both IL-6 and IL-10 had a graded dose response to exogenous murine VEGF. Murine thioglycolate induced peritoneal macrophages were used. Results expressed as the mean ± SEM. *Significant p-value (B) time course of cytokine production in THP-1 derived macrophages after VEGF stimulation. Exogenous VEGF at a dose of 1 μg/mL caused a sixfold induction of IL-6 at 2 h.
Fig. 6
Fig. 6
In vitro dose response and time course assay shows a reduction in inhibitory C/EBPβ at 2 h and complete loss of this isoform by 24 h. THP-1 derived macrophages were exposed to recombinant VEGF at a dose of 1 μg/mL and C/EBPβ protein expression analyzed by Western blot at 0.5, 2, and 24 h. All lanes were normalized for equal protein content.

References

    1. Bone RC, Sibbald WJ, Sprung CL. The ACCP-SCCM consensus conference on sepsis and organ failure. Chest. 1992;101(6):1481–1483.
    1. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med. 2003;348(16):1546–1554.
    1. Park WY, Goodman RB, Steinberg KP, Ruzinski JT, Radella F, II, Park DR, Pugin J, Skerrett SJ, Hudson LD, Martin TR. Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med. 2001;164(10 Pt 1):1896–1903.
    1. Eichacker PQ, Parent C, Kalil A, Esposito C, Cui X, Banks SM, Gerstenberger EP, Fitz Y, Danner RL, Natanson C. Risk and the efficacy of antiinflammatory agents: retrospective and confirmatory studies of sepsis. Am J Respir Crit Care Med. 2002;166(9):1197–1205.
    1. Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T. A nuclear factor for IL-6 expression NF-IL6 is a member of a C/EBP family. Embo J. 1990;9(6):1897–1906.
    1. Weiden M, Tanaka N, Qiao Y, Zhao BY, Honda Y, Nakata K, Canova A, Levy DE, Rom WN, Pine R. Differentiation of monocytes to macrophages switches the Mycobacterium tuberculosis effect on HIV-1 replication from stimulation to inhibition: Modulation of interferon response and CCAAT/enhancer binding protein beta expression. J Immunol. 2000;165(4):2028–2039.
    1. Gold JA, Parsey M, Hoshino Y, Hoshino S, Nolan A, Yee H, Tse DB, Weiden MD. CD40 contributes to lethality in acute sepsis: In vivo role for CD40 in innate immunity. Infect Immun. 2003;71(6):3521–3528.
    1. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes JC, Abraham JA. The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing. J Biol Chem. 1991;266(18):11947–11954.
    1. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9(6):669–676.
    1. Senger DR, Connolly DT, Van de Water L, Feder J, Dvorak HF. Purification and NH2-terminal amino acid sequence of guinea pig tumor-secreted vascular permeability factor. Cancer Res. 1990;50(6):1774–1778.
    1. Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J Cell Sci. 1995;108(Pt 6):2369–2379.
    1. Thickett DR, Armstrong L, Millar AB. A role for vascular endothelial growth factor in acute and resolving lung injury. Am J Respir Crit Care Med. 2002;166(10):1332–1337.
    1. Kaner RJ, Ladetto JV, Singh R, Fukuda N, Matthay MA, Crystal RG. Lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema. Am J Respir Cell Mol Biol. 2000;22(6):657–664.
    1. Meduri GU, Headley S, Kohler G, Stentz F, Tolley E, Umberger R, Leeper K. Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Plasma IL-1 beta and IL-6 levels are consistent and efficient predictors of outcome over time. Chest. 1995;107(4):1062–1073.
    1. Meduri GU, Kohler G, Headley S, Tolley E, Stentz F, Postlethwaite A. Inflammatory cytokines in the BAL of patients with ARDS. Persistent elevation over time predicts poor outcome. Chest. 1995;108(5):1303–1314.
    1. Nakahara H, Song J, Sugimoto M, Hagihara K, Kishimoto T, Yoshizaki K, Nishimoto N. Anti-interleukin-6 receptor antibody therapy reduces vascular endothelial growth factor production in rheumatoid arthritis. Arthritis Rheum. 2003;48(6):1521–1529.
    1. Wong AK, Alfert M, Castrillon DH, Shen Q, Holash J, Yancopoulos GD, Chin L. Excessive tumor-elaborated VEGF and its neutralization define a lethal paraneoplastic syndrome. Proc Natl Acad Sci U S A. 2001;98(13):7481–7486.
    1. Saishin Y, Takahashi K, Lima e Silva R, Hylton D, Rudge JS, Wiegand SJ, Campochiaro PA. VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier. J Cell Physiol. 2003;195(2):241–248.
    1. Holash J, Davis S, Papadopoulos N, Croll SD, Ho L, Russell M, Boland P, Leidich R, Hylton D, Burova E, et al. VEGF-Trap: A VEGF blocker with potent antitumor effects. Proc Natl Acad Sci USA. 2002;99(17):11393–11398.
    1. Wichterman KA, Baue AE, Chaudry IH. Sepsis and septic shock–a review of laboratory models and a proposal. J Surg Res. 1980;29(2):189–201.
    1. Voelkel NF, Cool C, Taraceviene-Stewart L, Geraci MW, Yeager M, Bull T, Kasper M, Tuder RM. Janus face of vascular endothelial growth factor: the obligatory survival factor for lung vascular endothelium controls precapillary artery remodeling in severe pulmonary hypertension. Crit Care Med. 2002;30(5 Suppl):S251–S256.
    1. Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med. 2003;348(2):138–150.
    1. Thickett DR, Armstrong L, Christie SJ, Millar AB. Vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome. Am J Respir Crit Care Med. 2001;164(9):1601–1605.
    1. Omori K, Naruishi K, Nishimura F, Yamada-Naruishi H, Takashiba S. High glucose enhances interleukin-6-induced vascular endothelial growth factor 165 expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in gingival fibroblasts. J Biol Chem. 2004;279(8):6643–6649.

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