Elevated levels of placental growth factor represent an adaptive host response in sepsis - PubMed (original) (raw)

Comparative Study

. 2008 Oct 27;205(11):2623-31.

doi: 10.1084/jem.20080398. Epub 2008 Oct 13.

Yoshiaki Okada, Guido Beldi, Shou-Ching Shih, Natalya Bodyak, Hitomi Okada, Peter M Kang, William Luscinskas, Simon C Robson, Peter Carmeliet, S Ananth Karumanchi, William C Aird

Affiliations

Comparative Study

Elevated levels of placental growth factor represent an adaptive host response in sepsis

Kiichiro Yano et al. J Exp Med. 2008.

Abstract

Recently, we demonstrated that circulating levels of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are increased in sepsis (Yano, K., P.C. Liaw, J.M. Mullington, S.C. Shih, H. Okada, N. Bodyak, P.M. Kang, L. Toltl, B. Belikoff, J. Buras, et al. 2006. J. Exp. Med. 203:1447-1458). Moreover, enhanced VEGF/Flk-1 signaling was shown to contribute to sepsis morbidity and mortality. We tested the hypothesis that PlGF also contributes to sepsis outcome. In mouse models of endotoxemia and cecal ligation puncture, the genetic absence of PlGF or the systemic administration of neutralizing anti-PlGF antibodies resulted in higher mortality compared with wild-type or immunoglobulin G-injected controls, respectively. The increased mortality associated with genetic deficiency of PlGF was reversed by adenovirus (Ad)-mediated overexpression of PlGF. In the endotoxemia model, PlGF deficiency was associated with elevated circulating levels of VEGF, induction of VEGF expression in the liver, impaired cardiac function, and organ-specific accentuation of barrier dysfunction and inflammation. Mortality of endotoxemic PlGF-deficient mice was increased by Ad-mediated overexpression of VEGF and was blocked by expression of soluble Flt-1. Collectively, these data suggest that up-regulation of PlGF in sepsis is an adaptive host response that exerts its benefit, at least in part, by attenuating VEGF signaling.

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Figures

Figure 1.

Figure 1.

PlGF levels in sepsis and survival studies in PlGF-deficient mice. (A, a) Plasma levels of PlGF in wild-type male FVB mice injected i.p. with 18 mg/kg LPS at the time points indicated. (b) Same as in a but in a CLP mouse model. (B) Shown are results of quantitative real-time analyses (mRNA copy number per 106 copies of 18S) of PlGF in organs from male FVB mice at various time points after i.p. injection with 18 mg/kg LPS. (C) In situ hybridization (ISH) and immunohistochemistry (IHC) for PlGF in the heart, lung, and liver of male FVB mice treated in the absence (nontreated) or presence of 18 mg/kg LPS at 12 h. The insets in d, h, and i show a higher magnification of a representative area from each field. Arrows indicate PlGF signal. Bar: 50 μm; (insets) 25 μm. (D, a) Survival curves for male PLGF+/+ (wild-type, WT) or PLGF−/− (knockout, KO) mice injected i.p. with 18 mg/kg LPS. (b) Survival curves for female PLGF+/+ (WT) or PLGF−/− (KO) mice injected i.p. with 18 mg/kg LPS. (c) Survival curves for wild-type male mice pretreated with anti-PlGF antibody (PlGF ab) or IgG (CTL ab) and injected i.p. with 18 mg/kg LPS. (d) Survival curves for male PLGF+/+ (WT) or PLGF−/− (KO) mice subjected to CLP. (e) Survival curves for female PLGF+/+ (WT) or PLGF−/− (KO) mice subjected to CLP. (f) Survival curves for wild-type male mice pretreated with anti-PlGF antibody (PlGF ab) or IgG (CTL ab) and subjected to CLP. Data in A and B are expressed as means + SD of at least three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.0001 compared with no treatment (0 h time point).

Figure 2.

Figure 2.

Effect of PlGF deficiency on tissue and/or circulating levels of PlGF, VEGF, sFlt-1, and IL-6. (A, a–d) PLGF+/+ (WT) or PLGF−/− (KO) male mice were injected i.p. with or without 16 mg/kg LPS. Blood samples were taken 24 h later and assayed for plasma levels of free PlGF, free VEGF, sFlt-1, and IL-6. (e–h) Same as in a–d, except that wild-type mice were pretreated with anti-PlGF antibody (Pab) or IgG (CTL) and injected i.p. with or without 16 mg/kg LPS. (B) PLGF+/+ (WT) or PLGF−/− male mice were injected i.p. with or without 16 mg/kg LPS. Mouse organs were assayed for VEGF protein levels by ELISA at the time points indicated. Data are expressed as means + SD of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.0001 compared with the respective untreated controls (and where indicated between LPS-treated PlGF-deficient and wild-type mice).

Figure 3.

Figure 3.

Effect of PlGF deficiency on cardiac function and vascular permeability in a mouse model of endotoxemia. (A, a and b) PLGF+/+ (WT) or PLGF−/− (KO) male mice were injected i.p. with or without 16 mg/kg LPS and were subjected to echocardiogram and electrocardiogram 24 h later. Shown are quantitative analyses for heart rate (HR) and fractional shortening (FS). (c and d) Same as in a and b except that wild-type mice were pretreated with anti-PlGF antibody (Pab) or IgG (CTL) and injected i.p. with or without 16 mg/kg LPS. (B, a) PLGF+/+ (WT, W) or PLGF−/− (KO, K) male mice were injected i.p. with or without 16 mg/kg LPS. 24 h later, the animals were injected i.v. with 0.1 ml 1% Evans blue dye. After 40 min, the mice were perfused, and the brain, lung, heart, liver, kidney, and spleen were harvested and incubated in formamide for 3 d to elute Evans blue dye. Shown is the quantitation of Evans blue extravasation (OD = 620 nm). (b) Same as in a except that wild-type mice were pretreated with anti-PlGF antibody (PlGF ab, P) or IgG (CTL IgG, C) and injected i.p. with or without 16 mg/kg LPS. Data are expressed as means + SD of three independent experiments. *, P < 0.05; and **, P < 0.01 compared with the respective untreated controls (and where indicated between PlGF-deficient and wild-type mice).

Figure 4.

Figure 4.

Effect of PlGF deficiency on tissue mRNA/protein levels of inflammatory and hemostatic markers in a mouse model of endotoxemia. PLGF+/+ (WT) or PLGF−/− (KO) male mice were injected i.p. with or without 16 mg/kg LPS. (A) Shown are the results of quantitative real-time PCR analyses (mRNA copy number per 106 copies of 18S) of ICAM-1, VCAM-1, E-selectin, P-selectin, COX-2, and PAI-1 in the heart, lung and liver at 24 h. Data are expressed as means + SD of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.0001 compared with untreated controls (and where indicated between PlGF-deficient and wild-type mice). (B) Double immunofluorescence staining for activation markers and CD31 in the liver of wild-type mice treated in the absence (WT) or presence of 16 mg/kg LPS (WT/L) and PlGF−/− mice treated with 16 mg/kg LPS (PKO/L) at 24 h. (a) ICAM-1 (green) and CD31 (red). (b) VCAM-1 (green) and CD31 (red). (c) E-selectin (green) and CD31 (red). (d) P-selectin (green) and CD31 (red). (e) COX-2 (red) and CD31 (green). (f) PAI-1 (red) and CD31 (green). Bars: 132 μm; (insets) 42 μm.

Figure 5.

Figure 5.

Effect of PlGF deficiency on liver phenotype in a mouse model of endotoxemia. PLGF+/+ (WT) or PLGF−/− (KO) male mice were injected i.p. with or without 16 mg/kg LPS. (A) Blood samples were taken at 24 h and assayed for plasma levels of alanine aminotransferase (ALT). (B) The liver was removed at 24 h and assayed for MPO activity. (C) The liver was removed at 24 h and stained for CD45. Data in A and B are expressed as means + SD of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.0001 compared with untreated controls (and where indicated between PlGF-deficient and wild-type mice. Bar, 50 μm.

Figure 6.

Figure 6.

Additional survival studies in mouse models of sepsis. Male PLGF+/+ (WT) or PLGF−/− mice were injected with Ad overexpressing GFP (GFP-ad) or PlGF (PlGF-ad; A), VEGF (VEGF-ad; B), or sFlt-1 (sFlt-ad; C). 3 d later, the animals were administered saline (control) or LPS i.p. and monitored for survival.

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