Hepatic dysfunction and thrombocytopenia induced by excess sFlt1 in mice lacking endothelial nitric oxide synthase - PubMed (original) (raw)

Hepatic dysfunction and thrombocytopenia induced by excess sFlt1 in mice lacking endothelial nitric oxide synthase

Yuji Oe et al. Sci Rep. 2018.

Abstract

Liver dysfunction is a major problem in patients with severe preeclampsia (PE), hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, or in patients receiving anti-vascular endothelial growth factor (VEGF) therapy. Excessive soluble fms-like tyrosine kinase 1 (sFlt1) that antagonizes VEGF has been implicated in the pathogenesis of PE. VEGF increases the expression of endothelial nitric oxide synthase (eNOS) and activates it. eNOS polymorphisms that cause reduced NO production are associated with PE. The aim of this study was to clarify the role on hepatic function by excess sFlt1 in the absence of eNOS gene product. We first overexpressed sFlt1 using adenovirus in eNOS -/- and eNOS +/+ mice. Excessive sFlt1 and lack of eNOS synergistically increased plasma levels of liver transaminases, exacerbated infiltration of inflammatory cells, elevated expression levels of cytokines in the liver, and aggravated oxidative stress and coagulation abnormalities. Lack of eNOS in the presence of excess sFlt1 also induced thrombocytopenia, whereas eNOS +/+ mice with excess sFlt1 alone showed no or modest liver phenotype. Taken together, excessive sFlt1 and lack of eNOS synergistically induce hepatic dysfunction and thrombocytopenia, suggesting a novel role for VEGF and nitric oxide signaling in hepatocyte-endothelial cross-talk in health and in liver injury states.

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Conflict of interest statement

S.A.K. is a coinventor of several patents related to angiogenic biomarkers that are held by BIDMC. S.A.K. reports serving as a consultant to Thermofisher Scientific, and has financial interest in Aggamin LLC.

Figures

Figure 1

Figure 1

Liver dysfunction induced by excessive sFlt1 in mice lacking eNOS. (a) Liver weight (Wt). (b) Liver Wt/body weight (BW). Excessive sFlt1 causes hepatomegaly. The levels of plasma Aspartate transaminase (AST) and Alanine transaminase (ALT) are severely increased in eNOS −/−; sFlt1 mice (c,d) n = 7–9. Data are shown as mean ± s.e.m. ANOVA or Kruskal-Wallis test.

Figure 2

Figure 2

Histological damage in the liver. (a) Representative photomicrographs of Hematoxylin Eosin (HE). Inflammatory foci (arrowheads), vacuolar degeneration, and necrosis are shown in the liver from eNOS −/−; sFlt1 mice. TUNEL (b) and immunohistochemistry against cleaved caspase 3 (c) in the liver. Scale bar indicates 100 µm. (d) The number of inflammatory foci is significantly increased in the liver from eNOS −/−; sFlt1 mice. (e) The score of ballooning hepatocytes. (f) Increased cleaved caspase 3 positive cells in the liver from eNOS −/−; sFlt1 mice. n = 5–8. Data are shown as box plot. ANOVA or Kruskal-Wallis test.

Figure 3

Figure 3

Inflammation in the liver. (a,b) Infiltrating neutrophils are visualized by Naphthol AS-D chloroacetate Esterase stain (blue). Scale bar indicates 100 µm. Number of neutrophil is increased by excessive sFlt1, which is further up-regulated by eNOS deletion. (c) The level of Mpo (myeloperoxidase) mRNA drastically increased in the liver from eNOS −/−; sFlt1 mice. (d) Expression of inflammation and pro-fibrotic related genes in the liver. N.S., not significant. n = 7–8. Data are shown as mean ± s.e.m or box plot. ANOVA or Kruskal-Wallis test.

Figure 4

Figure 4

Markers of oxidative stress and hypoxia in the liver. (a) Representative photomicrographs of immunohistochemistry against 4-hydroxy-2-nonenal (4HNE). (b) Strong immunoreactive 4HNE is shown in the liver from eNOS −/−; sFlt1 mice. (c) Representative photomicrographs of immunohistochemistry against HO-1. (d) Strong immunoreactive HO-1 is shown in the liver from eNOS −/−; sFlt1 mice. (e,f) The levels of Hmox1 and Nqo1 mRNA in the liver. (g) Representative photomicrographs of immunohistochemistry against Hypoxia inducible factor 1α (HIF1α) in the liver. n = 5–8. Data are shown as mean ± s.e.m or box plot. ANOVA or Kruskal-Wallis test.

Figure 5

Figure 5

Lipid metabolism in the liver. (a–c) Plasma triglyceride (TG), total cholesterol (TCho), and hepatic TG. (d) Expression of Fatty acid oxidation, lipogenesis, and lipoprotein clearance receptor related genes in the liver, which are down-regulated by excessive sFlt1. n = 7–8. Data are shown as mean ± s.e.m. ANOVA or Kruskal-Wallis test.

Figure 6

Figure 6

Fibrin deposition in the liver and thrombocytopenia. (a) Representative photomicrographs of immunohistochemistry against Fibrin. Scale bar indicates 100 µm. (b) Number of Fibrin thrombi is significantly increased in the liver from eNOS −/−; sFlt1 mice. (c–g) Data of blood count; platelets (c), white blood cells (d), red blood cells (e), hemoglobin (f), and hematocrit (g). Excessive sFlt1 combined eNOS deletion causes thrombocytopenia. N.S., not significant. n = 4–8. Data are shown as mean ± s.e.m or box plot. ANOVA or Kruskal-Wallis test.

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References

    1. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359–371. doi: 10.1038/nrm1911. - DOI - PubMed
    1. Walter TJ, Cast AE, Huppert KA, Huppert SS. Epithelial VEGF signaling is required in the mouse liver for proper sinusoid endothelial cell identity and hepatocyte zonation in vivo. Am J Physiol Gastrointest Liver Physiol. 2014;306:G849–862. doi: 10.1152/ajpgi.00426.2013. - DOI - PMC - PubMed
    1. Shimizu H, et al. Vascular endothelial growth factor secreted by replicating hepatocytes induces sinusoidal endothelial cell proliferation during regeneration after partial hepatectomy in rats. J Hepatol. 2001;34:683–689. doi: 10.1016/S0168-8278(00)00055-6. - DOI - PubMed
    1. Iacovelli R, et al. Incidence and relative risk of hepatic toxicity in patients treated with anti-angiogenic tyrosine kinase inhibitors for malignancy. Br J Clin Pharmacol. 2014;77:929–938. doi: 10.1111/bcp.12231. - DOI - PMC - PubMed
    1. Hod, T., Cerdeira, A. S. & Karumanchi, S. A. Molecular Mechanisms of Preeclampsia. Cold Spring Harb Perspect Med5, 10.1101/cshperspect.a023473 (2015). - PMC - PubMed

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