Surgery Today

, 41:1622 | Cite as

Roles of vascular endothelial growth factor and endothelial nitric oxide synthase during revascularization and regeneration after partial hepatectomy in a rat model

  • Daisuke Yoshida
  • Tomohiko Akahoshi
  • Hirofumi Kawanaka
  • Shohei Yamaguchi
  • Nao Kinjo
  • Akinobu Taketomi
  • Morimasa Tomikawa
  • Ken Shirabe
  • Yoshihiko Maehara
  • Makoto Hashizume
Original Article



Angiogenesis is an essential process in liver regeneration. Nitric oxide (NO) and vascular endothelial growth factor (VEGF) are the main regulators of normal and pathological angiogenesis. This study aimed to determine the roles of NO derived from endothelial nitric oxide synthase (eNOS) and VEGF in sinusoidal endothelial cell (SEC) proliferation during liver regeneration.


Sprague-Dawley rats underwent a 70% partial hepatectomy (PHx), and were euthanized 0, 24, 48, 72, or 168 h later. Liver regeneration and SEC proliferation were evaluated. The protein expression of VEGF and eNOS was examined by a Western blot analysis. The rats were also treated with the NO synthase inhibitor N G-nitro-l-arginine-methyl ester (L-NAME) to examine its effects on liver regeneration and SEC proliferation.


The proliferating cell nuclear antigen (PCNA) labeling index of hepatocytes was significantly increased at 24 h after PHx. The eNOS protein expression and NO production were significantly increased from 72 to 168 h. The expression of VEGF protein was significantly increased at 72 h. L-NAME significantly inhibited the increases in the liver mass and decreased the PCNA labeling index of hepatocytes at 24 h. L-NAME also inhibited the induction of VEGF protein at 72 h.


Endothelial NOS and VEGF coordinately regulate SEC proliferation during liver regeneration. Sinusoidal endothelial cell proliferation is necessary and is an important step in liver regeneration.

Key words

Liver regeneration Vascular endothelial growth factor Nitric oxide Endothelial nitric oxide synthase 


  1. 1.
    Fausto N, Laird AD, Webber EM. Liver regeneration. 2. Role of growth factors and cytokines in hepatic regeneration. FASEB J 1995;9:1527–1536.PubMedGoogle Scholar
  2. 2.
    Mochida S, Ishikawa K, Inao M, Shibuya M, Fujiwara K. Increased expressions of vascular endothelial growth factor and its receptors, flt-1 and KDR/flk-1, in regenerating rat liver. Biochem Biophys Res Commun 1996;226:176–179.PubMedCrossRefGoogle Scholar
  3. 3.
    Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276:60–66.PubMedCrossRefGoogle Scholar
  4. 4.
    Wack KE, Ross MA, Zegarra V, Sysko LR, Watkins SC, Stolz DB. Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver. Hepatology 2001;33:363–378.PubMedCrossRefGoogle Scholar
  5. 5.
    Kraizer Y, Mawasi N, Seagal J, Paizi M, Assy N, Spira G. Vascular endothelial growth factor and angiopoietin in liver regeneration. Biochem Biophys Res Commun 2001;287:209–215.PubMedCrossRefGoogle Scholar
  6. 6.
    Shimizu H, Miyazaki M, Wakabayashi Y, Mitsuhashi N, Kato A, Ito 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.PubMedCrossRefGoogle Scholar
  7. 7.
    Ross MA, Sander CM, Kleeb TB, Watkins SC, Stolz DB. Spatiotemporal expression of angiogenesis growth factor receptors during the revascularization of regenerating rat liver. Hepatology 2001;34:1135–1148.PubMedCrossRefGoogle Scholar
  8. 8.
    Blouin A, Bolender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol 1977;72:441–455.PubMedCrossRefGoogle Scholar
  9. 9.
    Sato T, El-Assal ON, Ono T, Yamanoi A, Dhar DK, Nagasue N. Sinusoidal endothelial cell proliferation and expression of angiopoietin/Tie family in regenerating rat liver. J Hepatol 2001;34:690–698.PubMedCrossRefGoogle Scholar
  10. 10.
    Tsurumi Y, Murohara T, Krasinski K, Chen D, Witzenbichler B, Kearney M, et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nat Med 1997;3:879–886.PubMedCrossRefGoogle Scholar
  11. 11.
    Snyder SH, Jaffrey SR. Vessels vivified by Akt acting on NO synthase. Nat Cell Biol 1999;1:E95–E96.PubMedCrossRefGoogle Scholar
  12. 12.
    Kimura H, Esumi H. Reciprocal regulation between nitric oxide and vascular endothelial growth factor in angiogenesis. Acta Biochim Pol 2003;50(1):49–59.PubMedGoogle Scholar
  13. 13.
    Obolenskaya M, Schulze-Specking A, Plaumann B, Frenzer K, Freudenberg N, Decker K. Nitric oxide production by cells isolated from regenerating rat liver. Biochem Biophys Res Commun 1994;204:1305–1311.PubMedCrossRefGoogle Scholar
  14. 14.
    Hortelano S, Dewez B, Genaro AM, Diaz-Guerra MJ, Bosca L. Nitric oxide is released in regenerating liver after partial hepatectomy. Hepatology 1995;21:776–786.PubMedGoogle Scholar
  15. 15.
    Ronco MT, Alvarez Mde L, Monti JA, Carrillo MC, Pisani GB, Lugano MC, et al. Role of nitric oxide increase on induced programmed cell death during early stages of rat liver regeneration. Biochim Biophys Acta 2004;1690:70–76.PubMedGoogle Scholar
  16. 16.
    Zeini M, Hortelano S, Traves PG, Gomez-Valades AG, Pujol A, Perales JC, et al. Assessment of a dual regulatory role for NO in liver regeneration after partial hepatectomy: protection against apoptosis and retardation of hepatocyte proliferation. FASEB J 2005;19:995–997.PubMedGoogle Scholar
  17. 17.
    Shimizu H, Miyazaki M, Ito H, Nakagawa K, Ambiru S, Kato A, et al. Mechanism of cold ischemia-reperfusion-induced graft injury after orthotopic liver transplantation in rats. Hepatogastroenterology 2001;48:216–219.PubMedGoogle Scholar
  18. 18.
    Arai M, Mochida S, Ohno A, Ogata I, Obama H, Maruyama I, et al. Blood coagulation equilibrium in rat liver microcirculation as evaluated by endothelial cell thrombomodulin and macrophage tissue factor. Thromb Res 1995;80:113–123.PubMedCrossRefGoogle Scholar
  19. 19.
    Ronco MT, Francés D, de Luján Alvarez M, Quiroga A, Monti J, Parody JP, et al. Vascular endothelial growth factor and nitric oxide in rat liver regeneration. Life Sci 2007;81(9):750–755.PubMedCrossRefGoogle Scholar
  20. 20.
    Rai RM, Lee FY, Rosen A, Yang SQ, Lin HZ, Koteish A, et al. Impaired liver regeneration in inducible nitric oxide synthase deficient mice. Proc Natl Acad Sci USA 1998;95(23):13829–13834.PubMedCrossRefGoogle Scholar
  21. 21.
    Zeini M, Hortelano S, Través PG, Gómez-Valadés AG, Pujol A, Perales JC, et al. Assessment of a dual regulatory role for NO in liver regeneration after partial hepatectomy: protection against apoptosis and retardation of hepatocyte proliferation. FASEB J 2005;19(8):995–997.PubMedGoogle Scholar
  22. 22.
    Higgins GM, Anderson RM. Experimental pathology of the liver: restoration of the liver of the white rat following surgical removal. Arch Pathol 1931;12:186–202.Google Scholar
  23. 23.
    Assy N, Spira G, Paizi M, Shenkar L, Kraizer Y, Cohen T, et al. Effect of vascular endothelial growth factor on hepatic regenerative activity following partial hepatectomy in rats. J Hepatol 1999;30:911–915.PubMedCrossRefGoogle Scholar
  24. 24.
    Selzner M, Clavien PA. Failure of regeneration of the steatotic rat liver: disruption at two different levels in the regeneration pathway. Hepatology 2000;31:35–42.PubMedCrossRefGoogle Scholar
  25. 25.
    Ishikawa K, Mochida S, Mashiba S, Inao M, Matsui A, Ikeda H, et al. Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis. Biochem Biophys Res Commun 1999;254:587–593.PubMedCrossRefGoogle Scholar
  26. 26.
    Yamaguchi S, Kawanaka H, Yoshida D, Maehara Y, Hashizume M. Splenic hemodynamics and decreased endothelial nitric oxide synthase in the spleen of rats with liver cirrhosis. Life Sci 2007;80:2036–2044.PubMedCrossRefGoogle Scholar
  27. 27.
    Anegawa G, Kawanaka H, Yoshida D, Konishi K, Yamaguchi S, Kinjo N, et al. Defective endothelial nitric oxide synthase signaling is mediated by rho-kinase activation in rats with secondary biliary cirrhosis. Hepatology 2008;47(3):966–977.PubMedCrossRefGoogle Scholar
  28. 28.
    Hall PA, Levison DA, Woods AL, Yu CC, Kellock DB, Watkins JA, et al. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 1990;162:285–294.PubMedCrossRefGoogle Scholar
  29. 29.
    Ijichi H, Taketomi A, Yoshizumi T, Uchiyama H, Yonemura Y, Soejima Y, et al. Hyperbaric oxygen induces vascular endothelial growth factor and reduces liver injury in regenerating rat liver after partial hepatectomy. J Hepatol 2006;45:28–34.PubMedCrossRefGoogle Scholar
  30. 30.
    Kurioka S, Koshimura K, Sugitani M, Murakami Y, Nishiki M, Kato Y. Analysis of urinary nitric oxide metabolites in healthy subjects. Endocr J 1999;46:421–428.PubMedCrossRefGoogle Scholar
  31. 31.
    Yamamoto M, Koshimura K, Sohmiya M, Murakami Y, Kato Y. Effect of erythropoietin on nitric oxide production in the rat hippocampus using in vivo brain microdialysis. Neuroscience 2004;128:163–168.PubMedCrossRefGoogle Scholar
  32. 32.
    Tomikawa M, Hashizume M, Higashi H, Ohta M, Sugimachi K. The role of spleen, platelets, and plasma hepatocyte growth factor activity on hepatic regeneration in rats. J Am Coll Surg 1996;182(1):12–16.PubMedGoogle Scholar
  33. 33.
    Shimabukuro R, Kawanaka H, Tomikawa M, Akahoshi T, Konishi K, Yoshida D, et al. Effect of thrombopoietin on platelet counts and liver regeneration after partial hepatectomy in a rat model. Surg Today 2009;39(12):1054–1059.PubMedCrossRefGoogle Scholar
  34. 34.
    Wang HH, Lautt WW. Evidence of nitric oxide, a flow-dependent factor, being a trigger of liver regeneration in rats. Can J Physiol Pharmacol 1998;76:1072–1079.PubMedCrossRefGoogle Scholar
  35. 35.
    Obolenskaya MYu, Vanin AF, Mordvintcev PI, Mülsch A, Decker K. EPR evidence of nitric oxide production by the regenerating rat liver. Biochem Biophys Res Commun 1994;202(1):571–576.PubMedCrossRefGoogle Scholar
  36. 36.
    Shah V, Haddad FG, Garcia-Cardena G, Frangos JA, Mennone A, Groszmann RJ, et al. Liver sinusoidal endothelial cells are responsible for nitric oxide modulation of resistance in the hepatic sinusoids. J Clin Invest 1997;100(11):2923–2930.PubMedCrossRefGoogle Scholar
  37. 37.
    Schoen JM, Wang HH, Minuk GY, Lautt WW. Shear stress-induced nitric oxide release triggers the liver regeneration cascade. Nitric Oxide 2001;5:453–464.PubMedCrossRefGoogle Scholar
  38. 38.
    Ronco MT, Frances D, de Lujan Alvarez M, Quiroga A, Monti J, Parody JP, et al. Vascular endothelial growth factor and nitric oxide in rat liver regeneration. Life Sci 2007;81:750–755.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2011

Authors and Affiliations

  • Daisuke Yoshida
    • 1
    • 2
  • Tomohiko Akahoshi
    • 2
  • Hirofumi Kawanaka
    • 2
  • Shohei Yamaguchi
    • 2
  • Nao Kinjo
    • 2
  • Akinobu Taketomi
    • 2
  • Morimasa Tomikawa
    • 2
  • Ken Shirabe
    • 2
  • Yoshihiko Maehara
    • 2
  • Makoto Hashizume
    • 1
  1. 1.Department of Disaster and Emergency MedicineKyushu UniversityFukuokaJapan
  2. 2.Department of Surgery and Science, Graduate School of Medical SciencesKyushu UniversityFukuokaJapan

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