Thrombin-triggered angiogenesis in rat brains following experimental intracerebral hemorrhage

Laboratory investigation

Restricted access

Object

Angiogenesis occurs after intracerebral hemorrhage (ICH). Thrombin mediates mitogenesis and survival in endothelial cells and induces angiogenesis. The present study aimed to clarify whether thrombin is involved in triggering ICH-related angiogenesis.

Methods

In the first part of the experiment, autologous blood (with or without hirudin) was injected to induce ICH. In the second part, rats received either 1 U (50 μl) thrombin or 50 μl 0.9% sterile saline. In both parts, 5-bromo-2-deoxyuridine (BrdU) was administered intraperitoneally. Brains were perfused to identify BrdU-positive/von Willebrand factor (vWF)–positive nuclei. The expression of hypoxia-inducible factor–1α (HIF-1α), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and Ang-2 was evaluated by immunohistochemistry and quantitative real-time reverse transcription polymerase chain reaction.

Results

After ICH, the number of BrdU-/vWF-positive nuclei increased until Day 14, and vessels positive for HIF-1α, VEGF, Ang-1, and Ang-2 were observed around the clot. Quantitative analysis showed that ICH upregulated expression of HIF-1α, VEGF, Ang-1, and Ang-2 notably compared with that in sham controls (p < 0.05). However, hirudin significantly inhibited these effects. After thrombin treatment, many BrdU-positive/vWF-positive nuclei and HIF-1α–, VEGF-, Ang-1– and Ang-2–positive vessels could be detected around the affected region.

Conclusions

Thrombin can induce angiogenesis in rat brains and may be an important trigger for ICH-related angiogenesis.

Abbreviations used in this paper:Ang = angiopoietin; BrdU = 5-bromo-2-deoxyuridine; CSU = Central South University; EC = endothelial cell; HIF-1α = hypoxia-inducible factor–1α; ICH = intracerebral hemorrhage; PAR-1 = protease-activated receptor–1; RT-PCR = reverse transcription polymerase chain reaction; VEGF = vascular endothelial growth factor; vWF = von Willebrand factor.
Article Information

Contributor Notes

Address correspondence to: Tao Tang, M.D., Ph.D., Institute of Integrative Medicine, Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China, 410008. email: falcontang@126.com.Please include this information when citing this paper: published online September 7, 2012; DOI: 10.3171/2012.8.JNS112152.
Headings
References
  • 1

    Andaluz NZuccarello M: Recent trends in the treatment of spontaneous intracerebral hemorrhage: analysis of a nationwide inpatient database. Clinical article. J Neurosurg 110:4034102009

    • Search Google Scholar
    • Export Citation
  • 2

    Bassus SHerkert OKronemann NGörlach ABremerich DKirchmaier CM: Thrombin causes vascular endothelial growth factor expression in vascular smooth muscle cells: role of reactive oxygen species. Arterioscler Thromb Vasc Biol 21:155015552001

    • Search Google Scholar
    • Export Citation
  • 3

    Carmeliet PCollen D: Molecular analysis of blood vessel formation and disease. Am J Physiol 273:5 Pt 2H2091H21041997

  • 4

    Caunt MHu LTang TBrooks PCIbrahim SKarpatkin S: Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis. Cancer Res 66:412541322006

    • Search Google Scholar
    • Export Citation
  • 5

    Croll SDWiegand SJ: Vascular growth factors in cerebral ischemia. Mol Neurobiol 23:1211352001

  • 6

    Dardik RLoscalzo JInbal A: Factor XIII (FXIII) and angiogenesis. J Thromb Haemost 4:19252006

  • 7

    Davis SAldrich THJones PFAcheson ACompton DLJain V: Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87:116111691996

    • Search Google Scholar
    • Export Citation
  • 8

    Dupuy EHabib ALebret MYang RLevy-Toledano STobelem G: Thrombin induces angiogenesis and vascular endothelial growth factor expression in human endothelial cells: possible relevance to HIF-1alpha. J Thromb Haemost 1:109611022003

    • Search Google Scholar
    • Export Citation
  • 9

    Fenton JW IIVillanueva GBOfosu FAMaraganore JM: Thrombin inhibition by hirudin: how hirudin inhibits thrombin. Haemostasis 21:Suppl 127311991

    • Search Google Scholar
    • Export Citation
  • 10

    Folkman JD'Amore PA: Blood vessel formation: what is its molecular basis?. Cell 87:115311551996

  • 11

    Grienberger HJPillai DRSchlachetzki FGruber MDittmar MS: Detection of free radicals by isolated perfusion of the rat brain following hemorrhagic stroke: a novel approach to cerebrovascular biomarker research. Exp Brain Res 206:3113172010

    • Search Google Scholar
    • Export Citation
  • 12

    Harrigan MR: Angiogenic factors in the central nervous system. Neurosurgery 53:6396612003

  • 13

    Herbert JMDupuy ELaplace MCZini JMBar Shavit RTobelem G: Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway. Biochem J 303:2272311994

    • Search Google Scholar
    • Export Citation
  • 14

    Hua YKeep RFGu YXi G: Thrombin and brain recovery after intracerebral hemorrhage. Stroke 40:3 SupplS88S892009

  • 15

    Jiang YWu JHua YKeep RFXiang JHoff JT: Thrombin-receptor activation and thrombin-induced brain tolerance. J Cereb Blood Flow Metab 22:4044102002

    • Search Google Scholar
    • Export Citation
  • 16

    Jiang YWu JKeep RFHua YHoff JTXi G: Hypoxiainducible factor-1alpha accumulation in the brain after experimental intracerebral hemorrhage. J Cereb Blood Flow Metab 22:6896962002

    • Search Google Scholar
    • Export Citation
  • 17

    Karamysheva AF: Mechanisms of angiogenesis. Biochemistry (Mosc) 73:7517622008

  • 18

    Koblizek TIWeiss CYancopoulos GDDeutsch URisau W: Angiopoietin-1 induces sprouting angiogenesis in vitro. Curr Biol 8:5295321998

    • Search Google Scholar
    • Export Citation
  • 19

    Kuehbacher AUrbich CDimmeler S: Targeting microRNA expression to regulate angiogenesis. Trends Pharmacol Sci 29:12152008

  • 20

    Laurén JGunji YAlitalo K: Is angiopoietin-2 necessary for the initiation of tumor angiogenesis?. Am J Pathol 153:133313391998

  • 21

    Lee KRKawai NKim SSagher OHoff JT: Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg 86:2722781997

    • Search Google Scholar
    • Export Citation
  • 22

    Lin TNWang CKCheung WMHsu CY: Induction of angiopoietin and Tie receptor mRNA expression after cerebral ischemia-reperfusion. J Cereb Blood Flow Metab 20:3873952000

    • Search Google Scholar
    • Export Citation
  • 23

    Liu JSchuff-Werner PSteiner M: Thrombin/thrombin receptor (PAR-1)-mediated induction of IL-8 and VEGF expression in prostate cancer cells. Biochem Biophys Res Commun 343:1831892006

    • Search Google Scholar
    • Export Citation
  • 24

    Liu MWu BWang WZLee LMZhang SHKong LZ: Stroke in China: epidemiology, prevention, and management strategies. Lancet Neurol 6:4564642007

    • Search Google Scholar
    • Export Citation
  • 25

    Livak KJSchmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:4024082001

    • Search Google Scholar
    • Export Citation
  • 26

    Maeda YHirano KKai YHirano MSuzuki SOSasaki T: Up-regulation of proteinase-activated receptor 1 and increased contractile responses to thrombin after subarachnoid haemorrhage. Br J Pharmacol 152:113111392007

    • Search Google Scholar
    • Export Citation
  • 27

    Markwardt F: The development of hirudin as an antithrombotic drug. Thromb Res 74:1231994

  • 28

    Pugh CWRatcliffe PJ: Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 9:6776842003

  • 29

    Qureshi AILing GSKhan JSuri MFMiskolczi LGuterman LR: Quantitative analysis of injured, necrotic, and apoptotic cells in a new experimental model of intracerebral hemorrhage. Crit Care Med 29:1521572001

    • Search Google Scholar
    • Export Citation
  • 30

    Qureshi AITuhrim SBroderick JPBatjer HHHondo HHanley DF: Spontaneous intracerebral hemorrhage. N Engl J Med 344:145014602001

    • Search Google Scholar
    • Export Citation
  • 31

    Rane SHe MSayed DVashistha HMalhotra ASadoshima J: Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circ Res 104:8798862009

    • Search Google Scholar
    • Export Citation
  • 32

    Richard DEBerra EPouyssegur J: Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells. J Biol Chem 275:26765267712000

    • Search Google Scholar
    • Export Citation
  • 33

    Rickles FRPatierno SFernandez PM: Tissue factor, thrombin, and cancer. Chest 124:3 Suppl58S68S2003

  • 34

    Smadja DMLaurendeau IAvignon CVidaud MAiach MGaussem P: The angiopoietin pathway is modulated by PAR-1 activation on human endothelial progenitor cells. J Thromb Haemost 4:205120582006

    • Search Google Scholar
    • Export Citation
  • 35

    Suri CMcClain JThurston GMcDonald DMZhou HOldmixon EH: Increased vascularization in mice overexpressing angiopoietin-1. Science 282:4684711998

    • Search Google Scholar
    • Export Citation
  • 36

    Tang TLiu XJZhang ZQZhou HJLuo JKHuang JF: Cerebral angiogenesis after collagenase-induced intracerebral hemorrhage in rats. Brain Res 1175:1341422007

    • Search Google Scholar
    • Export Citation
  • 37

    Tsopanoglou NEMaragoudakis ME: Role of thrombin in angiogenesis and tumor progression. Semin Thromb Hemost 30:63692004

  • 38

    Vaughan PJPike CJCotman CWCunningham DD: Thrombin receptor activation protects neurons and astrocytes from cell death produced by environmental insults. J Neurosci 15:538954011995

    • Search Google Scholar
    • Export Citation
  • 39

    Webb JDColeman MLPugh CW: Hypoxia, hypoxia-inducible factors (HIF), HIF hydroxylases and oxygen sensing. Cell Mol Life Sci 66:353935542009

    • Search Google Scholar
    • Export Citation
  • 40

    Xi GKeep RFHoff JT: Mechanisms of brain injury after intracerebral haemorrhage. Lancet Neurol 5:53632006

  • 41

    Xi GKeep RFHua YXiang JHoff JT: Attenuation of thrombin-induced brain edema by cerebral thrombin preconditioning. Stroke 30:124712551999

    • Search Google Scholar
    • Export Citation
  • 42

    Yamakawa MLiu LXDate TBelanger AJVincent KAAkita GY: Hypoxia-inducible factor-1 mediates activation of cultured vascular endothelial cells by inducing multiple angiogenic factors. Circ Res 93:6646732003

    • Search Google Scholar
    • Export Citation
  • 43

    Yang SSong SHua YNakamura TKeep RFXi G: Effects of thrombin on neurogenesis after intracerebral hemorrhage. Stroke 39:207920842008

    • Search Google Scholar
    • Export Citation
  • 44

    Zania PPapaconstantinou MFlordellis CSMaragoudakis METsopanoglou NE: Thrombin mediates mitogenesis and survival of human endothelial cells through distinct mechanisms. Am J Physiol Cell Physiol 294:C1215C12262008

    • Search Google Scholar
    • Export Citation
  • 45

    Zhang ZChopp M: Vascular endothelial growth factor and angiopoietins in focal cerebral ischemia. Trends Cardiovasc Med 12:62662002

    • Search Google Scholar
    • Export Citation
  • 46

    Zhou HTang TGuo CZhang HZhong JZheng J: Expression of Angiopoietin-1 and the receptor Tie-2 mRNA in rat brains following intracerebral hemorrhage. Acta Neurobiol Exp (Warsz) 68:1471542008

    • Search Google Scholar
    • Export Citation
  • 47

    Zhou HJZhang HNTang TZhong JHQi YLuo JK: Alteration of thrombospondin-1 and -2 in rat brains following experimental intracerebral hemorrhage. Laboratory investigation. J Neurosurg 113:8208252010

    • Search Google Scholar
    • Export Citation
TrendMD
Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 280 276 44
Full Text Views 162 98 2
PDF Downloads 117 56 2
EPUB Downloads 0 0 0
PubMed
Google Scholar