在血管生成的過程中，受到許多生長因子的影響，進而達成內皮細胞的增生、移動、管狀結構的形成以及血管穩定度的維持之目的，其中在血管的成熟與穩定度的維持部分，則是以Tie2接受器及其訊號傳遞路徑扮演著重要的角色。Tie2是個主要表現於內皮細胞表面的接受器，透過與其配體angiopoietin-1(Ang1)的結合以及訊號傳遞路徑，主要影響內皮細胞的重組、細胞間的交互作用、內皮細胞的存活以及內皮細胞與細胞外間質的交互作用。在先前研究中指出，在具有遺傳性靜脈發育不良症的家族當中，發現Tie2的第849個胺基酸發生Arginine變成Tryptophan的突變(Tie2-R849W)，此單一胺基酸的突變則會導致Tie2接受器的酪胺酸酶過度活化，意即不需其配體即可被活化。及至目前，對於Tie2-R849W所調控的訊號傳遞路徑還尚未釐清，我們利用靜脈形成不良症做為疾病模式，比較突變型的Tie2(R849W)與正常型的Tie2(WT)所調控的訊號傳遞路徑，盼能釐清導致靜脈發育不良症的致病機轉。在本研究中，我們成功地利用腺病毒運送系統(Anenovirus delivery system)將三種不同激酶活性的Tie2蛋白送入內皮細胞中大量表現，並發現在Tie2-R849W突變蛋白在缺乏配體刺激的狀況下，即可明顯促進STAT1上位於701位置的酪胺酸(Tyrosine)的磷酸化，以及磷酸化的STAT1能轉移到細胞核內的現象，除此之外，Tie2-R849W突變蛋白還能增強STAT5及MAPK家族成員(ERK1/2、JNK、p38)的基本磷酸化程度，半定量反轉錄聚合酶連鎖反應(Semi-quantitative RT-PCR)的結果指出，Tie2-R849W突變蛋白能增強(liposaccharides : LPS)促使IRF1和ICAM1的mRNA表現量增加的現象，但卻會減弱LPS對於VCAM1和E-selectin的刺激，在白血球黏附能力測試(Leucocyte adhesion assay)的部分，發現Tie2-R849W突變蛋白會降低白血球黏附至內皮細胞的能力。STAT1目前被指出是一種血管新生的負向調節者，可藉由降低MMP2的表現來達到抑制血管新生的效果，而在我們的研究中發現，Tie2-R849W突變蛋白不僅降低MMP2的酵素活性同時也降低的MMP2的mRNA表現量。綜合以上結果，我們可以得知透過持續過度活化的Tie2-R849W突變蛋白，可以引發與Tie2-WT不同的訊號傳遞路徑，如STAT和MAPK等，更多的問題仍尚待進一步的努力去釐清，以探討對於靜脈形成不良症的影響。

The formation of blood vessel is regulated by many growth factors and involved in endothelial cell proliferation, migration, tube formation and maintenance of blood vessel integrity. Among many mediators, Tie2 and its ligands play an important role in vessel maturation and maintenance of blood vessel integrity. Tie2, a receptor tyrosine kinase, is primarily expressed in vascular endothelium. One major ligand for Tie2 is angiopoietin 1(Ang1), which influences endothelial cell (EC) assembly, cell-cell interaction, EC survival and EC-extracellular matrix (ECM) interaction. The alteration of Tie2 at amino acid residue 849 from arginine to tryphtophan (Tie2-R849W) has been associated with familial Venous Malformations (VMs). So far, Tie2-R849W mediated signaling pathways remains unclear. By using VMs as a disease model, we compared the signaling pathways mediated by Tie2-WT and Tie2-R849W, and hoped to address the underlying mechanism responsible for VMs. We utilized adenoviral expressing vectors to overexpress Tie2 and its variants in HUVEC. We demonstrated that Tie2-R849W but neither wild-type counterpart nor kinase dead mutant (K855A) induced Tyr701 phosphorylation of STAT1 and a subsequent nuclear localization of phosphorylated STAT1 in a ligand-independent manner. Besides, Tie2-R849W also enhanced the basal phosphorylation of STAT5 and three MAPK family members, ERK1/2, JNK and p38-MAPK. Semi- quantitative RT-PCR analysis indicated that Tie2-R849W enhanced liposaccharides (LPS)-induced mRNA expression of IRF-1 and ICAM1 but attenuated the induction of VCAM1 and E-selectin mRNA. Leukocyte adhesion assay indicated that Tie2-R849W decreased the ability of leukocytes to adhere to HUVEC. STAT1, a newly identified negative regulator of angiogenesis, can reduce the expression of MMP2. Indeed, Tie2-R849W not only decreased the activity of MMP2 in the gelatin zymography assay but also decreased the mRNA expression of MMP2. Together, these data suggest that constitutive activated Tie2-R849W induced distinct signaling pathways from its wild type counterpart Tie2, including those mediated by STAT and MAPK members. More studies are warranted to address the exact role of these distinct signaling pathways in the VMs.

1.Vikkula M, Boon LM, Carraway KL 3rd, Calvert JT, Diamonti AJ, Goumnerov B,Pasyk KA, Marchuk DA, Warman ML, Cantley LC, Mulliken JB, Olsen BR. Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell. 1996 Dec 27; 87(7):1181-90.
2.Risau W, Lemmon V. Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis. Dev Biol. 1988 Feb; 125(2):441-50.
3.Risau W, Sariola H, Zerwes HG, Sasse J, Ekblom P, Kemler R, Doetschman T. Vasculogenesis and angiogenesis in embryonic- stem-cell-derived embryoid bodies. Development. 1988 Mar; 102(3):471-8.
4.Noden DM. Embryonic origins and assembly of blood vessels. Am Rev Respir Dis. 1989 Oct; 140(4):1097-103.
5.Thurston G, Suri C, Smith K, McClain J, Sato TN, Yancopoulos GD, McDonald DM. Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1. Science. 1999 Dec 24; 286(5449):2511-4.
6.Koblizek TI, Weiss C, Yancopoulos GD, Deutsch U, Risau W. Angiopoietin-1 induces sprouting angiogenesis in vitro. Curr Biol. 1998 Apr 23; 8(9):529-32.
7.Zhu WH, MacIntyre A, Nicosia RF. Regulation of angiogenesis by vascular endothelial growth factor and angiopoietin-1 in the rat aorta model: distinct temporal patterns of intracellular signaling correlate with induction of angiogenic sprouting. Am J Pathol. 2002 Sep; 161(3):823-30.
8.Chae JK, Kim I, Lim ST, Chung MJ, Kim WH, Kim HG, Ko JK, Koh GY. Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler Thromb Vasc Biol. 2000 Dec; 20(12):2573-8.
9.Shyu KG, Chang H, Isner JM. Synergistic effect of angiopoietin-1 and vascular endothelial growth factor on neoangiogenesis in hypercholesterolemic rabbit model with acute hindlimb ischemia. Life Sci. 2003 Jun 20; 73(5):563-79.
10.Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J, McDonald DM, Yancopoulos GD. Angiopoietin-1 protects the adult vasculature against plasma leakage. Nat Med. 2000 Apr; 6(4):460-3.
11.Nishishita T, Lin PC. Angiopoietin 1, PDGF-B, and TGF-beta gene regulation in endothelial cell and smooth muscle cell interaction. J Cell Biochem. 2004 Feb 15; 91(3):584-93.
12.Metheny-Barlow LJ, Tian S, Hayes AJ, Li LY. Direct chemotactic action of angiopoietin-1 on mesenchymal cells in the presence of VEGF. Microvasc Res. 2004 Nov; 68(3):221-30.
13.Dumont DJ, Yamaguchi TP, Conlon RA, Rossant J, Breitman ML. tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors. Oncogene. 1992 Aug; 7(8):1471-80.
14.Partanen J, Armstrong E, Makela TP, Korhonen J, Sandberg M, Renkonen R, Knuutila S, Huebner K, Alitalo K. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol Cell Biol. 1992 Apr; 12(4):1698-707.
15.Dumont DJ, Gradwohl GJ, Fong GH, Auerbach R, Breitman ML. The endothelial-specific receptor tyrosine kinase, tek, is a member of a new subfamily of receptors. Oncogene. 1993 May;8(5):1293-301.
16.Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V, Ryan TE, Bruno J, Radziejewski C, Maisonpierre PC, Yancopoulos GD. Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell. 1996 Dec 27; 87(7):1161-9.
17.Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, Radziejewski C, Compton D, McClain J, Aldrich TH, Papadopoulos N, Daly TJ, Davis S, Sato TN, Yancopoulos GD. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science. 1997 Jul 4; 277(5322):55-60.
18.Valenzuela DM, Griffiths JA, Rojas J, Aldrich TH, Jones PF, Zhou H, McClain J, Copeland NG, Gilbert DJ, Jenkins NA, Huang T, Papadopoulos N, Maisonpierre PC, Davis S, Yancopoulos GD. Angiopoietins 3 and 4: diverging gene counterparts in mice and humans. Proc Natl Acad Sci U S A. 1999 Mar 2; 96(5):1904-9.
19.Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD. Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 1996 Dec 27; 87(7):1171-80.
20.Holash J, Wiegand SJ, Yancopoulos GD. New model of tumor angiogenesis: dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene. 1999 Sep 20; 18(38):5356-62.
21.Lee HJ, Cho CH, Hwang SJ, Choi HH, Kim KT, Ahn SY, Kim JH, Oh JL, Lee GM, Koh GY. Biological characterization of angiopoietin-3 and angiopoietin-4. FASEB J. 2004 Aug; 18(11):1200-8.
22.Sato TN, Tozawa Y, Deutsch U, Wolburg-Buchholz K, Fujiwara Y, Gendron-Maguire M, Gridley T, Wolburg H, Risau W, Qin Y. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature. 1995 Jul 6; 376(6535):70-4.
23.Dumont DJ, Gradwohl G, Fong GH, Puri MC, Gertsenstein M, Auerbach A, Breitman ML. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev. 1994 Aug 15; 8(16):1897-909.
24.Hubbard SR, Till JH. Protein tyrosine kinase structure and function. Annu Rev Biochem. 2000; 69:373-98.
25.Jones N, Dumont DJ. The Tek/Tie2 receptor signals through a novel Dok-related docking protein, Dok-R. Oncogene. 1998 Sep 3; 17(9):1097-108.
26.Jones N, Master Z, Jones J, Bouchard D, Gunji Y, Sasaki H, Daly R, Alitalo K, Dumont DJ. Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site mediates cell survival and migration. J Biol Chem. 1999 Oct 22; 274(43):30896-905.
27.Huang L, Turck CW, Rao P, Peters KG. GRB2 and SH-PTP2: potentially important endothelial signaling molecules downstream of the TEK/TIE2 receptor tyrosine kinase. Oncogene. 1995 Nov 16; 11(10):2097-103.
28.Kim I, Ryu YS, Kwak HJ, Ahn SY, Oh JL, Yancopoulos GD, Gale NW, Koh GY. EphB ligand, ephrinB2, suppresses the VEGF- and angiopoietin 1-induced Ras/mitogen-activated protein kinase pathway in venous endothelial cells. FASEB J. 2002 Jul; 16(9):1126-8.
29.Yoon MJ, Cho CH, Lee CS, Jang IH, Ryu SH, Koh GY. Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells. Biochem Biophys Res Commun. 2003 Aug 15; 308(1):101-5
30.Kontos CD, Stauffer TP, Yang WP, York JD, Huang L, Blanar MA, Meyer T, Peters KG. Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt. Mol Cell Biol. 1998 Jul; 18(7):4131-40.
31.DeBusk LM, Hallahan DE, Lin PC. Akt is a major angiogenic mediator downstream of the Ang1/Tie2 signaling pathway. Exp Cell Res. 2004 Aug 1; 298(1):167-77.
32.Fujikawa K, de Aos Scherpenseel I, Jain SK, Presman E, Christensen RA, Varticovski L. Role of PI 3-kinase in angiopoietin-1-mediated migration and attachment-dependent survival of endothelial cells. Exp Cell Res. 1999 Dec 15; 253(2):663-72.
33.Hayes AJ, Huang WQ, Mallah J, Yang D, Lippman ME, Li LY. Angiopoietin-1 and its receptor Tie-2 participate in the regulation of capillary-like tubule formation and survival of endothelial cells. Microvasc Res. 1999 Nov; 58(3):224-37.
34.Audero E, Cascone I, Maniero F, Napione L, Arese M, Lanfrancone L, Bussolino F. Adaptor ShcA protein binds tyrosine kinase Tie2 receptor and regulates migration and sprouting but not survival of endothelial cells. J Biol Chem. 2004 Mar 26; 279(13):13224-33.
35.Hughes DP, Marron MB, Brindle NP. The antiinflammatory endothelial tyrosine kinase Tie2 interacts with a novel nuclear factor-kappaB inhibitor ABIN-2. Circ Res. 2003 Apr 4; 92(6):630-6.
36.Kim I, Moon SO, Park SK, Chae SW, Koh GY. Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression. Circ Res. 2001 Sep 14; 89(6):477-9.
37.Korpelainen EI, Karkkainen M, Gunji Y, Vikkula M, Alitalo K. Endothelial receptor tyrosine kinases activate the STAT signaling pathway: mutant Tie-2 causing venous malformations signals a distinct STAT activation response. Oncogene. 1999 Jan 7; 18(1):1-8.
38.Mulliken JB, Glowacki J. Hemangiomas and vascular malformations in infants and children: a classification based on endothelial characteristics. Plast Reconstr Surg. 1982 Mar; 69(3):412-22.
39.Vikkula M, Boon LM, Carraway KL 3rd, Calvert JT, Diamonti AJ, Goumnerov B, Pasyk KA, Marchuk DA, Warman ML, Cantley LC, Mulliken JB, Olsen BR. Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2. Cell. 1996 Dec 27; 87(7):1181-90.
40.Calvert JT, Riney TJ, Kontos CD, Cha EH, Prieto VG, Shea CR, Berg JN, Nevin NC, Simpson SA, Pasyk KA, Speer MC, Peters KG, Marchuk DA. Allelic and locus heterogeneity in inherited venous malformations. Hum Mol Genet. 1999 Jul; 8(7):1279-89.
41.Ihle JN. The Stat family in cytokine signaling. Curr Opin Cell Biol. 2001 Apr; 13(2):211-7.
42.Battle TE, Lynch RA, Frank DA. Signal transducer and activator of transcription 1 activation in endothelial cells is a negative regulator of angiogenesis. Cancer Res. 2006 Apr 1; 66(7):3649-57.
43.Morris PN, Dunmore BJ, Tadros A, Marchuk DA, Darland DC, D'Amore PA, Brindle NP. Functional analysis of a mutant form of the receptor tyrosine kinase Tie2 causing venous malformations. J Mol Med. 2005 Jan; 83(1):58-63.
44.Andl CD, Mizushima T, Oyama K, Bowser M, Nakagawa H, Rustgi AK. EGFR-induced cell migration is mediated predominantly by the JAK-STAT pathway in primary esophageal keratinocytes. Am J Physiol Gastrointest Liver Physiol. 2004 Dec; 287(6):G1227-37.
45.Munro JM, Pober JS, Cotran RS. Tumor necrosis factor and interferon-gamma induce distinct patterns of endothelial activation and associated leukocyte accumulation in skin of Papio anubis. Am J Pathol. 1989 Jul; 135(1):121-33.
46.Heremans H, Dijkmans R, Sobis H, Vandekerckhove F, Billiau A. Regulation by interferons of the local inflammatory response to bacterial lipopolysaccharide. J Immunol. 1987 Jun 15; 138(12):4175-9.
47.Johansen HK, Hougen HP, Rygaard J, Hoiby N. Interferon-gamma (IFN-gamma) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats. Clin Exp Immunol. 1996 Feb; 103(2):212-8.
48.Melrose J, Tsurushita N, Liu G, Berg EL. IFN-gamma inhibits activation-induced expression of E- and P-selectin on endothelial cells. J Immunol. 1998 Sep 1; 161(5):2457-64.
49.Goh KC, Haque SJ, Williams BR. p38 MAP kinase is required for STAT1 serine phosphorylation and transcriptional activation induced by interferons. EMBO J. 1999 Oct 15; 18(20):5601-8.