FGFR1 mediates recombinant thrombomodulin domain-induced angiogenesis.
OBJECTIVE
The recombinant epidermal growth factor-like domain plus the serine/threonine-rich domain of thrombomodulin (rTMD23) promotes angiogenesis and accelerates the generation of activated protein C (APC), which facilitates angiogenesis. The aim of this study was to elucidate the molecular mechanisms underlying the angiogenic activity of rTMD23.


RESULTS
We prepared rTMD23 and its mutants that did not possess the ability to promote APC generation and investigated their angiogenic activities in vitro and in vivo. rTMD23 mutants promoted proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro and induced neovascularization in vivo; these effects were similar to those exerted by wild-type rTMD23. To investigate its interaction with rTMD23, Type I fibroblast growth factor receptor (FGFR1) was precipitated along with syndecan-4 by rTMD23-conjugated Sepharose in human umbilical vein endothelial cells and FGFR1-expressing human embryonic kidney 293 cells. Additionally, the kinetics of the interaction between rTMD23 and FGFR1 were analysed using surface plasmon resonance. rTMD23-induced FGFR1 activation and tube formation were inhibited by an FGFR1-specific tyrosine kinase inhibitor, PD173074, or by knockdown of FGFR1 using siRNA technology. We observed an improvement in rat hindlimb recovery in an ischaemic model following rTMD23 treatment, and this was associated with increased neovascularization and FGFR1 phosphorylation.


CONCLUSIONS
rTMD23 induced angiogenesis via FGFR1, a process that is independent of the APC pathway.
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan.
Cheng-Hsiang
CH
Kuo
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan.
Meng-Chen
MC
Sung
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan.
Po-Ku
PK
Chen
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan.
Bi-Ing
BI
Chang
Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan.
Fang-Tzu
FT
Lee
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan.
Chia-Fong
CF
Cho
Division of Cardiovascular Surgery, Department of Surgery, National Cheng Kung University Hospital and College of Medicine, 138 Sheng-Li Road, Tainan 70403, Taiwan.
Tai-Tzu
TT
Hsieh
Division of Cardiovascular Surgery, Department of Surgery, National Cheng Kung University Hospital and College of Medicine, 138 Sheng-Li Road, Tainan 70403, Taiwan.
Yu-Ching
YC
Huang
Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan Department of Internal Medicine, Hospital and College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Yi-Heng
YH
Li
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan.
Guey-Yueh
GY
Shi
Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan Division of Cardiovascular Surgery, Department of Surgery, National Cheng Kung University Hospital and College of Medicine, 138 Sheng-Li Road, Tainan 70403, Taiwan luochwya@mail.ncku.edu.tw halnwu@mail.ncku.edu.tw.
Chwan-Yau
CY
Luo
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 701, Taiwan Cardiovascular Research Center, National Cheng Kung University, Tainan, Taiwan luochwya@mail.ncku.edu.tw halnwu@mail.ncku.edu.tw.
Hua-Lin
HL
Wu
D043925:Angiogenesis Inducing Agents / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D016510:Corneal Neovascularization / Q000209:etiology; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006614:Hindlimb / Q000098:blood supply; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007511:Ischemia / Q000188:drug therapy / Q000473:pathology / Q000503:physiopathology; D051379:Mice; D008807:Mice, Inbred BALB C; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D011486:Protein C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D053671:Syndecan-4 / Q000378:metabolism; D018180:Thrombomodulin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D043925:Angiogenesis Inducing Agents; C115711:PD 173074; D011486:Protein C; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011994:Recombinant Proteins; C508304:SDC4 protein, human; C508307:Sdc4 protein, rat; D053671:Syndecan-4; D018180:Thrombomodulin; C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1
Activated protein C; Angiogenesis; Syndecan-4; Thrombomodulin; Type I fibroblast growth factor receptor