The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen

Abstract

IntroductionAngiogenesis is involved in physiologic processes such as embryogenesis, wound healing, and the female reproductive cycle, and also contributes to the pathogenesis of numerous disorders, including atherosclerosis, ischemic disease, arthritis, and cancer. 1 Normally, angiogenesis is strictly controlled by the balance between angiogenic promoters and inhibitors.Thrombomodulin (TM) is a type I-glycosylated membrane protein composed of 5 distinct domains. At the NH 2 -terminal is a C-type lectin-like domain followed by 6 consecutively repeated epidermal growth factor (EGF)-like domains and an O-glycosylation site-rich domain. Connected to these extracellular domains is a transmembrane domain followed by a short cytoplasmic tail. 2 TM functions as part of the anticoagulant pathway; it binds thrombin on the cell surface and catalyzes proteolytic activation of protein C. 2 Soluble TM fragments have been detected in the medium of cultured TM-expressing cells 3 and human plasma and urine, 4-6 but the physiologic significance of the soluble TM fragments remains unclear. Our previous study showed that the recombinant EGF-like domain plus the O-glycosylation site-rich domain of TM (rTMD23) promotes angiogenesis in vitro and in vivo. 7 The multiple mechanisms underlying the anti-inflammatory activity of the recombinant lectin-like domain of TM have been demonstrated recently. [8][9][10][11] Lewis Y Ag (LeY) belongs to the type II Lewis Ag family, which is structurally related to the determinants of the ABH blood group system. LeY is expressed on the cell membrane along with phospholipids, 12 EGF receptors, 13 and mucins, 14 and its soluble form is up-regulated in the synovial fluid of human angiogenic rheumatoid arthritis. 15 LeY has been suggested to be a participant in cell-cell interaction, 16 arthritis, 15 and cancer. 17 In addition, ablation in endothelial cells of fucosyltransferase I, which is crucial for generation of the precursor of type II Lewis Ag, resulted in defective tube formation, suggesting the role of LeY in endothelial cell-cell contacts. 18 Previously, we demonstrated that recombinant lectin-like domain of TM (rTMD1) suppressed lipopolysaccharide-induced inflammation through interaction with LeY-conjugated lipopolysaccharide. 11 However, the biologic significance of the interaction between rTMD1 and LeY on the endothelial cell surface has never been investigated. In this study, the biologic function of LeY on endothelial cells and the antiangiogenic function of rTMD1 were investigated. In addition, recombinant adeno-associated virus (AAV) expression vector carrying TMD1 (AAV-TMD1) resulted in efficient suppression of angiogenesis with a single injection of AAV in mice. An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance ...

Thrombomodulin domain 1 ameliorates diabetic nephropathy in mice via anti-NF-κB/NLRP3 inflammasome-mediated inflammation, enhancement of NRF2 antioxidant activity and inhibition of apoptosis

“…However, studies by our team [13] and others [14] have shown that THBD also has a protein C-and thrombinindependent physiological function. In agreement with this notion, we recently demonstrated that recombinant THBDD1 (rTHBDD1 ) can inhibit an inflammatory response by neutralising lipopolysaccharide through binding to Lewis Y antigen [15] and has anti-angiogenesis effects on cultured HUVECS [16]. We also demonstrated that cardiomyocytes are protected from apoptosis by THBD treatment as a result of its effect on the balance of B cell leukaemia/lymphoma 2 (BCL-2) and BCL-2-associated X protein (BAX) levels [17].…”

“…In a recent study, we showed that administration of AAV serotype 2/8 carrying THBDD1, which was also used in the present study, can cause hepatic expression of THBDD1 and release of the protein into blood, thereby inhibiting angiogenesis in rats [16]. Other authors [23] have shown that recombinant AAV serotype 2/8 vectors are a promising tool for long-term tissue protein expression in vivo.…”

“…We injected the mice intravenously with a single dose of AAV or AAV-THBDD1 . This dose (10 11 genome copies) was chosen according to our previous study because it caused no detectable pathological effects in the mice [16] by the age of 32 weeks (week 0 after the AAV or AAV-THBDD1 injection), i.e. at a time point when hyperglycaemia and hyperglycosuria had already developed and just before the onset of proteinuria.…”

“…Preparation of rTHBDD1 protein and AAV-THBDD1 The production of rTHBDD1 and AAV serotype 2/8 vectors encoding human THBDD1 (amino acid 1-155) (AAV-THBDD1 ) has been described previously [16].…”

Thrombomodulin Protects Endothelial Cells From a Calcineurin Inhibitor–Induced Cytotoxicity by Upregulation of Extracellular Signal–Regulated Kinase/Myeloid Leukemia Cell-1 Signaling

“…27 On the other hand, TMD1 inhibited angiogenesis through interaction with Lewis Y antigen. 28 TMD1 dephosphorylated ERK and decreased levels of Mcl-1 in HUVECs ( Figure 6D). TMD1 possesses the distinct function in endothelial cells from other domains of TM.…”