Guey-Yueh Shi

Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response

Thrombomodulin (TM), a widely expressing glycoprotein originally identified in vascular endothelium, is an important cofactor in the protein C anticoagulant system. TM appears to exhibit anti-inflammatory ability through both protein C-dependent and -independent pathways. We presently have demonstrated that recombinant N-terminal lectinlike domain of TM (rTMD1) functions as a protective agent against sepsis caused by Gram-negative bacterial infections. rTMD1 caused agglutination of Escherichia coli and Klebsiella pneumoniae and enhanced the macrophage phagocytosis of these Gram-negative bacteria. Moreover, rTMD1 bound to the Klebsiella pneumoniae and lipopolysaccharide (LPS) by specifically interacting with Lewis Y antigen. rTMD1 inhibited LPS-induced inflammatory mediator production via interference with CD14 and LPS binding. Furthermore, rTMD1 modulated LPS-induced mitogen-activated protein kinase and nuclear factor-kappaB signaling pathway activations and inducible nitric oxide synthase expression in macrophages. Administration of rTMD1 protected the host by suppressing inflammatory responses induced by LPS and Gram-negative bacteria, and enhanced LPS and bacterial clearance in sepsis. Thus, rTMD1 can be used to defend against bacterial infection and inhibit LPS-induced inflammatory responses, suggesting that rTMD1 may be valuable in the treatment of severe inflammation in sepsis, especially in Gram-negative bacterial infections.

Evidence of human thrombomodulin domain as a novel angiogenic factor.

Background—Thrombomodulin is an anticoagulant, endothelial-cell-membrane glycoprotein. A recombinant thrombomodulin domain containing 6 epidermal growth factor–like structures exhibits mitogenic activity. This study explored the novel angiogenic effects of the recombinant domain using in vitro and in vivo models. Methods and Results—Human recombinant thrombomodulin containing 6 epidermal growth factor–like structures (TMD2) and TMD2 plus a serine and threonine-rich domain (TMD23) were prepared using the Pichia pastoris expression system. Combined with purified TMD2 or TMD23, thrombin effectively activated protein C. TMD23 had higher activity than TMD2 in stimulating DNA synthesis in cultured human umbilical vein endothelial cells. Additionally, TMD23 stimulated chemotactic motility and capillarylike tube formation in human umbilical vein endothelial cells, an effect mediated through phosphorylation of extracellular signal–regulated kinase 1/2 and p38 mitogen-activated protein kinase and the phosphatidylinositol-3 kinase/Akt/endothelial nitric oxide synthase pathway. TMD23 also stimulated endothelial cell expression of matrix metalloproteinases and plasminogen activators, which mediated extracellular proteolysis, leading to endothelial cell invasion and migration during angiogenesis. Furthermore, TMD23-containing implants in rat cornea induced ingrowth of new blood vessels from the limbus. With the murine angiogenesis assay, TMD23 not only induced neovascularization coinjected with Matrigel and heparin but also enhanced angiogenesis in Matrigel containing melanoma A2058 cells in nude mice. Conclusions—The recombinant thrombomodulin domain TMD23 enhanced the angiogenic response in vitro and in vivo, suggesting that thrombomodulin fragments may play a role in the formation of new vessels. These findings may provide a new therapeutic option for treating ischemic diseases.

The role of thrombomodulin lectin-like domain in inflammation

Thrombomodulin (TM) is a cell surface glycoprotein which is widely expressed in a variety of cell types. It is a cofactor for thrombin binding that mediates protein C activation and inhibits thrombin activity. In addition to its anticoagulant activity, recent evidence has revealed that TM, especially its lectin-like domain, has potent anti-inflammatory function through a variety of molecular mechanisms. The lectin-like domain of TM plays an important role in suppressing inflammation independent of the TM anticoagulant activity. This article makes an extensive review of the role of TM in inflammation. The molecular targets of TM lectin-like domain have also been elucidated. Recombinant TM protein, especially the TM lectin-like domain may play a promising role in the management of sepsis, glomerulonephritis and arthritis. These data demonstrated the potential therapeutic role of TM in the treatment of inflammatory diseases.

G-33A Mutation in the Promoter Region of Thrombomodulin Gene and Its Association With Coronary Artery Disease and Plasma Soluble Thrombomodulin Levels

Thrombomodulin is an endothelial glycoprotein that decreases thrombin activity and activates protein C. A recent study has shown that G-33A promoter mutation of the thrombomodulin gene occurs particularly in Asians. In this study, we analyzed the distribution of G-33A mutation in the promoter region of the thrombomodulin gene in the Chinese population and determined whether the mutation might be a risk for coronary artery disease (CAD). In addition, the influence of this mutation on plasma soluble thrombomodulin levels in patients with CAD was also examined. We studied 320 consecutive patients (mean age 63 years; 73% men) with CAD and 200 age- and sex-matched control subjects. Screening for thrombomodulin G-33A promoter mutation was conducted using polymerase chain reaction, single-strand conformation polymorphism, and direct deoxyribonucleic acid sequencing. The frequency of the G-33A mutation (GA+AA genotypes) was significantly higher in the CAD group (23.8% vs 15.5%, odds ratio [OR] 1.70, p = 0.031). Multiple logistic regression analysis showed that the mutation was an independent risk factor (OR 1.81, p = 0.016) for CAD, as was hypertension (OR 1.44, p = 0.040), diabetes mellitus (OR 2.50, p <0.001), and smoking (OR 2.15, p <0.001). In CAD patients with GG genotype, the soluble thrombomodulin level increased with the extent of CAD (36 +/- 15 vs 47 +/- 18 vs 55 +/- 36 ng/ml in 1-, 2-, or 3-vessel CAD, p <0.001). However, in CAD patients with G-33A mutation, there was no difference between the levels of soluble thrombomodulin (39 +/- 17 vs 37 +/- 15 vs 42 +/- 18 ng/ml, p = NS) in 1-, 2-, or 3-vessel CAD. Our observations suggest that there is a significant association of the G-33A mutation in thrombomodulin gene with CAD, and this mutation may influence the soluble thrombomodulin levels in patients with CAD.

Cytoprotective effect of reduced glutathione in arsenical-induced endothelial cell injury

The effect of four arsenic compounds on cultured endothelial cell isolated from bovine carotid arteries was studied. Only trivalent arsenicals (arsenic trioxide and sodium m-arsenite), but not pentavalent arsenicals (arsenic acid and p-arsenilic acid), induced significant cell injury. Since the intracellular reduced glutathione (GSH) plays an important role in detoxication in mammalian cells, its effect on arsenical-induced cell injury was then studied. Pretreatment of cells with 500 microM GSH not only resulted in several-fold increase in the intracellular level of GSH but also effectively protected them against the injury caused by arsenic trioxide. After a pretreatment of cells with GSH for 3 h, the intracellular GSH reached a plateau. A longer pretreatment for 24 h still kept GSH at a very significant high level. The cell injury induced by arsenic trioxide was protected by GSH, and then cellular biosynthesis of PGI2 in culture was also increased. The cytoprotective effect and the stimulatory effect on PGI2 production, where both were dose-dependent on GSH, were in a strict reverse relationship. Aspirin treatment inhibited the PGI2 biosynthesis induced by GSH in the arsenic trioxide-induced cell injury, and significantly reduced the cytoprotective effect induced by GSH. These results suggest that the marked stimulation of endogenous PGI2 biosynthesis by GSH is the mechanism of the latters cytoprotective effect on arsenic trioxide-induced endothelial cell injury.

Platelet-activating factor-acetylhydrolase A379V (exon 11) gene polymorphism is an independent and functional risk factor for premature myocardial infarction

Summary.  Background: Oxidation of low density lipoproteins is an initial step of atherogenesis that generates pro‐inflammatory phospholipids, including platelet‐activating factor (PAF). PAF is degraded by PAF‐acetylhydrolase (PAF‐AH), which has been postulated to be a risk factor for myocardial infarction (MI). The role of PAF‐AH for the onset of premature MI is unclear. Methods: Polymorphisms located in putatively functional regions were investigated in a cohort of patients having premature MI onset prior to 46 years of age (n = 200) and a sex‐age‐matched control group (n = 200). The activity of PAF‐AH and coronary angiograms were evaluated for the severity of coronary atherosclerosis. Results: The V allele of A379V (exon 11) polymorphism on PAF‐AH gene was more frequent in patients with premature MI (P = 0.001). This V allele polymorphism was also associated with a lower activity of plasma PAF‐AH and a more complex coronary atherosclerosis (p Trends <0.05). Multiple logistic regression analysis showed that this polymorphism was an independent risk factor (Odds Ratio [OR] 1.66, 95% CI 1.14.1 to 5.80, P = 0.008) as well as smoking (OR 3.72, 95% CI 1.77 to 9.28, P = 0.001), diabetes mellitus (OR 2.25, 95% CI 1.40 to 5.32, P = 0.007) and hypertension (OR 1.88, 95% CI 1.25 to 5.36, P = 0.003) for the onset of premature MI. Conclusion: We conclude that a functional and significant association between the A379V polymorphism on exon 11 of PAF‐AH gene and premature MI exists in this Taiwanese population. This polymorphism is significantly associated with the PAF‐AH activity and the severity of coronary atherosclerosis.

Functional mutation in the promoter region of thrombomodulin gene in relation to carotid atherosclerosis

Thrombomodulin is an important endothelial anticoagulant protein that decreases thrombin activity and activates protein C. Our recent study has shown that the G-33A promoter mutation of thrombomodulin gene is associated with coronary artery disease. This study was conducted to determine whether the G-33A mutation in the promoter region of thrombomodulin gene is a genetic risk factor for ischemic stroke or carotid atherosclerosis. The functional significance of this mutation was also evaluated. We recruited 333 patients (mean age 64 years, 59% male) with ischemic stroke and 257 age- and sex-matched controls. In all study participants, carotid atherosclerosis was assessed by Duplex scanning, and thrombomodulin G-33A promoter mutation was detected by single-strand conformation polymorphism. Luciferase reporter gene assay was used to assess the influence of this mutation on thrombomodulin promoter activity. There was no significant difference in the thrombomodulin G-33A mutation frequency (GA+AA genotypes) between the stroke and the control groups (18.3 vs. 24. 1%, P=0.105). The G-33A mutation frequency was also similar between the study participants with and without carotid atherosclerosis (22.2 vs. 19.8%, P=0.550). When only younger subjects (age </=60 years) were included in the analysis, however, we found the mutation occurred more frequently in participants with carotid atherosclerosis (33.3 vs. 17.3%, odds ratio [OR]=2.38, 95% confidence interval [CI]=1.16-4.90, P=0.027). Multiple logistic regression analyses showed that only diabetes mellitus (OR=3.11, 95% CI=1.33-7.30, P=0.009) and G-33A mutation (OR=2.46, 95% CI=1.14-5.29, P=0.021) were associated independently with carotid atherosclerosis in younger subjects. As assessed by luciferase reporter gene assays, the contructs bearing the G-33A mutation showed a significant decrease (36+/-12%) in transcriptional activity in comparison with the wild type constructs. Our findings suggest that G-33A mutation reduces the thrombomodulin promoter activity and is associated with carotid atherosclerosis in younger subjects.

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

Aims/hypothesisChronic inflammatory processes have been increasingly shown to be involved in the pathogenesis of diabetes and diabetic nephropathy. Recently, we demonstrated that a lectin-like domain of thrombomodulin (THBD), which is known as THBD domain 1 (THBDD1) and which acts independently of protein C activation, neutralised an inflammatory response in a mouse model of sepsis. Here, therapeutic effects of gene therapy with adeno-associated virus (AAV)-carried THBDD1 (AAV-THBDD1) were tested in a mouse model of type 2 diabetic nephropathy.MethodsTo assess the therapeutic potential of THBDD1 and the mechanisms involved, we delivered AAV-THBDD1 (1011 genome copies) into db/db mice and tested the effects of recombinant THBDD1 on conditionally immortalised podocytes.ResultsA single dose of AAV-THBDD1 improved albuminuria, renal interstitial inflammation and glomerular sclerosis, as well as renal function in db/db mice. These effects were closely associated with: (1) inhibited activation of the nuclear factor κB (NF-κB) pathway and the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome; (2) promotion of nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation; and (3) suppression of mitochondria-derived apoptosis in the kidney of treated mice.Conclusions/interpretationAAV-THBDD1 gene therapy resulted in improvements in a model of diabetic nephropathy by suppressing the NF-κB–NLRP3 inflammasome-mediated inflammatory process, enhancing the NRF2 antioxidant pathway and inhibiting apoptosis in the kidney.

Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing.

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

The 161TT genotype in the exon 6 of the peroxisome-proliferator-activated receptor γ gene is associated with premature acute myocardial infarction and increased lipid peroxidation in habitual heavy smokers

PPAR (peroxisome-proliferator-activated receptor) is a nuclear receptor. Activation of PPARgamma by its ligands could modulate gene transcription, thereby leading to multiple anti-atherogenic and fibrinolytic effects. However, the association between the 161T allele in exon 6 of the PPARgamma gene and premature AMI (acute myocardial infarction) is not clear. We recruited 146 patients with premature AMI (onset age < or =50 years) and 146 controls. The C161T polymorphism was examined using PCR and restriction-fragment-length polymorphism. Plasma levels of Ab-ox-LDL (antibody against oxidized low-density lipoprotein) were measured in 27 male smokers, whose genotypes have been identified. The frequency of the PPARgamma TT genotype among patients with AMI was significantly higher than that in controls [13% compared with 5.5%; OR (95% CI) 2.7, (1.1-6.5), where OR and CI are odds ratio and confidence interval respectively]. This association was not observed in CC or CT genotypes. Using multivariate logistic regression analyses, we found that the homozygous TT genotype [OR (95% CI), 3.1 (1.2-7.9)], smoking [OR (95% CI), 3.5, (2.1-6.0)], hypertension [OR (95% CI), 3.6, (1.9-6.9)] and diabetes mellitus [OR (95% CI), 3.5 (1.5-8.4)] were independent risk factors for premature AMI. Plasma levels of Ab-ox-LDL were significantly higher in healthy volunteers with the TT genotype compared with those with the CC genotype (49.3+/-18.1 compared with 24.2+/-15.2 units/l respectively; P=0.02). Therefore in our study we observed an association between the PPARgamma 161 TT genotype and premature AMI. Lipid peroxidation was significantly influenced by the 161T allele.