Peng Zhang1,2, Kunmei Gong1,2, Lili Zhang1,2, Le Xiao1,2, Shikui Guo1,2, Yu Li1,2, Jian Zhang3,2. 1. Department of General Surgery, the First People's Hospital of Yunnan Province, Kunming, China. 2. The Affiliated Hospital of Kunming University of Science and Technology, Kunming, China. 3. Department of General Surgery, the First People's Hospital of Yunnan Province, Kunming, China - zhangg143@163.com.
Abstract
BACKGROUND: Studies have shown that the thrombomodulin gene (THBD) c.1418C>T polymorphism is associated with a variety of cardiovascular diseases. However, the study of THBD c.1418C>T polymorphism in deep vein thrombosis (DVT) is rare. This study aimed to reveal the correlation between the THBD c.1418C>T mutation and the occurrence of DVT, and to reveal partial molecular mechanism of endothelial progenitor cells (EPCs) participating in the onset of DVT. METHODS: Whole blood samples of patients with lower extremity DVT (N.=100) and normal volunteers (N.=100) were collected to analyze the distribution of genotype of THBD c.1418C>T polymorphism using PCR and DNA sequencing. The pCMV6-entry vectors containing wild-type (WT) or mutated THBD cDNA (p. Ala473Val) were transfected into bone marrow derived EPCs. And the successful transfection of recombinant THBD and the stable expression of p. Ala473Val variant were determined by ELISA, respectively. Wound healing assay and Transwell migration assay were used to determine the migration ability of EPCs, and the cell angiogenesis ability was determined by tube formation assay. Western blotting was used to detect the expression level of related proteins. RESULTS: The frequencies of CC, CT and TT genotypes were 56%, 36%, 8% in patients with lower extremity DVT and 72%, 25%, 3% in controls group, respectively, and THBD c.1418C>T polymorphism was related with increased risk of DVT, especially in women. High level of p. Ala473Val variant inhibited the EPCs migration, the p. Ala473Val variant significantly decreased the activation of protein C and the expressions of VEGFRs and MMP1, MMP2, MMP3. Furthermore, p. Ala473Val variant also weaken the angiogenesis of EPCs and decreased the expression level of VE-cadherin, Flk-1, eNOS, and TIE-2. CONCLUSIONS: THBD c.1418C>T polymorphism is related with the lower extremity DVT, this may partially because of the inhibition of migration and angiogenesis of EPCs.
BACKGROUND: Studies have shown that the thrombomodulin gene (THBD) c.1418C>T polymorphism is associated with a variety of cardiovascular diseases. However, the study of THBD c.1418C>T polymorphism in deep vein thrombosis (DVT) is rare. This study aimed to reveal the correlation between the THBD c.1418C>T mutation and the occurrence of DVT, and to reveal partial molecular mechanism of endothelial progenitor cells (EPCs) participating in the onset of DVT. METHODS: Whole blood samples of patients with lower extremity DVT (N.=100) and normal volunteers (N.=100) were collected to analyze the distribution of genotype of THBD c.1418C>T polymorphism using PCR and DNA sequencing. The pCMV6-entry vectors containing wild-type (WT) or mutated THBD cDNA (p. Ala473Val) were transfected into bone marrow derived EPCs. And the successful transfection of recombinant THBD and the stable expression of p. Ala473Val variant were determined by ELISA, respectively. Wound healing assay and Transwell migration assay were used to determine the migration ability of EPCs, and the cell angiogenesis ability was determined by tube formation assay. Western blotting was used to detect the expression level of related proteins. RESULTS: The frequencies of CC, CT and TT genotypes were 56%, 36%, 8% in patients with lower extremity DVT and 72%, 25%, 3% in controls group, respectively, and THBD c.1418C>T polymorphism was related with increased risk of DVT, especially in women. High level of p. Ala473Val variant inhibited the EPCs migration, the p. Ala473Val variant significantly decreased the activation of protein C and the expressions of VEGFRs and MMP1, MMP2, MMP3. Furthermore, p. Ala473Val variant also weaken the angiogenesis of EPCs and decreased the expression level of VE-cadherin, Flk-1, eNOS, and TIE-2. CONCLUSIONS: THBD c.1418C>T polymorphism is related with the lower extremity DVT, this may partially because of the inhibition of migration and angiogenesis of EPCs.