Expression of tissue transglutaminase in human umbilical vein endothelial cells
This study investigated the expression and activity of tissue transglutaminase (tTG) in human umbilical vein endothelial cells (HUVEC) and vessel wall. tTG was located in the SMC and sub-endothelium of normal vessels. Cross-linking activity was also in this area. Vessels with atherosclerotic plaque showed increased staining for tTG and cross-links. Positive staining for tTG was located in the SMC, neointima, macrophages and the fibrous cap. Most cross-linking activity was observed in the fibrous cap, and cross-linking was observed around macrophages and smooth muscle cells. Cross-linking activity was also observed with incorporation of a labelled cross-linking substrate into vessel sections. Free tTG could be extracted from the vessel wall. HUVEC expressed 10 g tTG/mg total protein. tTG was detected in cell lysate and extracellular matrix, but not in the culture supernatant. Thrombin up-regulated tTG expression at both the mRNA and protein level. Optimal up-regulation was at a thrombin concentration of 1 U/ml The up-regulation by thrombin was dependent on thrombin activity, and was mediated through the thrombin receptor, protease-activated receptor 1 (PAR-1). Cross-linking activity was also increased after thrombin treatment, measured with a microtitre plate assay and an in situ assay. The specific activity of tTG increased after thrombin treatment. Thrombin treatment increased the level of tTG in the HUVEC ECM. Treatment of HUVEC with PMA reduced the expression of tTG mRNA, reduced the level of tTG protein, but increased the tTG cross-linking activity compared to untreated cells.