Benedicte Stavik1, Sverre Holm2, Sandra Espada3, Nina Iversen4, Bjørnar Sporsheim5, Vigdis Bjerkeli6, Tuva Børresdatter Dahl7, Per Morten Sandset8, Mona Skjelland9, Terje Espevik5, Grethe Skretting10, Bente Halvorsen11. 1. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Department of Haematology, Oslo University Hospital Rikshospitalet, Oslo, Norway. Electronic address: benedicte.stavik@rr-research.no. 2. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Hospital for Rheumatic Diseases, Lillehammer, Norway. 3. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Department of Haematology, Oslo University Hospital Rikshospitalet, Oslo, Norway; Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway. 4. Department of Medical Genetics, Oslo University Hospital Rikshospitalet, Oslo, Norway. 5. Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway. 6. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 7. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway. 8. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Department of Haematology, Oslo University Hospital Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway. 9. Department of Neurology, Oslo University Hospital Rikshospitalet, Oslo, Norway. 10. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Department of Haematology, Oslo University Hospital Rikshospitalet, Oslo, Norway. 11. Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo, Norway; Institute of Clinical Medicine, University of Oslo, Oslo, Norway; K.G. Jebsen Inflammatory Research Centre, University of Oslo, Oslo, Norway.
Abstract
INTRODUCTION: Tissue factor (TF) pathway inhibitor (TFPI) is the physiological inhibitor of TF induced blood coagulation and two isoforms exists, TFPIα and TFPIβ. In atherosclerotic plaques, TFPI may inhibit TF activity and thrombus formation, which is the main cause of ischemic stroke in carotid artery disease. We aimed to identify the isoforms of TFPI present in human carotid plaques and potential sources of TFPI. MATERIALS AND METHODS: Human atherosclerotic plaques from carotid endarterectomies were used for mRNA and immunohistochemistry analyses. hPBMCs isolated from buffy coats and THP-1 cells were differentiated and polarized into M1 or M2 macrophages, and subsequently cultured with or without cholesterol crystals (CC). mRNA and protein expression were measured with qRT-PCR and ELISA, respectively, and procoagulant activity was assessed using a two-stage chromogenic assay. RESULTS: TFPIα and TFPIβ mRNA levels were significantly increased in carotid plaques, whereas TF levels were unchanged as compared to healthy arteries. Antibodies against total TFPI showed elevated levels compared to antibodies against free TFPIα, both by immunohistochemical and ELISA detection in plaques. The antibody against total TFPI also co-localized with CD68 and the M1 and M2 markers CD80 and CD163, respectively. The TFPI mRNA expression was elevated and the procoagulant activity was decreased in M2 compared to M1 polarized human macrophages. TFPI was present in early foam cell formation and CC treatment increased the TFPI mRNA expression even further in M2 macrophages. CONCLUSIONS: Our data indicate that both isoforms of TFPI are present in advanced plaques and that anti-inflammatory M2 macrophages may be a potential source of TFPI.
INTRODUCTION:Tissue factor (TF) pathway inhibitor (TFPI) is the physiological inhibitor of TF induced blood coagulation and two isoforms exists, TFPIα and TFPIβ. In atherosclerotic plaques, TFPI may inhibit TF activity and thrombus formation, which is the main cause of ischemic stroke in carotid artery disease. We aimed to identify the isoforms of TFPI present in human carotid plaques and potential sources of TFPI. MATERIALS AND METHODS:Humanatherosclerotic plaques from carotid endarterectomies were used for mRNA and immunohistochemistry analyses. hPBMCs isolated from buffy coats and THP-1 cells were differentiated and polarized into M1 or M2 macrophages, and subsequently cultured with or without cholesterol crystals (CC). mRNA and protein expression were measured with qRT-PCR and ELISA, respectively, and procoagulant activity was assessed using a two-stage chromogenic assay. RESULTS:TFPIα and TFPIβ mRNA levels were significantly increased in carotid plaques, whereas TF levels were unchanged as compared to healthy arteries. Antibodies against total TFPI showed elevated levels compared to antibodies against free TFPIα, both by immunohistochemical and ELISA detection in plaques. The antibody against total TFPI also co-localized with CD68 and the M1 and M2 markers CD80 and CD163, respectively. The TFPI mRNA expression was elevated and the procoagulant activity was decreased in M2 compared to M1 polarized human macrophages. TFPI was present in early foam cell formation and CC treatment increased the TFPI mRNA expression even further in M2 macrophages. CONCLUSIONS: Our data indicate that both isoforms of TFPI are present in advanced plaques and that anti-inflammatory M2 macrophages may be a potential source of TFPI.