Paola Altieri1, Maria Bertolotto2, Patrizia Fabbi1, Elena Sportelli3, Manrico Balbi4, Francesco Santini3, Claudio Brunelli5, Marco Canepa4, Fabrizio Montecucco6, Pietro Ameri7. 1. Laboratory of Cardiovascular Biology, Department of Internal Medicine, University of Genova, Genova, Italy. 2. Department of Internal Medicine, University of Genova, Genova, Italy; First Clinic of Internal Medicine, IRCCS Ospedale Policlinico San Martino, Genova, Italy. 3. Department of Diagnostic and Surgical Sciences, University of Genova, Genova, Italy; Cardiovascular Surgery Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy. 4. Cardiovascular Disease Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy. 5. Laboratory of Cardiovascular Biology, Department of Internal Medicine, University of Genova, Genova, Italy; Cardiovascular Disease Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy. 6. Department of Internal Medicine, University of Genova, Genova, Italy; Centre of Excellence for Biomedical Research (CEBR), University of Genova, Genova, Italy; First Clinic of Internal Medicine, IRCCS Ospedale Policlinico San Martino, Genova, Italy. 7. Laboratory of Cardiovascular Biology, Department of Internal Medicine, University of Genova, Genova, Italy; Cardiovascular Disease Unit, IRCCS Ospedale Policlinico San Martino, Genova, Italy. Electronic address: pietroameri@unige.it.
Abstract
BACKGROUND: Data with animal cells and models suggest that thrombin activates cardiac fibroblasts (Fib) to myofibroblasts (myoFib) via protease-activated receptor 1 (PAR1) cleavage, and in this way promotes adverse atrial remodeling and, thereby, atrial fibrillation (AF). OBJECTIVE: Here, we explored the effects of thrombin on human atrial Fib and whether they are antagonized by the clinically available direct thrombin inhibitor, dabigatran. METHODS: Fib isolated from atrial appendages of patients without AF undergoing elective cardiac surgery were evaluated for PAR expression and treated with thrombin with or without dabigatran. PAR1 cleavage, downstream signaling and myoFib markers were investigated by immunofluorescence and Western blot. Collagen synthesis, activity of matrix metalloprotease (MMP)-2 and proliferation were assessed by Picro-Sirius red staining, gelatinolytic zymography and BrdU incorporation, respectively. Fib function was studied as capability to contract a collagen gel and stimulate the chemotaxis of peripheral blood monocytes from healthy volunteers. RESULTS: Primary human atrial Fib expressed PAR1, while levels of the other PARs were very low. Thrombin triggered PAR1 cleavage and phosphorylation of ERK1/2, p38 and Akt, elicited a switch to myoFib enriched for αSMA, fibronectin and type I collagen, and induced paracrine/autocrine transforming growth factor beta-1, cyclooxygenase-2, endothelin-1 and chemokine (C-C motif) ligand 2 (CCL2); conversely, MMP-2 activity decreased. Thrombin-primed cells displayed enhanced proliferation, formed discrete collagen-containing cellular nodules, and stimulated the contraction of a collagen gel. Furthermore, their conditioned medium caused monocytes to migrate. All these effects were prevented by dabigatran. CONCLUSION: These results with human cells complete the knowledge about thrombin actions on cardiac Fib and strengthen the translational potential of the emerging paradigm that pharmacological blockade of thrombin may counteract molecular and cellular events underlying AF.
BACKGROUND: Data with animal cells and models suggest that thrombin activates cardiac fibroblasts (Fib) to myofibroblasts (myoFib) via protease-activated receptor 1 (PAR1) cleavage, and in this way promotes adverse atrial remodeling and, thereby, atrial fibrillation (AF). OBJECTIVE: Here, we explored the effects of thrombin on human atrial Fib and whether they are antagonized by the clinically available direct thrombin inhibitor, dabigatran. METHODS: Fib isolated from atrial appendages of patients without AF undergoing elective cardiac surgery were evaluated for PAR expression and treated with thrombin with or without dabigatran. PAR1 cleavage, downstream signaling and myoFib markers were investigated by immunofluorescence and Western blot. Collagen synthesis, activity of matrix metalloprotease (MMP)-2 and proliferation were assessed by Picro-Sirius red staining, gelatinolytic zymography and BrdU incorporation, respectively. Fib function was studied as capability to contract a collagen gel and stimulate the chemotaxis of peripheral blood monocytes from healthy volunteers. RESULTS: Primary human atrial Fib expressed PAR1, while levels of the other PARs were very low. Thrombin triggered PAR1 cleavage and phosphorylation of ERK1/2, p38 and Akt, elicited a switch to myoFib enriched for αSMA, fibronectin and type I collagen, and induced paracrine/autocrine transforming growth factor beta-1, cyclooxygenase-2, endothelin-1 and chemokine (C-C motif) ligand 2 (CCL2); conversely, MMP-2 activity decreased. Thrombin-primed cells displayed enhanced proliferation, formed discrete collagen-containing cellular nodules, and stimulated the contraction of a collagen gel. Furthermore, their conditioned medium caused monocytes to migrate. All these effects were prevented by dabigatran. CONCLUSION: These results with human cells complete the knowledge about thrombin actions on cardiac Fib and strengthen the translational potential of the emerging paradigm that pharmacological blockade of thrombin may counteract molecular and cellular events underlying AF.
Authors: Anke C Fender; Sonja Kleeschulte; Svenja Stolte; Katja Leineweber; Markus Kamler; Johannes Bode; Na Li; Dobromir Dobrev Journal: Basic Res Cardiol Date: 2020-01-07 Impact factor: 17.165
Authors: Paola Altieri; Maria Bertolotto; Patrizia Fabbi; Elena Sportelli; Manrico Balbi; Francesco Santini; Claudio Brunelli; Marco Canepa; Fabrizio Montecucco; Pietro Ameri Journal: Data Brief Date: 2018-05-25