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A novel pathological role of hnRNPA1 in vascular smooth muscle cell functions and neointima hyperplasia
Abstracts / Atherosclerosis 263 (2017) e1ee28
Oral Communications: Endothelium and SMC Vascuar Biology CO4:1. MYOSIN-X IS REQUIRED FOR ICAM-1-RICH ENDOTHELIAL FILOPODIA THROUGH THE SMALL GTPASE CDC42 AND PAK4 TO FUNCTIONALLY SUPPORT LEUKOCYTE ADHESION Jeffrey Kroon1, Antje Schaefer2, Mark Hoogenboezem2, Floris Van € mblad4, Kees Hoeben5, Jacco Van Alphen2, Peter Hordijk3, Staffan Stro 6 1 Rhenen , Erik Stroes , Jaap Van Buul2. 1 Academic Medical Center, Department of Vascular Medicine, Amsterdam, The Netherlands; 2 Sanquin Research and Landsteiner Laboratory, Department of Molecular Cell Biology, Amsterdam, The Netherlands; 3 VU University Medical Center, Department of Physiology, Amsterdam, The Netherlands; 4 Karolinska Institutet, Department of Biosciences and Nutrition, Novum, Sweden; 5 Academic Medical Center, Department of Electron Microscopy, Amsterdam, The Netherlands; 6 Hubrecht Institute-KNAW & University Medical Center, Cancer Genomics Netherlands, Utrecht, The Netherlands Aim: Leukocyte transendothelial migration is one of the key hallmarks of atherosclerosis. Multiple signalling events between leukocytes and the endothelium ensure that immune cells only emigrate out of the bloodstream at sites of inflammation. Although several mechanistic details involved in leukocyte adhesion and transmigration have been uncovered during the recent years, the vascular molecular mechanisms that drives this process still shows significant gaps in our understanding. Inflamed endothelial cells express small finger-like protrusions that stick out into the lumen. However, the function and regulation of these structures remains unclear. Methods: By using both Scanning Electron Microscopy together with live cell imaging we aim to elucidate the molecular mechanism behind the formation of these structures. Results: We present evidence that these endothelial finger-like protrusions, rich in ICAM-1- and F-actin are in fact filopodia and function as adhesive platforms for leukocytes to bind to but not for actual diapedesis. The formation of these structures depends on the motor function of Myosin-X and the activity of the small GTPase Cdc42. Functionally, endothelial filopodia require PAK4 as a downstream Cdc42 target to serve as adhesive structures for leukocytes to adhere to. By intravital maging of vessels from GFP-transgenic knock-in mice during leukocyte extravasation and ex vivo en face imaging of inflamed murine vessels, we identified intraluminal structures that resemble the filopodia phenotype. Conclusions: Together, our data show for the first time the functionality of the ICAM-1-Cdc42-Myosin-X-PAK4-F-actin signaling axis in the formation of apical filopodia on the surface of inflamed endothelium to regulate the adhesion of leukocytes.
CO4:2. ROLE OF THE NUCLEAR RECEPTOR REV-ERB-ALPHA DEVELOPMENT OF VASCULAR CALCIFICATION
IN
THE
Pourcet, Lise Ferri, Jonathan Mathilde Zecchin, Benoit phane Delhaye, Bart Staels, He le ne Duez. INSERM U1011, Vanhoutte, Ste Institut Pasteur de Lille, Lille, France Aim: In late stage of atherosclerosis, vascular inflammation triggers vascular calcification which results from the imbalance between the activators and inhibitors of calcification. We have shown that the nuclear receptor Rev-erb-alpha protects from the development of atherosclerosis. However, its role in vascular calcification has not been elucidated yet. Methods: To determine whether Rev-erb-alpha is implicated in the development of vascular calcification, we compared atherosclerostic plaque calcification in 1.5 year-old rev-erbalpha-/- and rev-erbalpha+/+ mice in a pro-atherogenic LDLr-/- genetic background. The size of calcified areas was quantified after alizarin red staining on heart sections surrounding the aortic sinus. To uncover the molecular mechanisms by which Rev-erbalpha might be involved in the development of vascular calcification, we performed qPCR analysis of the expression of calcification-related genes in key cells involved in this process, namely primary aortic smooth muscle
e19
cells differentiated into osteoblasts and bone marrow-derived macrophages differentiated into osteoclasts. Results: rev-erbalpha-/- LDLr-/- mice show larger calcification areas compared to rev-erbalpha+/+ LDLr-/- mice. Consistently, calcium deposition, quantified after alizarin red staining, is increased in Rev-erbalphadeficient osteoblast-like cells compared to control cells. The expression of osteogenic markers such as osteocalcin and osteopontin and the inhibitor of calcification mgp is modulated in the absence of Rev-erb-alpha. In Reverbalpha-deficient osteoclast-like cells, the expression of osteoclast markers such as cathepsin k, mmp-9 and rank is also enhanced. Conclusions: These results indicate that Rev-erb-alpha may protect from the development of vascular calcification. The study of the mechanisms involved therein is in progress.
CO4:3. A NOVEL PATHOLOGICAL ROLE OF HNRNPA1 IN VASCULAR SMOOTH MUSCLE CELL FUNCTIONS AND NEOINTIMA HYPERPLASIA Qingzhong Xiao. William Harvey Research Institute, Barts and The London Sch of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom Aim: Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) plays a critical role in gene expression. However, little is known about the functional involvements of hnRNPA1 in VSMC functions and neointima hyperplasia. In this study, we attempted to investigate the functional roles of hnRNPA1 in the contexts of VSMC functions, injury-induced vessel remodelling and human atherosclerotic lesions, and the molecular mechanisms involved. Methods: Primary VSMCs, normal and diseased human arteries and carotid arterial injury animal model were used to investigate potential involvements of hnRNPA1 in VSMC functions and neointima hyperplasia. Results: hnRNPA1 expression levels were consistently modulated during VSMC phenotype switching and neointimal lesion formation induced by wire injury. Functional studies showed that VSMC-specific gene expression, proliferation and migration were closely controlled by hnRNPA1. Our data shows that hnRNPA1 exerts its effects on VSMC functions through modulating IQ motif containing GTPase activating protein 1 (IQGAP1). Mechanistically, hnRNPA1 regulates IQGAP1 mRNA degradation through two mechanisms: upregulating microRNA-124 (miR-124) and AU-rich element. Further evidence suggests that hnRNPA1 up-regulates miR-124 through regulating miR-124 biogenesis, and that IQGAP1 is the authentic target gene of miR-124. Importantly, ectopic overexpression of hnRNPA1 greatly reduced VSMC proliferation, and inhibited neointima formation in wire-injured carotid arteries. Finally, lower expression levels of hnRNPA1 and miR-124, while higher expression levels of IQGAP1, were observed in human atherosclerotic lesions. Conclusions: Our data show that hnRNPA1 is a critical regulator of VSMC functions/behaviours and neointima hyperplasia, and the hnRNPA1/miR124/IQGAP1 regulatory axis represents a novel therapeutic target for the prevention of cardiovascular diseases.
CO4:4. ROLE OF ENDOTHELIAL IKB KINASE 2 IN ATHEROSCLEROSIS Salzmann, Mario Kuttke, Jose Marion Mussbacher, Manuel € sel, Alice Assinger, Johannes Schmid. Institute of Basilio, Bastian Ho Vascular Biology and Thrombosis Research, Vienna, Austria Aim: The transcription factor NF-kB has a key role in inflammation and is also an important regulator of genes involved in coagulation and thrombosis. NF-kB is activated by a number of signaling pathways that converge in the majority of cases at the level of IkB kinase 2 (IKK2). Our study focusses on the role of constitutive active IKK2 (caIKK2) in a conditional transgene mouse model to mimic chronic inflammation specifically in endothelial cells and to test a potential aggravating impact on the onset of atherosclerosis. Methods: Mice bearing inducible, aortic-EC-specific Cre recombinase on an ApoE-deficient background were crossed with a strain expressing
Oral Communications: Endothelium and SMC Vascuar Biology CO4:1. MYOSIN-X IS REQUIRED FOR ICAM-1-RICH ENDOTHELIAL FILOPODIA THROUGH THE SMALL GTPASE CDC42 AND PAK4 TO FUNCTIONALLY SUPPORT LEUKOCYTE ADHESION Jeffrey Kroon1, Antje Schaefer2, Mark Hoogenboezem2, Floris Van € mblad4, Kees Hoeben5, Jacco Van Alphen2, Peter Hordijk3, Staffan Stro 6 1 Rhenen , Erik Stroes , Jaap Van Buul2. 1 Academic Medical Center, Department of Vascular Medicine, Amsterdam, The Netherlands; 2 Sanquin Research and Landsteiner Laboratory, Department of Molecular Cell Biology, Amsterdam, The Netherlands; 3 VU University Medical Center, Department of Physiology, Amsterdam, The Netherlands; 4 Karolinska Institutet, Department of Biosciences and Nutrition, Novum, Sweden; 5 Academic Medical Center, Department of Electron Microscopy, Amsterdam, The Netherlands; 6 Hubrecht Institute-KNAW & University Medical Center, Cancer Genomics Netherlands, Utrecht, The Netherlands Aim: Leukocyte transendothelial migration is one of the key hallmarks of atherosclerosis. Multiple signalling events between leukocytes and the endothelium ensure that immune cells only emigrate out of the bloodstream at sites of inflammation. Although several mechanistic details involved in leukocyte adhesion and transmigration have been uncovered during the recent years, the vascular molecular mechanisms that drives this process still shows significant gaps in our understanding. Inflamed endothelial cells express small finger-like protrusions that stick out into the lumen. However, the function and regulation of these structures remains unclear. Methods: By using both Scanning Electron Microscopy together with live cell imaging we aim to elucidate the molecular mechanism behind the formation of these structures. Results: We present evidence that these endothelial finger-like protrusions, rich in ICAM-1- and F-actin are in fact filopodia and function as adhesive platforms for leukocytes to bind to but not for actual diapedesis. The formation of these structures depends on the motor function of Myosin-X and the activity of the small GTPase Cdc42. Functionally, endothelial filopodia require PAK4 as a downstream Cdc42 target to serve as adhesive structures for leukocytes to adhere to. By intravital maging of vessels from GFP-transgenic knock-in mice during leukocyte extravasation and ex vivo en face imaging of inflamed murine vessels, we identified intraluminal structures that resemble the filopodia phenotype. Conclusions: Together, our data show for the first time the functionality of the ICAM-1-Cdc42-Myosin-X-PAK4-F-actin signaling axis in the formation of apical filopodia on the surface of inflamed endothelium to regulate the adhesion of leukocytes.
CO4:2. ROLE OF THE NUCLEAR RECEPTOR REV-ERB-ALPHA DEVELOPMENT OF VASCULAR CALCIFICATION
IN
THE
Pourcet, Lise Ferri, Jonathan Mathilde Zecchin, Benoit phane Delhaye, Bart Staels, He le ne Duez. INSERM U1011, Vanhoutte, Ste Institut Pasteur de Lille, Lille, France Aim: In late stage of atherosclerosis, vascular inflammation triggers vascular calcification which results from the imbalance between the activators and inhibitors of calcification. We have shown that the nuclear receptor Rev-erb-alpha protects from the development of atherosclerosis. However, its role in vascular calcification has not been elucidated yet. Methods: To determine whether Rev-erb-alpha is implicated in the development of vascular calcification, we compared atherosclerostic plaque calcification in 1.5 year-old rev-erbalpha-/- and rev-erbalpha+/+ mice in a pro-atherogenic LDLr-/- genetic background. The size of calcified areas was quantified after alizarin red staining on heart sections surrounding the aortic sinus. To uncover the molecular mechanisms by which Rev-erbalpha might be involved in the development of vascular calcification, we performed qPCR analysis of the expression of calcification-related genes in key cells involved in this process, namely primary aortic smooth muscle
e19
cells differentiated into osteoblasts and bone marrow-derived macrophages differentiated into osteoclasts. Results: rev-erbalpha-/- LDLr-/- mice show larger calcification areas compared to rev-erbalpha+/+ LDLr-/- mice. Consistently, calcium deposition, quantified after alizarin red staining, is increased in Rev-erbalphadeficient osteoblast-like cells compared to control cells. The expression of osteogenic markers such as osteocalcin and osteopontin and the inhibitor of calcification mgp is modulated in the absence of Rev-erb-alpha. In Reverbalpha-deficient osteoclast-like cells, the expression of osteoclast markers such as cathepsin k, mmp-9 and rank is also enhanced. Conclusions: These results indicate that Rev-erb-alpha may protect from the development of vascular calcification. The study of the mechanisms involved therein is in progress.
CO4:3. A NOVEL PATHOLOGICAL ROLE OF HNRNPA1 IN VASCULAR SMOOTH MUSCLE CELL FUNCTIONS AND NEOINTIMA HYPERPLASIA Qingzhong Xiao. William Harvey Research Institute, Barts and The London Sch of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom Aim: Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) plays a critical role in gene expression. However, little is known about the functional involvements of hnRNPA1 in VSMC functions and neointima hyperplasia. In this study, we attempted to investigate the functional roles of hnRNPA1 in the contexts of VSMC functions, injury-induced vessel remodelling and human atherosclerotic lesions, and the molecular mechanisms involved. Methods: Primary VSMCs, normal and diseased human arteries and carotid arterial injury animal model were used to investigate potential involvements of hnRNPA1 in VSMC functions and neointima hyperplasia. Results: hnRNPA1 expression levels were consistently modulated during VSMC phenotype switching and neointimal lesion formation induced by wire injury. Functional studies showed that VSMC-specific gene expression, proliferation and migration were closely controlled by hnRNPA1. Our data shows that hnRNPA1 exerts its effects on VSMC functions through modulating IQ motif containing GTPase activating protein 1 (IQGAP1). Mechanistically, hnRNPA1 regulates IQGAP1 mRNA degradation through two mechanisms: upregulating microRNA-124 (miR-124) and AU-rich element. Further evidence suggests that hnRNPA1 up-regulates miR-124 through regulating miR-124 biogenesis, and that IQGAP1 is the authentic target gene of miR-124. Importantly, ectopic overexpression of hnRNPA1 greatly reduced VSMC proliferation, and inhibited neointima formation in wire-injured carotid arteries. Finally, lower expression levels of hnRNPA1 and miR-124, while higher expression levels of IQGAP1, were observed in human atherosclerotic lesions. Conclusions: Our data show that hnRNPA1 is a critical regulator of VSMC functions/behaviours and neointima hyperplasia, and the hnRNPA1/miR124/IQGAP1 regulatory axis represents a novel therapeutic target for the prevention of cardiovascular diseases.
CO4:4. ROLE OF ENDOTHELIAL IKB KINASE 2 IN ATHEROSCLEROSIS Salzmann, Mario Kuttke, Jose Marion Mussbacher, Manuel € sel, Alice Assinger, Johannes Schmid. Institute of Basilio, Bastian Ho Vascular Biology and Thrombosis Research, Vienna, Austria Aim: The transcription factor NF-kB has a key role in inflammation and is also an important regulator of genes involved in coagulation and thrombosis. NF-kB is activated by a number of signaling pathways that converge in the majority of cases at the level of IkB kinase 2 (IKK2). Our study focusses on the role of constitutive active IKK2 (caIKK2) in a conditional transgene mouse model to mimic chronic inflammation specifically in endothelial cells and to test a potential aggravating impact on the onset of atherosclerosis. Methods: Mice bearing inducible, aortic-EC-specific Cre recombinase on an ApoE-deficient background were crossed with a strain expressing