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      A Methodology for Concomitant Isolation of Intimal and Adventitial Endothelial Cells from the Human Thoracic Aorta

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          Abstract

          Background

          Aortic diseases are diverse and involve a multiplicity of biological systems in the vascular wall. Aortic dissection, which is usually preceded by aortic aneurysm, is a leading cause of morbidity and mortality in modern societies. Although the endothelium is now known to play an important role in vascular diseases, its contribution to aneurysmal aortic lesions remains largely unknown. The aim of this study was to define a reliable methodology for the isolation of aortic intimal and adventitial endothelial cells in order to throw light on issues relevant to endothelial cell biology in aneurysmal diseases.

          Methodology/Principal Findings

          We set up protocols to isolate endothelial cells from both the intima and the adventitia of human aneurysmal aortic vessel segments. Throughout the procedure, analysis of cell morphology and endothelial markers allowed us to select an endothelial fraction which after two rounds of expansion yielded a population of >90% pure endothelial cells. These cells have the features and functionalities of freshly isolated cells and can be used for biochemical studies. The technique was successfully used for aortic vessel segments of 20 patients and 3 healthy donors.

          Conclusions/Significance

          This simple and highly reproducible method allows the simultaneous preparation of reasonably pure primary cultures of intimal and adventitial human endothelial cells, thus providing a reliable source for investigating their biology and involvement in both thoracic aneurysms and other aortic diseases.

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          Most cited references 49

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          Phenotypic heterogeneity of the endothelium: II. Representative vascular beds.

          Endothelial cells, which form the inner cellular lining of blood vessels and lymphatics, display remarkable heterogeneity in structure and function. This is the second of a 2-part review on the phenotypic heterogeneity of blood vessel endothelial cells. The first part discusses the scope, the underlying mechanisms, and the diagnostic and therapeutic implications of phenotypic heterogeneity. Here, these principles are applied to an understanding of organ-specific phenotypes in representative vascular beds including arteries and veins, heart, lung, liver, and kidney. The goal is to underscore the importance of site-specific properties of the endothelium in mediating homeostasis and focal vascular pathology, while at the same time emphasizing the value of approaching the endothelium as an integrated system.
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            A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association.

            The compositions of lesion types that precede and that may initiate the development of advanced atherosclerotic lesions are described and the possible mechanisms of their development are reviewed. While advanced lesions involve disorganization of the intima and deformity of the artery, such changes are absent or minimal in their precursors. Advanced lesions are either overtly clinical or they predispose to the complications that cause ischemic episodes; precursors are silent and do not lead directly to complications. The precursors are arranged in a temporal sequence of three characteristic lesion types. Types I and II are generally the only lesion types found in children, although they may also occur in adults. Type I lesions represent the very initial changes and are recognized as an increase in the number of intimal macrophages and the appearance of macrophages filled with lipid droplets (foam cells). Type II lesions include the fatty streak lesion, the first grossly visible lesion, and are characterized by layers of macrophage foam cells and lipid droplets within intimal smooth muscle cells and minimal coarse-grained particles and heterogeneous droplets of extracellular lipid. Type III (intermediate) lesions are the morphological and chemical bridge between type II and advanced lesions. Type III lesions appear in some adaptive intimal thickenings (progression-prone locations) in young adults and are characterized by pools of extracellular lipid in addition to all the components of type II lesions.
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              Lessons on the pathogenesis of aneurysm from heritable conditions.

              Aortic aneurysm is common, accounting for 1-2% of all deaths in industrialized countries. Early theories of the causes of human aneurysm mostly focused on inherited or acquired defects in components of the extracellular matrix in the aorta. Although several mutations in the genes encoding extracellular matrix proteins have been recognized, more recent discoveries have shown important perturbations in cytokine signalling cascades and intracellular components of the smooth muscle contractile apparatus. The modelling of single-gene heritable aneurysm disorders in mice has shown unexpected involvement of the transforming growth factor-β cytokine pathway in aortic aneurysm, highlighting the potential for new therapeutic strategies.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 November 2015
                2015
                : 10
                : 11
                Affiliations
                [1 ]Université de Bordeaux, Bordeaux, France
                [2 ]INSERM, U1045, Bordeaux, France
                [3 ]Assistance Publique-Hôpitaux de Paris, Hôpital Bichat, Paris, France
                [4 ]SFR TransBioMed, Bordeaux, France
                [5 ]Service de chirurgie cardiaque et vasculaire, Hôpital Haut-L’Evêque, Pessac, France
                INSERM, FRANCE
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AL EG. Performed the experiments: AL VV SL PS EG. Analyzed the data: AL VV EG. Contributed reagents/materials/analysis tools: XR FM. Wrote the paper: AL PS EG.

                Article
                PONE-D-15-28271
                10.1371/journal.pone.0143144
                4658207
                26599408

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

                Page count
                Figures: 6, Tables: 2, Pages: 24
                Product
                Funding
                This work was funded by Fondation Lefoulon-Delalande (to AL), Fondation pour la Recherche Médicale (to AL), and Agence Nationale de la Recherche, ANR-2010-BLAN-1237-01 (to EG).
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                Research Article
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                All relevant data are within the paper and its Supporting Information files.

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