Identification and Functional Response of "Interstitial Cells of Cajal"-like Cells from Rat Mesenteric Artery
This thesis is a contribution to the field of interstitial cells of Cajal-like cells (ICC-LCs) in the vasculature. Indeed, cells with irregular shapes and numerous long thin filaments, morphologically similar to the gastrointestinal ICCs, have been observed in the wall of some blood vessels. These ICC-LCs are distinct from the other cell types present in the arterial wall: smooth muscle cells (SMCs), endothelial cells (ECs), fibroblasts, inflammatory cells and pericytes. However, no clear physiological role has been yet determined for ICC-LCs in the vascular wall. The aim of this thesis is to identify and to characterize the functional response of ICC-LCs in rat mesenteric arteries. This study is composed of two main experimental parts involving rat mesenteric arteries, freshly dispersed and primary cultured cells from those arteries: an investigation allowing the identification of ICC-LCs and a study on the functional characterization of these cells. In the first part of this work, we identify ICC-LCs morphologically and histologically in three different environments: isolated artery, freshly dispersed cells and primary cultured cells from the arterial wall. For this purpose, we develop methods to enrich freshly dispersed cells in ICC-LCs and to primary culture them from arterial explants. Similar to ICCs but contrary to SMCs, ICC-LCs are positively stained by methylene blue. At this stage, we develop a procedure to link, for the first time, positive blue cells among the arteries and freshly dispersed cells. Using immunohistochemistry techniques, we demonstrate that cells morphologically resembling to methylene blue positive cells are also positive for ICCs and ICC-LCs markers a-smooth muscle actin and desmin. Furthermore, ICC-LCs' expression of connexin 40 gap junctional protein, c-Kit tyrosine kinase receptors and the higher expression of vimentin in ICC-LCs compared to SMCs, allow a clear discrimination between these two cell types. To conclude the first part of the thesis on the identification of ICC-LCs, we prove that ICC-LCs are able to contract in response to high depolarization, and that SMCs' contraction is significantly higher than that of ICC-LCs. In the second part of this thesis we investigate ICC-LCs at the functional level by studying their free calcium concentration responses to agonists. We first show that freshly dispersed ICC-LCs are responsive to depolarization in a way that differs from the SMCs' one. Then we demonstrate for the first time a physiological difference between ICC-LCs and SMCs. Indeed, freshly dispersed ICC-LCs respond, in term of variations of cytosolic free calcium concentration, to a certain number of vasoactive molecules but not to exogenous ATP and [arginine]8-vasopressin (AVP), contrary to SMCs. Furthermore, we search to gain insight into the calcium behavior of ICC-LCs located in the arterial wall. As far as we know, this is the first time that a calcium study of ICC-LCs is performed directly on the vascular tissue. We are able to identify calcium-dye loaded ICC-LCs in the arterial tissue and record their calcium dynamics, which is then compared to that of SMCs in control conditions as well as when stimulated with vasoactive molecules. We show that ICC-LCs behavior differs if the studied arteries present, or not, vasomotion. In addition, their response to vasoactive agonists is similar to the one observed in freshly dispersed cells. In conclusion, we demonstrate that ICC-LCs are present in the rat mesenteric arterial wall. The identification of ICC-LCs at three levels of organization "isolated artery, freshly dispersed and primary-cultured cells" provides a model to study ICC-LCs and to investigate their physiological role. Furthermore, calcium imaging appears to be a helpful tool to study their responses to agonists in order to characterize them at the functional level.
Keywords: ICC-like cells ; rat mesenteric arteries ; smooth muscle cells ; cytosolic free calcium concentration variations ; vasomotion ; cellules ressemblant aux cellules interstitielles de Cajal ; cellules musculaires lisses ; variations de la concentration en calcium intracellulaire ; vasomotionThèse École polytechnique fédérale de Lausanne EPFL, n° 4966 (2011)
Programme doctoral Biotechnologie et Génie biologique
Faculté des sciences de la vie
Institut de physique des systèmes biologiques
Laboratoire de biophysique cellulaire
Record created on 2011-01-06, modified on 2016-08-09