Intercellular ultrafast calcium wave in vascular smooth muscle cells

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is controled by cytosolic Ca2+ concentration ([Ca2+]i) which propagates as Ca2+ waves over a significant distance along the vessel. In the first part, we have characterized an intercellular ultrafast Ca2+ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15mm/s. Combining of precise alignment of cells on a micro-fabricated substrate with fast Ca2+ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca2+]i dynamics and membrane potential events along the network of cells. The rate of [Ca2+]i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca2+ channels (VOCCs) inhibition suppressed the ultrafast Ca2+ wave. The second part is focused on the electrophysiology aspects of the mechanically-induced membrane depolarization. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. The spreading velocity of the membrane depolarization had a similar magnitude than the velocity of the ultrafast Ca2+ wave. In the last part, we tested the hypothesis that the ultrafast Ca2+ wave results from cell membrane depolarization propagation with the implementation of a mathematical model. The theoretical model reproduced qualitatively and quantitatively the main experimental findings that supported the conclusions of this thesis. Together, these results demonstrate that an electrotonic spread of the cell membrane depolarization drives a rapid Ca2+ entry from the external medium through VOCCs, modeled as an ultrafast Ca2+ wave. This wave may trigger and drive slower Ca2+ waves observed ex vivo and in vivo.

Ultrafast Ca2+ wave in cultured vascular smooth muscle cells aligned on a micropatterned surface

Jean-Louis Bény, Jean-Jacques Meister, Jairo Camilo Quijano Perez, Benoît Vianay

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca2+ concentration (Ca2+) which propagates as Ca2+ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca2+ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCI stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca2+ imaging and intracellular membrane potential recording, allowed us to analyze rapid Ca2+ dynamics and membrane potential events along the network of cells. The rate of Ca2+ increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca2+ channels (VOCCs) inhibition suppressed the ultrafast Ca2+ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca2+ entry from the external medium through VOCCs, modeled as an ultrafast Ca2+ wave. This wave may trigger and drive slower Ca2+ waves observed ex vivo and in vivo. (C) 2013 Elsevier Ltd. All rights reserved.

Elsevier2013

Identification and Functional Response of "Interstitial Cells of Cajal"-like Cells from Rat Mesenteric Artery

Aurélie Formey de Saint Louvent

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.

EPFL2011

Contraction of myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity

Boris Hinz, Michael David Martin

During wound healing and fibrocontractive diseases fibroblasts acquire a smooth muscle cell-like phenotype by differentiating into contractile force generating myofibroblasts. We examined whether regulation of myofibroblast contraction in granulation tissue is dominated by Ca2+-induced phosphorylation of myosin light chain kinase or by Rho/Rho kinase (ROCK)-mediated inhibition of myosin light chain phosphatase, similar to that of cultured myofibroblasts. Strips of granulation tissue obtained from rat granuloma pouches were stimulated with endothelin-1 (ET-1), serotonin, and angiotensin-II and isometric force generation was measured. We here investigated ET-1 in depth, because it was the only agonist that produced a long-lasting and strong response. The ROCK inhibitor Y27632 completely inhibited ET-1-promoted contraction and the phosphatase inhibitor calyculin elicited contraction in the absence of any other agonists, suggesting that activation of the Rho/ROCK/myosn light chain phosphatase pathway is critical in regulating in vivo myofibroblast contraction. Membrane depolarization with K+ also stimulated a long-lasting contraction of granulation tissue; however, the amount of force generated was significantly less compared to ET-1. Moreover, K+-induced contraction was inhibited by Y27632. These results are consistent with inhibition of myosin light chain phosphatase by the Rho/ROCK signaling pathway, which would account for the long-duration contraction of myofibroblasts necessary for wound closure.

Wiley-Blackwell2006

Identification and Functional Response of "Interstitial Cells of Cajal"-like Cells from Rat Mesenteric Artery

Aurélie Formey de Saint Louvent

EPFL2011