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.

Identification and functional response of interstitial Cajal-like cells from rat mesenteric artery

Jean-Louis Bény, Lara Buscemi Estefanell, Aurélie Formey de Saint Louvent, Jean-Jacques Meister

Cells with irregular shapes, numerous long thin filaments, and morphological similarities to the gastrointestinal interstitial cells of Cajal (ICCs) have been observed in the wall of some blood vessels. These ICC-like cells (ICC-LCs) do not correspond to the other cell types present in the arterial wall: smooth muscle cells (SMCs), endothelial cells, fibroblasts, inflammatory cells, or pericytes. However, no clear physiological role has as yet been determined for ICC-LCs in the vascular wall. The aim of this study has been to identify and characterize the functional response of ICC-LCs in rat mesenteric arteries. We have observed ICC-LCs and identified them morphologically and histologically in three different environments: isolated artery, freshly dispersed cells, and primary-cultured cells from the arterial wall. Like ICCs but unlike SMCs, ICC-LCs are positively stained by methylene blue. Cells morphologically resembling methylene-blue-positive cells are also positive for the ICC and ICC-LC markers alpha-smooth muscle actin and desmin. Furthermore, the higher expression of vimentin in ICC-LCs compared with SMCs allows a clear discrimination between these two cell types. At the functional level, the differences observed in the variations of cytosolic free calcium concentration of freshly dispersed SMCs and ICC-LCs in response to a panel of vasoactive molecules show that ICC-LCs, unlike SMCs, do not respond to exogenous ATP and Arginine-vasopressin.

Springer-Verlag2011

A theoretical model of the cellular calcium dynamics and vasomotion in muscular arteries

Michèle Koenigsberger

Muscular arteries possess the ability to control actively their lumen by altering the tone of the smooth muscle cells of the arterial wall, a property which is vital for a large number of the hemodynamical functions of the body. Arterial contraction is due to an increase in the smooth muscle cytosolic calcium concentration. Abnormalities in the contractile mechanisms of arteries contribute to a variety of cardiovascular diseases such as hypertension. The understanding of the mechanisms of vascular smooth muscle functions can therefore contribute to a better diagnosis and treatment of such diseases. Muscular arteries, arterioles and capillaries display also slow rhythmic diameter variations, called vasomotion, independent on other rhythms in the body (cardiac, respiratory, circadian). It is well established that vasomotion is due to the contractile activity of vascular smooth muscle cells, but its underlying mechanisms are not well understood. The aim of the present thesis is to develop a theoretical model to get a better understanding of vasomotion in muscular arteries and arterioles. Many experimental studies have shown that arterial smooth muscle cells respond with cytosolic calcium rises to stimulation by substances causing arterial contraction (vasoconstrictors). A low vasoconstrictor concentration gives rise to asynchronous "all or none" calcium rises (calcium flashes) in few cells. With a higher vasoconstrictor concentration, the number of smooth muscle cells responding in this way increases (recruitment). When all cells are recruited, synchronous calcium oscillations are observed that generate arterial contraction and vasomotion. We show that these phenomena of recruitment and synchronization naturally emerge from a theoretical model of a population of smooth muscle cells coupled through their gap junctions. The effects of electrical, calcium and inositol 1,4,5-trisphosphate couplings are studied. In our model, the asynchronous calcium flashes arise from stochastic opening of channels. The calcium oscillations result from a dynamic system instability and can be synchronized by gap junctional coupling. Our model is validated by published in vitro experiments obtained on rat mesenteric arterial segments. Several experimental studies report that cells presenting only a transient calcium increase when freshly dispersed may present calcium oscillations when they are coupled. Such observations suggest that the role of gap junctions is not only to coordinate calcium oscillations of adjacent cells. Gap junctions may also be important to generate oscillations. To address this point, we study in more detail the properties of electrically coupled smooth muscle cells. We show that the effect of electrical coupling may not only be to synchronize the calcium oscillations in smooth muscle cells, as intuitively expected. A bifurcation analysis in the case of two cells reveals that electrical coupling can cause the calcium oscillations to be synchronous or asynchronous. In a larger population of smooth muscle cells, electrical coupling may result in the formation of groups of cells presenting synchronous calcium oscillations. The elements of one group may be distant from each other. Moreover, our results highlight a general mechanism by which gap junctional electrical coupling can give rise to out of phase calcium oscillations in smooth muscle cells that are non oscillating when uncoupled. Even though it is now established that vasomotion is induced by synchronous calcium oscillations of smooth muscle cells, the role of the endothelium for vasomotion is still unclear and controversial. Some experimental studies claim that the endothelium is necessary for synchronization and vasomotion, whereas others report rhythmic diameter oscillations in absence of endothelium. Moreover, endothelium derived factors have been shown to abolish vasomotion by desynchronizing the calcium signals in smooth muscle cells. By modeling the calcium dynamics of a population of smooth muscle cells coupled to a population of endothelial cells, we analyze the effects of a smooth muscle vasoconstrictor stimulation on endothelial cells and the feedback effects of endothelium derived factors. Our results show that the endothelium essentially decreases the calcium level in smooth muscle cells and may move these cells from a steady state to an oscillatory domain, and vice versa. In the oscillatory domain, a population of coupled smooth muscle cells exhibits synchronous calcium oscillations. Outside this domain, the coupled smooth muscle cells present only irregular calcium flashes. Our findings provide explanations for the published contradictory experimental observations. Smooth muscle and endothelial cells in the arterial wall are exposed to mechanical stress. Indeed, blood flow induces intraluminal pressure variations and shear stress. An increase in pressure may induce a vessel contraction, a phenomenon known as the myogenic response. Vasomotion has also been shown to be modulated by pressure changes. To get a better understanding of the effect of stress and in particular pressure on vasomotion, we propose a model of a blood vessel describing the calcium dynamics in a coupled population of smooth muscle cells and endothelial cells and the consequent vessel diameter variations. We show that a rise in pressure increases the calcium concentration. This may either induce or abolish vasomotion, or increase its frequency depending on the initial conditions. In our model the myogenic response is less pronounced for large arteries than for small arteries and occurs at higher values of pressure if the arterial wall is thickened. Our results are in agreement with experimental observations concerning a broad range of vessels. In vitro different techniques are used to study the calcium dynamics and contraction/relaxation mechanisms on arteries. Most experimental studies use either an isometric (i.e. the arterial diameter is kept constant) or an isobaric (i.e. the intraluminal pressure is kept constant) setup. However none of these setups correspond to the in vivo situation. In vivo, a blood vessel is neither isobaric nor isometric nor isotonic (i.e. the wall tension is kept constant), since it is continuously submitted to intraluminal pressure variations arising from heart beat. We determine theoretically whether results may be considerably different depending on the experimental setup (isometric, isobaric, or isotonic) used. We show that the vasoconstrictor sensitivity is higher in isometric than isobaric or isotonic conditions, in agreement with experimental observations. The threshold vasoconstrictor concentration necessary for vasomotion is therefore higher in isobaric than isometric setups. The model suggests that isometric conditions may generate the coexistence of multiple stable states. The contraction is less pronounced in isotonic than in isobaric conditions, in agreement with experimental findings.

EPFL2006

Calcium Dynamics and Intercellular Communication in Arterial Smooth Muscle Cells in Vitro

Nadia Halidi

Tissue blood flow is controlled by changes in the diameter of the arteries and arterioles through coordinated contraction and relaxation of smooth muscle cells (SMCs) within the vascular wall. The contraction of SMCs is primarily regulated by the intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]i, in response to stimuli, can propagate from cell to cell, as an intercellular Ca2+ wave along the vessel wall and can activate the process of contraction. The aim of this thesis is to elucidate the mechanisms underlying intercellular Ca2+ wave propagation between SMCs. In the first part of this thesis, we used A7r5 cells, a rat aortic SMC line, loaded with the fluorescent Ca2+ dye Fluo-4 to study intercellular Ca2+ wave propagation. Local mechanical stimulation evoked a Ca2+ wave in the stimulated cell that failed to propagate to neighboring cells. Using primary cultured rat mesenteric smooth muscle cells (pSMCs) instead, intercellular Ca2+ wave propagation was observed. To understand the difference in junctional communication between A7r5 and pSMCs, we investigated the expression of connexin37 (Cx37), Cx40, Cx43 and Cx45. RNA and protein analysis demonstrated that Cx40 – in contrast to A7r5 cells – is not expressed in pSMCs. To confirm that coexpression of Cx40 and Cx43 interfered with junctional communication, we used 6B5N cells, a clone of A7r5 cells with a higher Cx43:Cx40 expression ratio. Junctional communication, assessed by transfer of Lucifer Yellow and propagation of Ca2+ waves, was comparable between 6B5N cells and pSMCs. In addition, Ca2+ wave propagation was inhibited with the connexin-mimetic peptide 43Gap 26, that targets Cx43. Our results demonstrate that Cx43 gap junctions are primarily involved in mediating intercellular Ca2+ waves between pSMCs, and the coexpression of Cx43 with Cx40 may interfere with Cx43 gap junction formation, affecting cell-cell communication. In the second part of this thesis, we applied the microcontact printing technique to culture pSMCs on collagen lines. The aligned arrangement of the cells facilitates the observation of Ca2+ wave progression from one cell to another. To induce a Ca2+ wave, a single pSMC was locally stimulated with a micropipette (transientmechanical stimulation) or by microejection of KCl. Mechanical stimulation evoked two distinct Ca2+ waves: 1) a fast wave (∼2 mm/s) that propagated to all observed neighbouring cells, and 2) a slow wave (∼20 µm/s), that was most often limited in propagation to the first cell. KCl induced only fast Ca2+ waves of the same velocity as the mechanically-induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors, showed that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca2+ influx, whereas, the slow wave was due to Ca2+ release primarily through IP3 receptors. Together, these results suggest a mechanism by which intercellular Ca2+ waves can propagate between SMCs of the arterial wall.