Intercellular calcium dynamics in smooth muscle cells

Muscular arteries are able to actively modify their diameter by modulating the tone of the smooth muscle cells located within the arterial wall. A small variation of lumen diameter has a large influence on blood flow as well as on arterial pressure. The cardiovascular system pressure is mainly regulated by the resistance arteries and arterioles. Most of these arteries show cyclic variations of their diameter. This phenomenon is known as vasomotion and is in no way linked to any other physiological cycle like heartbeat or respiratory cycles. This phenomenon can be observed as well in vivo as in vitro. The arterial diameter oscillations are attributed to the contractile activity of the smooth muscle cells which is directly linked to their intracellular calcium concentration. Although observed since years, the mechanisms underlying vasomotion remain not well understood. It is known that arterial vasomotion is modified by apparition of abnormalities in the contractile mechanisms of arteries linked to cardiovascular diseases like hypertension. A better understanding of this phenomenon could therefore contribute to better treatments or diagnosis of such diseases. This thesis is divided in two parts which have each the goal to clear up a definite aspect of arterial vasomotion. The experiments have been performed in vitro on resistance arteries: the rat mesenteric arteries. These arteries have been observed under confocal fluorescence microscope allowing to quantify the intracellular calcium concentration in smooth muscle cells. In the first part of this work, we investigate the role played by the endothelium on arterial vasomotion. So far, contradictory results have been related in the literature about the role of the endothelium in the onset and maintenance of vasomotion. Even the elementary question of knowing if vasomotion can arise without an intact endothelium is quite controversial. To understand how the endothelium may either abolish or promote vasomotion, we have stimulated rat mesenteric arterial strips with a vasoconstrictor: the phenylephrine. Our results show that the endothelium is not necessary for vasomotion. However, when the endothelium is removed the phenylephrine concentration needed to induce vasomotion is lower and the rhythmic contractions occur for a narrower range of phenylephrine concentrations. By selectively inhibiting the endothelium-derived relaxing products, we demonstrate that these substances may either induce or abolish vasomotion. On the one hand, when the strip is tonically contracted in a non-oscillating state, an endothelium-derived relaxation may induce vasomotion. On the other hand, if the strip displays vasomotion, a relaxation may induce a transition to a non-oscillating state with a small contraction. In conclusion, our findings clarify the role of the endothelium on vasomotion and reconcile the seemingly contradictory observations reported in the literature. In the second part of this thesis we are interested in a vasomotion property not studied until now which is the propagation of vasomotion along arterial segments. The aim of this study is first to characterize these contraction waves, then in a second part to define what are the mechanisms allowing this propagation. We have stimulated rat mesenteric arterial strips with phenylephrine to obtain vasomotion and observed that the contraction waves are linked to intercellular calcium waves. A velocity of about 100 µm/s was measured for the two kinds of waves and this velocity was independent of the presence of the endothelium on the strip. To investigate the calcium wave propagation mechanisms, we have elaborated a method allowing a phenylephrine stimulation of a small area of the strip. No calcium propagation could be induced by this local stimulation when the strip was in its resting state. However, if a low phenylephrine concentration was added on the whole strip, local phenylephrine stimulations induced calcium waves spreading over a finite distance of almost 400 µm. The calcium wave velocity induced by local stimulation was identical to the velocity observed during vasomotion. This suggests that the propagation mechanisms are similar in the two cases. Using inhibitors of gap junctions and of voltage operated calcium channels, we have shown that the intercellular calcium wave propagation depends on the propagation of the smooth muscle cells depolarization. Finally, the results allow to propose a model of the mechanisms underlying the propagation of intercellular calcium waves along arterial segments during vasomotion.

Calcium dynamics and intercellular communication in arterial smooth muscle cells

Mathieu Lamboley

This thesis is a contribution to the field of cellular communication in arteries during vasomotion, i.e. rhythmic and spontaneous diameter oscillations of arteries. Investigating how individual smooth muscle cells (SMCs) and endothelial cells (ECs) calcium variations interact to induce an arterial response is a key to understanding the physical mechanisms leading to contraction and vasomotion. This study is composed of two main experimental parts using rat mesenteric arteries: a study on recruitment and synchronization of SMCs and an analysis on SMCs-ECs communication in arterial strips. The introduction is an overview of the mechanism leading to arterial vasomotion. We focused on the calcium signaling between SMCs, between ECs and between SMCs and ECs. In the chapter "Material and methods", we present the different experimental techniques we developed or improved in order to study calcium signaling in SMCs and/or ECs. For this purpose, we used arterial strips or cannulated arteries using a confocal microscope or a conventional microscope. Calcium concentration were measured using fluorescent dyes. These methods allow the correlation of calcium oscillations of individual SMCs, of individual ECs or both, together with mean calcium variations and arterial contraction. In chapter 3, we investigated the behavior of individual SMCs in order to determine if all cells presented the same variations of calcium concentration as the mean calcium variations or if vasomotion results of an unequal contribution of each SMC. Arterial strips were stimulated by increasing concentration of vasoconstrictors phenylephrine (PE) or potassium chloride (KCl). We revealed that the number of SMCs presenting calcium variations and their synchronization depend on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells present asynchronous calcium variations and no local contraction is detected. Recruitment of cells is complete and synchronous at medium concentration, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous oscillations and a fully contracted arterial strip, whereas high concentration of KCl leads to a sustained non-oscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling artery contraction and vasomotion. In chapter 4, we investigated the three main possible ways to synchronize the recruitment of SMCs when stimulating with PE and KCl. We tested the importance of calcium voltage-gated channels (VOCs) and thus the electrical communication, by using different inhibitors of the VOCs. We also tested the importance of stretch-activated channels (SACs) by inhibiting contraction of individual SMCs. Finally, we verified the importance of gap junctions and thus of chemical and/or electrical coupling. We applied a gap junction inhibitor and we also performed microinjection of fluorescent dyes in single SMCs. Our results provide evidence that under PE the synchronization of the SMCs recruitment occurred electrically through gap junctions. Under KCl, every SMC was simultaneously depolarized and calcium entered the cell simultaneously through VOCs. This leads to a global [Ca2+]i increase. When we studied the recruitment and synchronization of the SMCs under PE, we sometimes observed intercellular calcium waves propagating between SMCs in a direction corresponding to the arterial axis. In chapter 5, we used a tracking process to quantify these intercellular waves. We found that intercellular calcium waves propagating along the strip were linked to the local contraction variations. The velocity of both waves are similar (~60 µm/s). This method of tracking is particularly powerful for the study of cellular communication in arteries. It has the advantage of allowing the correlation of calcium with contraction dynamics. The role of the endothelium for vasomotion is matter of debate. SMCs and ECs are electrically and chemically coupled allowing exchange of intercellular signaling. In chapter 6, we investigated heterocellular communication at the cellular level in order to understand how SMCs can influence ECs calcium dynamics. SMCs of the arterial strip were stimulated by KCl and by PE. We verified that PE and KCl did not directly act on the calcium concentration of ECs. Depending on vasoconstrictor concentration, calcium concentration increased in some ECs 5 to 11 s after that a calcium concentration increase was observed in SMCs. The existence of this time interval suggests a chemical coupling between SMCs and ECs through gap junctions. Most probable chemical mediators are either calcium or inositol 1,4,5-trisphosphate (IP3). To discriminate between calcium and IP3 diffusion, first a phospholipase C inhibitor was applied to prevent IP3 production in response to the calcium concentration increase in SMCs. Under this inhibitor and KCl, all SMCs presented a global and synchronous calcium concentration increase, but no calcium concentration variations in ECs were detected. Secondly, 2-Aminoethoxydiphenylborate, an inhibitor of IP3-induced calcium release, reduced the number of flashing ECs by 75±3 %. The number of flashing ECs was also significantly reduced when palmitoleic acid, a gap junction uncoupler was added. Our results were compatible with a heterocellular communication based on IP3 diffusion through gap junctions from SMCs to ECs. The last chapter summarizes our findings and provides our conclusions with some perspectives.

EPFL2005

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

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