Recruitment of smooth muscle cells and arterial vasomotion

Investigating the recruitment and synchronization of smooth muscle cells (SMCs) is the key to understanding the physical mechanisms leading to contraction and spontaneous diameter oscillations of arteries, called vasomotion. We improved a method that allows the correlation of calcium oscillations (flashing) of individual SMCs with mean calcium variations and arterial contraction using confocal microscopy. Endothelium-stripped rat mesenteric arteries were cut open, loaded with dual calcium fluorescence probes, and stimulated by increasing concentrations of the vasoconstrictors phenylephrine (PE) and KCl. We found that the number and synchronization of flashing cells depends on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells flash asynchronously and no local contraction is detected. At medium concentration, recruitment of cells is complete and synchronous, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous calcium oscillations and fully contracted vessels, whereas high concentration of KCl leads to a sustained nonoscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling rat mesenteric artery contraction and 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

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Jean-Louis Bény, Jean-Jacques Meister

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.

Elsevier2004