Regulation of myofibroblast activities: Calcium pulls some strings behind the scene

Myofibroblast-induced remodeling of collagenous extracellular matrix is a key component of our body's strategy to rapidly and efficiently repair damaged tissues; thus myofibroblast activity is considered crucial in assuring the mechanical integrity of vital organs and tissues after injury. Typical examples of beneficial myofibroblast activities are scarring after myocardial infarct and repair of damaged connective tissues including dermis, tendon, bone, and cartilage. However, deregulation of myofibroblast contraction causes the tissue deformities that characterize hypertrophic scars as well as organ fibrosis that ultimately leads to heart, lung, liver and kidney failure. The phenotypic features of the myofibroblast, within a spectrum going from the fibroblast to the smooth muscle cell, raise the question as to whether it regulates contraction in a fibroblast- or muscle-like fashion. In this review, we attempt to elucidate this point with a particular focus on the role of calcium signaling. We suggest that calcium plays a central role in myofibroblast biological activity not only in regulating contraction but also in mediating intracellular and extracellular mechanical signals, structurally organizing the contractile actin-myosin cytoskeleton, and establishing lines of intercellular communication. (C) 2010 Elsevier Inc. All rights reserved.

Coordination of Myofibroblast Contraction in Vitro

Lysianne Follonier Castella

The aim of this thesis is to elucidate the common mechanisms that regulate myofibroblast contraction and intercellular communication, with a particular focus on the role of Ca2+ signalling. Synthesis and remodelling of collagenous matrix by myofibroblasts are key elements in the healing process of injured organs. Deregulation of myofibroblast activity and excessive remodelling result in hypertrophic scarring and organ fibrosis. Myofibroblasts are specialised fibroblasts with particularly contractile stress fibres and features that resemble smooth muscle cells. Although myofibroblast contraction is acknowledged being responsible for wound closure and tissue contracture, the underlying regulation mechanisms are unclear. In particular, it remains elusive to what extent changes in the cytosolic Ca2+, a central regulator of smooth muscle contraction, regulate myofibroblast activities. Previous studies have revealed that cultured fibroblasts and myofibroblasts exhibit spontaneous cytosolic Ca2+ oscillations but a link with cell contraction has not been established. In the first part of this thesis, we are testing the hypothesis that cytosolic Ca2+ oscillations regulate myofibroblast contraction. For this, we use analysis of Ca2+ dynamics with fluorescent indicators, tracking of stress-fibre-linked microbeads on the myofibroblast surface and quantification of subcellular pulling events with atomic force microscopy. This combined approach demonstrates that myofibroblasts exhibit periodic (∼100 seconds) Ca2+ oscillations that control small (∼400 nm) and weak (∼100 pN) contractions. Using deformable culture substrate to assess cell isometric tension, we further demonstrate that myofibroblast contraction is regulated at two levels: variations of intracellular Ca2+ mediate contractions of dorsal stress fibres whereas the Ca2+-independent Rho kinase pathway is responsible for maintaining overall isometric cell tension. In the second part of this thesis, we investigate whether the observed subcellular contractions and Ca2+ oscillations may play a role in coordinating the activities between directly contacting myofibroblasts. Rational for this hypothesis is that myofibroblasts in densely populated wound granulation tissue form two types of intercellular junctions: adherens junctions that mechanically couple stress fibres between cells, and gap junctions that provide electrochemical coupling. Both junctions have been shown to play a role in modulating the contraction of wound tissue and of myofibroblast collagen gel cultures but their precise roles are still elusive. To fill this gap in knowledge, we analyse the coordination of Ca2+ oscillations between adjacent fibroblasts and myofibroblasts, respectively. Intercellular communication is modulated with pharmacological treatments acting on either mechanical or electrochemical coupling, as well as on the cell's contractile apparatus and mechanosensitive membrane channels. Our results demonstrate that stress-fibre associated adherens junctions mechanically coordinate myofibroblast activities within a cell population. This mechanism implies that the transmission of contractile events from one cell induces a Ca2+ influx through mechanosensitive ion channels in its neighbour and there triggers a new contraction. Based on these results we suggest that, at the tissue level, myofibroblasts remodel collagen by Ca2+-dependent micro-contractile events that add up to macroscopic tissue contracture, whereas Rho kinase maintains cell isometric tension. Mechanical coupling via adherens junctions may simultaneously improve the remodelling of cell-dense tissue by coordinating the activity of myofibroblasts.

EPFL2010

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