The myofibroblast matrix: implications for tissue repair and fibrosis

Myofibroblasts, and the extracellular matrix ( ECM ) in which they reside, are critical components of wound healing and fibrosis. The ECM , traditionally viewed as the structural elements within which cells reside, is actually a functional tissue whose components possess not only scaffolding characteristics, but also growth factor, mitogenic, and other bioactive properties. Although it has been suggested that tissue fibrosis simply reflects an ‘exuberant’ wound‐healing response, examination of the ECM and the roles of myofibroblasts during fibrogenesis instead suggest that the organism may be attempting to recapitulate developmental programmes designed to regenerate functional tissue. Evidence of this is provided by the temporospatial re‐emergence of embryonic ECM proteins by fibroblasts and myofibroblasts that induce cellular programmatic responses intended to produce a functional tissue. In the setting of wound healing (or physiological fibrosis), this occurs in a highly regulated and exquisitely choreographed fashion which results in cessation of haemorrhage, restoration of barrier integrity, and re‐establishment of tissue function. However, pathological tissue fibrosis, which oftentimes causes organ dysfunction and significant morbidity or mortality, likely results from dysregulation of normal wound‐healing processes or abnormalities of the process itself. This review will focus on the myofibroblast ECM and its role in both physiological and pathological fibrosis, and will discuss the potential for therapeutically targeting ECM proteins for treatment of fibrotic disorders.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94657/1/path4104.pd

Coordination of Myofibroblast Contraction in Vitro

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

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

A New Lock-step Mechanism of Matrix Remodelling Based on Subcellular Contractile Events

Myofibroblasts promote tissue contractures during fibrotic diseases. To understand how spontaneous changes in the intracellular calcium concentration, [Ca2+](i), contribute to myofibroblast contraction, we analysed both [Ca2+](i) and subcellular contractions. Contractile events were assessed by tracking stress-fibre-linked microbeads and measured by atomic force microscopy. Myofibroblasts exhibit periodic (similar to 100 seconds) [Ca2+](i) oscillations that control small (similar to 400 nm) and weak (similar to 100 pN) contractions. Whereas depletion of [Ca2+](i) reduces these microcontractions, cell isometric tension is unaffected, as shown by growing cells on deformable substrates. Inhibition of Rho-and ROCK-mediated Ca2+-independent contraction has no effect on microcontractions, but abolishes cell tension. On the basis of this two-level regulation of myofibroblast contraction, we propose a single-cell lock-step model. Rho- and ROCK-dependent isometric tension generates slack in extracellular matrix fibrils, which are then accessible for the low-amplitude and high-frequency contractions mediated by [Ca2+](i). The joint action of both contraction modes can result in macroscopic tissue contractures of similar to 1 cm per month

The Mechanical Environment Modulates Intracellular Calcium Oscillation Activities of Myofibroblasts

Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair

Restricting cell size decreases [Ca²⁺]_i frequency.

<p>A) SCMF were grown on microcontact-printed FN square islets of 100–10,000 μm², immunostained for F-actin, and imaged by confocal microscopy. A composite was produced by stitching images from different cells on the same substrate, containing all square sizes. Scale bar = 500 μm. B) Cells spreading on FN islets of 2,500 and 4,900 μm² were immunostained for F-actin (red), vinculin (green), and FN (blue). Scale bar = 250 μm. C) Representative fluorescence ratios (Em₃₄₀/Em₃₈₀) were recorded over time on Fura-2-loaded cells, stimulated with increasing concentrations of endothelin-1 (ET-1). D) Distribution fits of [Ca²⁺]_i oscillation periods are displayed for cells grown on 2,500 and 4,900 μm² islands (n_exp = 18–25, n_cells = 24–29) and treated with 50 nM ET-1.</p

Disrupting actin stress fibers decreases [Ca²⁺]_i oscillation frequency.

<p>SCMF were grown for 2 days on FN-coated coverslips. A) Cells fixed before (left panel) and 30 min after Cytochalasin D treatment were immunostained for F-actin (red), vinculin (green) and nuclei (blue). Scale bar = 50 μm. B) Representative fluorescence ratio (Em₃₄₀/Em₃₈₀) of a Fura-2-loaded cell over time is shown. Fluorescence was recorded for 15 min before and 15 min after 30 min treatment with Cytochalasin D (15 μM) or vehicle (DMSO) only (C). D) [Ca²⁺]_i oscillation period was calculated before Cytochalasin D treatment and plotted against the period after treatment for the same cell. Any point above the diagonal indicates a period decrease after addition of the drug (n_exp =9, n_cells = 25).</p

Decreasing cell adhesion decreases [Ca²⁺]_i oscillation frequency.

<p>SCMF were grown for 2 days on glass coverslips, coated with PLL at 5.0 μg/cm², 0.5 μg/cm², or with FN. A) Cells were immunostained for F-actin (red), vinculin (green) and nuclei (blue). Scale bar = 50 μm. B) Representative fluorescence ratios (Em₃₄₀/Em₃₈₀) were recorded over time on Fura-2-loaded cells. C) [Ca²⁺]_i period distribution fits are displayed and maxima highlighted with dotted lines (n_exp = 29–34, n_cells = 68-93). D) SCMF area (n = 3; mean±SD) and E) the length of vinculin-positive focal adhesions were quantified from fluorescence staining (n = 3; mean±SEM, *p≤0.05, ***p≤0.001), F) Period distribution fit maxima were translated into oscillation frequency (peaks/min) and expressed as a function of the mean SCMF focal adhesion lengths on differently adhesive substrates.</p

Increasing the E-modulus of silicone substrates increases [Ca²⁺]_i oscillation frequency.

<p>SCMF were cultured on FN-coated silicone substrates produced with E-moduli of 5, 15, and 50 kPa for 2 days. A) Cells were immunostained for α-SMA (red), F-actin (green) and nuclei (blue). Scale bar = 50 μm. B) Representative fluorescence ratios (Em₃₄₀/Em₃₈₀) were recorded over time on Fura-2-loaded cells of each stiffness group. C) The dominant periods of regular oscillations were determined and pooled into a histogram that was fitted following a generalized extreme value distribution. D) [Ca²⁺]_i period distribution fits of 5 kPa, 15 kPa and 50 kPa groups are displayed and maxima highlighted with dotted lines (n_exp≥14, n_cells≥44). E) Period distribution fit maxima were translated into oscillation frequency (peaks/min) and expressed as a function of the Young's E-modulus of silicone substrates.</p