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THE LABORATORY OF CELL BIOPHYSICS – CELL CONTRACTILITY
Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
Dr Boris Hinz

STUDYING the contractile activity of non-muscle cells is one of three research areas (Cell Contractility, Cell
Motility, Calcium Dynamics) explored in the Laboratory of Cell Biophysics (http://ipmc.epfl.ch/LCB). The research of my ‘Contractility’ team of Biologists, Physicists and Engineers (Figure 1) is situated at the interface of cell and molecular biology, biophysics, bioengineering and clinical research. It is our aim to understand the molecular mechanisms that lead to myofibroblast formation and that control its fibrotic activity with the vision to offer novel
strategies counteracting myofibroblast malfunction.

Of clinical relevance are the retractile phenomena caused by excessive myofibroblast activity that characterize
the vast majority of fibrocontractive diseases. This includes fibrosis affecting vital organs, such as heart, liver, kidney and lung and fibrotic phenomena reducing life quality in scleroderma, hypertrophic scars (remarkably severe after large burn wounds), chronic asthma and Dupuytren’s disease. Moreover, myofibroblasts at the tumor invasion front are activated by the transformed epithelium to stimulate tumor growth and invasion by promoting angiogenesis and tumor cell migration. In tissue bioengineering considerable effort is required to prevent myofibroblast formation, either arising from mesenchymal stem cells that are implanted to repair tissues or at the interface between the implant and the host connective tissue. But don’t get fooled – the myofibroblast is not necessarily the ‘bad guy’ as it contributes to physiological wound healing and its absence has been associated with the development of chronic wounds.

Figure 1: The Hinz group.
The Cell Contractility team of the Laboratory of Cell Biophysics was testing
alternative resources for funding and new lab coats in 2006.
Quite successfully – Michael Schumacher resigned.

Cell Biophysics in tissue repair
After tissue injury and during tissue remodeling, reparative fibroblasts are not only in contact with an altered chemical milieu but face a dramatically changed mechani-cal microenvironment. Having spent most of their lives shielded by a protective extracellular matrix (ECM) these cells now become exposed to considerable stress. To cope with this new challenge and to resist, they respond by developing tension on their own and by building up the contractile machinery manifested as stress fibers. The force developed by initially formed cytoplasmic actin stress fibers is limited and further increasing stress stimulates de novo expression of a-smooth muscle actin (a-SMA) and thus allows generation of superior tension. Pharmacological targeting of a specific sequence in the a-SMA protein inhibits this contractile activity and reduces scar formation in animal experiments.

Figure 2: Myofibroblast junctions. Cultured myofibroblasts
connect a-SMA-positive stress fibers (red) at sites of cell-cell
(blue) and cell-matrix adhesions (green). Bar: 20 am.

To prevent formation of the myofibroblast in the first place, we propose to target their stress-sensors, in other words to render them blind for mechanical inputs. In particular, we characterize how mechanical tension regulates the formation and function of cell-cell and cell-matrix contacts (Figure 2). Both structures transmit intracellular tensile stress to the extracellular environment and at the same time serve as mechano-sensing organelles. Rather than summarizing our published work for which you may consult our latest reviews and the attached reference list, I will give an overview of the methods that are established in the laboratory and on recent studies that have just been submitted.

Mechanical stress perceived from the ECM is translated into myofibroblast differentiation
Myofibroblast ECM adhesions exhibit a specific molecular composition that differs from that of normal fibroblasts and that is influenced by applied stress. Recently, we have shown that the level of intracellular tension is directly controlled by the size of ECM adhesions, which in turn is determined by the stiffness of the substrate. We identified a-SMA as a mechano-sensitive protein that only exerts its contractile function when stress fibers experience an external resistance corresponding to the elastic modulus of fibrotic tissue.

To address these questions we use engineering technologies (or abuse as the engineers may feel):

1. Microcontact printing, a conventional method in micro- technology, is used to create adhesive fibronectin islands forcing myofibroblasts to form only ECM contacts with defined size and shape (Figure 3A) and to attain defined spreading area; one esthetically interesting side-effect is the creation of square cells (Figure 3B).
2. Material science is employed to produce silicone culture substrates with tunable compliance in the range of tissue stiffness.
3. We use atomic force microscopy, originally developed to test molecular material properties, to determine the stiffness of wound granulation tissue (Figure 3C) and of cultured cells (Figure 3D).
4. To measure the stress exerted at individual ECM adhesions, we implant regular arrays of fluorescent markers in the surface of compliant substrates and analyze the distortion pattern caused by myofibroblast contraction (Figure
   3E). A simplified version is the ‘wrinkling’ silicone substrate that allows direct assessment of cell force development by conventional transmission light microscopy combined with epifluorescence (Figure 3F). We have now developed a standardized protocol to produce such wrinkling elastomers and make the technique available for the average clinical or biological laboratory.

Mechanical and chemical signals converge to produce a fibrogenic phenotype
Although researchers tend to (and need to) concentrate on selected aspects of complex biological processes, cells do not. It would be too ambitious to state that only mechanical factors drive myofibroblast differentiation as well as it is oversimplified to attribute all fibrogenic effects to the action of growth factors. The truth – as always – lies somewhere in between and expression of a-SMA demands both, a mechanically restrained environment and the action of transforming growth factor beta (TGFß1).

One intriguing new hypothesis of how mechanical stress induces myofibroblast differentiation is by modulating the activity of TGFß1 that is secreted into the ECM as a large latent complex. In a submitted work we describe a novel mechanism by which myofibroblasts activate TGFa1 from self-generated stores in the ECM. This process requires three key elements:

1. high contraction mediated by a-SMA stress fibers,
2. incorporation of latent TGFß1 into a mechano-resistant ECM, and
3. transmission of stress fiber contraction to the latent TGFß1 complex via integrins. Inducing contraction of myofibroblasts

and of isolated cytoskeletons releases active TGFß1 from the ECM; this is inhibited by antagonizing integrins and by reducing ECM compliance. On the other hand, stretching latent TGFß1-conditioned ECM in the presence of mechanically apposing stress fibers induces immediate activation of TGFß1. In vivo, the TGFß1 downstream targets Smad2 and Smad3 exhibit higher activation in stressed compared with relaxed wound granulation tissues, despite similar levels of total TGFß1 and its receptor. We propose mechanical activation of TGFß1 as a crucial checkpoint in the progression of fibrosis by restricting autocrine maintenance of the myofibroblast phenotype to a sufficiently remodeled and stiffened ECM.

Myofibroblasts communicate with each other – small talk through big junctions
Physical intercellular contacts gain increasing importance in the highly cellularized contractile wound granulation tissue and in fibrotic lesions. We have recently shown that myofibroblasts couple stress fibers at sites of neo-formed cell-to-cell adherens junctions (AJs) with specific molecular properties. We are currently investigating two possible functions of myofibroblast-specific AJs: 1) establishing mechanical communication to coordinate myofibroblast contraction between cells that compete for the same remodeled ECM and 2) informing individual myofibroblasts about the number of similar partners. The latter supposedly provides signals to induce myofibroblast regression and termination of contracture.

Figure 3: A panel of fancy biophysics methods. (A-B): Focal adhesions (vinculin, red) link a-SMA-positive stress fibers (green) to microcontact-printed small 10x1.5 µm islets (A) and large 100x100 µm islands (B) of fibronectin (blue). (C-D): Atomic force microscopy ‘rigidity maps’ of rat wound granulation tissue sections (C) and of cultured myofibroblasts (D) represent stiff sample regions with bright values. Note the stiffer wall of small vessels (C, lower right) compared with the surrounding myofibroblast-populated tissue and the stiff stress fibers of cultured cells (D). (E): Myofibroblasts expressing GFPtagged ECM adhesion protein (paxillin, green) deform compliant silicone substrates that have been ‘tattooed’ with red fluorescent position markers. Close-up shows marker position before (red) and after (blue) cell relaxation. (F): Novel highly deformable silicone substrates permit simultaneous visualization of cell contraction by assessing ‘wrinkles’ (arrowheads) with phase-contrast microscopy, stress fibers (F-actin, red; a-SMA, green) and ECM adhesions (vinculin, blue). Bars: 10 µm.

Evidence for a coordinating role of mechanical cell coupling comes from the observation that inhibition of myofibroblast AJs with specific peptides reduces ECM contraction by cell populations. Like smooth muscle cells, cultured myofibroblasts exhibit spontaneous and periodic intracellular Ca2+ transients that are coordinated between contacting cells. We propose that single cell contraction triggers Ca2+ influx into the neighboring cell by exerting a mechanical stimulus via cell-cell junctions; this evokes a contractile response feeding back to the first cell. Inhibition of mechanical junctions (but not of gap junctions), destruction of stress fibers, inhibition of cell contraction, and blocking of mechano-sensitive Ca2+ channels all desynchronize Ca2+ transients. To withstand the significantly higher stress generated by neo-incorporation of a-SMA into stress fibers, AJs of cultured differentiated myofibroblasts increase in size. Moreover, N-cadherin, the major transmembrane linker expressed in fibroblasts, becomes replaced by OB-cadherin (cadherin-11). By assessing native cadherin bonds formed between living fibroblasts with atomic force microscopy we found that OB-cadherin bonds resist approximately 2-fold higher forces compared with N-cadherin on the single molecule level.

Novel concepts for standard tissue culture
In addition to our genuine myofibroblast research we join our biological know-how with the engineering expertise and methodology accessible at the Swiss Federal Institute of Technology in Lausanne to develop new strategies for standard cell culture. Despite their obvious advantages, plastic culture dishes by no means provide the mechanical boundary conditions that define cells in tissue. We and others drive the idea to using soft materials for cell culture; e.g., we completely abolish myofibroblast development simply by decreasing substrate stiffness despite abundant presence of fibrogenic TGFß1. Other groups recently committed mesenchymal stem cells to different lineages by changing matrix stiffness at constant chemical culture conditions and defined the ‘optimal’ substrate compliance for growth of different cell types. We will soon publish a straight-forward method to create silicone-based cell culture substrates with elastic modulus ranging from 1.0- 5,000 kPa.

Figure 4: A novel cell culture expansion device - the‘Cellerator’.
Accomplished by a highly elastic new membrane the iris-mechanism of the Cellerator
allows computer-controlled culture surface expansion by ~10- times.
This permits gradual increase in cell number at constant cell density without passing.
The device is equally suitable to perform mechanical stimulation experiments
and to induce large cell/protein deformations.

We further collaborate with EPFL spin-off Cytomec GmbH (www.cytomec.com) which commercializes novel
cell culture devices based on transparent and high y extendable culture surfaces. When placed in an iris-like expansion
device the culture surface area may be increased (under computer control) up to 10-fold; this reduces the need for traumatic cell passaging by ~3-times. We show that the high compliance of the culture surface membrane prohibits myofibroblast differentiation either from cultured fibroblasts or from mesenchymal stem cells. It is one of our missions to make these new approaches and ideas accessible for the standard laboratory.

References
Reviews and book chapters

Hinz, B. 2007. Formation and function of the myofibroblast
during tissue repair. J Invest Dermatol. 127:
526–37.
Schurch, W., T.A. Seemayer, B. Hinz, and G. Gabbiani.
2007. Myofibroblast. In Histology for Pathologists.
S.E. Mills, editor. Lippincott-Williams & Wilkins
Pub., Philadelphia, U.S.A. 123–164.
Hinz, B., S.H. Phan, V.J. Thannickal, A. Galli, M.L.
Bochaton-Piallat, and G. Gabbiani. 2007. The
myofibroblast: one function, multiple origins. Am J
Pathol. (submitted).
Hinz, B. 2006. Masters and servants of the force: The
role of matrix adhesions in myofibroblast force
perception and transmission. Eur J Cell Biol. 85:
175–81.
Hinz, B., and G. Gabbiani. 2003. Cell-matrix and cellcell
contacts of myofibroblasts: role in connective
tissue remodeling. Thromb Haemost. 90: 993–1002.
Hinz, B., and G. Gabbiani. 2003. Mechanisms of force
generation and transmission by myofibroblasts. Curr
Opin Biotechnol. 14: 538–46.
Tomasek, J.J., G. Gabbiani, B. Hinz, C. Chaponnier, and
R.A. Brown. 2002. Myofibroblasts and mechano–
regulation of connective tissue remodelling. Nat Rev
Mol Cell Biol. 3: 349–63.
Selected published manuscripts:
Clement, S., B. Hinz, V. Dugina, G. Gabbiani, and C.
Chaponnier. 2005. The N-terminal Ac-EEED
sequence plays a role in {alpha}-smooth-muscle actin
incorporation into stress fibers. J Cell Sci. 118:
1395–404.
Goffin, J.M., P. Pittet, G. Csucs, J.W. Lussi, J.J. Meister,
and B. Hinz. 2006. Focal adhesion size controls
tension-dependent recruitment of alpha-smooth
muscle actin to stress fibers. J Cell Biol. 172:259–68.
Hinz, B., W. Alt, C. Johnen, V. Herzog, and H.W. Kaiser.
1999. Quantifying lamella dynamics of cultured cells by
SACED, a new computer- assisted motion
analysis. Exp. Cell Res. 251: 234–43.
Hinz, B., G. Celetta, J.J. Tomasek, G. Gabbiani, and C.
Chaponnier. 2001a. Alpha-smooth muscle actin
expression upregulates fibroblast contractile activity.
Mol Biol Cell. 12: 2730–41.
Hinz, B., V. Dugina, C. Ballestrem, B. Wehrle-Haller,
and C. Chaponnier. 2003. Alpha-smooth muscle
actin Is crucial for focal adhesion maturation in
myofibroblasts. Mol Biol Cell. 14: 2508–2519.
Hinz, B., G. Gabbiani, and C. Chaponnier. 2002. The
NH2-terminal peptide of alpha-smooth muscle actin
inhibits force generation by the myofibroblast in
vitro and in vivo. J Cell Biol. 157: 657–63.
Hinz, B., D. Mastrangelo, C.E. Iselin, C. Chaponnier,
and G. Gabbiani. 2001b. Mechanical tension
controls granulation tissue contractile activity and
myofibroblast differentiation. Am J Pathol. 159:
1009–20.
Hinz, B., P. Pittet, J. Smith-Clerc, C. Chaponnier, and
J.J. Meister. 2004. Myofibroblast development is
characterized by specific cell—cell adherens junctions.
Mol Biol Cell. 15: 4310–20.
Montjovent, M.O., L. Mathieu, B. Hinz, L.L. Applegate,
P.-E. Bourban, P.Y. Zambelli, J.A. Manson, and D.P.
Pioletti. 2005. Biocompatibility of bioresorbable
PLA composite scaffolds with human fetal bone
cells. Tissue Engineering. 11: 1640–1649.
Ng, C.P., B. Hinz, and M.A. Swartz. 2005. Interstitial
fluid flow induces myofibroblast differentiation and
collagen alignment in vitro. J Cell Sci. 118: 4731–9.
Peters, T., A. Sindrilaru, B. Hinz, R. Hinrichs, A. Menke,
E.A. Al-Azzeh, K. Holzwarth, T. Oreshkova, H.
Wang, D. Kess, B. Walzog, S. Sulyok, C.
Sunderkotter, W. Friedrich, M. Wlaschek, T. Krieg,
and K. Scharffetter-Kochanek. 2005. Wound-healing
defect of CD18(-/-) mice due to a decrease in TGFbeta1
and myofibroblast differentiation. Embo J. 24:
3400–10.
Ronty, M.J., S.K. Leivonen, B. Hinz, A. Rachlin, C.A.
Otey, V.M. Kahari, and O.M. Carpen. 2006.
Isoform-Specific Regulation of the Actin-Organizing
Protein Palladin during TGF-beta1-Induced
Myofibroblast Differentiation. J Invest Dermatol.
126: 2387–96.
Shephard, P., B. Hinz, S. Smola-Hess, J.J. Meister, T.
Krieg, and H. Smola. 2004. Dissecting the roles of
endothelin, TGF-beta and GM-CSF on
myofibroblast differentiation by keratinocytes.
Thromb Haemost. 92: 262–74.
Thorey, I.S., B. Hinz, A. Hoeflich, S. Kaesler, P. Bugnon,
M. Elmlinger, R. Wanke, E. Wolf, and S. Werner.
2004. Transgenic mice reveal novel activities of
growth hormone in wound repair, angiogenesis, and
myofibroblast differentiation. J Biol Chem. 279:
26674–84.
Tomasek, J.J., M.B. Vaughan, B.P. Kropp, G. Gabbiani,
M.D. Martin, C.J. Haaksma, and B. Hinz. 2006.
Contraction of myofibroblasts in granulation tissue
is dependent on Rho/Rho kinase/myosin light chain
phosphatase activity. Wound Repair Regen. 14: 313–
20.
Upcoming work – submitted
Pittet, P., B. Hinz. et al.: Formation of OB-cadherin-type
junctions establishes mechano-resistant coupling
between myofibroblasts.
Wipff, P.-J., B. Hinz. et al.: Myofibroblast contraction
activates TGF-a1 from extracellular matrix depots in
a stress-controlled autocrine loop.
Garonna, A., B. Hinz. et al.: Novel silicone culture
substrates allow conjunct analysis of cell forces,
protein localization and expression: a new wrinkle in
an old face.