To achieve this a custom-made heater had to be made to use on the AFM. ... ring-heater and 1.9 as the ideal position using the electro slider. For more details on.
12 Force mapping of Vascular Smooth Muscle Cells with Atomic Force Microscopy: influence of norepinephrine on local stiffness distribution
Internship report BMTE04.30
Institution: Professor at Osaka University: Supervisor at Osaka University: Professor at home university: Supervisor at home university:
Osaka University, Japan Graduate school of engineering science K. Hayashi, PhD H. Miyazaki, PhD F.P.T. Baaijens, PhD C.V.C. Bouten, PhD
Robert van Lith Department of Biomedical Engineering Division of Soft Tissue Biomechanics & Engineering Eindhoven University of Technology
Eindhoven, March 2004
Abstract Mechanical properties of vascular smooth muscle cells (VSMCs) obtained from a rabbit thoracic aorta were studied using the force mapping technique available on an atomic force microscope (AFM). Correlation of higher stiffness structures with fibrous protrusions apparent in topographical images was evaluated and the influence of adding a contractile agent to the cells was investigated. Furthermore, parameter b and c were used as main indices of mechanical properties, being the rate of stiffness change and initial stiffness, respectively. VSMCs showed a stiffness at the highest point of the cell close to that of endothelial cells in the medial wall of aortic bifurcations, being about 1.2 nN/µm, but parameter b was much lower than in endothelial cells. Moreover, the forceindentation curves of the VSMCs showed a wider scattering pattern. Parameter c seemed to decrease after addition of norepinephrine (NE), while parameter b remained constant. Also, the distribution of both parameters appeared more homogenous. This may indicate a more deformable state and thus a lower stiffness to facilitate contraction, but an unequivocal conclusion could not be agreed upon, due also to the low number of samples. The results however, encourage the progress of experiments in this area to further elucidate this remarkable behaviour.
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Index INDEX ..................................................................................................................................................1 INTRODUCTION .................................................................................................................................2 Outline of study ...............................................................................................................................2
MATERIALS AND METHODS ..........................................................................................................4 Isolation of cells ..............................................................................................................................4 Experimental setup .........................................................................................................................4 Atomic Force Microscopy ................................................................................................................6 Force volume imaging .....................................................................................................................6 Data analysis of the force-deflection curves .................................................................................7 Cytoskeletal observation ................................................................................................................8
RESULTS ............................................................................................................................................9 Fibrous protrusions .........................................................................................................................9 Contracted versus normal cells ....................................................................................................10
DISCUSSION ...................................................................................................................................12 Fibrous protrusions vs. local stiffness .........................................................................................12 Force-indentation curves ..............................................................................................................12 Mechanical properties ..................................................................................................................13 Effect of norepinephrine ...............................................................................................................13
CONCLUSION ..................................................................................................................................14 REFERENCES ....................................................................................................................................15 APPENDIX I ......................................................................................................................................17 Extraction of a piece of the thoracic aorta ...................................................................................17 Materials .........................................................................................................................17 Aorta Extraction Protocol ...............................................................................................17
APPENDIX II ....................................................................................................................................18 Enzymatic digestion ......................................................................................................................18 Materials .........................................................................................................................18 Enzymatic Digestion Protocol ........................................................................................18 Protocol for making HBSS(E) ........................................................................................18
APPENDIX III ...................................................................................................................................19 Immunofluorescence microscopy ................................................................................................19 Materials .........................................................................................................................19 Staining Protocol ............................................................................................................19
APPENDIX IV ...................................................................................................................................20 Custom-made culture dish ...........................................................................................................20 Dish preparation .............................................................................................................20
APPENDIX V ....................................................................................................................................21 Heater Fabrication .........................................................................................................................21 Attempt 1 ........................................................................................................................21 Outcome .........................................................................................................................21 Attempt 2 ........................................................................................................................21 Outcome .........................................................................................................................21 Attempt 3 ........................................................................................................................21 Outcome .........................................................................................................................21 Attempt 4 ........................................................................................................................22 Outcome .........................................................................................................................22 Attempt 5 ........................................................................................................................22 Outcome .........................................................................................................................22
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Introduction Recently it has become known that the mechanical properties of vascular smooth muscle cells (VSMCs) are closely related to their physiological function within the arterial wall (Ingber et al., 1995; Wang, 1998). The dynamical changes of structure, morphology and orientation in response to mechanical stimuli are related to cellular functions and are reflected by cellular mechanics (Birukov et al., 1995; Dartsch et al., 1986; Gong et al., 2003; Jakkaraju et al., 2003). Therefore it is important to study the mechanical properties to fully understand the underlying principles of arterial wall behaviour. Also, since current insights are such that underlying cell structures like the cytoskeleton, with in particular actin filaments and stress fibers, determine the shape of cells and their mechanical properties (Goldmann, 2000; Sato et al., 1996; Trickey et al., 2004), the exact relationship between cytoskeletal elements and the cellular properties need to be investigated to further elucidate the underlying mechanisms of atherogenesis. The atomic force microscope (AFM) (Binnig, 1986) has recently evolved from an imaging device towards a more elaborate function, since now it has become clear that qualitative measurements of local elasticity are possible (Tao et al., 1992; Weisenhorn et al., 1993). Radmacher was the first to introduce the term Force Mapping for making two-dimensional maps of the sample’s elasticity by recording force curves while the AFM is raster scanned across the sample (Radmacher et al., 1994). These force curves can be used to calculate local elasticities by using specific models to fit the force curves, for example the Hertz model (Hertz, 1882). Many researchers has already used this technique on endothelial cells (Miyazaki et al., 1999), fibroblasts (Haga et al., 2000; Kawabata et al., 2001; Rotsch et al., 2000), osteoblasts (Domke et al., 2000), platelets (Radmacher et al., 1996), airway smooth muscle cells (An et al., 2002), epithelial cells (Bhadriraju et al., 2002) or bacteria (Schaer-Zammaretti et al., 2003), focussing on changes in stiffness during migration, drug-induced changes or positional differences. However, to our knowledge, no study has ever investigated the local elastic properties of VSMCs and correlation with cytoskeletal elements, let alone tried to identify changes in local elasticity of VSMCs when triggered to contract. The networked structures underlying the plasma membrane, of which actin presumably is one, are at present considered to account for the resistance against the force exerted by the cantilever tip, the apical stress fibers serving as elements that strengthen the cellular architecture. In this present study therefore a distinction will be made between the local stiffness distribution in VSMCs in a contracted state and cells that are not, as measured by the AFM, since surveying the behaviour of actin filaments as well as cell stiffness under various conditions in particular is the main goal. Furthermore the correlation between elastic properties of VSMCs and one of their cytoskeletal main structures, the actin filaments, is studied by using the AFM to obtain maps of the spatial distribution of elasticity and using the confocal laser scanning microscope (CLSM) to identify actin filaments in the cell by using immunofluorescence microscopy.
Outline of study After isolation of VSMCs following extraction, cells are seeded on custom-made dishes and cultured in Dulbecco’s modified eagle’s medium (DMEM) for 3-7 days. Then, after a visual check that cells still contained the contractile phenotype, since phenotypic modulation involves structural changes in cytoskeletal organization (Worth et al., 2001), force mapping experiments are conducted on the cells. Doing this, first of all a good image of the region of interest, which is the nuclear and perinuclear area, is made in contact mode and secondly images are made by using
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the force volume mode of the AFM, as defined by the manufacturer. In this mode, the sample is raster-scanned over the surface while obtaining force-deflection curves at every pixel, resulting in a map of local mechanical properties after extracting relevant parameters from the force-deflection curves. Both normal VSMCs and contracted VSMCs (achieved by adding norepinephrine (NE)) are investigated. When AFM experiments have finished, the cells are fixed and specimens are prepared for CLSM experiments. Again the region of interest, found by using a grid on the bottom side of the dish, is subject of the experiment.
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Materials and Methods Isolation of cells Vascular smooth muscle cells from the proximal part of the thoracic aorta of matured female Japanese white rabbits were obtained by the enzymatic digestion method (Appendix II) after aseptical extraction (Appendix I). In brief, a proximal part of about 20 mm length was extracted from the thoracic aorta and stored temporarily in a HBSS (LC). Subsequently, the aortic piece was cut opened lengthwise, after which the endothelial cells were removed by gently scraping with a spatula. Then, the medial layer was separated from the adventitia at the intimal side by carefully peeling it off with a pair of forceps. As a final step in the procedure, the medial pieces were cut into small rectangular pieces of about 1 mm length with a pair of surgical blades. To isolate the contractile VSMC, the pieces were transferred to a flask together with 5 ml of HBSS(+), modified by adding trypsin solution, 10 mg collagenase (Sigma) and 7.87 mg elastase (Worthington), and stored in a thermostatic bath at 37° C while shaken at a frequency of 2 Hz for the period of 90 minutes. After 45 minutes gentle pipetting was performed for 30 seconds. The isolated VSMCs were cultured (Chamley-Campbell et al., 1979) on collagencoated coverslips in several custom-made culture dishes (Appendix IV) for 3-7 days, depending on the adhesion behaviour of the cells. Before AFM imaging could take place, the medium was removed, the dish was rinsed with HBSS three times to remove death, floating cells and finally 5 ml of HBSS was added.
Experimental setup Since the object of these experiments involves investigating the differences in mechanical properties between VSMC in passive and active states (i.e. normal and contracted state with contractile phenotype), physiological conditions should be approached as far as possible. In this case it means that the temperature of the medium in which the cells are emerged has to be kept at a temperature of about 36.5° Celsius (as in an incubator), to facilitate contraction of the VSMCs and to increase the life-span of the cells, of which the latter is desirable because of the time-consuming experiments on the AFM. To achieve this a custom-made heater had to be made to use on the AFM. First attempts involved using only a NiCr-wire attached to the bottom surface of the AFM-stage with silicon-glue, through which a current was induced. However, this proved to heat the stage insufficiently. Using an Aluminium ring (Al-ring) that fit exactly around a dish, with a NiCr-wire incorporated, made a second attempt. This method was abandoned because of a discontinuous heat-distribution within the dish. Lastly a combination was sought which showed to be sufficient. The NiCr-heater at the bottom surface of the stage is used first to elevate the temperature of the stage. By using an electro slider, a relatively large amount of current can be sent through the wire. A glass-stage, made from several microscopic glass plates, is placed on top of the stage. A more continuous heat distribution is hereby warranted since the whole bottom surface of the dish is heated (instead of part of the dish unheated, i.e. the centre part). Finally, the Al-ring is used for the final temperature elevation to be sufficient. Testing of the system for deciding on the right amount of current needed through the wire yielded values of 1.2 ampere for the glass-heater, 0.6 ampere for the ring-heater and 1.9 as the ideal position using the electro slider. For more details on the process of heater fabrication see appendix V. The reliability of the designed heater system was checked in the following way: on 5 points in the dish, a thermocouple was used to monitor the temperature distribution in the dish for a period of 1 hour, after allowing the heater to stabilize for about 1.5
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hours. Within the hour of measuring, no significant change in temperature was observed as well as a maximum temperature gradient within the dish of about 2 degrees, indicating a sufficient functioning of the heater system (data not shown). Every 30 minutes the medium in the dish will be replaced to prevent the build-up of salts and minerals in the dish due to evaporation of the medium. Trial experiments on adding NE brought to light some practical difficulties of exchanging the solution inside the dish without touching and/or moving the dish. This led eventually to the following experimental setup:
Figure 1: Experimental setup for AFM experiments on living cells that allows for adding NE or other vaso-active agents and replacing medium without moving the culture dish. Top image reflects the complete setup while the bottom image shows a close-up.
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The micromanipulators allow us to lower the bent micropipette very precisely to the bottom of the dish. In this way, the pipette can be placed slightly above the dish’s surface. A three-way valve connected to two syringes makes it possible to exchange medium without actually touching the setup.
Atomic Force Microscopy A commercial AFM having a xy scan range of 100 micrometer and a z scan range of 6 micrometer, type Bioscope (Veeco, Santa Barbara, CA) was used for the present study. The Bioscope consists basically out of an AFM mounted on an inverted optical microscope that facilitates the search for regions of interest. The used cantilevers were silicon-nitride and V-shaped with a force constant of 0.06 N/m (manufacturer’s value). Cells were scanned first at a scan range of 50-100 micrometer in contact mode, while not exceeding a contact force of 1 nN for prevention of cell scraping, to obtain a clear overview, necessary for detecting a relevant region of interest. After adjusting the AFM for acquiring a decent height image, an area of 30x30 micrometer was scanned in contact mode, zoomed in on the nuclear and perinuclear regions (see figure 2). A height image was typically taken with frequency of 0.2-0.5 Hz and with a lateral resolution of 256 lines per image. Figure 2: zooming in on the region of interest, the nuclear and perinuclear area, to only a surface of 30 x 30 micrometer
Force volume imaging Force volume imaging, also called force mapping (Radmacher et al., 1996), involves raster scanning a surface while collecting force-deflection curves on every point in a two-dimensional scan (x-y). After completion of a force map, one will have obtained data, providing the opportunity to flip through maps of the cell to observe the force the cantilever was sensing corresponding to each particular height. Hence the term ‘force volume’, as used by Veeco company. It was typically performed with a frequency of 10 Hz at a resolution of 64x64 pixels with 64 data points for each curve, again on a 30x30 micrometer area. Z scan size was held at 2 micrometer and the relative trigger mode was used with a threshold of 100 nm. When using cells in a contracted state, adaptations had to be made to the protocol. A typical experimental session on a single cell (i.e. overview height image, zoomed height image and a force volume image) takes about 40 minutes, while a VSMC can only keep its contracted state for a maximum time span of 15 minutes. It was decided that after obtaining an overview height image, the zoomed in image would be taken of a smaller area, namely 15x15 micrometer. When the force-volume image of that same area was taken, the HBSS was replaced by HBSS with 10-5 M norephinephrine (NE) and another force-volume image was taken after allowing the cells to contract for 5 minutes. By abandoning the overview image and the zoomed in image after adding NE a slight drawback in control was accepted. Also, instead of 64x64 pixels, only 32x32 pixels were used, thereby decreasing the amount of time needed for a complete force-volume image with 75 %. Because of the parallel reduction of the scan size to 15x15 micrometer, still the same resolution as in the earlier experiments is achieved. The amount of time needed for a force-volume
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image is reduced by the above-mentioned alterations to only 5-8 minutes. The extra time span between the end of scanning and the end of contraction was desirable for the procedure of executing the fixing- and staining-protocol.
Data analysis of the force-deflection curves The slope of a force-deflection curve is already an indicator for the stiffness of a sample. The steeper it is, the stiffer the sample. An infinitely stiff sample will yield a deflection/force value of unity (deflection d= movement of piezo in z direction), the force being linear related to the deflection according to the relation F=kd with k the spring constant of the cantilever. In the case of a soft sample like a VSMC however, the cantilever will indent the surface, leading to a deflection that has a smaller value than the movement of the piezo (d=z-indentation δ). The movement difference of the measured value on a VSMC at a certain deflection value compared to the one on a infinitely stiff substrate (like glass) is equal to the indentation of the cell (Weisenhorn et al., 1993).
Figure 3: Diagram of two forcedistance curves of a soft and a hard material. The deflection d is proportional to the applied force F for undeformed samples: F=kd (k is the spring constant). The straight falling line represents a hard sample, where the distance move downward is equal to the deflection of the cantilever. Here, the applied force is: F=kzAD. If the sample is soft, however, the deflection is less because of indentation of the sample. The indentation is equal to zAB and thus a force-indentation graph can be calculated. Courtesy of Weisenhorn (Weisenhorn et al 1993)
Thus a force-indentation curve can be calculated (see fig.3), with which the mechanical properties of the VSMC can be evaluated (for a more detailed description of force-curve analysis see (Domke et al., 1998)) by fitting the force-indentation curve to the following relation, as described previously by Miyazaki et al. (Miyazaki et al., 1999), by using a Levenberg-Marquardt algorithm:
F = a(e b⋅δ − 1)
(1)
With a and b being constants.
dF = b⋅F + c dδ
(2)
Is the expression for the slope of the force-indentation curve. In this study, focus was laid on the parameters b and c (=ab), depicting rate of modulus change and initial stiffness, respectively. Parameter b is related to the structural inhomogeneity apparent in cells and its change, as induced by stress. Parameter a is related to the locational change of inhomogeneity inside a cell, but the latter will not be subject of interest in the present study.
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The widely used Hertz model for fitting the force-indentation curves (Haga et al., 2000; Rotsch et al., 2000; Wojcikiewicz et al., 2004) was not used, since the assumption of the investigated material being a homogenous one is not met. Clearly, the cytoskeletal elements inside the cell have very different stiffnesses from the rest of the cell.
Cytoskeletal observation Right after the AFM-experiments, the cells are fixed by adding 3.7 % formaldehyde solution to the dish. After this, subsequent addition of 0.1 % Triton X-100 to permeabilize the cell’s membrane and FITC-phalloidin was performed and the cells were left to incubate for 20 minutes. Between every two steps, the cells were rinsed 3 times with PBS. After having added slowfadeTM A and C (Leiden, Netherlands), the middle part of the dish is covered with a coverslip and by using paraffin the two coverslips are sealed together. Lastly the center part of the dish is cut with a custommade tool. After this the actin elements can be observed by means of a Confocal Laser Scanning Microscope (CLSM) combined with an inverted microscope. For more information on the exact p[rocedure see Appendix III. With the CLSM 1000 slices are examined, with which a 3-dimensional image can be reconstructed.
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Results Fibrous protrusions First of all, images taken in force volume mode right away indicated that the fibrous protrusions, as observed in height and deflections images of cells, represent much stiffer elements inside the cell. This is confirmed by the colour maps of the distribution of the two main parameters b and c, the rate of stiffness change and initial stiffness, respectively (see fig. 4). Clearly visible is that especially the parameter c has a strong tendency towards reflecting the fibrous protrusions. Parameter b however, also seems to coincide with these fiber like elements. These results are similar to results as obtained by Rotsch and Nagayama (Nagayama et al., 2001; Rotsch et al., 2000). They observed in fibroblasts that the fibrous structures are in fact stiffer elements in the elasticity maps.
Figure 4: Topographical images (A: height and B: deflection) of a VSMC with corresponding elasticity map (C) and distribution of parameter b (D) and parameter c (E). Clearly visible is the correlation between fibrous protrusions in the topographical images and stiffer fiber-like elements in the elasticity map. Indicated by the white arrows is a portion that shows up in the topographical images (deflection) as causing less deflection and in the elasticity maps as softer (E) but also as having a lower value for parameter b (D).
The highest point of each cell, as selected after visual inspection, was used for examination of the force-indentation curves. The highest points of uncontracted cells yielded a fairly wide variety of force-indentation curves (see fig. 5), what could indicate a certain variability in cellular stiffness, even when the cells are harvested from identical locations in close proximity of one another.
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Figure 5: Averaged force-indentation curves of VSMCs obtained from the highest point of the cells.
Contracted versus normal cells Cells that were selected for contraction experiments were allowed to react to the NE for 5 minutes. Then, the experiments were being done as described earlier. Again, the highest point was selected (white arrow in fig. 6) and nine pixels around that point were used to average the force-indentation curves. These averaged highest-point curves were used from each cell to derive the averaged force-indentation curves for uncontracted and contracted cells. A group of eight cells that were only used for experiments without contraction served as controls. After cells where administered NE, parameter maps of b and c show a parameter distribution that appears to become more homogenous over the whole surface, as well as an apparent marked decrease in initial stiffness around the nuclear region.
Fig. 6: Parameter distributions of a typical VSMC while still uncontracted and in a contracted state. Left parameter b in the two states and on the right side parameter c, being the rate of stiffness change and initial stiffness, respectively. The white arrow indicates the highest point of the cell, which clearly suffers from a decreasing initial stiffness when contracted.
The averaged force-indentation curve became less steep (see fig. 7), again indicating a decrease in stiffness. The averaged curve from the control group show that the cells used in the NE-experiment expressed a behaviour that was initially similar to the control group. The value of parameters b and c did not change significantly after addition of NE (fig.7). However, the parameter c seems to have an inclination to decrease, as would be expected by the obtained force-indentation curve. The low number of samples prevents us from drawing any relevant conclusions on this matter, though.
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Figure 7: The upper graph depicts averaged force-indentation curves from the highest points of cells in an uncontracted state (green, n=4) and of the same cells after addition of norephinephrine (mint).As a control group data is used from VSMCs that were not used for contractionexperiments (blue, n=8). Curves were first averaged over several pixels at the highest point of each cell and then averaged for each cell. Control group and uncontracted cells show remarkable similarity, indicating identical mechanical properties, hence justifying comparing data of uncontracted cells with all uncontracted cells. The lower graph shows the averaged value of parameters b and c at the highest point of the cell. As already indicated by the upper graph, parameter c tends to decrease under the influence of norepinephrine.
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Discussion Elasticity measurements have been implemented in cellular research in the last couple of years to address mechanical properties of cells in combination with their cytoskeletal structure, which presumably accounts for motility, morphology and structural integrity of living cells (among others). Especially the tensegrity model as originally proposed by Ingber in 1993 (Ingber, 1993) is a conceptual model on correlation between dynamic behaviour of cytoskeletal elements and elasticity of cells that serves as the underlying principle for these research interests (Costa et al., 2002; Heidemann et al., 1999; Wang et al., 2004). Several groups have made efforts in this area to clarify the properties of particular cells with respect to (local) elasticity, of which especially fibroblasts have proven to be a popular cell type. They have been the object of many studies on elasticity, for example to investigate the influence of druginduced changes in internal structure (Rotsch et al., 2000), local stiffness change during migration (Nagayama et al., 2001) and correlation of local stiffness with internal structures (Haga et al., 2000). Also endothelial cells (Miyazaki et al., 1999) and osteoblasts (Domke et al., 2000) have been used for experiments with the AFM on local stiffness. Smooth muscle cells however, have been far less used for similar experiments. Human airway smooth muscle cells (HASM) have been investigated on stiffness and prestress correlation with a twisting cytometry technique (Wang et al., 2002), whereas VSMCs have been studied on tensile properties (Matsumoto et al., 2000; Miyazaki et al., 2003). To our knowledge though, up till now no studies have been published on local stiffness of VSMCs by using the force volume mode of the AFM, and in particular not on stiffness changes caused by contraction of the cell.
Fibrous protrusions vs. local stiffness The present study shows again that fibrous structures as observed by topography images can be clearly attributed to stress fibres, namely actin filaments. This observation was consistent with ones made previously by Rotsch (Rotsch et al., 2000), Kawabata (Kawabata et al., 2001) and Haga (Haga et al., 2000). Moreover, parameters b and c both show a strong correspondence with the fibre like elements. Hence it can be concluded that these elements have a strong influence on local stiffness of living cells, similar to results obtained by Bhadriraju (Bhadriraju et al., 2002). Immunofluorescence experiments were difficult to perform and only once fibres could be observed. This was probably caused by an insufficient ability of the staining agent to enter the cells. Possibly the use of saponin instead of Triton-X in the future to permeabilize the cells will be an improvement. Future studies to correlate actin (re)organization with the cells parameters are taken in consideration.
Force-indentation curves The force-indentation curves show a fairly wide scattering among cells, even when observing averaged curves from highest area of the cells. This scattering was wider than the one observed by Miyazaki (Miyazaki et al., 1999) on endothelial cells. Although it might be due to a natural variability among VSMCs that is not present in endothelial cells to such an extent, the reason for this remains unclear. Another possibility could be an imprecise determination of the exact point of maximum height in the cells. More research on VSMCs is desirable though to establish a true insight in this scattering phenomenon.
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Mechanical properties The stiffness as expressed by the parameter c was about 1.2 nN/µm, a stiffness close to the one of endothelial cells in the medial wall observed by Miyazaki in 1999. In a more recent publication by the same Miyazaki however, the stiffness of contractile VSMCs was shown to be 170 nN/µm, differing a factor 100 from our results. Their method however was based on tensile testing in the direction of the fibre like elements, while force mapping can be looked upon as testing in the perpendicular direction to that respect, which would explain the factor difference. Parameter b however, is about 1 µm-1, a much lower value than in endothelial cells, which was 3-4 µm-1, indicating the presence of a significant difference in mechanical characteristics between the two cell types.
Effect of norepinephrine As mentioned before, no studies have been done using the force mapping technique on evaluating stiffness changes in VSMCs caused by contraction. Wang (Wang et al., 2002) performed a study in which they investigated the stiffness change with oscillating magnetic twisting cytometry after addition of histamine. This contractile agonist clearly caused an increase in stiffness. Karl (Karl et al., 1998) also reported an increase in elasticity upon cell contraction, however mainly in the cell periphery. In our study we could observe an opposite effect, which is also contrary to our hypothesis. Although this could be caused by the difference in cell type used, also the different technique used in determining cellular stiffness could be of influence. The latter opinion is strengthened by the results obtained by Nagayama (Nagayama et al., 2001). They observed a drastic decrease in stiffness during migration of a living fibroblast, when the cell should be in a contracted state to facilitate movement. The small number of specimens used in the present study forces us to refrain from drawing conclusions on this matter. Also it should not be ruled out that the cells were not yet in a completely contracted state but still in the contracting phase, which may be a highly deformable reorganization phase, thereby being less stiff (Nagayama et al., 2001). This mechanism was proposed by Nagayama in a slightly dissimilar context, but seems appropriate in this case as well. The parameter b did not exhibit a tendency to change after addition of NE and since Miyazaki et al. (Miyazaki et al., 1999) experienced a parameter b whose value was independent of the location in the arterial wall, it is suggested that the parameter b depends only on cell type. However, exact physiology is yet unexplained. The more homogenous distribution of both parameters is not well understood and requires more research to elucidate A final comment should be made here about the custom-made heater unit that was used. Although the temperature distribution was sufficient, the influence of the small heat gradient was not investigated. Possibly small flowing patterns caused by this temperature gradient affect the cells and measurements. Furthermore, a heating unit that also regulates the concentrations of gases such as oxygen and carbon dioxide was not designed, but surely would be able to simulate the natural environment of the cells even more. A sealed box with flow possibilities was considered, but the sensitivity of the piezo to humidity made us abandon such a concept, together with the lack of belief in necessity of gas regulation. Clearly, some circumstances are still not ideal and therefore we encourage other groups to reconsider alternatives that are capable of regulating all the physiological factors.
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Conclusion Local stiffness of VSMCs was investigated by using a mechanical model instead of the Hertz model. Both the parameter b and c show a clear correlation with the distribution of fibre like elements that show up in the topographical images, being actin filaments. Also for the first time the effect of a contractile agonist on the two parameters of VSMCs delivered by this model was investigated by using the force mapping technique. The addition of NE brought about a more homogenous distribution of both parameters and an apparent decrease in initial stiffness as depicted by parameter c. More studies are needed however to verify these observations. Parameter b did not change after triggering contraction.
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Appendix I Extraction of a piece of the thoracic aorta Materials -surgical gloves -30 ml of HBSS(+) -surgical blade -chest opener -L-shaped tool -pair of forceps -4-0 sutures -5 ml HBSS(HC) -pair of scissors -pair of special scissors -cutter Aorta Extraction Protocol 1. Put on plastic gloves 2. Prepare Al-foil with tools besides rabbit and put 30 ml of HBSS(+) in 100 mm dish. 3. Clean rabbits chest with disinfectant and sacrifice rabbit with overdose of pentobarbital. 4. Put on surgical gloves and cut chest bone right above lowest rib bone. 5. Cut along ribs until enough space for the chest opener has been made and open chest, in the meanwhile cutting the attached fleeces slightly. 6. While using the L-tool to create more space, gently (!) remove connective tissue. 7. When aorta is freed, tie with sutures at both sides, finishing at the abdominal side. 8. Cut the aorta, again finishing at the abdominal side. 9. Wash the aortic segment in the HBSS(+) and transfer to the tube with HBSS(HC)
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Appendix II Enzymatic digestion Materials -pair of forceps -scissors -3x 100 mm dish -spatula -2x surgical blades -sieve -HBSS(LC) -25 ml flask -5 ml HBSS(E) -pipette -DMEM(+) -EDTA-trypsin solution -HBSS(+) -HBSS(-) -HBSS(HC) -collagenase -elastase -trypsin inhibitor Enzymatic Digestion Protocol 1. Open the aortic segment lengthwise in the bottom piece of a 100 mm dish with a small amount of HBSS(LC). 2. Remove endothelial cells by gently scraping with a spatula and wash segment twice in HBSS(LC). 3. Transfer segment to a top side of a 100 mm dish and detach intimal side media layer from the adventitia by using a pair of forceps. 4. Transfer medial piece(s) to the bottom part of a new 100 mm dish and wash three times in HBSS(LC). 5. Cut medial piece(s) in small rectangular pieces (app. 0.5 x 0.5 mm) with a pair of surgical blades in a top piece of the new 100 mm dish with a small amount of HBSS(LC). 6. Put the aortic pieces in a 25 ml flask with 5 ml of HBSS(E) and incubate them in a shaker at 37° Celsius at 2 Hz for 60 minutes (if necessary 30 minutes more). 7. Filter large tissue debris out of solution by pipetting through a 250 µm sieve into one or two 60 mm dishes. 8. Observe with microscope. Then transfer medium with cells to 15 ml tube and centrifuge at 800 rpm for 7 minutes at 4 Celsius. The remaining dish(es) are refilled with 4 ml DMEM(+) and placed in incubator. 9. After centrifuging, suspend cells in 8 ml of DMEM(+) and divide over two 60 mm dishes. Place in incubator after observing. Protocol for making HBSS(E) Add 10 mg collagenase Thand en and 7.87 mg of elastase (Worthington) to 5 ml of HBSS(LC) and 50 µl of Trypsin inhibitor. After shaking, the solution needs to be sterilized by using a syringe and syringe sterilizer.
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Appendix III Immunofluorescence microscopy Materials -PBS -0.1 % Triton-X 100 -3.7 % Formaldehyde -slow fade A + C -FITC-phalloidin -PBS+1 % DMSO -paraffin wax Staining Protocol 1. Wash VSMCs three times with 2 ml of PBS 2. Add 2 ml of PBS + 3.7 % formaldehyde and cells are left for 10 minutes at room temperature 3. Add 2 ml of PBS and leave cells for 5 minutes. Repeat this procedure three times. 4. Add 2 ml of PBS + 0.1 % Triton-X and leave cells 30 seconds at room temperature. 5. Repeat step 3. 6. Add 0.1 ml FITC-Phalloidin, adjusted with 110 µl PBS + 1 % DMSO to 12.5 mg/l and cells are left at room temperature for 20 minutes. Light is shielded from the dish with Al-foil and room is dimmed as much as possible. 7. Repeat step 3 8. Blot PBS with water-absorbing paper and put drop of slow-fade on cells (A + C, Leiden). 9. Cover cells with cover glass and seal with paraffin wax.
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Appendix IV Custom-made culture dish Dish preparation Especially for the purpose of these experiments, plastic culture dishes (60 mm, IWAKI) were modified to fit the needs. A circular part of about 20 mm diameter in the centre of the dish was removed with a custom-made heating apparatus, where after a cover glass (24x24 mm, Iwaki) was glued on the inside of the dish with silicon glue, with a grid placed on the bottom of the cover glass (used for retrieving identical cells on AFM and CLSM). After thorough cleaning and sterilization, the glass part was covered with a collagen coating of 200 microliter and sterilized in an EOG sterilizer and aerated to allow the remains of ethylene oxide, what could inhibit the adhesion of the cells to the dish, to disappear. Hereafter stored for later use
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Appendix V Heater Fabrication Attempt 1 On the bottom side of the large metal stage, a NiCr-wire was folded every centimetre and placed in a circular structure. Silicon sheets provided the interface between the two metals, together with silicon glue. Both ends of the wired where then connected with a voltage generator to supply the necessary current. Outcome Failure because of insufficient capacity Attempt 2 The second attempt was based on a circular heater placed around the dish. To achieve this, a tight-fit Aluminium-ring was made. On the outer side of the ring, a gutter was made of about half the width of the ring and half the height, in which the heating element could be placed. The heating element in this case was made of a NiCr-wire that was inserted in a silicon rubber tube and subsequently coiled. This coiled element was then placed in the gutter on the outer side of the ring and again connected to a voltage generator. Outcome Still insufficient capacity Discontinuous heat distribution
Figure A.1: first attempt for a heating unit. Black indicates the part were the NiCr-wire was attached. Upper images are side view, lower images are bottom view.
Attempt 3 To achieve enough heat dissipation to the dish, a small circular wire in the same fashion as attempt 1 was placed on the bottom side of the smaller metal AFM-stage, directly below the center of the dish. Outcome Enough capacity Very bad heat distribution
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Attempt 4 To achieve a homogenous distribution, the concept of a custom-made stage for the dish was thought of. A small glass stage, provided with NiCr heating wires from sideways should guarantee a heat distribution that is homogenous throughout the glass plate. Outcome Good distribution Insufficient capacity due to bad heat dissipation in glass Figure A2: Fourth attempt for a heating unit. Black indicates the three separate NiCr-wire parts. Side view.
Attempt 5 A combination of three separate heaters was sought to achieve a homogenous distribution as well as enough capacity. The Al-ring, the NiCr wires on the large stage and also the glass plate are all connected to separate voltage generators and can be adjusted when desirable. To achieve a sufficient heat supply an extra NiCr-wire was added to the bottom stage. Outcome Sufficient capacity Reasonable distribution Figure A3: Final setup.Upper image indicates side view, lower image depicts a bottom view.
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