A model for estimating traction force magnitude reveals differential regulation of actomyosin activity and matrix adhesion number in response to smooth muscle cell spreading

Decreased aortic compliance is associated with ageing and vascular disease, including atherosclerosis and hypertension. Ultimately, changes in aortic compliance are driven by altered ECM composition however, recent findings have identified a cellular component to decreased aortic compliance observed in ageing and hypertension. Vascular smooth muscle cells (VSMCs) line the blood vessel wall and VSMC contraction regulates vascular tone and contributes to aortic compliance. Mechanical cues derived from the ECM influence VSMC function, yet whether ECM rigidity influences VSMC force generation remains unclear. In this study, we describe the relationship between VSMC spreading, traction force magnitude and matrix rigidity. Importantly, we show that spreading predicts integrated traction force (integrated-TF) magnitude independently of matrix rigidity. Using linear regression analysis, we have generated a model for calculating integrated-TF from VSMC area. This model closely predicts the integrated traction force measured by live VSMC traction force microscopy. Vinculin staining analysis revealed that spreading strongly correlated with adhesion number per VSMC, suggesting that increased VSMC integrated-TF was due to enhanced matrix anchor points. Further analysis revealed that calculated integrated-TF per adhesion was reduced by matrix rigidity, however, adhesion number/μm2 increased, resulting in the average integrated-TF/μm2 remaining unaltered. As a result, the integrated-TF/VSMC spreading relationship is independent of matrix rigidity. Therefore, our study has identified and validated a novel model to predict and understand the mechanisms influencing VSMC traction force magnitude.


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Running Head: VSMC spreading predicts traction force phenotype. This synthetic phenotype is prevalent in vascular disease, including 83 atherosclerosis and aneurysm (10,11). Phenotypic modulation is associated with 84 changes in VSMC contractile protein expression and morphology (12); however, we 85 still do not fully understand whether the ability of VSMCs to generate actomyosin 86 derived-force is altered by VSMC phenotypic modulation. 87 Microenvironment rigidity transmits 'outside-in' forces to VSMCs and this 88 process is dependent on adhesions that convey force between the ECM and 89 cytoskeleton (13-16). VSMCs and other cell types respond to outside-in signals by 90 exerting actomyosin based contractile forces on the matrix (inside-out forces) that 91 scale with ECM stiffness (13,15,16 Our study suggests that VSMC traction force is tightly regulated by a compensatory 113 mechanism involving differential actomyosin and adhesion regulation.

Polyacrylamide hydrogel preparation and Traction Force Microscopy
126 Hydrogels were prepared as described previously (25,26). A JPK Nanowizard-127 3 atomic force microscope was used to confirm hydrogel stiffness as described 128 previously (25). VSMCs were seeded onto 12kPa hydrogels containing 0.5µm red 129 fluorescent (580/605) FluoSpheres (Invitrogen). Imaging was performed using a Nikon 130 Eclipse Ti-E live cell imaging system to capture 20x magnification images before and 131 after cell lysis by the addition of 0.5% Tx-100. Drift was corrected using the ImageJ 132 StackReg plugin and traction force was calculated using the ImageJ plugin described Phalloidin was used to stain F-actin and DAPI was used to visualise cell nuclei. All 158 images were captured at 20x magnification using a Leica SP2 laser scanning confocal 159 microscope. Cell area and aspect ratio was measured using the ImageJ open source 160 analysis software (28). pMLC mean intensity and total intensity were measured using 161 the ROI function in ImageJ. Focal adhesion number and size were measured in 162 ImageJ, as described previously (28).   The above data shows that matrix rigidity enhances both VSMC spreading and 197 integrated traction force magnitude. VSMC spreading has previously been reported to 198 correlate with traction force magnitude on soft 1kPa hydrogels (29). We next 199 speculated that matrix rigidity influences the relationship between VSMC spreading 200 and traction force magnitude. Analysis revealed that a moderate correlation between 201 VSMC spreading and integrated-TF magnitude existed on both 12kPa (R 2 = 0.4510) 202 and 72kPa (R 2 = 0.6415) hydrogels ( Figure 1D). Importantly, the relationship between 203 VSMC spreading and integrated-TF was not significantly altered by matrix rigidity, 204 suggesting that changes in traction force magnitude were driven by changes in VSMC 205 spreading.

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As the VSMC spreading/integrated-TF magnitude relationship was 207 independent of matrix rigidity, we hypothesised that integrated-TF could be calculated 208 from VSMC area. To test this idea, we combined the data from the 12kPa and 72kPa 209 hydrogels and calculated the gradient of the trend line ( Figure 1E). The formula 210 generated was used to calculate integrated-TF magnitude from the measured VSMC 211 area. As expected, the spread of calculated data closely resembled that of measured 212 integrated-TF magnitude ( Figures 1F and G). To test this hypothesis further, we used 213 an independent aortic VSMC isolate. TFM was performed to experimentally measure 214 VSMC traction force. The measured data confirmed that bead displacement was 215 reduced whereas integrated-TF magnitude was enhanced by matrix rigidity, 216 recapitulating the above findings (Figures 2A-C). Similar to our above findings, the 217 integrated-TF/spreading relationship for isolate-2 displayed no significant difference 218 between 12kPa (R 2 = 0.4818) and 72kPa (R 2 = 0.6325) ( Figure 2D). The estimated 219 integrated-TF was independently calculated from VSMC area, using the trend line 220 formula generated from isolate-1. Comparison of the measured and calculated 221 integrated-TF revealed no significant difference between the two data sets ( Figure 2E).

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Next, we sought to refine our model and compared the measured integrated-223 TF/spreading relationship between the two independent isolates. Both isolates 224 displayed a similar integrated-TF/spreading relationship ( Figure 2F), and as there was 225 no significant difference between the two isolates, we combined all the data and 226 calculated the gradient of the trend line ( Figure 2G). This combined formula was used 227 to calculate the integrated-TF of both isolates. Comparison revealed that the 228 measured and calculated integrated-TF, were in close agreement with no significant 229 difference between these data sets ( Figures 2H and I).

MLC phosphorylation strongly correlates with calculated integrated-TF
233 magnitude 234 To further confirm that VSMC spreading and calculated integrated-TF 235 correlated, we used an independent marker of actomyosin activity. We next performed 236 immunofluorescence microscopy to assay phosphorylated myosin light chain (pMLC) 237 levels in VSMCs grown on 12kPa and 72kPa hydrogels. Surprisingly, analysis 238 revealed that pMLC mean intensity was reduced on 72kPa compared to 12kPa 239 hydrogels, however, the total pMLC intensity remained unchanged ( Figures 3A-C). were not significantly different between 12kPa and 72kPa hydrogels for the 246 relationship between total pMLC total intensity and VSMC spreading/calculated 247 integrated-TF magnitude. This suggests that total pMLC levels was also independent 248 of matrix stiffness. The above data shows that suggests that increased VSMC spreading was not 253 stimulating enhanced actomyosin-generated forces. We predicted a role for VSMC 254 matrix adhesions, so we performed IF analysis of VSMCs stained with an antibody 255 raised to the matrix-adhesion protein vinculin. Analysis revealed that VSMCs grown 256 on 72kPa hydrogels possessed an increased number of matrix adhesions and larger 257 matrix adhesions compared to VSMCs grown on 12kPa hydrogels ( Figure 4A-C). This 258 data shows that VSMC matrix adhesions are altered by matrix rigidity. Next, using 259 formula 2, the integrated-TF was calculated from the cell area data and confirmed that 260 cell area and calculated integrated-TF were enhanced by matrix rigidity 261 (Supplementary Figure 3 and Figure 4D). We next examined the relationship between 262 VSMC spreading/calculated integrated-TF magnitude and vinculin organisation. (30). Matrix rigidity has been shown to promote VSMC proliferation, suggesting that 283 microenvironment rigidity influences VSMC phenotype (30). Matrix rigidity is also 284 known to influence VSMC morphology and traction force magnitude however, the 285 mechanisms driving these processes remain to be defined (31, 32). In agreement with 286 previous findings using immature embryonic aortic VSMCs, we show that VSMC 287 traction force magnitude and spreading are enhanced by matrix rigidity. However, this 288 previous study used hydrogels between 10kPa and 25kPa (32). These rigidities are 289 within the normal range of adult aortic rigidity (4). Our study is the first to examine this 290 phenomenon in mature adult VSMCs at a rigidity more akin to disease. In other cell 291 types, matrix rigidity enhances the relationship between traction force magnitude and 292 cell spreading (33). However, we show that this relationship in adult VSMCs is 293 independent of matrix rigidity in two independent VSMC isolates. Furthermore, we 294 have generated a model for estimating integrated-TF magnitude from VSMC 295 spreading data. TFM is a time consuming and specialised technique that can only be 296 performed on live cells. We have used our model to estimate integrated-TF magnitude 297 using fixed VSMC data. Importantly, we show that in a population of smooth muscle 298 cells there is no significant difference in between measured and calculated integrated-299 TF.

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Although increased VSMC spreading stimulated integrated-TF magnitude, 301 TF/µm 2 remained unaltered by both matrix rigidity and spreading. This suggests that 302 actomyosin-generated force that is transferred to the ECM by VSMCs is unaffected by 303 spreading. However, pMLC analysis revealed that matrix rigidity reduces pMLC levels, between VSMCs and the ECM. As a result, the integrated-TF/VSMC spreading 317 relationship is matrix rigidity independent. This suggests that the force/µm 2 exerted by 318 VSMCs on the ECM is tightly regulated. We propose that matrix rigidity triggers a 319 compensatory mechanism to preserve force/µm 2 that potentially protects VSMCs from    The raw/processed data required to reproduce these findings cannot be shared at this 370 time due to technical or time limitations. 371 372