These studies demonstrate that the ECM microenvironment of leiomyoma cells is characterized by increased mechanical stress. Here we extend the results of our previous study (Rogers et al., 2008
) to show that the viscoelastic properties of the ECM contributes substantially to the increased tissue stiffness of leiomyoma. Since the viscoelastic properties of the ECM are complex, it is possible that the interstitial fluid may alter the repulsive forces of the GAGs allowing them to collapse or inflate. Additional studies will be needed to discern how the complex ECM of leiomyoma and its molecular rearrangement contributes to the observed changes in viscoelasticity. Interestingly, in this environment characterized by increased stress, we noted that leiomyoma cells had an attenuated response to mechanical cues compared to myometrial cells as shown by: 1) reduced levels of active RhoA to acute strain; 2) failure to respond to cyclic stresses in a cell re-orientation assay; and 3) an attenuated response to substrates of varied stiffness. Leiomyoma cells did respond normally to LPA-mediated activation of RhoA, but only when the cells were cultured on a flexible substrate. Collectively, the flndings are consistent with the conclusion that mechanical signaling is attenuated in leiomyoma cells.
We noted a four-fold increase in both the pseudo-dynamic modulus and the peak strain in leiomyoma tissue relative to patient-matched myometrium (). Using a confined compression chamber with a porous paten, we observed a much higher modulus than in prior tests conducted on unconfined samples with a non-porous piston (Rogers et al., 2008
). This increased modulus is, in part, likely explained by the contribution of both the fluid phase and solid phase of the tissue. Not only does the rich fluid component of leiomyoma contribute to its bulk (Okuda et al., 2008
), but similar to articular cartilage (Cohen et al., 1998
; Ateshian et al., 2003
; Park et al., 2004
), the fluid phase contributes to the viscoelastic properties of fibroids, contributing to large interstitial pressurized forces. For example, after testing bovine cartilage in a confined compression chamber, Soltz and Ateshian (Soltz and Ateshian, 2000
) concluded that cartilage dynamic stiffness was derived primarily from flow-dependent viscoelasticity as predicted by the linear biphasic theory and that interstitial fluid pressurization is the fundamental mechanism of cartilage load support. Our findings support the notion that leiomyomata are tumors composed of large amounts of aberrant ECM (Malik et al., 2010
) and that cells within the tumor continue to grow and proliferate (Peddada et al., 2008
) while exposed to increased viscoelastic forces.
Changes in the mechanical properties of a tissue and the cellular microenvironment have been shown to contribute to tumor formation in other organ systems and in experimental models (Ingber, 2008
; Butcher et al., 2009
). The concept that changes in the cellular microenvironment could contribute to tumorigenesis were first suggested by experiments of Bischoff and Bryson (Bischoff and Bryson, 1964
) where tumor formation was observed after implanting a rigid piece of metal or plastic, as opposed to the same material as a powder. Alterations to the ECM structure also appear to play a central role in tumor formation and in the tumor cell’s ability to sense and respond to the altered physical environment (Weaver et al., 1997
; Paszek et al., 2005
; Ghosh et al., 2008
). The findings reported here, together with our previous data (Rogers et al., 2008
), suggest that the mechanical properties of leiomyoma are a key feature of these tumors, and may contribute to their growth, but further studies will be needed to assess whether growth of a specific leiomyoma is correlated to its stiffness. One limitation of the studies presented is that the viscoelastic properties of a tissue are complex, especially in a tissue containing ECM consisting of numerous proteins and glycoproteins all of which may contribute to mechanical behavior. In this report, we have focused on characterization of the differences between leiomyoma and uterine muscle, especially differences in Rho signaling based on our prior report, but a more detailed assessment of the rheological differences between the cells such as reported for other tissue types (Stamenović, 2008
) remains to be performed.
Notably, leiomyoma differ from other tumors in that some grow to several centimeters in size. Each uterine leiomyoma represents a monoclonal process, but within a single uterus different tumors arise from different cells, such that within a uterus multiple clones may be represented (Ligon and Morton, 2000
). Within one uterus some tumors may grow, while others may undergo a reduction in size (Peddada et al., 2008
). Recent reports of assessment of the elastic modulus in vivo (Stewart et al., 2011
) may represent a clinical application of our findings to assess the stiffness in vivo and explore a possible correlation with growth or senescence of an individual leiomyoma.
The establishment of a tumor microenvironment by leiomyoma cells characterized by increased viscoelastic forces begs the question: is mechanical signaling altered in leiomyoma cells? The results indicate that myometrial cells responded to perturbation of the extracellular mechanical stresses as expected; but by three different measures of mechanosensing, leiomyoma cells appeared to have an attenuated response relative to myometrial cells. Specifically, leiomyoma cells failed to reorient perpendicularly to the applied uniaxial strain direction, had an attenuated RhoA activation response to uniaxial strain, and showed a diminished ability to change morphology in response to altered substrate stiffness. In contrast to these three observations which suggest an impaired response to extracellular mechanical cues, on the extremely rigid polystyrene plates with an estimated stiffness of 2–4 GPa (Paszek et al., 2005
), leiomyoma cells demonstrated increased basal levels of active RhoA relative to myometrial cells. These observations could be considered contradictory. We interpret the increase in the basal
levels of RhoA on the polystyrene substrate may reflect prior adaptation of the leiomyoma cells to a very stiff microenvironment. However, with each dynamic
mechanical challenge, leiomyoma cells were not as adroit in their response, suggesting a fundamental alteration exists in communication between the external mechanical forces and the ability of the actin cytoskeleton to reorganize via RhoA. The findings suggest that mechanical signaling in leiomyoma cells is fundamentally altered, because in all 4 assays involving external mechanical cues, leiomyoma cells responded abnormally.
One plausible explanation for the seemingly contradictory results is that leiomyoma cells have become fundamentally adapted to their very stiff microenvironment, and are insensitive to more moderate and subtle mechanical cues. Stated differently, the cell response to mechanical stimulation could be down-regulated through feedback mechanisms, although the mechanisms responsible remain unknown. In support of this explanation, and contrary to the findings of Ghosh and colleagues (Ghosh et al., 2008
) for capillary endothelial cells, the fundamental alteration in leiomyoma cells was not ROCK-dependent, as demonstrated by the finding that leiomyoma cells pre-treated Y27632 prior to uniaxial straining remained largely unchanged (). In further support of this explanation, leiomyoma cells contain increased levels of the Rho-GEF AKAP13 (Rogers et al., 2008
), and knockdown of AKAP13 differentially affected leiomyoma cells, compared to myometrial cells (Owen et al., 2010
). Thus, the results are consistent with the notion that leiomyoma cells have undergone a specific adaptation to their stiff microenvironment that is not ROCK-dependent, is associated with increased levels of Rho-GEF, and this adaptation persists in tissue culture. Additional experiments will be needed to unravel the specific changes associated with the mechanotransduction response of leiomyoma cells.
In conclusion, these results reveal that the increased stiffness and elastic moduli demonstrated in leiomyomata is accompanied by an altered mechanosensory response characterized by attenuated levels of active RhoA. A further understanding of mechanotransduction as it relates to leiomyomata may explain why some leiomyoma grow and others do not, and could help to guide future treatments for this very prevalent pelvic tumor.