Recent studies have suggested that extracellular matrix rigidity regulates cancer invasiveness, including the formation of cellular invadopodial protrusions; however, the relevant mechanical range is unclear. Here, we used a combined analysis of tissue-derived model basement membrane (BM) and stromal matrices and synthetic materials to understand how substrate rigidity regulates invadopodia. Urinary bladder matrix-BM (UBM-BM) was found to be a rigid material with elastic moduli of 3-8 MPa, as measured by atomic force microscopy and low-strain tensile testing. Stromal elastic moduli were ∼6-fold lower, indicating a more compliant material. Using synthetic substrates that span kPa-GPa moduli, we found a peak of invadopodia-associated extracellular matrix degradation centered around 30 kPa, which also corresponded to a peak in invadopodia/cell. Surprisingly, we observed another peak in invadopodia numbers at 2 GPa as well as gene expression changes that indicate cellular sensing of very high moduli. Based on the measured elastic moduli of model stroma and BM, we expected to find more invadopodia formation on the stroma, and this was verified on the stromal versus BM side of UBM-BM. These data suggest that cells can sense a wide range of rigidities, up into the GPa range. Furthermore, there is an optimal rigidity range for invadopodia activity that may be limited by BM rigidity.

Objective Dystrophic calcific nodule formation in vitro involves differentiation of aortic valve interstitial cells (AVICs) into a myofibroblast phenotype. Interestingly, inhibition of the kinase MEK1/2 prevents calcific nodule formation despite leading to myofibroblast activation of AVICs, indicating the presence of an additional mechanotransductive component required for calcific nodule morphogenesis. In this study, we assess the role of TGF-β1-induced cadherin-11 expression in calcific nodule formation. Methods and Results As shown previously, porcine AVICs treated with TGF-β1 prior to cyclic strain exhibit increased myofibroblast activation and significant calcific nodule formation. In addition to an increase in contractile myofibroblast markers, TGF-β1 treated AVICs exhibit significantly increased expression of cadherin-11. This expression is inhibited by the addition of U0126, a specific MEK1/2 inhibitor. The role of increased cadherin-11 is revealed through a wound assay, which demonstrates increased intercellular tension in TGF-β1 treated AVICs possessing cadherin-11. Furthermore, when siRNA is used to knockdown cadherin-11, calcific nodule formation is abrogated, indicating that robust cell-cell connections are necessary in generating tension for calcific nodule morphogenesis. Finally, we demonstrate enrichment of cadherin-11 in human calcified leaflets. Conclusions These results indicate the necessity of cadherin-11 for dystrophic calcific nodule formation, which proceeds through an Erk1/2 dependent pathway.

The role of cancer-associated fibroblasts (CAFs) as regulators of tumor progression, specifically vascular growth, has only recently been described. CAFs are thought to be more mechanically active but how this trait may alter the tumor microenvironment is poorly understood. We hypothesized that enhanced mechanical activity of CAFs, as regulated by the Rho/ROCK pathway, contributes to increased blood vessel growth. Using a 3D in vitro tissue model of vasculogenesis, we observed increased vascularization in the presence of breast cancer CAFs compared to normal breast fibroblasts. Further studies indicated this phenomenon was not simply a result of enhanced soluble signaling factors, including vascular endothelial growth factor (VEGF), and that CAFs generated significantly larger deformations in 3D gels compared to normal fibroblasts. Inhibition of the mechanotransductive pathways abrogated the ability of CAFs to deform the matrix and suppressed vascularization. Finally, utilizing magnetic microbeads to mechanically stimulate mechanically-inhibited CAFs showed partial rescue of vascularization. Our studies demonstrate enhanced mechanical activity of CAFs may play a crucial and previously unappreciated role in the formation of tumor-associated vasculature which could possibly offer potential novel targets in future anti-cancer therapies.

Although artificial prostheses for diseased heart valves have been around for several decades, viable heart valve replacements have yet to be developed due to their complicated nature. The majority of research in heart valve replacement technology seeks to improve decellularization techniques for porcine valves or bovine pericardium as an effort to improve current clinically used valves. The drawback of clinically used valves is that they are nonviable and thus do not grow or remodel once implanted inside patients. This is particularly detrimental for pediatric patients, who will likely need several reoperations over the course of their lifetimes to implant larger valves as the patient grows. Due to this limitation, additional biomaterials, both synthetic and natural in origin, are also being investigated as novel scaffolds for tissue engineered heart valves, specifically for the pediatric population. Here, we provide a brief overview of valves in clinical use as well as of the materials being investigated as novel tissue engineered heart valve scaffolds. Additionally, we focus on natural-based biomaterials for promoting cell behavior that is indicative of the developmental biology process that occurs in the formation of heart valves in utero, such as epithelial-to-mesenchymal transition or transformation (EMT). By engineering materials that promote native developmental biology cues and signaling, while also providing mechanical integrity once implanted, a viable tissue engineered heart valve may one day be realized. A viable tissue engineered heart valve, capable of growing and remodeling actively inside a patient, could reduce risks and complications associated with current valve replacement options and improve overall quality of life in the thousands of patients who received such valves each year, particularly for children.

Objective Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder, and controversy exists as to whether it is primarily a dystrophic or osteogenic process in vivo. In this study, we sought to clarify the mechanism of CAVD by assessing a genetic mutation, Notch1 heterozygosity, which leads to CAVD with 100% penetrance in humans. Approach and Results Murine immortalized Notch1+/− aortic valve interstitial cells (AVICs) were isolated and expanded in vitro. Molecular signaling of wild type (WT) and Notch1+/− AVICs were compared to identify changes in pathways that have been linked to CAVD – TGFβ/BMP, MAPK and PI3K/Akt – and assessed for calcification potential. Additionally, AVIC mechanobiology was studied in a physiologically relevant, dynamic mechanical environment (10% cyclic strain) to investigate differences in responses between the cell types. We found that Notch1+/− AVICs resembled a myofibroblast-like phenotype expressing higher amounts of cadherin-11, a known mediator of dystrophic calcification, and decreased Runx2, a known osteogenic marker. We determined that cadherin-11 expression is regulated by Akt activity, and inhibition of Akt phosphorylation significantly reduced cadherin-11 expression. Moreover, in the presence of cyclic strain, Notch1+/− AVICs exhibited significantly upregulated phosphorylation of Akt at Ser473 and smooth muscle α-actin (αSMA) expression, indicative of a fully activated myofibroblast. Finally, these Notch1 mediated alterations led to enhanced dystrophic calcific nodule formation. Conclusions This study presents novel insights in our understanding of Notch1-mediated CAVD by demonstrating that the mutation leads to AVICs that are fully activated myofibroblasts, resulting in dystrophic, but not osteogenic, calcification.