Background: Aortic valve stenosis (AVS) is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain under explored. Methods: Hydrogel scaffolds were designed to recapitulate key aspects of the valve tissue microenvironment and serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to pro-fibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA-sequencing was used to define pathways involved in driving sex-dependent activation. Interventions using small molecule inhibitors and small interfering RNA (siRNA) transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. Results: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker, alpha-smooth muscle actin (α-SMA), compared to male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-SMA stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase (RhoA/ROCK) signaling as a potential driver of this sex-dependent myofibroblast activation. Further, we found that genes that escape X-chromosome inactivation, such as BMX and STS (encoding for Bmx non-receptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation via RhoA/ROCK signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation via RhoA/ROCK signaling. Conclusions: Together, in vivo and in vitro results confirm sex-dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent AVS progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of AVS and help guide sex-based precision therapies.

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Valvular interstitial cells (VICs) are responsible for the maintenance of the extracellular matrix in heart valve leaflets and, in response to injury, activate from a quiescent fibroblast to a wound healing myofibroblast phenotype. Under normal conditions, myofibroblast activation is transient, but the chronic presence of activated VICs can lead to valve diseases, such as fibrotic aortic valve stenosis, for which non-surgical treatments remain elusive. We monitored the porcine VIC response to exogenously delivered fibroblast growth factor 2 (FGF-2; 100 ng/ml), transforming growth factor beta 1 (TGF-β1; 5 ng/ml), or a combination of the two while cultured within 3D matrix metalloproteinase (MMP)-degradable 8-arm 40 kDa poly(ethylene glycol) hydrogels that mimic aspects of the aortic valve. Here, we aimed to investigate VIC myofibroblast activation and subsequent contraction or the reparative wound healing response. To this end, VIC morphology, proliferation, gene expression related to the myofibroblast phenotype [alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF)] and matrix remodeling [collagens (COL1A1 and COL3) and MMP1], and contraction assays were used to quantify the cell response. Treatment with FGF-2 resulted in increased cellular proliferation while reducing the myofibroblast phenotype, as seen by decreased expression of CTGF and α-SMA, and reduced contraction relative to untreated control, suggesting that FGF-2 encourages a reparative phenotype, even in the presence of TGF-β1. TGF-β1 treatment predictably led to an increased proportion of VICs exhibiting the myofibroblast phenotype, indicated by the presence of α-SMA, increased gene expression indicative of matrix remodeling, and bulk contraction of the hydrogels. Functional contraction assays and biomechanical analyses were performed on VIC encapsulated hydrogels and porcine aortic valve tissue explants to validate these findings.

OpenBU http://open.bu.edu Mechanical Engineering BU Open Access Articles 2019-09 Quantifying heart valve interstitial cell contractile state using highly tunable poly(ethylene glyc...

BACKGROUND: Hyaluronic acid (HA) is a polysaccharide present in almost all animal tissues, in which it carries out important biological functions, among them, the protection of the joints by lubricating them and dampening the tension in them. OBJECTIVE: This study compares the viscoelastic properties of several commercial preparations of HA, to determine their suitability for use as viscosupplementation therapy in joint pathology (osteoarthritis). METHODS: 4 HA hydrogels: Durolane®, Synocrom_Forte_One®, Synvisc_One® and Viscoplus_Matrix® and 4 HA solutions: Ostenil®, Ostenil_Plus®, Viscoplus_Gel® and Orthovisc® were analyzed to compare their viscoelatsic rheological parameters using an oscillatory-rotational rheometer. RESULTS: With respect to the 4 HA hydrogels, comparison of crossover frequencies allowed division into two main groups: Synvisc_One® and Viscoplus_Matrix®, with crossover frequencies in the order of magnitude of 10−2 Hz, while Synocrom_Forte_One® and Durolane® showed crossover frequencies on the order of 10−1 Hz. Only one of the 4 HA solutions, Viscoplus_Gel®, showed a crossover frequency on the order of 10−2, whereas Ostenil_Plus® and Orthovisc® showed crossover frequencies on the order of 10−1, and Ostenil® remained as a predominantly viscous fluid for frequencies as high as 4.8 Hz. CONCLUSIONS: The viscoelastic properties of the HA preparations can be ordered according to the values of G ∗ (the rigidity, or vector sum of the elastic modulus G ′ and the viscous modulus G ′′) at both transition points (0.5 and 2.5 Hz) as follows: Viscoplus_Matrix®  > Viscoplus_Gel®  > Durolane®  > Synocrom_Forte_One®  > Ostenil_Plus®  > Synvisc_One®  > Orthovisc®  > Ostenil®.