Bone marrow derived human mesenchymal stem cells (hMSCs) are a promising cell source for regenerative therapies; however, ex vivo expansion is often required to achieve clinically useful cells numbers. Recent results reveal that when MSCs are cultured in stiff microenvironments, their regenerative capacity can be altered in a manner that is dependent on time (e.g., a mechanical dosing analogous to a chemical one). It is hypothesized that epigenomic modifications are involved in storing these mechanical cues, regulating gene expression, and ultimately leading to a mechanical memory. Using hydrogels containing an allyl sulfide cross‐linker and a radical‐mediated addition‐fragmentation chain transfer process, in situ softened hMSC‐laden hydrogels at different time points are achieved and the effects of short‐term and long‐term mechanical dosing on epigenetic modifications in hMSCs are quantified. Results show that histone acetylation and chromatin organization adapt rapidly after softening and can be reversible or irreversible depending on time of exposure to stiff microenvironments. Furthermore, epigenetic modulators are differentially expressed depending on the culture history. Collectively, these experiments suggest that epigenetic remodeling can be persistent and might be a memory keeper.

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Cells sense mechanical cues from the extracellular matrix to regulate cellular behavior and maintain tissue homeostasis. The nucleus has been implicated as a key mechanosensor and can directly influence chromatin organization, epigenetic modifications, and gene expression. Dysregulation of nuclear mechanosensing has been implicated in several diseases, including bone degeneration. Here, we exploit photostiffening hydrogels to manipulate nuclear mechanosensing in human mesenchymal stem cells (hMSCs) in vitro. Results show that hMSCs respond to matrix stiffening by increasing nuclear tension and causing an increase in histone acetylation via deactivation of histone deacetylases (HDACs). This ultimately induces osteogenic fate commitment. Disrupting nuclear mechanosensing by disconnecting the nucleus from the cytoskeleton up-regulates HDACs and prevents osteogenesis. Resetting HDAC activity back to healthy levels rescues the epigenetic and osteogenic response in hMSCs with pathological nuclear mechanosensing. Notably, bone from patients with osteoarthritis displays similar defective nuclear mechanosensing. Collectively, our results reveal that nuclear mechanosensing controls hMSC osteogenic potential mediated by HDAC epigenetic remodeling and that this cellular mechanism is likely relevant to bone-related diseases.

The transcatheter aortic valve replacement (TAVR) procedure has emerged as a minimally invasive treatment for patients with aortic valve stenosis (AVS). However, alterations in serum factor composition and biological activity after TAVR remain unknown. Here, we quantified the systemic inflammatory effects of the TAVR procedure and hypothesized that alterations in serum factor composition would modulate valve and cardiac fibrosis. Serum samples were obtained from patients with AVS immediately before their TAVR procedure (pre-TAVR) and about 1 month afterward (post-TAVR). Aptamer-based proteomic profiling revealed alterations in post-TAVR serum composition, and ontological analysis identified inflammatory macrophage factors implicated in myofibroblast activation and deactivation. Hydrogel biomaterials used as valve matrix mimics demonstrated that post-TAVR serum reduced myofibroblast activation of valvular interstitial cells relative to pre-TAVR serum from the same patient. Transcriptomics and curated network analysis revealed a shift in myofibroblast phenotype from pre-TAVR to post-TAVR and identified p38 MAPK signaling as one pathway involved in pre-TAVR–mediated myofibroblast activation. Post-TAVR serum deactivated valve and cardiac myofibroblasts initially exposed to pre-TAVR serum to a quiescent fibroblast phenotype. Our in vitro deactivation data correlated with patient disease severity measured via echocardiography and multimorbidity scores, and correlations were dependent on hydrogel stiffness. Sex differences in cellular responses to male and female sera were also observed and may corroborate clinical observations regarding sex-specific TAVR outcomes. Together, alterations in serum composition after TAVR may lead to an antifibrotic fibroblast phenotype, which suggests earlier interventions may be beneficial for patients with advanced AVS to prevent further disease progression.

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.

Animals alter their reproductive programs to accommodate changes in nutrient availability, yet the connections between known nutrient-sensing systems and reproductive programs are underexplored, and whether there is a mechanism that senses nucleotide levels to coordinate germline proliferation is unknown. We established a model system in which nucleotide metabolism is perturbed in both the nematode Caenorhabditis elegans (cytidine deaminases) and its food (Escherichia coli); when fed food with a low uridine/thymidine (U/T) level, germline proliferation is arrested. We provide evidence that this impact of U/T level on the germline is critically mediated by GLP-1/Notch and MPK-1/MAPK, known to regulate germline mitotic proliferation. This germline defect is suppressed by hyperactivation of glp-1 or disruption of genes downstream from glp-1 to promote meiosis but not by activation of the IIS or TORC1 pathways. Moreover, GLP-1 expression is post-transcriptionally modulated by U/T levels. Our results reveal a previously unknown nucleotide-sensing mechanism for controlling reproductivity.

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Mechanobiological cues influence chondrocyte biosynthesis and are often used in tissue engineering applications to improve the repair of articular cartilage in load‐bearing joints. In this work, the biophysical effects of an applied dynamic compression on chondrocytes encapsulated in viscoelastic hydrazone covalent adaptable networks (CANs) is explored. Here, hydrazone CANs exhibit viscoelastic loss tangents ranging from (9.03 ± 0.01) 10–4 to (1.67 ± 0.09) 10–3 based on the molar percentages of alkyl‐hydrazone and benzyl‐hydrazone crosslinks. Notably, viscoelastic alkyl‐hydrazone crosslinks improve articular cartilage specific gene expression showing higher SOX9 expression in free swelling hydrogels and dynamic compression reduces hypertrophic chondrocyte markers (COL10A1, MMP13) in hydrazone CANs. Interestingly, dynamic compression also improves matrix biosynthesis in elastic benzyl‐hydrazone controls but reduces biosynthesis in viscoelastic alkyl‐hydrazone CANs. Additionally, intermediate levels of viscoelastic adaptability demonstrate the highest levels of matrix biosynthesis in hydrazone CANs, demonstrating on average 70 ± 4 µg of sulfated glycosaminoglycans per day and 31 ± 3 µg of collagen per day over one month in dynamic compression bioreactors. Collectively, the results herein demonstrate the role of matrix adaptability and viscoelasticity on chondrocytes in hydrazone CANs during dynamic compression, which may prove useful for tissue engineering applications in load‐bearing joints.