Osteopontin activity modulates sex-specific calcification in engineered valve tissue mimics.

Patients with aortic valve stenosis (AVS) have sexually dimorphic phenotypes in their valve tissue, where male valvular tissue adopts a calcified phenotype and female tissue becomes more fibrotic. The molecular mechanisms that regulate sex-specific calcification in valvular tissue remain poorly understood. Here, we explored the role of osteopontin (OPN), a pro-fibrotic but anti-calcific bone sialoprotein, in regulating the calcification of female aortic valve tissue. Recognizing that OPN mediates calcification processes, we hypothesized that aortic valvular interstitial cells (VICs) in female tissue have reduced expression of osteogenic markers in the presence of elevated OPN relative to male VICs. Human female valve leaflets displayed reduced and smaller microcalcifications, but increased OPN expression relative to male leaflets. To understand how OPN expression contributes to observed sex dimorphisms in valve tissue, we employed enzymatically degradable hydrogels as a 3D cell culture platform to recapitulate male or female VIC interactions with the extracellular matrix. Using this system, we recapitulated sex differences observed in human tissue, specifically demonstrating that female VICs exposed to calcifying medium have smaller mineral deposits within the hydrogel relative to male VICs. We identified a change in OPN dynamics in female VICs in the presence of calcification stimuli, where OPN deposition localized from the extracellular matrix to perinuclear regions. Additionally, exogenously delivered endothelin-1 to encapsulated VICs increased OPN gene expression in male cells, which resulted in reduced calcification. Collectively, our results suggest that increased OPN in female valve tissue may play a sex-specific role in mitigating mineralization during AVS progression.

Extracellular matrix stiffness controls cardiac valve myofibroblast activation through epigenetic remodeling.

Aortic valve stenosis (AVS) is a progressive fibrotic disease that is caused by thickening and stiffening of valve leaflets. At the cellular level, quiescent valve interstitial cells (qVICs) activate to myofibroblasts (aVICs) that persist within the valve tissue. Given the persistence of myofibroblasts in AVS, epigenetic mechanisms have been implicated. Here, we studied changes that occur in VICs during myofibroblast activation by using a hydrogel matrix to recapitulate different stiffnesses in the valve leaflet during fibrosis. We first compared the chromatin landscape of qVICs cultured on soft hydrogels and aVICs cultured on stiff hydrogels, representing the native and diseased phenotypes respectively. Using assay for transposase-accessible chromatin sequencing (ATAC-Seq), we found that open chromatin regions in aVICs were enriched for transcription factor binding motifs associated with mechanosensing pathways compared to qVICs. Next, we used RNA-Seq to show that the open chromatin regions in aVICs correlated with pro-fibrotic gene expression, as aVICs expressed higher levels of contractile fiber genes, including myofibroblast markers such as alpha smooth muscle actin (αSMA), compared to qVICs. In contrast, chromatin remodeling genes were downregulated in aVICs compared to qVICs, indicating qVICs may be protected from myofibroblast activation through epigenetic mechanisms. Small molecule inhibition of one of these remodelers, CREB Binding Protein (CREBBP), prevented qVICs from activating to aVICs. Notably, CREBBP is more abundant in valves from healthy patients compared to fibrotic valves. Our findings reveal the role of mechanical regulation in chromatin remodeling during VIC activation and quiescence and highlight one potential therapeutic target for treating AVS.

Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

BACKGROUND: Aortic valve stenosis 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 underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic 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 with small molecule inhibitors and 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 α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase 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 through Rho-associated protein kinase 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 aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Matters of the heart: Cellular sex differences.

Nearly all cardiovascular diseases show sexual dimorphisms in prevalence, presentation, and outcomes. Until recently, most clinical trials were carried out in males, and many animal studies either failed to identify the sex of the animals or combined data obtained from males and females. Cellular sex in the heart is relatively understudied and many studies fail to report the sex of the cells used for in vitro experiments. Moreover, in the small number of studies in which sex is reported, most of those studies use male cells. The observation that cells from males and females are inherently different is becoming increasingly clear - either due to acquired differences from hormones and other factors or due to intrinsic differences in genotype (XX or XY). Because of the likely contribution of cellular sex differences in cardiac health and disease, here, we explore differences in mammalian male and female cells in the heart, including the less-studied non-myocyte cell populations. We discuss how the heart's microenvironment impacts male and female cellular phenotypes and vice versa, including how secretory profiles are dependent on cellular sex, and how hormones contribute to sexually dimorphic phenotypes and cellular functions. Intracellular mechanisms that contribute to sex differences, including gene expression and epigenetic remodeling, are also described. Recent single-cell sequencing studies have revealed unexpected sex differences in the composition of cell types in the heart which we discuss. Finally, future recommendations for considering cellular sex differences in the design of bioengineered in vitro disease models of the heart are provided.

Cardiac Fibroblasts Mediate a Sexually Dimorphic Fibrotic Response to ß-Adrenergic Stimulation.

Background Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored. Methods and Results Isoproterenol (ISO) was administered to rats as a model for chronic ß-adrenergic receptor (ß-AR)-mediated cardiovascular disease. ISO-treated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, ß-AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of ß1- and ß2-ARs, whereas expression of both receptors decreased in female CFs. Moreover, ISO-treated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs. Conclusions Chronic in vivo ß-AR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher ß-AR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury.

Nuclear mechanosensing drives chromatin remodelling in persistently activated fibroblasts.

Fibrotic disease is caused by the continuous deposition of extracellular matrix by persistently activated fibroblasts (also known as myofibroblasts), even after the resolution of the injury. Using fibroblasts from porcine aortic valves cultured on hydrogels that can be softened via exposure to ultraviolet light, here we show that increased extracellular stiffness activates the fibroblasts, and that cumulative tension on the nuclear membrane and increases in the activity of histone deacetylases transform transiently activated fibroblasts into myofibroblasts displaying condensed chromatin with genome-wide alterations. The condensed structure of the myofibroblasts is associated with cytoskeletal stability, as indicated by the inhibition of chromatin condensation and myofibroblast persistence after detachment of the nucleus from the cytoskeleton via the displacement of endogenous nesprins from the nuclear envelope. We also show that the chromatin structure of myofibroblasts from patients with aortic valve stenosis is more condensed than that of myofibroblasts from healthy donors. Our findings suggest that nuclear mechanosensing drives distinct chromatin signatures in persistently activated fibroblasts.

Mechanobiological Interactions between Dynamic Compressive Loading and Viscoelasticity on Chondrocytes in Hydrazone Covalent Adaptable Networks for Cartilage Tissue Engineering.

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.

Tumor necrosis factor-α promotes and exacerbates calcification in heart valve myofibroblast populations.

Pro-inflammatory cytokines play critical roles in regulating valvular interstitial cell (VIC) phenotypic changes that can cause heart valve fibrosis and calcification. Tumor necrosis factor alpha (TNF-α) is a cytokine known to influence VIC behavior and has been reported at high levels in calcified valves ex vivo. We sought to understand the specific effects of TNF-α on VIC phenotypes (eg, fibroblast, profibrotic activated myofibroblasts) and its link with heart valve disorders. We characterize human aortic valve tissue from patients with valve disorders and identify a high variability of fibrotic and calcific markers between tissues. These results motivated in vitro studies to explore the effects of TNF-α on defined VIC fibroblasts and profibrotic activated myofibroblasts, induced via FGF-2 and TGF-ß1 treatment. Using 3D hydrogels to culture VICs, we measure the effect of TNF-α (0.1-10 ng/mL) on key markers of fibrosis (eg, αSMA, COL1A1) and calcification (eg, RUNX2, BMP2, and calcium deposits). We observe calcification in TNF-α-treated VIC activated myofibroblasts and identify the MAPK/ERK signaling cascade as a potential pathway for TNF-α mediated calcification. Conversely, VIC fibroblasts respond to TNF-α with decreased calcification. Treatment of VIC profibrotic activated myofibroblast populations with TNF-α leads to increased calcification. Our in vitro findings correlate with findings in diseased human valves and highlight the importance of understanding the effect of cytokines and signaling pathways on specific VIC phenotypes. Finally, we reveal MAPK/ERK as a potential pathway involved in VIC-mediated matrix calcification with TNF-α treatment, suggesting this pathway as a potential pharmaceutical target for aortic valve disease.

Collagen networks within 3D PEG hydrogels support valvular interstitial cell matrix mineralization.

Enzymatically degradable hydrogels were designed for the 3D culture of valvular interstitial cells (VICs), and through the incorporation of various functionalities, we aimed to investigate the role of the tissue microenvironment in promoting the osteogenic properties of VICs and matrix mineralization. Specifically, porcine VICs were encapsulated in a poly(ethylene glycol) hydrogel crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinker (KCGPQG↓IWGQCK) and formed via a thiol-ene photoclick reaction in the presence or absence of collagen type I to promote matrix mineralization. VIC-laden hydrogels were treated with osteogenic medium for up to 15 days, and the osteogenic response was characterized by the expression of RUNX2 as an early marker of an osteoblast-like phenotype, osteocalcin (OCN) as a marker of a mature osteoblast-like phenotype, and vimentin (VIM) as a marker of the fibroblast phenotype. In addition, matrix mineralization was characterized histologically with Von Kossa stain for calcium phosphate. Osteogenic response was further characterized biochemically with calcium assays, and physically via optical density measurements. When the osteogenic medium was supplemented with calcium chloride, OCN expression was upregulated and mineralization was discernable at 12 days of culture. Finally, this platform was used to screen various drug therapeutics that were assessed for their efficacy in preventing mineralization using optical density as a higher throughput readout. Collectively, these results suggest that matrix composition has a key role in supporting mineralization deposition within diseased valve tissue.

Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment.

Background Cardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling. Methods and Results Myofibroblast activation was modulated in vitro by combining stiffness cues with TGFß1 (transforming growth factor ß 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFß1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGFß1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy. Conclusions Culturing CFs on stiff substrates, treating with TGFß1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGFß1 caused myocyte hypertrophy, most likely through insulin growth factor 1.

Nuclear mechanosensing controls MSC osteogenic potential through HDAC epigenetic remodeling.

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.

Viscoelasticity of hydrazone crosslinked poly(ethylene glycol) hydrogels directs chondrocyte morphology during mechanical deformation.

Chondrocyte deformation influences disease progression and tissue regeneration in load-bearing joints. In this work, we found that viscoelasticity of dynamic covalent crosslinks temporally modulates the biophysical transmission of physiologically relevant compressive strains to encapsulated chondrocytes. Chondrocytes in viscoelastic alky-hydrazone hydrogels demonstrated (91.4 ± 4.5%) recovery of native rounded morphologies during mechanical deformation, whereas primarily elastic benzyl-hydrazone hydrogels significantly limited morphological recovery (21.2 ± 1.4%).

Three-dimensional encapsulation of adult mouse cardiomyocytes in hydrogels with tunable stiffness.

Numerous diseases, including those of the heart, are characterized by increased stiffness due to excessive deposition of extracellular matrix proteins. Cardiomyocytes continuously adapt their morphology and function to the mechanical changes of their microenvironment. Because traditional cell culture is conducted on substrates that are many orders of magnitude stiffer than any environment encountered by a cardiomyocyte in health or disease, alternate culture systems are necessary to model these processes in vitro. Here, we employ photo-clickable thiol-ene poly(ethylene glycol) (PEG) hydrogels for three-dimensional cell culture of adult mouse cardiomyocytes. PEG hydrogels serve as versatile biocompatible scaffolds, whose stiffness can be precisely tuned to mimic physiological and pathological microenvironments. Compared to traditional culture, adult cardiomyocytes encapsulated in PEG hydrogels exhibited longer survival and preserved sarcomeric and T-tubular architecture. Culture in PEG hydrogels of varying stiffnesses regulated the subcellular localization of the mechanosensitive transcription factor, YAP, in adult cardiomyocytes, indicating PEG hydrogels offer a versatile platform to study the role of mechanical cues in cardiomyocyte biology.

Transcatheter aortic valve replacements alter circulating serum factors to mediate myofibroblast deactivation.

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.

Extended Exposure to Stiff Microenvironments Leads to Persistent Chromatin Remodeling in Human Mesenchymal Stem Cells.

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.

FGF-2 inhibits contractile properties of valvular interstitial cell myofibroblasts encapsulated in 3D MMP-degradable hydrogels.

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-ß1; 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-ß1. TGF-ß1 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.

Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling in C. elegans.

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.