There is a growing interest in materials that can dynamically change their properties in the presence of cells to study mechanobiology. Herein, we exploit the 365 nm light mediated 4+4 ...photodimerization of anthracene groups to develop cytocompatible PEG‐based hydrogels with tailorable initial moduli that can be further stiffened. A hydrogel formulation that can stiffen from 10 to 50 kPa, corresponding to the stiffness of a healthy and fibrotic heart, respectively, was prepared. This system was used to monitor the stiffness‐dependent localization of NFAT, a downstream target of intracellular calcium signaling using a reporter in live cardiac fibroblasts (CFbs). NFAT translocates to the nucleus of CFbs on stiffening hydrogels within 6 h, whereas it remains cytoplasmic when the CFbs are cultured on either 10 or 50 kPa static hydrogels. This finding demonstrates how dynamic changes in the mechanical properties of a material can reveal the kinetics of mechanoresponsive cell signaling pathways that may otherwise be missed in cells cultured on static substrates.
Materials with on‐demand tunable properties can be used to study changes in cellular behavior in response to their microenvironments. Anthracene photodimerization enables a simple, but well‐defined and cytocompatible strategy to stiffen PEG‐based hydrogels. By using these hydrogels, it was found that NFAT, a downstream target of calcium signaling, responds to changes in hydrogel modulus on short timescales (80 min to <24 h) in cardiac fibroblasts.
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 TGFβ1 (transforming growth factor β 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFβ1, 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 TGFβ1 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 TGFβ1, 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 TGFβ1 caused myocyte hypertrophy, most likely through insulin growth factor 1.
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.
There is a growing interest in materials that can dynamically change their properties in the presence of cells to study mechanobiology. Herein, we exploit the 365 nm light mediated 4+4 ...photodimerization of anthracene groups to develop cytocompatible PEG‐based hydrogels with tailorable initial moduli that can be further stiffened. A hydrogel formulation that can stiffen from 10 to 50 kPa, corresponding to the stiffness of a healthy and fibrotic heart, respectively, was prepared. This system was used to monitor the stiffness‐dependent localization of NFAT, a downstream target of intracellular calcium signaling using a reporter in live cardiac fibroblasts (CFbs). NFAT translocates to the nucleus of CFbs on stiffening hydrogels within 6 h, whereas it remains cytoplasmic when the CFbs are cultured on either 10 or 50 kPa static hydrogels. This finding demonstrates how dynamic changes in the mechanical properties of a material can reveal the kinetics of mechanoresponsive cell signaling pathways that may otherwise be missed in cells cultured on static substrates.
Materialien mit justierbaren Eigenschaften werden zur Untersuchung von Veränderungen im Zellverhalten als Reaktion auf ihre Mikroumgebung verwendet. Eine zytokompatible Strategie zur Versteifung von PEG‐basierten Hydrogelen basiert auf der Photodimerisierung von Anthracen. Durch die Verwendung dieser Hydrogele wurde festgestellt, dass NFAT, ein nachgeordnetes Ziel der Calcium‐Signalkaskade, in kardialen Fibroblasten auf Veränderungen des Hydrogelmoduls innerhalb von 80 min bis <24 h reagiert.
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 β-adrenergic receptor (β-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, β-AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of β1- and β2-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 β-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 β-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.
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