FRET Sensor-Modified Synthetic Hydrogels for Real-Time Monitoring of Cell-Derived Matrix Metalloproteinase Activity using Fluorescence Lifetime Imaging.

Matrix remodeling plays central roles in a range of physiological and pathological processes and is driven predominantly by the activity of matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) proteins. Our understanding of how MMPs regulate cell and tissue dynamics is often incomplete as in vivo approaches are lacking and many in vitro strategies cannot provide high-resolution, quantitative measures of enzyme activity in situ within tissue-like 3D microenvironments. Here, we incorporate a Förster resonance energy transfer (FRET) sensor of MMP activity into fully synthetic hydrogels that mimic many properties of the native ECM. We then use fluorescence lifetime imaging to provide a real-time, fluorophore concentration-independent quantification of MMP activity, establishing a highly accurate, readily adaptable platform for studying MMP dynamics in situ. MCF7 human breast cancer cells encapsulated within hydrogels highlight the detection of MMP activity both locally, at the sub-micron level, and within the bulk hydrogel. Our versatile platform may find use in a range of biological studies to explore questions in the dynamics of cancer metastasis, development, and tissue repair by providing high-resolution, quantitative and in situ readouts of local MMP activity within native tissue-like environments.

Injectable PEG Hydrogels with Tissue-Like Viscoelasticity Formed through Reversible Alendronate-Calcium Phosphate Crosslinking for Cell-Material Interactions.

Synthetic hydrogels provide controllable 3D environments, which can be used to study fundamental biological phenomena. The growing body of evidence that cell behavior depends upon hydrogel stress relaxation creates a high demand for hydrogels with tissue-like viscoelastic properties. Here, a unique platform of synthetic polyethylene glycol (PEG) hydrogels in which star-shaped PEG molecules are conjugated with alendronate and/or RGD peptides, attaining modifiable degradability as well as flexible cell adhesion, is created. Novel reversible ionic interactions between alendronate and calcium phosphate nanoparticles, leading to versatile viscoelastic properties with varying initial elastic modulus and stress relaxation time, are identified. This new crosslinking mechanism provides shear-thinning properties resulting in differential cellular responses between cancer cells and stem cells. The novel hydrogel system is an improved design to the other ionic crosslink platforms and opens new avenues for the development of pathologically relevant cancer models, as well as minimally invasive approaches for cell delivery for potential regenerative therapies.

Characteristics of traumatic brain injury models: from macroscopic blood flow changes to microscopic mitochondrial changes.

Controlled cortical impingement is a widely accepted method to induce traumatic brain injury to establish a traumatic brain injury animal model. A strike depth of 1 mm at a certain speed is recommended for a moderate brain injury and a depth of > 2 mm is used to induce severe brain injury. However, the different effects and underlying mechanisms of these two model types have not been proven. This study investigated the changes in cerebral blood flow, differences in the degree of cortical damage, and differences in motor function under different injury parameters of 1 and 2 mm at injury speeds of 3, 4, and 5 m/s. We also explored the functional changes and mitochondrial damage between the 1 and 2 mm groups in the acute (7 days) and chronic phases (30 days). The results showed that the cerebral blood flow in the injured area of the 1 mm group was significantly increased, and swelling and bulging of brain tissue, increased vascular permeability, and large-scale exudation occurred. In the 2 mm group, the main pathological changes were decreased cerebral blood flow, brain tissue loss, and cerebral vasospasm occlusion in the injured area. Substantial motor and cognitive impairments were found on day 7 after injury in the 2 mm group; at 30 days after injury, the motor function of the 2 mm group mice recovered significantly while cognitive impairment persisted. Transcriptome sequencing showed that compared with the 1 mm group, the 2 mm group expressed more ferroptosis-related genes. Morphological changes of mitochondria in the two groups on days 7 and 30 using transmission electron microscopy revealed that on day 7, the mitochondria in both groups shrank and the vacuoles became larger; on day 30, the mitochondria in the 1 mm group became larger, and the vacuoles in the 2 mm group remained enlarged. By analyzing the proportion of mitochondrial subgroups in different groups, we found that the model mice had different patterns of mitochondrial composition at different time periods, suggesting that the difference in the degree of damage among traumatic brain injury groups may reflect the mitochondrial changes. Taken together, differences in mitochondrial morphology and function between the 1 and 2 mm groups provide a new direction for the accurate classification of traumatic brain injury. Our results provide reliable data support and evaluation methods for promoting the establishment of standard mouse controlled cortical impingement model guidelines.

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling.

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation.

Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A.

PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to ß-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca2+ sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive. Here, we specifically altered the phosphorylation level of cTnI in heart muscle cells and characterized the structural and functional effects at different levels of background phosphorylation of cMyBP-C and with two different SLs. We found Ser22/23 bisphosphorylation of cTnI was indispensable for the enhancement of length-dependent activation by PKA, as was cMyBP-C phosphorylation. This high level of coordination between cTnI and cMyBP-C may suggest coupling between their regulatory mechanisms. Further evidence for this was provided by our finding that cardiac troponin (cTn) can directly interact with cMyBP-C in vitro, in a phosphorylation- and Ca2+-dependent manner. In addition, bisphosphorylation at Ser22/Ser23 increased Ca2+ sensitivity at long SL in the presence of endogenously phosphorylated cMyBP-C. When cMyBP-C was dephosphorylated, bisphosphorylation of cTnI increased Ca2+ sensitivity and decreased cooperativity at both SLs, which may translate to deleterious effects in physiological settings. Our results could have clinical relevance for disease pathways, where PKA phosphorylation of cTnI may be functionally uncoupled from cMyBP-C phosphorylation due to mutations or haploinsufficiency.

Cancer-associated fibroblast-derived exosomal miR-18b promotes breast cancer invasion and metastasis by regulating TCEAL7.

Studies have shown that cancer-associated fibroblasts (CAFs) play an irreplaceable role in the occurrence and development of tumors. Therefore, exploring the action and mechanism of CAFs on tumor cells is particularly important. In this study, we compared the effects of CAFs-derived exosomes and normal fibroblasts (NFs)-derived exosomes on breast cancer cells migration and invasion. The results showed that exosomes from both CAFs and NFs could enter into breast cancer cells and CAFs-derived exosomes had a more enhancing effect on breast cancer cells migration and invasion than NFs-derived exosomes. Furthermore, microRNA (miR)-18b was upregulated in CAFs-derived exosomes, and CAFs-derived exosomes miR-18b can promote breast cancer cell migration and metastasis by specifically binding to the 3'UTR of Transcription Elongation Factor A Like 7 (TCEAL7). The miR-18b-TCEAL7 pathway promotes nuclear Snail ectopic activation by activating nuclear factor-kappa B (NF-κB), thereby inducing epithelial-mesenchymal transition (EMT) and promoting cell invasion and metastasis. Moreover, CAFs-derived exosomes miR-18b could promote mouse xenograft model tumor metastasis. Overall, our findings suggest that CAFs-derived exosomes miR-18b promote nuclear Snail ectopic by targeting TCEAL7 to activate the NF-κB pathway, thereby inducing EMT, invasion, and metastasis of breast cancer. Targeting CAFs-derived exosome miR-18b may be a potential treatment option to overcome breast cancer progression.

Selectively Cross-Linked Tetra-PEG Hydrogels Provide Control over Mechanical Strength with Minimal Impact on Diffusivity.

Synthetic hydrogels formed from poly(ethylene glycol) (PEG) are widely used to study how cells interact with their extracellular matrix. These in vivo-like 3D environments provide a basis for tissue engineering and cell therapies but also for research into fundamental biological questions and disease modeling. The physical properties of PEG hydrogels can be modulated to provide mechanical cues to encapsulated cells; however, the impact of changing hydrogel stiffness on the diffusivity of solutes to and from encapsulated cells has received only limited attention. This is particularly true in selectively cross-linked "tetra-PEG" hydrogels, whose design limits network inhomogeneities. Here, we used a combination of theoretical calculations, predictive modeling, and experimental measurements of hydrogel swelling, rheological behavior, and diffusion kinetics to characterize tetra-PEG hydrogels' permissiveness to the diffusion of molecules of biologically relevant size as we changed polymer concentration, and thus hydrogel mechanical strength. Our models predict that hydrogel mesh size has little effect on the diffusivity of model molecules and instead predicts that diffusion rates are more highly dependent on solute size. Indeed, our model predicts that changes in hydrogel mesh size only begin to have a non-negligible impact on the concentration of a solute that diffuses out of hydrogels for the smallest mesh sizes and largest diffusing solutes. Experimental measurements characterizing the diffusion of fluorescein isothiocyanate (FITC)-labeled dextran molecules of known size aligned well with modeling predictions and suggest that doubling the polymer concentration from 2.5% (w/v) to 5% produces stiffer gels with faster gelling kinetics without affecting the diffusivity of solutes of biologically relevant size but that 10% hydrogels can slow their diffusion. Our findings provide confidence that the stiffness of tetra-PEG hydrogels can be modulated over a physiological range without significantly impacting the transport rates of solutes to and from encapsulated cells.

CCL18 promotes the invasion and metastasis of breast cancer through Annexin A2.

Chemokine (C­C motif) ligand 18 (CCL18) is derived from breast tumor­associated macrophages (TAMs), which are primarily a macrophage subpopulation with an M2 phenotype. CCL18 binds to its receptor, PYK2 N­terminal domain interacting receptor 1 (Nir1), and promotes tumor progression and metastasis by inducing epithelial­mesenchymal transition (EMT) via the PI3K/Akt/GSK3ß/Snail signaling pathway in breast cancer cells. Recent research shows that Annexin A2 (AnxA2) plays a significant role in the invasion, metastasis, angiogenesis, proliferation, F­actin polymerization and multidrug resistance to chemotherapy of breast cancer. The present study aimed to elucidate the molecular mechanisms by which CCL18 promotes breast cancer progression through AnxA2 which are not fully understood. Western blot analysis showed that the expression of AnxA2 was upregulated in highly invasive breast cancer cell lines and invasive ductal carcinoma. Furthermore, through chemotaxis, scratch, Matrigel invasion, and spontaneous metastasis assays, it was demonstrated that AnxA2 enhanced the invasion of breast cancer cells and the metastasis of human breast cancer cells to lungs of SCID mice with CCL18 stimulation. Cellular F­actin measurement assay showed that reduction of AnxA2 suppressed CCL18­induced F­actin polymerization though phosphorylation of integrin ß1 in breast cancer cells. Immunofluorescence and western blot analysis revealed that AnxA2 promoted CCL18­induced EMT via the PI3K/Akt/GSK3ß/Snail signaling pathway, and LY294002 inhibited the phosphorylation of AnxA2 in vitro. In brief, AnxA2, as a downstream molecule of Nir 1 binding to CCL18, promotes invasion and metastasis by EMT through the PI3K/Akt/GSK3ß/Snail signaling pathway in breast cancer. This study suggests that AnxA2 is a potential anti­invasion/metastasis target for therapeutic intervention in breast cancer.

Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle.

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure.

Reversible Covalent Binding to Cardiac Troponin C by the Ca2+-Sensitizer Levosimendan.

The binding of Ca2+ to cardiac troponin C (cTnC) triggers contraction in heart muscle. In the diseased heart, the myocardium is often desensitized to Ca2+, which leads to impaired contractility. Therefore, compounds that sensitize cardiac muscle to Ca2+ (Ca2+-sensitizers) have therapeutic promise. The only Ca2+-sensitizer used regularly in clinical settings is levosimendan. While the primary target of levosimendan is thought to be cTnC, the molecular details of this interaction are not well understood. In this study, we used mass spectrometry, computational chemistry, and nuclear magnetic resonance spectroscopy to demonstrate that levosimendan reacts specifically with cysteine 84 of cTnC to form a reversible thioimidate bond. We also showed that levosimendan only reacts with the active, Ca2+-bound conformation of cTnC. Finally, we propose a structural model of levosimendan bound to cTnC, which suggests that the Ca2+-sensitizing function of levosimendan is due to stabilization of the Ca2+-bound conformation of cTnC.

Myosin binding protein-C activates thin filaments and inhibits thick filaments in heart muscle cells.

Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.