bims-ecemfi Biomed News
on ECM and fibroblasts
Issue of 2024–06–02
27 papers selected by
Badri Narayanan Narasimhan, University of California, San Diego



  1. ACS Appl Mater Interfaces. 2024 May 31.
      The inherent extracellular matrix (ECM) originating from a specific tissue impacts the process of vascularization, specifically vascular network formation (VNF) orchestrated by endothelial cells (ECs). The specific contribution toward these processes of ECM from highly disparate organs such as the skin and lungs remains a relatively unexplored area. In this study, we compared VNF and ECM remodeling mediated by microvascular ECs within gel, lung, and combinations thereof (hybrid) ECM hydrogels. Irrespective of the EC source, the skin-derived ECM hydrogel exhibited a higher propensity to drive and support VNF compared to both lung and hybrid ECM hydrogels. There were distinct disparities in the physical properties of the three types of hydrogels, including viscoelastic properties and complex architectural configurations, including fiber diameter, pore area, and numbers among the fibers. The hybrid ECM hydrogel properties were unique and not the sum of the component ECM parts. Furthermore, cellular ECM remodeling responses varied with skin ECM hydrogels promoting matrix metalloproteinase 1 (MMP1) secretion, while hybrid ECM hydrogels exhibited increased MMP9, fibronectin, and collagen IV deposition. Principal component analysis (PCA) indicated that the influence of a gel's mechanical properties on VNF was stronger than the biochemical composition. These data indicate that the organ-specific properties of an ECM dictate its capacity to support VNF, while intriguingly showing that ECs respond to more than just the biochemical constituents of an ECM. The study suggests potential applications in regenerative medicine by strategically selecting ECM origin or combinations to manipulate vascularization, offering promising prospects for enhancing wound healing through pro-regenerative interventions.
    Keywords:  ECM hydrogel; biomechanics; endothelial cells; extracellular matrix; vascularization
    DOI:  https://doi.org/10.1021/acsami.4c05864
  2. bioRxiv. 2024 May 18. pii: 2024.05.17.594765. [Epub ahead of print]
      As the cytoskeleton sustains cell and tissue forces, it incurs physical damage that must be repaired to maintain mechanical homeostasis. The LIM-domain protein zyxin detects force-induced ruptures in actin-myosin stress fibers, coordinating downstream repair factors to restore stress fiber integrity through unclear mechanisms. Here, we reconstitute stress fiber repair with purified proteins, uncovering detailed links between zyxin's force-regulated binding interactions and cytoskeletal dynamics. In addition to binding individual tensed actin filaments (F-actin), zyxin's LIM domains form force-dependent assemblies that bridge broken filament fragments. Zyxin assemblies engage repair factors through multi-valent interactions, coordinating nucleation of new F-actin by VASP and its crosslinking into aligned bundles by ɑ-actinin. Through these combined activities, stress fiber repair initiates within the cores of micron-scale damage sites in cells, explaining how these F-actin depleted regions are rapidly restored. Thus, zyxin's force-dependent organization of actin repair machinery inherently operates at the network scale to maintain cytoskeletal integrity.
    Keywords:  Actin cytoskeleton; LIM domain; mechanosensing; mechanotranduction; stress fiber; zyxin
    DOI:  https://doi.org/10.1101/2024.05.17.594765
  3. Commun Biol. 2024 May 29. 7(1): 658
      The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.
    DOI:  https://doi.org/10.1038/s42003-024-06366-4
  4. Adv Healthc Mater. 2024 May 28. e2401719
      A high density of macrophages in the ovarian cancer microenvironment is associated with disease progression and poor outcomes. Understanding cancer-macrophage interaction mechanisms that establish this pro-tumorigenic microenvironment is critical for developing macrophage-targeted therapies. Here, we utilize three-dimensional microfluidic assays and patient-derived xenografts to define the role of cancer-derived CSF1 on macrophage infiltration dynamics towards ovarian cancer cells. We demonstrate that multiple ovarian cancer models promote the infiltration of macrophages into a 3D extracellular matrix in vitro in a cell density-dependent manner. Macrophages exhibit directional migration and increased migration speed under both direct interactions with cancer cells embedded within the matrix, and paracrine crosstalk with cancer cells seeded in an independent microchannel. We also found that platinum-based chemotherapy increases macrophage recruitment and the levels of cancer cell-derived CSF1. Targeting CSF1 signaling under baseline or chemotherapy-treatment conditions reduced the number of infiltrated macrophages. We further show that results obtained with our 3D microfluidic model reflect the recruitment profiles of macrophages in patient-derived xenografts in vivo. These results highlight the role of CSF1 signaling in establishing macrophage-rich ovarian cancer microenvironments, as well as the utility of microfluidic models in recapitulating 3D tumor ecosystems and dissecting cancer-macrophage signaling. This article is protected by copyright. All rights reserved.
    Keywords:  3D migration; cell‐cell signaling; chemotherapy; microfluidics; ovarian cancer
    DOI:  https://doi.org/10.1002/adhm.202401719
  5. Matrix Biol Plus. 2024 Jun;22 100148
      The pancreatic islet is surrounded by ECM that provides both biochemical and mechanical cues to the islet β-cell to regulate cell survival and insulin secretion. Changes in ECM composition and mechanical properties drive β-cell dysfunction in many pancreatic diseases. While several studies have characterized changes in islet insulin secretion with changes in substrate stiffness, little is known about the mechanotransduction signaling driving altered islet function in response to mechanical cues. We hypothesized that increasing matrix stiffness will lead to insulin secretion dysfunction by opening the mechanosensitive ion channel Piezo1 and disrupting intracellular Ca2+ dynamics in mouse and human islets. To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D. Our results indicate that increased scaffold stiffness causes insulin secretion dysfunction mediated by increases in Ca2+ influx and altered Ca2+ dynamics via opening of the mechanosensitive Piezo1 channel. Additionally, inhibition of Piezo1 rescued glucose-stimulated insulin secretion (GSIS) in islets in stiff scaffolds. Overall, our results emphasize the role mechanical properties of the islet microenvironment plays in regulating function. It also supports further investigation into the modulation of Piezo1 channel activity to restore islet function in diseases like type 2 diabetes (T2D) and pancreatic cancer where fibrosis of the peri-islet ECM leads to increased tissue stiffness and islet dysfunction.
    Keywords:  Extracellular matrix; Insulin secretion; Mechanotransduction; Pancreatic islet; Piezo1; Stiffness
    DOI:  https://doi.org/10.1016/j.mbplus.2024.100148
  6. bioRxiv. 2024 May 16. pii: 2024.05.13.594007. [Epub ahead of print]
      Placental organoid models are a promising platform to study human placental development and function. Organoid systems typically use naturally derived hydrogel extracellular matrices (ECM), resulting in batch-to-batch variability that limits experimental reproducibility. As an alternative, synthetic ECM-mimicking hydrogel matrices offer greater consistency and control over environmental cues. Here, we generated trophoblast stem cell-derived placental organoids using poly(ethylene glycol) (PEG) hydrogels with tunable degradability and placenta-derived ECM cues to evaluate trophoblast differentiation relative to Matrigel and two-dimensional (2D) culture controls. Our data demonstrate that PEG hydrogels support trophoblast viability and metabolic function comparable to gold standard Matrigel. Additionally, phenotypic characterization via proteomic analysis revealed that PEG and Matrigel matrices drive syncytiotrophoblast and extravillous trophoblast-dominant placental organoid phenotypes, respectively. Further, three-dimensional (3D) environments promoted greater integrin expression and ECM production than 2D culture. This study demonstrates that engineered 3D culture environments can be used to reliably generate placental organoids and guide trophoblast differentiation.
    DOI:  https://doi.org/10.1101/2024.05.13.594007
  7. Adv Healthc Mater. 2024 May 30. e2401603
      The ability to promote three-dimensional (3D) self-organization of induced pluripotent stem cells into complex tissue structures called organoids presents new opportunities for the field of developmental biology. Brain organoids have been used to investigate principles of neurodevelopment and neuropsychiatric disorders and serve as a drug screening and discovery platform. However, brain organoid cultures are currently limited by a lacking ability to precisely control their extracellular environment. Here, we employed 3D bioprinting to generate a high-throughput, tunable, and reproducible scaffold for controlling organoid development and patterning. Additionally, our approach supports the co-culture of organoids and vascular cells in a custom architecture containing interconnected endothelialized channels. Printing fidelity and mechanical assessments confirm that fabricated scaffolds closely match intended design features and exhibit stiffness values reflective of the developing human brain. Using organoid growth, viability, cytoarchitecture, proliferation, and transcriptomic benchmarks, we found that organoids cultured within the bioprinted scaffold long-term are healthy and have expected neuroectodermal differentiation. Lastly, we confirmed that the endothelial cells in printed channel structures can migrate towards and infiltrate into the embedded organoids. This work demonstrates a tunable 3D culturing platform that can be used to create more complex and accurate models of human brain development and underlying diseases. This article is protected by copyright. All rights reserved.
    Keywords:  3D bioprinting; brain organoids; extracellular matrix; induced pluripotent stem cells; vasculature
    DOI:  https://doi.org/10.1002/adhm.202401603
  8. Adv Healthc Mater. 2024 May 29. e2400472
      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, we have created a unique platform of synthetic PEG hydrogels in which star-shaped PEG molecules were conjugated with alendronate and/or RGD peptides, attaining modifiable degradability as well as flexible cell adhesion. We identified novel reversible ionic interactions between alendronate and calcium phosphate nanoparticles, leading to versatile viscoelastic properties with varying initial elastic modulus and stress relaxation time. 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. This article is protected by copyright. All rights reserved.
    Keywords:  hydrogel; poly(ethylene glycol); self‐healing; stress relaxation; viscoelastic
    DOI:  https://doi.org/10.1002/adhm.202400472
  9. Cancer Cell Int. 2024 May 31. 24(1): 190
       BACKGROUND: Cancer-associated fibroblasts (CAFs) are the major cellular component of the tumor microenvironment and are known to affect tumor growth and response to various treatments. This study was undertaken to investigate the crosstalk between tumor-matched or unmatched CAFs and head and neck squamous cell carcinoma (HNSCC) cells regarding tumor growth and treatment response.
    METHODS: Three HNSCC cell lines (LK0412, LK0902 and LK0923), were cocultured in 2D or in 3D with their tumor-matched CAFs, site matched CAFs from other tumors or normal oral fibroblasts (NOFs). Cell proliferation was assessed as the amount of Ki67 positive cells/ spheroid area in formalin-fixed- paraffin-embedded 3D spheroids stained with Ki67 antibody. Viability after seven days of cisplatin treatment was measured with CellTiter-Glo 3D Viability Assay. The mRNA expression of CAF-associated markers (ACTA2, COL1A2, FAP, PDGFRα, PDGFRβ, PDPN, POSTN and S100A4) in CAFs before and after coculture with tumor cells as well as mRNA expression of CAF-induced genes (MMP1, MMP9 and FMOD) in tumor cells separated from CAFs after co-culture was measured with RT-qPCR. The expression of selected protein biomarkers was validated with immunohistochemistry based on previous mRNA expression results.
    RESULTS: The proliferation of the LK0412 and LK0902 tumor spheroids varied significantly when cocultured with different CAFs and NOFs as shown by Ki-67 positive cells. RT‒qPCR analysis revealed different molecular profile of the analyzed HNSCC-derived CAFs concerning the expression of CAF-associated markers. The interaction between CAFs and HNSCC cells was more pronounced after coculture with unmatched CAFs as shown by changes in mRNA expression pattern of CAF-specific markers. Additionally, the unmatched CAFs significantly upregulated the mRNA expression of MMP1, MMP9 and FMOD in tumor cells compared to tumor-matched CAFs.
    CONCLUSION: Our results indicate that tumor-matched CAFs are unique for each tumor and affect the proliferation and the gene/protein expression of tumor cells in a distinct manner. The interaction between tumor unmatched CAFs and HNSCC cells in the tumor spheroids is associated with significant changes in the mRNA expression of CAF-specific markers and significant increases in FMOD and MMP9 in tumor cells compared to when cocultured with tumor-matched CAFs. Taken together, our results show how important the selection of CAFs is to get a reliable in vitro model that mimics the patients' tumor.
    Keywords:  3D cultures; Cancer-associated fibroblasts; Cisplatin; Drug response; Head and neck squamous cell carcinoma; Proliferation
    DOI:  https://doi.org/10.1186/s12935-024-03388-0
  10. bioRxiv. 2024 May 17. pii: 2024.05.14.593924. [Epub ahead of print]
      Animal cells build actin-based surface protrusions to enable biological activities ranging from cell motility to mechanosensation to solute uptake. Long-standing models of protrusion growth suggest that actin filament polymerization provides the primary mechanical force for "pushing" the plasma membrane outward at the distal tip. Expanding on these actin-centric models, our recent studies used a chemically inducible system to establish that plasma membrane-bound myosin motors, which are abundant in protrusions and accumulate at the distal tips, can also power robust filopodial growth. How protrusion resident myosins coordinate with actin polymerization to drive elongation remains unclear, in part because the number of force generators and thus, the scale of their mechanical contributions remain undefined. To address this gap, we leveraged the SunTag system to count membrane-bound myosin motors in actively growing filopodia. Using this approach, we found that the number of myosins is log-normally distributed with a mean of 12.0 ± 2.5 motors [GeoMean ± GeoSD] per filopodium. Together with unitary force values and duty ratio estimates derived from biophysical studies for the motor used in these experiments, we calculate that a distal tip population of myosins could generate a time averaged force of ∼tens of pN to elongate filopodia. This range is comparable to the expected force production of actin polymerization in this system, a point that necessitates revision of popular physical models for protrusion growth.
    SIGNIFICANCE STATEMENT: This study describes the results of in-cell molecular counting experiments to define the number of myosin motors that are mechanically active in growing filopodia. This data should be used to constrain future physical models of the formation of actin-based protrusions.
    DOI:  https://doi.org/10.1101/2024.05.14.593924
  11. J Mech Behav Biomed Mater. 2024 May 22. pii: S1751-6161(24)00218-2. [Epub ahead of print]156 106586
      Both mechanical and adhesion properties of cancer cells are complex and reciprocally related to migration, invasion, and metastasis with large cell deformation. Therefore, we evaluated these properties for human cervical cancer cells (HeLa) simultaneously using our previously developed micro tensile tester system. For efficient evaluation, we developed image analysis software to modify the system. The software can analyze the tensile force in real time. The modified system can evaluate the tensile stiffness of cells to which a large deformation is applied, also evaluate the adhesion strength of cancer cells that adhered to a culture substrate and were cultured for several days with their adhesion maturation. We used the modified system to simultaneously evaluate the stiffness of the cancer cells to which a large deformation was applied and their adhesion strength. The obtained results revealed that the middle phase of tensile stiffness and adhesion force of the microtubule-depolymerized group treated with colchicine (an anti-cancer drug) (stiffness, 13.4 ± 7.5 nN/%; adhesion force, 460.6 ± 258.2 nN) were over two times larger than those of the control group (stiffness, 5.0 ± 3.5 nN/%; adhesion force, 168.2 ± 98.0 nN). Additionally, the same trend was confirmed with the detailed evaluation of cell surface stiffness using an atomic force microscope. Confocal fluorescence microscope observations showed that the stress fibers (SFs) of colchicine-treated cells were aligned in the same direction, and focal adhesions (FAs) of the cells developed around both ends of the SFs and aligned parallel to the developed direction of the SFs. There was a possibility that the microtubule depolymerization by the colchicine treatment induced the development of SFs and FAs and subsequently caused an increment of cell stiffness and adhesion force. From the above results, we concluded the modified system would be applicable to cancer detection and anti-cancer drug efficacy tests.
    Keywords:  Cancer cell; Cell biomechanics; Mechanical properties; Mechanobiology; Micromanipulation
    DOI:  https://doi.org/10.1016/j.jmbbm.2024.106586
  12. Acta Biomater. 2024 May 25. pii: S1742-7061(24)00259-9. [Epub ahead of print]
      Self-assembling peptide-based hydrogels have become a highly attractive scaffold for three-dimensional (3D) in vitro disease modeling as they provide a way to create tunable matrices that can resemble the extracellular matrix (ECM) of various microenvironments. Alzheimer's disease (AD) is an exceptionally complex neurodegenerative condition; however, our understanding has advanced due to the transition from two-dimensional (2D) to 3D in vitro modeling. Nonetheless, there is a current gap in knowledge regarding the role of amyloid structures, and previously developed models found long-term difficulty in creating an appropriate model involving the ECM and amyloid aggregates. In this report, we propose a multi-component self-assembling peptide-based hydrogel scaffold to mimic the amyloid-beta (β) containing microenvironment. Characterization of the amyloid-β-mimicking hydrogel (Col-HAMA-FF) reveals the formation of β-sheet structures as a result of the self-assembling properties of phenylalanine (Phe, F) through π-π stacking of the residues, thus mimicking the amyloid-β protein nanostructures. We investigated the effect of the amyloid-β-mimicking microenvironment on healthy neuronal progenitor cells (NPCs) compared to a natural-mimicking matrix (Col-HAMA). Our results demonstrated higher levels of neuroinflammation and apoptosis markers when NPCs were cultured in the amyloid-like matrix compared to a natural brain matrix. Here, we provided insights into the impact of amyloid-like structures on NPC phenotypes and behaviors. This foundational work, before progressing to more complex plaque models, provides a promising scaffold for future investigations on AD mechanisms and drug testing. STATEMENT OF SIGNIFICANCE: In this study, we engineered two multi-component hydrogels: one to mimic the natural extracellular matrix (ECM) of the brain and one to resemble an amyloid-like microenvironment using a self-assembling peptide hydrogel. The self-assembling peptide mimics β-amyloid fibrils seen in amyloid-β protein aggregates. We report on the culture of neuronal progenitor cells within the amyloid-mimicking ECM scaffold to study the impact through marker expressions related to inflammation and DNA damage. This foundational work, before progressing to more complex plaque models, offers a promising scaffold for future investigations on AD mechanisms and drug testing. This is suitable for your readership as it fits into the scope of a hypothesis-driven design of biomaterials for modeling and understanding biological interactions.
    Keywords:  Alzheimer's disease; amyloid fibrils; hydrogel; neuronal cells; peptide self-assembly
    DOI:  https://doi.org/10.1016/j.actbio.2024.05.020
  13. bioRxiv. 2024 May 14. pii: 2024.05.13.593760. [Epub ahead of print]
      Aortic valve stenosis (AVS) is a progressive disease wherein males develop valve calcification relative to females that develop valve fibrosis. Valvular interstitial cells (VICs) aberrantly activate to myofibroblasts during AVS, driving the fibrotic valve phenotype in females. Myofibroblasts further differentiate into osteoblast-like cells and secrete calcium nanoparticles, driving valve calcification in males. We hypothesized the lysine demethylase UTY (ubiquitously transcribed tetratricopeptide repeat containing, Y-linked) decreases methylation uniquely in response to nanoparticle cues in the valve extracellular matrix to promote an osteoblast-like phenotype. Here, we describe a bioinspired hydrogel cell culture platform to interrogate how nanoscale cues modulate sex-specific methylation states in VICs activating to myofibroblasts and osteoblast-like cells. We found UTY (ubiquitously transcribed tetratricopeptide repeat containing, Y-linked) modulates VIC phenotypes in response to nanoscale cues uniquely in males. Overall, we reveal a novel role of UTY in the regulation of calcification processes in males during AVS progression.
    DOI:  https://doi.org/10.1101/2024.05.13.593760
  14. Eur Phys J E Soft Matter. 2024 May 27. 47(5): 36
      Fibrous networks such as collagen are common in biological systems. Recent theoretical and experimental efforts have shed light on the mechanics of single component networks. Most real biopolymer networks, however, are composites made of elements with different rigidity. For instance, the extracellular matrix in mammalian tissues consists of stiff collagen fibers in a background matrix of flexible polymers such as hyaluronic acid (HA). The interplay between different biopolymer components in such composite networks remains unclear. In this work, we use 2D coarse-grained models to study the nonlinear strain-stiffening behavior of composites. We introduce a local volume constraint to model the incompressibility of HA. We also perform rheology experiments on composites of collagen with HA. Theoretically and experimentally, we demonstrate that the linear shear modulus of composite networks can be increased by approximately an order of magnitude above the corresponding moduli of the pure components. Our model shows that this synergistic effect can be understood in terms of the local incompressibility of HA, which acts to suppress density fluctuations of the collagen matrix with which it is entangled.
    DOI:  https://doi.org/10.1140/epje/s10189-024-00422-x
  15. Int Ophthalmol. 2024 May 25. 44(1): 229
       BACKGROUND: The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-β2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment.
    METHODS: Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-β2 (5 ng/mL). PAE (300 μM) was added to serum-starved confluent cultures of HTM cells for 2 h, followed by incubation with TGF-β2 for 22 h. SB-431542, a TGF-β receptor inhibitor (10 μM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-β2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells.
    RESULTS: PAE attenuated TGF-β2-induced oxidative stress and suppressed TGF-β2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-β2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-β2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells.
    CONCLUSION: PAE alleviates TGF-β2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.
    Keywords:  Extracellular matrix deposition; Paeoniflorin; Primary open angle glaucoma; Smad2/3; TGF-β2
    DOI:  https://doi.org/10.1007/s10792-024-02917-0
  16. Soft Matter. 2024 May 31.
      We formulate and characterize silicone gels near the gelation threshold with tunable refractive index, 1.4 < n < 1.49, and small viscoelastic moduli, G'∼1 Pa, for use in traction force microscopy. The near-critical gels have low-frequency storage plateau moduli between 50 Pa and 1 Pa, with loss moduli that are more than fifty times lower at low frequencies. The gels are linearly elastic up to strains of at least 50%. The refractive index of the gel is tuned to eliminate spherical aberrations during confocal imaging thereby minimizing signal loss when imaging through thick gel substrates. We also develop an index-matched colloidal particle, stabilized by a silicone brush, that can be dispersed throughout the gel. These particles can be used to determine the deformation of the gel. The combination of mechanical and optical properties of these near-critical gels extends the lower limit of stresses that can be measured with traction force microscopy to single mPa values, while minimizing optical aberrations.
    DOI:  https://doi.org/10.1039/d4sm00016a
  17. Sci Adv. 2024 May 31. 10(22): eadn0235
      The ability of cells to organize into tissues with proper structure and function requires the effective coordination of proliferation, migration, polarization, and differentiation across length scales. Skeletal muscle is innately anisotropic; however, few biomaterials can emulate mechanical anisotropy to determine its influence on tissue patterning without introducing confounding topography. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively polarize in the stiffest direction. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of the same chemical formulation. These findings provide valuable insights for designing biomaterials that mimic anisotropic microenvironments and underscore the importance of stiffness anisotropy in orchestrating tissue morphogenesis.
    DOI:  https://doi.org/10.1126/sciadv.adn0235
  18. JACS Au. 2024 May 27. 4(5): 2041-2049
      The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of actin networks and changes in their dynamics are related to various diseases, from actin myopathies to uncontrolled cell growth and tumorigenesis. Importantly, the biophysical and mechanical properties of actin and its assemblies are deeply intertwined with the biological functions of the cytoskeleton. Novel tools to study actin and its associated biophysical features are, therefore, of prime importance. Here we develop a new approach which exploits fluorescence lifetime imaging microscopy (FLIM) and environmentally sensitive fluorophores termed molecular rotors, acting as quantitative microviscosity sensors, to monitor dynamic viscoelastic properties of both actin structures and lipid membranes. In order to reproduce a minimal actin cortex in synthetic cell models, we encapsulated and attached actin networks to the lipid bilayer of giant unilamellar vesicles (GUVs). Using a cyanine-based molecular rotor, DiSC2(3), we show that different types of actin bundles are characterized by distinct packing, which can be spatially resolved using FLIM. Similarly, we show that a lipid bilayer-localized molecular rotor can monitor the effects of attaching cross-linked actin networks to the lipid membrane, revealing an increase in membrane viscosity upon actin attachment. Our approach bypasses constraints associated with existing methods for actin imaging, many of which lack the capability for direct visualization of biophysical properties. It can therefore contribute to a deeper understanding of the role that actin plays in fundamental biological processes and help elucidate the underlying biophysics of actin-related diseases.
    DOI:  https://doi.org/10.1021/jacsau.4c00237
  19. Biomed Mater. 2024 May 30.
      As the structural basis of connective and load-bearing tissues, collagen fibers with orientation play an important role in the mechanical properties and physiological and biochemical functions of the tissues, but viable methods for preparing scaffolds with highly oriented collagenous structure still need to be further studied. In this study, pure collagen was used as printing ink to 3D printing. Harnessing oriented collagen fiber structure by 3D printing for promoting mechanical and osteogenic properties of scaffolds. The scaffolds with different printed angles and thicknesses were prepared to fit the bone defect site and realize personalized customization. The orientation assembly of collagen fibers was promoted by shear force action of 3D printing, the regular arrangement of collagen fibers and stabilization of fiber structure were promoted by pH adjustment and glutaraldehyde cross-linking, and the collagen fibers were mineralized by cyclic mineralization method. The microscopic morphology of fiber arrangement in the scaffolds were investigated by scanning electron microscopy. Results demonstrated that collagen fibers were changed from non-oriented to oriented after 3D printing. And the tensile modulus of the scaffolds with oriented collagen fibers was nine times higher than that of the scaffolds with non-oriented fibers. Moreover, the effects of oriented collagen fibers on the proliferation, differentiation and mineralization of MC3T3-E1 cells were studied by CCK-8 assay, live/dead cell staining, alkaline phosphatase activity test, and Alizarin red staining. The results indicated that cell proliferation, differentiation and mineralization were significantly promoted by oriented collagen fibers, and the cells proliferated directionally in the direction of the fibers. Taken together, mineralized collagen fiber scaffolds with oriented collagen fibers have great potential in bone tissue engineering applications.
    Keywords:  3D printing; mineralized collagen fiber; oriented collagen fibers; tensile modulus
    DOI:  https://doi.org/10.1088/1748-605X/ad5244
  20. bioRxiv. 2024 May 13. pii: 2024.05.09.593171. [Epub ahead of print]
      Endothelia cells respond to mechanical force by stimulating cellular signaling, but how these pathways are linked to elevations in cell metabolism and whether metabolism supports the mechanical response remains poorly understood. Here, we show that application of force to VE-cadherin stimulates liver kinase B1 (LKB1) to activate AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. VE-cadherin stimulated AMPK increases eNOS activity and localization to the plasma membrane as well as reinforcement of the actin cytoskeleton and cadherin adhesion complex, and glucose uptake. We present evidence for the increase in metabolism being necessary to fortify the adhesion complex, actin cytoskeleton, and cellular alignment. Together these data extend the paradigm for how mechanotransduction and metabolism are linked to include a connection to vasodilation, thereby providing new insight into how diseases involving contractile, metabolic, and vasodilatory disturbances arise.
    DOI:  https://doi.org/10.1101/2024.05.09.593171
  21. bioRxiv. 2024 May 18. pii: 2024.05.15.594354. [Epub ahead of print]
      Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patients' overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8 + T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-β-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8 + T cells activation. Mechanistically, these CAFs downregulated components of the TGF-β pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer.
    Statement of Significance: Here we uncovered the pro-tumor roles of the synaptic protein NGL-1 in the tumor microenvironment of pancreatic cancer, defining a new target that simultaneously modulates tumor cell, fibroblast, and immune cell functions. This study reports a new pathway where NGL-1 controls TGF-β, AP-1 transcription factor members and NFAT1, modulating the immunosuppressive microenvironment in pancreatic cancer. Our findings highlight NGL-1 as a new stromal immunomodulator in pancreatic cancer.
    DOI:  https://doi.org/10.1101/2024.05.15.594354
  22. Biofabrication. 2024 May 31.
      Embedded bioprinting is an emerging technology for precise deposition of cell-laden or cell-only bioinks to construct tissue like structures. Bioink is extruded or transferred into a yield stress hydrogel or a microgel support bath allowing print needle motion during printing and providing temporal support for the printed construct. Although this technology has enabled creation of complex tissue structures, it remains a challenge to develop a support bath with user-defined extracellular mimetic cues and their spatial and temporal control. This is crucial to mimic the dynamic nature of the native tissue to better regenerate tissues and organs. To address this, we present a bioprinting approach involving printing of a photocurable viscous support layer and bioprinting of a cell-only or cell-laden bioink within this viscous layer followed by brief exposure to light to partially crosslink the support layer. This approach does not require shear thinning behavior and is suitable for a wide range of photocurable hydrogels to be used as a support. It enables multi-material printing to spatially control support hydrogel heterogeneity including temporal delivery of bioactive cues (e.g., growth factors), and precise patterning of dense multi-cellular structures within these hydrogel supports. Here, dense stem cell aggregates are printed within methacrylated hyaluronic acid-based hydrogels with patterned heterogeneity to spatially modulate human mesenchymal stem cell osteogenesis. This study has significant impactions on creating tissue interfaces (e.g., osteochondral tissue) in which spatial control of ECM properties for patterned stem cell differentiation is crucial.
    Keywords:  bioinks; bone regeneration; embedded printing; human adult mesenchymal stem cells; stem cell differentiation; tissue engineering
    DOI:  https://doi.org/10.1088/1758-5090/ad52f1
  23. ACS Appl Bio Mater. 2024 May 28.
      Fibroblastic reticular cells (FRCs) are stromal cells (SCs) that can be isolated from lymph node (LN) biopsies. Studies have shown that these nonhematopoietic cells have the capacity to shape and regulate adaptive immunity and can become a form of personalized cell therapy. Successful translational efforts, however, require the cells to be formulated as injectable units, with their native architecture preserved. The intrinsic reticular organization of FRCs, however, is lost in the monolayer cultures. Organizing FRCs into three-dimensional (3D) clusters would recapitulate their structural and functional attributes. Herein, we report a scaffolding method based on the self-assembling peptide (SAP) EAKII biotinylated at the N-terminus (EAKbt). Cross-linking with avidin transformed the EAKbt fibrils into a dense network of coacervates. The combined forces of fibrillization and bioaffinity interactions in the cross-linked EAKbt likely drove the cells into a cohesive 3D reticula. This facile method of generating clustered FRCs (clFRCs) can be completed within 10 days. In vitro clFRCs attracted the infiltration of T cells and rendered an immunosuppressive milieu in the cocultures. These results demonstrate the potential of clFRCs as a method for stromal cell delivery.
    Keywords:  3D cell culture; T cell tolerance; cell therapy; injectable scaffold; lymph node; lymph node stromal cells; self-assembling peptide; spheroids
    DOI:  https://doi.org/10.1021/acsabm.4c00331
  24. Front Mol Neurosci. 2024 ;17 1376997
      The location of the axon initial segment (AIS) at the junction between the soma and axon of neurons makes it instrumental in maintaining neural polarity and as the site for action potential generation. The AIS is also capable of large-scale relocation in an activity-dependent manner. This represents a form of homeostatic plasticity in which neurons regulate their own excitability by changing the size and/or position of the AIS. While AIS plasticity is important for proper functionality of AIS-containing neurons, the cellular and molecular mechanisms of AIS plasticity are poorly understood. Here, we analyzed changes in the AIS actin cytoskeleton during AIS plasticity using 3D structured illumination microscopy (3D-SIM). We showed that the number of longitudinal actin fibers increased transiently 3 h after plasticity induction. We further showed that actin polymerization, especially formin mediated actin polymerization, is required for AIS plasticity and formation of longitudinal actin fibers. From the formin family of proteins, Daam1 localized to the ends of longitudinal actin fibers. These results indicate that active re-organization of the actin cytoskeleton is required for proper AIS plasticity.
    Keywords:  AIS plasticity; actin; axon initial segment (AIS); formin; plasticity; super-resolution
    DOI:  https://doi.org/10.3389/fnmol.2024.1376997
  25. ArXiv. 2024 May 13. pii: arXiv:2405.07899v1. [Epub ahead of print]
      The mechanisms leading cells to acquire a fitness advantage and establish themselves in a population are paramount to understanding the development and growth of cancer. Although there are many works that study separately either the evolutionary dynamics or the mechanics of cancer, little has been done to couple evolutionary dynamics to mechanics. To address this question, we study a confluent model of tissue using a Self-Propelled Voronoi (SPV) model with stochastic growth rates that depend on the mechanical variables of the system. The SPV model is an out-of-equilibrium model of tissue derived from an energy functional that has a jamming/unjamming transition between solid-like and liquid-like states. By considering several scenarios of mutants invading a resident population in both phases, we determine the range of parameters that confer a fitness advantage and show that the preferred area and perimeter are the most relevant ones. We find that the liquid-like state is more resistant to invasion and show that the outcome of the competition can be determined from the simulation of a non-growing mixture. Moreover, a mean-field approximation can accurately predict the fate of a mutation affecting mechanical properties of a cell. Our results can be used to infer evolutionary dynamics from tissue images, understand cancer-suppressing effects of tissue mechanics, and even search for mechanics-based therapies.
  26. Int J Med Sci. 2024 ;21(7): 1307-1320
      Transforming growth factor-β (TGF-β) is strongly associated with the cell adhesion signaling pathway in cell differentiation, migration, etc. Mechanistically, TGF-β is secreted in an inactive form and localizes to the extracellular matrix (ECM) via the latent TGF-β binding protein (LTBP). However, it is the release of mature TGF-β that is essential for the activation of the TGF-β signaling pathway. This progress requires specific integrins (one of the main groups of cell adhesion molecules (CAMs)) to recognize and activate the dormant TGF-β. In addition, TGF-β regulates cell adhesion ability through modulating CAMs expression. The aberrant activation of the TGF-β signaling pathway, caused by abnormal expression of key regulatory molecules (such as Smad proteins, certain transcription factors, and non-coding RNAs), promotes tumor invasive and metastasis ability via epithelial-mesenchymal transition (EMT) during the late stages of tumorigenesis. In this paper, we summarize the crosstalk between TGF-β and cell adhesion signaling pathway in cancer and its underlying molecular mechanisms.
    Keywords:  TGF-β signaling pathway; cancer; cell adhesion signaling pathway
    DOI:  https://doi.org/10.7150/ijms.96274
  27. Front Biosci (Landmark Ed). 2024 May 16. 29(5): 192
      Chronic kidney disease (CKD) is a disorder that causes changes in both the structure and function of the kidneys, causing complications such as hypertension, edema, and oliguria. Renal fibrosis is also a common pathological feature of CKD. Matrix metalloproteinases (MMPs) are endopeptidases that degrade extracellular matrix (ECM) proteins. The proteinase domain consists of a zinc ion in the active site, which contributes to its stabilization with another zinc and three calcium structural ions. Many cellular processes are controlled by MMPs, such as cell-cell interactions and various signaling pathways, while they are also involved in degrading substrates on cell surfaces. Tissue inhibitors of metalloproteinases (TIMPs) are key regulators of metalloproteinases, and both are involved in regulating cell turnover, the regulation, and the progression of fibrosis and apoptosis in the tissue. MMPs play a role in renal fibrosis, such as the tubular cell epithelial-mesenchymal transition (TEM), activation of resident fibroblasts, endothelial-mesenchymal transition (EndoMT), and pericyte-myofibroblast transdifferentiation. This review aims to show the mechanisms through which MMPs contribute to renal fibrosis, paying particular attention to MMP-9 and the epithelial-mesenchymal transition.
    Keywords:  TIMPs; apoptosis; cell signaling; chronic kidney disease; renal fibrosis; renal tubular epithelial cells
    DOI:  https://doi.org/10.31083/j.fbl2905192