Matrix Molecules Research Articles

Reciprocal sharing of extracellular proteases and extracellular matrix molecules facilitates Bacillus subtilis biofilm formation.

Extracellular proteases are a class of public good that support growth of Bacillus subtilis when nutrients are in a polymeric form. Bacillus subtilis biofilm matrix molecules are another class of public good that are needed for biofilm formation and are prone to exploitation. In this study, we investigated the role of extracellular proteases in B. subtilis biofilm formation and explored interactions between different public good producer strains across various conditions. We confirmed that extracellular proteases support biofilm formation even when glutamic acid provides a freely available nitrogen source. Removal of AprE from the NCIB 3610 secretome adversely affects colony biofilm architecture, while sole induction of WprA activity into an otherwise extracellular protease-free strain is sufficient to promote wrinkle development within the colony biofilm. We found that changing the nutrient source used to support growth affected B. subtilis biofilm structure, hydrophobicity and architecture. We propose that the different phenotypes observed may be due to increased protease dependency for growth when a polymorphic protein presents the sole nitrogen source. We however cannot exclude that the phenotypic changes are due to alternative matrix molecules being made. Co-culture of biofilm matrix and extracellular protease mutants can rescue biofilm structure, yet reliance on extracellular proteases for growth influences population coexistence dynamics. Our findings highlight the intricate interplay between these two classes of public goods, providing insights into microbial social dynamics during biofilm formation across different ecological niches.

Diamino-functionalized metal-organic framework for selective capture of gold ions

Adsorptive recovery of valuable gold (Au) ions from wastes is vital but still challenged, especially regarding adsorption capacity and selectivity. A novel M − 3,5-DABA metal-organic framework (MOF) adsorbent was prepared via anchoring 3,5-diaminobenzoic acid (3,5-DABA) molecule in the MOF-808 matrix. Benefiting from the positive charge property, dense amino groups (3.2 mmol g−1) and high porosity, the adsorption capacity of M − 3,5-DABA reaches 1391.5 mg g−1 (pH = 2.5) and adsorption equilibrium is attained in 5 min. This amino-based material shows excellent selectivity towards various metal ions, evading the poor selectivity problem of classical thiol groups (e.g. for Ag+, Cu2+, Pb2+ and Hg2+ ions). In addition, the regeneration was easily achieved via using a hydrochloric acid-thiourea eluent. Experimental analysis and density functional theory (DFT) calculation show the amino group works as a reductant for Au(III) ions and meanwhile acts as an active site for adsorbing Au(III) ions together with the μ-OH group. Thus, M − 3,5-DABA can act as a potential adsorbent for Au(III) ions, and our work offers a viable strategy to construct novel MOF-based adsorbents.

Dental pulp mesenchymal stem cells-response to fibrin hydrogel reveals ITGA2 and MMPs expression

Regenerative endodontic procedures (REP) aim at reestablishing tooth vitality by replacing the irreversibly damaged dental pulp removed by the dental practitioner with a new functional one. The current treatment of advanced caries relies on the replacement of the inflamed or necrosed dental pulp with an inert filling material. This leads to a functional but non-vital tooth, which lacks the ability to sense dental tissue damage, and to protect from further bacterial attack. Therapeutic strategies inspired by tissue engineering called REP propose to regenerate a fully functional dental pulp directly in the canal space. Promising results were obtained using dental pulp mesenchymal stem cells (DP-MSCs) in combination with bio-inspired artificial and temporary 3D hydrogels made of extracellular matrix molecules such as collagen and fibrin biomacromolecules. However, the uncontrolled mechanisms of DP regeneration from DP-MSCs in 3D biomacromolecules fail to regenerate a fully functional DP and can induce fibrotic scarring or mineralized tissue formation to a non-negligible extent. The lack of knowledge regarding the early molecular mechanisms initiated by DP-MSCs seeded in ECM-made hydrogels is a scientific lock for REP. In this study, we investigated the early DP-MSC–response in a 3D fibrin hydrogel. DP-MSCs isolated from human third molars were cultured for 24 h in the fibrin hydrogel. The differential transcript levels of extracellular and cell surface genes were screened with 84-gene PCR array. Out of the 84 genes screened, 9 were found to be overexpressed, including those coding for the integrin alpha 2 subunit, the collagenase MMP1 and stromelysins MMP3, MMP10 and MMP12. Over-expression of ITGA2 was confirmed by RT-qPCR. The expression of alpha 2 integrin subunit protein was assessed over time by immunoblot and immunofluorescence staining. The increase in the transcript level of MMP1, MMP3, MM10 and MMP12 was confirmed by RT-qPCR. The overexpression of MMP1 and 3 at the protein level was assessed by immunoblot. MMP3 expression by DP-MSCs was observed by immunofluorescence staining. This work demonstrates overexpression of ITGA2 and of MMP1, 3, 10 and 12 by DP-MSCs cultured in a fibrin hydrogel. The main preliminary extracellular and cell surface response of the DP-MSCs to fibrin hydrogel seems to rely on a ITGA2/MMP3 axis. Further investigations are needed to precisely decipher the role of this axis in dental pulp tissue building. Nevertheless, this work identifies extracellular and cell surface molecules that could be potential checkpoints to be targeted to guide proper dental pulp tissue regeneration.

Lysophosphatidic Acid Modulates TGF-β2-Induced Biological Phenotype in Human Conjunctival Fibroblasts

Background: Although lysophosphatidic acid (LPA) is known to have multiple pathophysiological roles, its contributions to ocular tissues, especially conjunctival fibrogenesis, remain to be elucidated. Methods: To study this issue, the effects of LPA on transforming growth factor-β2 (TGF-β2)-induced fibrogenesis of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblasts (HconF) were examined by the following analyses: (1) planar proliferation determined by transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran permeability measurements, (2) real-time metabolic analyses, (3) measurements of the size and stiffness of 3D spheroids, and (4) mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: LPA had no effect on TGF-β2-induced increase in the planar proliferation of HconF cells. LPA induced a more quiescent metabolic state in 2D HconF cells, but this metabolic suppression by LPA was partially blunted in the presence of TGF-β2. In contrast, LPA caused a substantial decrease in the hardness of 3D HconF spheroids independently of TGF-β2. In agreement with these different LPA-induced effects between 2D and 3D cultured HconF cells, mRNA expressions of ECM and their modulators were differently modulated. Conclusion: The findings that LPA induced the inhibition of both TGF-β2-related and -unrelated subepithelial proliferation of HconF cells may be clinically applicable.

Combined effects of matrix stiffness and obesity-associated signaling directs progressive phenotype in PANC-1 pancreatic cancer cells in vitro.

Obesity is a leading risk factor of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor disease prognosis and outcomes. Retrospective studies have identified this link, but interactions surrounding obesity and PDAC are still unclear. Research has shifted to contributions of fibrosis (desmoplasia) on malignancy, which involves increased deposition of collagens and other extracellular matrix (ECM) molecules and increased ECM crosslinking, all of which contribute to increased tissue stiffening. However, fibrotic stiffening is underrepresented as a model feature in current PDAC models. Fibrosis is shared between PDAC and obesity, and can be leveraged for in vitro model design, as current animal obesity models of PDAC are limited in their ability to isolate individual components of fibrosis to study cell behavior. In the current study, methacrylated type I collagen (PhotoCol®) was photo-crosslinked to pathological stiffness levels to recapitulate fibrotic ECM stiffening. PANC-1 cells were encapsulated within PhotoCol®, and the tumor-tissue constructs were prepared to represent normal (healthy) (∼600 Pa) and pathological (∼2000 Pa) tissues. Separately, human mesenchymal stem cells were differentiated into adipocytes representing lean (2D differentiation) and obese fat tissue (3D collagen matrix differentiation), and conditioned media was applied to PANC-1 tumor-tissue constructs. Conditioned media from obese adipocytes showed increased vimentin expression, a hallmark of invasiveness and progression, that was not seen after exposure to media from lean adipocytes or control media. Characterization of the obese adipocyte secretome suggested that some PANC-1 differences may arise from increased interleukin-8 and -10 compared to lean adipocytes. Additionally, high matrix stiffness associated induced an amoeboid morphology in PANC-1 cells that was not present at low stiffness. Amoeboid morphology is an accessory to epithelial-to-mesenchymal transition and is used to navigate complex ECM environments. This plasticity has greater implications for treatment efficacy of metastatic cancers. Overall, this work 1) highlights the importance of investigating PDAC-obesity interactions to study the effects on disease progression and persistence, 2) establishes PhotoCol® as a matrix material that can be leveraged to study amoeboid morphology and invasion in PDAC, and 3) emphasizes the importance of integrating both biophysical and biochemical interactions associated within both pathologies for in vitro PDAC models.

The strongest protein binder is surprisingly labile.

Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.

PHOTOCHEMISTRY OF RADICAL CATIONS OF SOME ORGANIC OXYGEN-CONTAINING COMPOUNDS IN FROZEN MATRICES

The review is dedicated to the photochemical reactions of radical cations (RC) of various organic oxygen-containing compounds stabilized in low-temperature matrices. The nature of RC and the products of their photochemical reactions has been established via quantum chemistry, electron paramagnetic resonance (EPR) and low-temperature optical spectroscopy. Depending on the structure of the precursor molecule, various mechanisms of photoconversion arise for these RC: charge transfer to matrix molecules, hydrogen atom and proton transfer, isomerization, dissociation. This study allowed us to posit that there is no correlation between the structure of the molecule of the precursor molecule and the variety of available phototransformation channels for the corresponding RC in frozen matrices.

Unraveling the efficacy of verbascoside in thwarting MRSA pathogenicity by targeting sortase A

In the fight against hospital-acquired infections, the challenge posed by methicillin-resistant Staphylococcus aureus (MRSA) necessitates the development of novel treatment methods. This study focused on undermining the virulence of S. aureus, especially by targeting surface proteins crucial for bacterial adherence and evasion of the immune system. A primary aspect of our approach involves inhibiting sortase A (SrtA), a vital enzyme for attaching microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) to the bacterial cell wall, thereby reducing the pathogenicity of S. aureus. Verbascoside, a phenylethanoid glycoside, was found to be an effective SrtA inhibitor in our research. Advanced fluorescence quenching and molecular docking studies revealed a specific interaction between verbascoside and SrtA, pinpointing the critical active sites involved in this interaction. This molecular interaction significantly impedes the SrtA-mediated attachment of MSCRAMMs, resulting in a substantial reduction in bacterial adhesion, invasion, and biofilm formation. The effectiveness of verbascoside has also been demonstrated in vivo, as shown by its considerable protective effects on pneumonia and Galleria mellonella (wax moth) infection models. These findings underscore the potential of verbascoside as a promising component in new antivirulence therapies for S. aureus infections. By targeting crucial virulence factors such as SrtA, agents such as verbascoside constitute a strategic and potent approach for tackling antibiotic resistance worldwide.Key points• Verbascoside inhibits SrtA, reducing S. aureus adhesion and biofilm formation.• In vivo studies demonstrated the efficacy of verbascoside against S. aureus infections.• Targeting virulence factors such as SrtA offers new avenues against antibiotic resistance.Graphical abstract

In-situ enforcing molecular diffusion to functionalize hollow fiber carbon membranes enables efficient CO2 separations

Microporous carbon membranes with tunable micropores are attractive materials for CO2 separations. Tailoring the gas transport channels is an effective strategy to achieve high separation performance, while it remains challenging due to the lack of control over sub-nanopores. Herein, we present an in-situ molecular functionalization strategy wherein the confined sub-nanopore properties are designed by enforcing molecules to diffuse over pores and functionalize the pore affinity. By enforcing oxygen-functionalization, a strong CO2 affinity between the carbon matrix and CO2 molecules is generated, which facilitated CO2 transport, thereby enhancing the CO2 permeability by ∼4.7-fold and without sacrificing molecular sieving ability for gas mixtures, like CO2/N2 and CO2/CH4. The functionalization mechanism was confirmed using reactive force field molecular dynamics (ReaxFF-MD) simulations. Furthermore, this strategy, which was directly applied to hollow fiber membrane modules, provides a facile and scalable approach, and expands the currently limited library of penetrative micropore tailoring of microporous membranes.

Asporin expression is associated with human atherosclerotic plaque integrity

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): This work was supported by the Swedish Research Council, the Swedish Heart and Lung Foundation, Skåne University Hospital funds Background Calcification serves as a clinical marker of atherosclerotic plaque burden. Recent studies have also shed light on its intricate association with plaque vulnerability, likely depend on calcification structures and the surrounding environment. Asporin (ASPN) is a member of the small-leucine-rich proteoglycans that contains a unique and well-conserved stretch of aspartate (D) residues, which mimic the role of glycosaminoglycans. Via its D-repeat, ASPN can interact with calcium and several other extracellular matrix molecules, thus influencing collagen mineralization and TGF-β signaling. ASPN expression has been shown to be higher in asymptomatic compared to symptomatic atherosclerotic plaques, suggesting a potential protective role. However, the role of ASPN in maintaining plaque integrity remains unknown. Purpose Here we aimed to explore the role of ASPN in atherosclerosis, by investigating if ASPN was expressed in human carotid plaques and by determining if ASPN is associated with risk for postoperative cardiovascular events and death. Methods ASPN and TGF-β protein levels were measured by ELISA and Milliplex in 176 human plaque tissue homogenates obtained from the Carotid Plaque Imaging Project (CPIP) biobank. Plaque tissue sections were stained for ASPN, destabilizing (CD68, oil red O, glycophorin A) and stabilizing plaque components (Movat, α-actin). Plaque tissue RNA expression levels were assessed in 82 plaques using bulk RNA sequencing. Plaque levels of phosphate, elastin and collagen were measured using ELISA. Results ASPN protein levels were higher in plaques from asymptomatic patients compared to symptomatic patients (p=0.011). ASPN RNA levels correlated positively to RNA levels of the smooth muscle cell markers ACTA2, MYH11 and TAGLN. Moreover, protein levels correlated with plaque levels of phosphate (r=0.258, p=0.001), elastin (r=0.423, p<0.001), collagen (r=0.248, p=0.002) and stability factors like the TGF-β family members, TGF-β1 (r=0.176, p=0.035), TGF-β2 (r=0.581, p<0.001) and TGF-β3 (r=0.396, p<0.001). Furthermore, plaque ASPN protein concentration, correlated inversely with a histological plaque vulnerability index (r=-0.265, p=0.0029), which is ratio between the plaque area of destabilizing (macrophages, lipids, intra-plaque hemorrhage) and stabilizing plaque components (collagen and smooth muscle cells). Additionally, above median ASPN levels were associated with lower risk for postoperative cardiovascular events (p=0.008) and death (p=0.009). Conclusion Our findings suggest that elevated levels of ASPN might have a crucial role in maintaining plaque stability, affecting plaque calcification.

Targeting F-actin stress fibers to suppress the dedifferentiated phenotype in chondrocytes

Actin is a central mediator of the chondrocyte phenotype. Monolayer expansion of articular chondrocytes on tissue culture polystyrene, for cell-based repair therapies, leads to chondrocyte dedifferentiation. During dedifferentiation, chondrocytes spread and filamentous (F-)actin reorganizes from a cortical to a stress fiber arrangement causing a reduction in cartilage matrix expression and an increase in fibroblastic matrix and contractile molecule expression. While the downstream mechanisms regulating chondrocyte molecular expression by alterations in F-actin organization have become elucidated, the critical upstream regulators of F-actin networks in chondrocytes are not completely known. Tropomyosin (TPM) and the RhoGTPases are known regulators of F-actin networks. The main purpose of this study is to elucidate the regulation of passaged chondrocyte F-actin stress fiber networks and cell phenotype by the specific TPM, TPM3.1, and the RhoGTPase, CDC42. Our results demonstrated that TPM3.1 associates with cortical F-actin and stress fiber F-actin in primary and passaged chondrocytes, respectively. In passaged cells, we found that pharmacological TPM3.1 inhibition or siRNA knockdown causes F-actin reorganization from stress fibers back to cortical F-actin and causes an increase in G/F-actin. CDC42 inhibition also causes formation of cortical F-actin. However, pharmacological CDC42 inhibition, but not TPM3.1 inhibition, leads to the re-association of TPM3.1 with cortical F-actin. Both TPM3.1 and CDC42 inhibition, as well as TPM3.1 knockdown, reduces nuclear localization of myocardin related transcription factor, which suppresses dedifferentiated molecule expression. We confirmed that TPM3.1 or CDC42 inhibition partially redifferentiates passaged cells by reducing fibroblast matrix and contractile expression, and increasing chondrogenic SOX9 expression. A further understanding on the regulation of F-actin in passaged cells may lead into new insights to stimulate cartilage matrix expression in cells for regenerative therapies.

Identification of established and novel extracellular matrix components in glioblastoma as targets for angiogenesis and prognosis.

Glioblastomas (GBM) are aggressive tumors known for their heterogeneity, rapid proliferation, treatment resistance, and extensive vasculature. Angiogenesis, the formation of new vessels, involves endothelial cell (EC) migration and proliferation. Various extracellular matrix (ECM) molecules regulate EC survival, migration, and proliferation. Culturing human brain EC (HBMEC) on GBM-derived ECM revealed a decrease in EC numbers compared to controls. Through in silico analysis, we explored ECM gene expression differences between GBM and brain normal glia cells and the impact of GBM microenvironment on EC ECM transcripts. ECM molecules such as collagen alpha chains (COL4A1, COL4A2, p < 0.0001); laminin alpha (LAMA4), beta (LAMB2), and gamma (LAMC1) chains (p < 0.0005); neurocan (NCAN), brevican (BCAN) and versican (VCAN) (p < 0.0005); hyaluronan synthase (HAS) 2 and metalloprotease (MMP) 2 (p < 0.005); MMP inhibitors (TIMP1-4, p < 0.0005), transforming growth factor beta-1 (TGFB1) and integrin alpha (ITGA3/5) (p < 0.05) and beta (ITGB1, p < 0.0005) chains showed increased expression in GBM. Additionally, GBM-influenced EC exhibited elevated expression of COL5A3, COL6A1, COL22A1 and COL27A1 (p < 0.01); LAMA1, LAMB1 (p < 0.001); fibulins (FBLN1/2, p < 0.01); MMP9, HAS1, ITGA3, TGFB1, and wingless-related integration site 9B (WNT9B) (p < 0.01) compared to normal EC. Some of these molecules: COL5A1/3, COL6A1, COL22/27A1, FBLN1/2, ITGA3/5, ITGB1 and LAMA1/B1 (p < 0.01); NCAN, HAS1, MMP2/9, TIMP1/2 and TGFB1 (p < 0.05) correlated with GBM patient survival. In conclusion, this study identified both established and novel ECM molecules regulating GBM angiogenesis, suggesting NCAN and COL27A1 are new potential prognostic biomarkers for GBM.

Effect of surface engineering of nanofillers using plasma-treated water for carbon dioxide capture in mixed matrix membranes: Experimental and molecular dynamics

This work introduces an innovative approach to enhance carbon dioxide (CO2) capture performance in mixed-matrix membranes (MMMs) by incorporating plasma-treated ceria (CeO2) nanoparticles into a polyurethane (PU)-based membrane. Functionalization of CeO2 by means of plasma aimed at improving interfacial interactions with both polymer matrix and CO2 gas molecules, with the consequent enhancement of CeO2 nanoparticles dispersion within the matrix and improvement of gas separation performance. Based on the results of the gas separation tests, the PU membrane containing 5 wt% plasma-treated CeO2 exhibited a CO2 permeability of 51.2 barrer and a CO2/N2 ideal selectivity of approximately 76.5, demonstrating a 26.1% increase in CO2/N2 ideal selectivity compared to the pristine PU membrane. However, a further increase in filler content to 10 wt% led to nanoparticle agglomeration, resulting in a decrease in selectivity due to the formation of non-selective pathways through the membrane. Molecular dynamics (MD) simulations corroborated these experimental findings, providing additional insights into the interaction mechanisms between the polymer matrix and the functionalized nanoparticles.

Scalable fabrication of red Room-Temperature phosphorescent materials through molecular doping strategy for versatile applications

Room-temperature phosphorescence (RTP) materials renowned for their long emission lifetime have garnered tremendous research interest not only for elucidating their underlying mechanisms but also for exploring their potential applications in anti-counterfeiting, encryption, and bioimaging. Nevertheless, the RTP color of most organic materials has been limited to blue, green, and yellow. In this study, a series of red RTP materials have been successfully fabricated at a large scale through a straightforward molecular doping strategy. Remarkably, the doped materials exhibited red RTP at 622 nm and 678 nm with an ultralong lifetime of 373 ms and an efficiency of 5.21 %, which was among the best performances reported for organic RTP materials. The systematic investigation unveiled that the efficient RTP emanated from the robust interactions between the emitter and matrix molecules as well as the formation of D/A exciplexes. Furthermore, the red RTP materials could be easily manufactured into diverse forms, including fine powders, aqueous inks, and nanoparticles. Their multifunctional applications in fingerprint recognition, encryption, and live bioimaging were demonstrated. With distinctive advantages such as red RTP, ultralong lifetime, scalable production, and cost-effectiveness, these RTP materials pave the way for the extensive utilization of organic RTP materials in various engineering sectors.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC–MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

Binding requirements for latent transforming growth factor Beta2 activation

Although the mechanism for activation of latent TGFβ1 and TGFβ3 is understood to involve the binding of the TGFβ propeptide (LAP) to both an integrin and an insoluble substrate, the activation of latent TGFβ2 has been unclear because the TGFβ2 LAP does not have the classical integrin binding sequence found in the other two TGFβ isoform LAPs. To assess the potential requirement for covalent linkage with a matrix or cell surface protein for the activation of latent TGFβ2, we generated mice in which the TGFβ2 Cys residue predicted to be involved in binding was mutated to Ser (Tgfb2C24S). We reasoned that, if covalent interaction with a second molecule is required for latent TGFβ2 activation, mutant mice should display a Tgfb2 null (Tgfb2−/−)-like phenotype. Tgfb2C24S mice closely phenocopy Tgfb2−/− mice with death in utero between E18 and P1 and with congenital heart and kidney defects similar to those described for Tgfb2−/− mice. The mutant latent TGFβ2 is secreted at levels similar to WT, yet TGFβ signaling monitored as nuclear pSmad2 is suppressed. We conclude that, like latent TGFβ1, latent TGFβ2 activation requires binding to an immobilized matrix or plasma membrane molecule.

Presence of XX Sex Chromosome Complement Increases Aortic Circumferential Stiffness in Female Mice

Elastin and collagen uniquely adapt to the mechanical demands of high pulsatile pressure in the vascular system in conjunction with the extracellular matrix (ECM). Elastic fiber networks and collagen are affected by component disorganization, leading to an array of vascular diseases and compromised mechanical behavior. It is shown that women than men have accelerated arterial stiffening. Previously, we have shown increased axial and circumferential linearized material stiffness in the carotid arteries of female than male wildtype mice. Whether sex hormones interact with sex chromosomes is unknown. Therefore, we hypothesize that sex hormones interact with sex chromosomes to increase aortic circumferential stiffening in female than male mice. In this study, we used gonadal intact 15-week-old four core genotype mice (n=4-6/group) comprising female (F) and male (M) mice with either XX or XY sex chromosome complement (SCC). Using a tissue puller, we assessed the stress-strain and displacement-force relationship of the abdominal aorta. Briefly, the aorta was cut into 3mm rings and pulled apart to a range of 1300 μm with a velocity of 35 μm/sec. To determine the load-bearing effect of collagen and elastin, collagenase type II (16 units) and elastase (5 units) were used to digest the aortic ring before pulling. Part of the aortic ring was digested for a PCR array of extracellular matrix and adhesion molecules. We observed a leftward shift of stress-strain in XXF than in XXM (P=0.03). A leftward shift of stress-strain was also indicated in XXF than in XYM mice (P=0.004). Displacement-force tests showed a similar effect with a leftward shift in XXF than XXM (P=0.01). A leftward shift of displacement force was also indicated in XXF than in XYM (P=0.004). Aorta digestion with elastase reversed the phenotype, showing a leftward shift of displacement force in XXM than in XXF (P=0.02). Collagenase digestion removed the observed sex differences in stress-strain and displacement force tests. The PCR array comparing XXF and XXM showed increased fold change in Adamts5 (4.2), Emilin (2.2), Icam1 (2.4), and Thbs2 (3). We also observed the downregulation of Cntn1 (0.4), Cntnna1 (0.5), Ecm1 (0.3), and Fbln1 (4). Our data suggested that XX sex chromosome complement, and ECM genes contribute to the structural stiffness of female mice aorta. Our study will contribute to understanding connective tissue diseases, including arterial stiffness, aortic aneurysms, Marfan and Danlos syndrome. HL105400 and HL155841. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HIF-1α-mediated LAMC1 overexpression is an unfavorable predictor of prognosis for glioma patients: evidence from pan-cancer analysis and validation experiments

BackgroundLaminin subunit gamma-1 (LAMC1) is a major extracellular matrix molecule involved in the tumor microenvironment. Knowledge of the biological features and clinical relevance of LAMC1 in cancers remains limited.MethodsWe conducted comprehensive bioinformatics analysis of LAMC1 gene expression and clinical relevance in pan-cancer datasets of public databases and validated LAMC1 expression in glioma tissues and cell lines. The association and regulatory mechanism between hypoxia inducible factor-1α (HIF-1α) and LAMC1 expression were explored.ResultsLAMC1 expression in most cancers in The Cancer Genome Atlas (TCGA) including glioma was significantly higher than that in normal tissues, which had a poor prognosis and were related to various clinicopathological features. Data from the Chinese Glioma Genome Atlas also showed high expression of LAMC1 in glioma associated with poor prognoses. In clinical glioma tissues, LAMC1 protein was highly expressed and correlated to poor overall survival. LAMC1 knockdown in Hs683 glioma cells attenuated cell proliferation, migration, and invasion, while overexpression of LAMC1 in U251 cells leads to the opposite trend. Most TCGA solid cancers including glioma showed enhancement of HIF-1α expression. High HIF-1α expression leads to adverse prognosis in gliomas, besides, HIF-1α expression was positively related to LAMC1. Mechanistically, HIF-1α directly upregulated LAMC1 promotor activity. Hypoxia (2% O2)-treated Hs683 and U251 cells exhibited upregulated HIF-1α and LAMC1 expression, which was significantly attenuated by HIF-1α inhibitor YC-1 and accompanied by attenuated cell proliferation and invasion.ConclusionsHigh expression of LAMC1 in some solid tumors including gliomas suggests a poor prognosis. The hypoxic microenvironment in gliomas activates the HIF-1α/LAMC1 signaling, thereby promoting tumor progression. Targeted intervention on the HIF-1α/LAMC1 signaling attenuates cell growth and invasion, suggesting a new strategy for glioma treatment.

Hyaluronan-based hydrogel delivering glucose to mesenchymal stem cells intended to treat osteoarthritis

Mesenchymal stem cell (MSC) therapy shows promise in regenerative medicine. For osteoarthritis (OA), MSCs delivered to the joint have a temporal window in which they can secrete growth factors and extracellular matrix molecules, contributing to cartilage regeneration and cell proliferation. However, upon injection in the non-vascularized joint, MSCs lacking energy supply, starve and die too quickly to efficiently deliver enough of these factors. To feed injected MSCs, we developed a hyaluronic acid (HA) derivative, where glucose is covalently bound to hyaluronic acid. To achieve this, the glucose moiety in 4-aminophenyl-β-D-glucopyranoside was linked to the HA backbone through amidation. The hydrogel was able to deliver glucose in a controlled manner using a trigger system based on hydrolysis catalyzed by endogenous ß-glucosidase. This led to glucose release from the hyaluronic acid backbone inside the cell. Indeed, our hydrogel proved to rescue starvation and cell mortality in a glucose-free medium. Our approach of adding a nutrient to the polymer backbone in hydrogels opens new avenues to deliver stem cells in poorly vascularized, nutrient-deficient environments, such as osteoarthritic joints, and for other regenerative therapies.

Generation of human alveolar epithelial type I cells from pluripotent stem cells

Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here, we engineer a human invitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, enriched in these cells. Next, we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an invitro model of human alveolar epithelial differentiation and a potential source of human AT1s.