Corrigendum to Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction [Research and Practice in Thrombosis and Haemostasis Volume 8, Issue 1, January 2024, 102322].

[This corrects the article DOI: 10.1016/j.rpth.2024.102322.].

Salmonella-driven intestinal edema in mice is characterized by tensed fibronectin fibers.

Intestinal edema is a common manifestation of numerous gastrointestinal diseases and is characterized by the accumulation of fluid in the interstitial space of the intestinal wall. Technical advances in laser capture microdissection and low-biomass proteomics now allow us to specifically characterize the intestinal edema proteome. Using advanced proteomics, we identify peptides derived from antimicrobial factors with high signal intensity, but also highlight major contributions from the blood clotting system, extracellular matrix (ECM) and protease-protease inhibitor networks. The ECM is a complex fibrillar network of macromolecules that provides structural and mechanical support to the intestinal tissue. One abundant component of the ECM observed in Salmonella-driven intestinal edema is the glycoprotein fibronectin, recognized for its structure-function interplay regulated by mechanical forces. Using mechanosensitive staining of fibronectin fibers reveals that they are tensed in the edema, despite the high abundance of proteases able to cleave fibronectin. In contrast, fibronectin fibers increasingly relax in other cecal tissue areas as the infection progresses. Co-staining for fibrin(ogen) indicates the formation of a provisional matrix in the edema, similar to what is observed in response to skin injury, while collagen staining reveals a sparse and disrupted collagen fiber network. These observations plus the absence of low tensional fibronectin fibers and the additional finding of a high number of protease inhibitors in the edema proteome could indicate a critical role of stretched fibronectin fibers in maintaining tissue integrity in the severely inflamed cecum. Understanding these processes may also provide valuable functional diagnostic markers of intestinal disease progression in the future.

Contractility defects hinder glycoprotein VI-mediated platelet activation and affect platelet functions beyond clot contraction.

Background: Active and passive biomechanical properties of platelets contribute substantially to thrombus formation. Actomyosin contractility drives clot contraction required for stabilizing the hemostatic plug. Impaired contractility results in bleeding but is difficult to detect using platelet function tests. Objectives: To determine how diminished myosin activity affects platelet functions, including and beyond clot contraction. Methods: Using the myosin IIA-specific pharmacologic inhibitor blebbistatin, we modulated myosin activity in platelets from healthy donors and systematically characterized platelet responses at various levels of inhibition by interrogating distinct platelet functions at each stage of thrombus formation using a range of complementary assays. Results: Partial myosin IIA inhibition neither affected platelet von Willebrand factor interactions under arterial shear nor platelet spreading and cytoskeletal rearrangements on fibrinogen. However, it impacted stress fiber formation and the nanoarchitecture of cell-matrix adhesions, drastically reducing and limiting traction forces. Higher blebbistatin concentrations impaired platelet adhesion under flow, altered mechanosensing at lamellipodia edges, and eliminated traction forces without affecting platelet spreading, α-granule secretion, or procoagulant platelet formation. Unexpectedly, myosin IIA inhibition reduced calcium influx, dense granule secretion, and platelet aggregation downstream of glycoprotein (GP)VI and limited the redistribution of GPVI on the cell membrane, whereas aggregation induced by adenosine diphosphate or arachidonic acid was unaffected. Conclusion: Our findings highlight the importance of both active contractile and passive crosslinking roles of myosin IIA in the platelet cytoskeleton. They support the hypothesis that highly contractile platelets are needed for hemostasis and further suggest a supportive role for myosin IIA in GPVI signaling.

Relaxed fibronectin: a potential novel target for imaging endometriotic lesions.

BACKGROUND: Endometriosis is characterized by the ectopic occurrence of endometrial tissue. Though considered benign, endometriotic lesions possess tumor-like properties such as tissue invasion and remodeling of the extracellular matrix. One major clinical hurdle concerning endometriosis is its diagnosis. The diagnostic modalities ultrasound and MRI are often unable to detect all lesions, and a clear correlation between imaging and clinical symptoms is still controversial. Therefore, it was our aim to identify a potential target to image active endometriotic lesions. RESULTS: For our studies, we employed the preclinical radiotracer [111In]In-FnBPA5, which specifically binds to relaxed fibronectin-an extracellular matrix protein with key functions in homeostasis that has been implicated in the pathogenesis of diseases such as cancer and fibrosis. We employed this tracer in biodistribution as well as SPECT/CT studies in mice and conducted immunohistochemical stainings on mouse uterine tissue as well as on patient-derived endometriosis tissue. In biodistribution and SPECT/CT studies using the radiotracer [111In]In-FnBPA5, we found that radiotracer uptake in the myometrium varies with the estrous cycle of the mouse, leading to higher uptake of [111In]In-FnBPA5 during estrogen-dependent phases, which indicates an increased abundance of relaxed fibronectin when estrogen levels are high. Finally, immunohistochemical analysis of patient samples demonstrated that there is preferential relaxation of fibronectin in the proximity of the endometriotic stroma. CONCLUSION: Estrous cycle stages characterized by high estrogen levels result in a higher abundance of relaxed fibronectin in the murine myometrium. This finding together with a first proof-of-concept study employing human endometriosis tissues suggests that relaxed fibronectin could be a potential target for the development of a diagnostic radiotracer targeting endometriotic lesions. With [111In]In-FnBPA5, the matching targeting molecule is in preclinical development.

Advancing the Design of Artificial Nano-organelles for Targeted Cellular Detoxification of Reactive Oxygen Species.

Artificial organelles (AnOs) are in the spotlight as systems to supplement biochemical pathways in cells. While polymersome-based artificial organelles containing enzymes to reduce reactive oxygen species (ROS) are known, applications requiring control of their enzymatic activity and cell-targeting to promote intracellular ROS detoxification are underexplored. Here, we introduce advanced AnOs where the chemical composition of the membrane supports the insertion of pore-forming melittin, enabling molecular exchange between the AnO cavity and the environment, while the encapsulated lactoperoxidase (LPO) maintains its catalytic function. We show that H2O2 outside AnOs penetrates through the melittin pores and is rapidly degraded by the encapsulated enzyme. As surface attachment of cell-penetrating peptides facilitates AnOs uptake by cells, electron spin resonance revealed a remarkable enhancement in intracellular ROS detoxification by these cell-targeted AnOs compared to nontargeted AnOs, thereby opening new avenues for a significant reduction of oxidative stress in cells.

External Mechanical Stability Regulates Hematoma Vascularization in Bone Healing Rather than Endothelial YAP/TAZ Mechanotransduction.

Bone fracture healing is regulated by mechanobiological cues. Both, extracellular matrix (ECM) deposition and microvascular assembly determine the dynamics of the regenerative processes. Mechanical instability as by inter-fragmentary shear or compression is known to influence early ECM formation and wound healing. However, it remains unclear how these external cues shape subsequent ECM and microvascular network assembly. As transcriptional coactivators, the mechanotransducers yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) translate physical cues into downstream signaling events, yet their role in sprouting angiogenesis into the hematoma after injury is unknown. Using bone healing as model system for scar-free regeneration, the role of endothelial YAP/TAZ in combination with tuning the extrinsic mechanical stability via fracture fixation is investigated. Extrinsically imposed shear across the gap delayed hematoma remodeling and shaped the morphology of early collagen fiber orientations and microvascular networks, suggesting that enhanced shear increased the nutrient exchange in the hematoma. In contrast, endothelial YAP/TAZ deletion has little impact on the overall vascularization of the fracture gap, yet slightly increases the collagen fiber deposition under semi-rigid fixation. Together, these data provide novel insights into the respective roles of endothelial YAP/TAZ and extrinsic mechanical cues in orchestrating the process of bone regeneration.

Longevity interventions modulate mechanotransduction and extracellular matrix homeostasis in C. elegans.

Dysfunctional extracellular matrices (ECM) contribute to aging and disease. Repairing dysfunctional ECM could potentially prevent age-related pathologies. Interventions promoting longevity also impact ECM gene expression. However, the role of ECM composition changes in healthy aging remains unclear. Here we perform proteomics and in-vivo monitoring to systematically investigate ECM composition (matreotype) during aging in C. elegans revealing three distinct collagen dynamics. Longevity interventions slow age-related collagen stiffening and prolong the expression of collagens that are turned over. These prolonged collagen dynamics are mediated by a mechanical feedback loop of hemidesmosome-containing structures that span from the exoskeletal ECM through the hypodermis, basement membrane ECM, to the muscles, coupling mechanical forces to adjust ECM gene expression and longevity via the transcriptional co-activator YAP-1 across tissues. Our results provide in-vivo evidence that coordinated ECM remodeling through mechanotransduction is required and sufficient to promote longevity, offering potential avenues for interventions targeting ECM dynamics.

Transglutaminase 2 has higher affinity for relaxed than for stretched fibronectin fibers.

Transglutaminase 2 (TG2) plays a vital role in stabilizing extracellular matrix (ECM) proteins through enzymatic crosslinking during tissue growth, repair, and inflammation. TG2 also binds non-covalently to fibronectin (FN), an essential component of the ECM, facilitating cell adhesion, migration, proliferation, and survival. However, the interaction between TG2 and fibrillar FN remains poorly understood, as most studies have focused on soluble or surface-adsorbed FN or FN fragments, which differ in their conformations from insoluble FN fibers. Using a well-established in vitro FN fiber stretch assay, we discovered that the binding of a crosslinking enzyme to ECM fibers is mechano-regulated. TG2 binding to FN is tuned by the mechanical tension of FN fibers, whereby TG2 predominantly co-localizes to low-tension FN fibers, while fiber stretching reduces their affinity for TG2. This mechano-regulated binding relies on the proximity between the N-terminal ß-sandwich and C-terminal ß-barrels of TG2. Crosslinking mass spectrometry (XL-MS) revealed a novel TG2-FN synergy site within TG2's C-terminal ß-barrels that interacts with FN regions located outside of the canonical gelatin binding domain, specifically FNI2 and FNIII14-15. Combining XL-MS distance restraints with molecular docking revealed the mechano-regulated binding mechanism between TG2 and modules FNI7-9 by which mechanical forces regulate TG2-FN interactions. This highlights a previously unrecognized role of TG2 as a tension sensor for FN fibers. This novel interaction mechanism has significant implications in physiology and mechanobiology, including how forces regulate cell adhesion, spreading, migration, phenotype modulation, depending on the tensional state of ECM fibers. Data are available via ProteomeXchange with identifier PXD043976.

Molecular dynamics of JUNO-IZUMO1 complexation suggests biologically relevant mechanisms in fertilization.

JUNO-IZUMO1 binding is the first known physical link created between the sperm and egg membranes in fertilization, however, how this initiates sperm-egg fusion remains elusive. As advanced structural insights will help to combat the infertility crisis, or advance fertility control, we employed all-atom Molecular Dynamics (MD) to derive dynamic structural insights that are difficult to obtain experimentally. We found that the hydrated JUNO-IZUMO1 interface is composed of a large set of short-lived non-covalent interactions. The contact interface is destabilized by strategically located point mutations, as well as by Zn2+ ions, which shift IZUMO1 into the non-binding "boomerang" conformation. We hypothesize that the latter might explain how the transient zinc spark, as released after sperm entry into the oocyte, might contribute to block polyspermy. To address a second mystery, we performed another set of simulations, as it was previously suggested that JUNO in solution is unable to bind to folate despite it belonging to the folate receptor family. MD now suggests that JUNO complexation with IZUMO1 opens up the binding pocket thereby enabling folate insertion. Our MD simulations thus provide crucial new hypotheses how the dynamics of the JUNO-IZUMO1 complex upon solvation might regulate fertility.

Extracellular matrix remodelling in dental pulp tissue of carious human teeth through the prism of single-cell RNA sequencing.

Carious lesions are bacteria-caused destructions of the mineralised dental tissues, marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue. While major molecular players in tooth decay have been uncovered during the past years, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. In this study we used single-cell RNA sequencing analysis, supplemented with immunostaining, to generate a comprehensive single-cell atlas of the pulp of carious human teeth. Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the pulp of healthy human teeth. Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters. These changes include the upregulation of genes encoding extracellular matrix (ECM) components, including COL1A1 and Fibronectin (FN1), and the enrichment of the fibroblast cluster with myofibroblasts. The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses. Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues, compared to the healthy ones. The present data demonstrate molecular, cellular and biomechanical alterations in the pulp of human carious teeth, indicative of extensive ECM remodelling, reminiscent of fibrosis observed in other organs. This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.

Growth factors and mechano-regulated reciprocal crosstalk with extracellular matrix tune the keratocyte-fibroblast/myofibroblast transition.

Improper healing of the cornea after injury, infections or surgery can lead to corneal scar formation, which is associated with the transition of resident corneal keratocytes into activated fibroblasts and myofibroblasts (K-F/M). Myofibroblasts can create an extracellular matrix (ECM) niche in which fibrosis is promoted and perpetuated, resulting in progressive tissue opacification and vision loss. As a reversion back to quiescent keratocytes is essential to restore corneal transparency after injury, we characterized how growth factors with demonstrated profibrotic effects (PDGF, FGF, FBS, TGFß1) induce the K-F/M transition, and whether their withdrawal can revert it. Indeed, the upregulated expression of αSMA and the associated changes in cytoskeletal architecture correlated with increases in cell contractility, fibronectin (Fn) and collagen matrix density and Fn fiber strain, as revealed by 2D cell culture, nanopillar cellular force mapping and a FRET-labeled Fn tension probe. Substrate mechanosensing drove a more complete K-F/M transition reversal following growth factor withdrawal on nanopillar arrays than on planar glass substrates. Using decellularized ECM scaffolds, we demonstrated that the K-F/M transition was inhibited in keratocytes reseeded onto myofibroblast-assembled, and/or collagen-1-rich ECM. This supports the presence of a myofibroblast-derived ECM niche that contains cues favoring tissue homeostasis rather than fibrosis.

How the mechanobiology orchestrates the iterative and reciprocal ECM-cell cross-talk that drives microtissue growth.

Controlled tissue growth is essential for multicellular life and requires tight spatiotemporal control over cell proliferation and differentiation until reaching homeostasis. As cells synthesize and remodel extracellular matrix, tissue growth processes can only be understood if the reciprocal feedback between cells and their environment is revealed. Using de novo-grown microtissues, we identified crucial actors of the mechanoregulated events, which iteratively orchestrate a sharp transition from tissue growth to maturation, requiring a myofibroblast-to-fibroblast transition. Cellular decision-making occurs when fibronectin fiber tension switches from highly stretched to relaxed, and it requires the transiently up-regulated appearance of tenascin-C and tissue transglutaminase, matrix metalloprotease activity, as well as a switch from α5ß1 to α2ß1 integrin engagement and epidermal growth factor receptor signaling. As myofibroblasts are associated with wound healing and inflammatory or fibrotic diseases, crucial knowledge needed to advance regenerative strategies or to counter fibrosis and cancer progression has been gained.

Nucleolin promotes angiogenesis and endothelial metabolism along the oncofetal axis in the human brain vasculature.

Glioblastomas are among the deadliest human cancers and are highly vascularized. Angiogenesis is dynamic during brain development, almost quiescent in the adult brain but reactivated in vascular-dependent CNS pathologies, including brain tumors. The oncofetal axis describes the reactivation of fetal programs in tumors, but its relevance in endothelial and perivascular cells of the human brain vasculature in glial brain tumors is unexplored. Nucleolin is a regulator of cell proliferation and angiogenesis, but its roles in the brain vasculature remain unknown. Here, we studied the expression of Nucleolin in the neurovascular unit in human fetal brains, adult brains, and human gliomas in vivo as well as its effects on sprouting angiogenesis and endothelial metabolism in vitro. Nucleolin is highly expressed in endothelial and perivascular cells during brain development, downregulated in the adult brain, and upregulated in glioma. Moreover, Nucleolin expression correlated with glioma malignancy in vivo. In culture, siRNA-mediated Nucleolin knockdown reduced human brain endothelial cell (HCMEC) and HUVEC sprouting angiogenesis, proliferation, filopodia extension, and glucose metabolism. Furthermore, inhibition of Nucleolin with the aptamer AS1411 decreased brain endothelial cell proliferation in vitro. Mechanistically, Nucleolin knockdown in HCMECs and HUVECs uncovered regulation of angiogenesis involving VEGFR2 and of endothelial glycolysis. These findings identify Nucleolin as a neurodevelopmental factor reactivated in glioma that promotes sprouting angiogenesis and endothelial metabolism, characterizing Nucleolin as an oncofetal protein. Our findings have potential implications in the therapeutic targeting of glioma.

Infiltrating CD8+ T cells and M2 macrophages are retained in tumor matrix tracks enriched in low tension fibronectin fibers.

Tracks rich in matrix and cells, as described in several cancer types, have immunosuppressive functions and separate tumor nests and stroma, yet their origin is unknown. Immunostainings of cryosections from mouse breast tumors show that these tracks are bordered by an endothelial-like basement membrane, filled with fibers of collagen adjacent to tenascin-C (TNC) and low-tension fibronectin (Fn) fibers. While present in early-stage tumors and maturing with time, tracks still form under TNC KO conditions, however, host (not tumor cell)-derived TNC is important for track maturation. Tumor infiltrating leukocytes (mostly M2 macrophages and CD8+ T cells) are retained in tracks of early-stage tumors. Following track maturation, retained tumor infiltrating leukocyte (TIL) numbers get reduced and more CD8+ TIL enter the tumor nests in the absence of TNC. As these tracks are enriched with platelets and fibrinogen and have a demarcating endothelial-like basement membrane often adjacent to endothelial cells, this suggests a role of blood vessels in the formation of these tracks. The Fn fiber tension probe FnBPA5 colocalizes with TNC and immune cells in the tracks and shows decreased binding in tracks lacking TNC. Consequently, FnBPA5 can serve as probe for tumor matrix tracks that have immune suppressive properties.

Blockage of lamin-A/C loss diminishes the pro-inflammatory macrophage response.

Mutations and defects in nuclear lamins can cause major pathologies, including inflammation and inflammatory diseases. Yet, the underlying molecular mechanisms are not known. We now report that the pro-inflammatory activation of macrophages, as induced by LPS or pathogenic E. coli, reduces Lamin-A/C levels thereby augmenting pro-inflammatory gene expression and cytokine secretion. We show that the activation of bone-marrow-derived macrophages (BMDMs) causes the phosphorylation and degradation of Lamin-A/C, as mediated by CDK1 and Caspase-6, respectively, necessary for upregulating IFN-ß expression. Enhanced IFN-ß expression subsequently increases pro-inflammatory gene expression via the IFN-ß-STAT axis. Pro-inflammatory gene expression was also amplified in the complete absence of Lamin-A/C. Alternatively, pharmacological inhibition of either Lamin-A/C phosphorylation or degradation significantly downregulated pro-inflammatory gene expression, as did the targeting of IFN-ß-STAT pathway members, i.e. phospho-STAT1 and phospho-STAT3. As Lamin-A/C is a previously unappreciated regulator of the pro-inflammatory macrophage response, our findings suggest novel opportunities to treat inflammatory diseases.

Dual MVK cleavable linkers effectively reduce renal retention of 111In-fibronectin-binding peptides.

BACKGROUND: Previously, we have exploited bacterial adhesins-derived fibronectin-binding peptides (FnBPs) for targeting mechanically altered fibronectin (Fn) fibrils within the cancer-associated extra-cellular matrix (ECM). However, despite the ability of FnBP probes to visualize pathological lesions, when labeled with metallic radionuclides and administered for targeted imaging, they exhibit high and persistent retention of radioactivity within the kidneys. Intending to overcome this issue towards a future translation of FnBPs to the clinic, the goal of the present study was to reduce the renal retention of 111In-labelled FnBPs employing dual renal brush border membrane (BBM) enzyme-sensitive Met-Val-Lys-based linkers, enabling a rapid washout of radioactivity from the kidneys. METHODS: Three maleimide-activated NOTA-conjugated brush border-enzyme cleavable linkers equipped with either single or dual consecutive MVK-based cleavable moieties were designed and synthesized. Their respective NOTA-MVK-based FnBPA5.1 conjugates were obtained by means of maleimide-thiol mediated conjugation at the N-terminus of the Fn-binding sequence, radiolabeled with indium-111, and further evaluated in vitro and in vivo in comparison to the control [111In]In-FnBPA5.1. RESULTS: The linker equipped with two MVK sites displayed a two-fold more effective cleavage rate than the single MVK featuring linker in vitro, as revealed by the quantification of the released Met-containing radiometabolites. SPECT/CT imaging and biodistribution studies of the series of FnBPA5.1 radioconjugates performed at 24 h post-injection (p.i.) confirmed the in vitro results, indicating that the renal retention of 111In-labelled FnBPs can be significantly lowered through the interposition of a single MVK-based sequence between the Fn-targeting moiety and the chelating unit (52.75 ± 9.79 vs 92.88 ± 4.85 % iA/g, P < 0.001), and even further reduced by the addition of a second one (down to 34.82 ± 6.04, P < 0.001), with minor influence on the biodistribution in other organs, such as tumors. CONCLUSIONS: In summary, we report here promising 111In-labelled FnBP radiotracers equipped with dual MVK-based cleavable linkers leading to a more effective reduction of renal retention and improved tumor-to-kidney ratios compared to the single MVK-featuring derivative. Our dual MVK strategy is a crucial step towards the clinical translation of mechano-sensory FnBPs and might as well be adopted for other radiopharmaceuticals suffering from persistent renal retention of radioactivity.

Mechanoimmunology: Are inflammatory epigenetic states of macrophages tuned by biophysical factors?

Many inflammatory diseases that are responsible for a majority of deaths are still uncurable, in part as the underpinning pathomechanisms and how to combat them is still poorly understood. Tissue-resident macrophages play pivotal roles in the maintenance of tissue homeostasis, but if they gradually convert to proinflammatory phenotypes, or if blood-born proinflammatory macrophages persist long-term after activation, they contribute to chronic inflammation and fibrosis. While biochemical factors and how they regulate the inflammatory transcriptional response of macrophages have been at the forefront of research to identify targets for therapeutic interventions, evidence is increasing that physical factors also tune the macrophage phenotype. Recently, several mechanisms have emerged as to how physical factors impact the mechanobiology of macrophages, from the nuclear translocation of transcription factors to epigenetic modifications, perhaps even DNA methylation. Insight into the mechanobiology of macrophages and associated epigenetic modifications will deliver novel therapeutic options going forward, particularly in the context of increased inflammation with advancing age and age-related diseases. We review here how biophysical factors can co-regulate pro-inflammatory gene expression and epigenetic modifications and identify knowledge gaps that require urgent attention if this therapeutic potential is to be realized.

Endothelial-Smooth Muscle Cell Interactions in a Shear-Exposed Intimal Hyperplasia on-a-Dish Model to Evaluate Therapeutic Strategies.

Intimal hyperplasia (IH) represents a major challenge following cardiovascular interventions. While mechanisms are poorly understood, the inefficient preventive methods incentivize the search for novel therapies. A vessel-on-a-dish platform is presented, consisting of direct-contact cocultures with human primary endothelial cells (ECs) and smooth muscle cells (SMCs) exposed to both laminar pulsatile and disturbed flow on an orbital shaker. With contractile SMCs sitting below a confluent EC layer, a model that successfully replicates the architecture of a quiescent vessel wall is created. In the novel IH model, ECs are seeded on synthetic SMCs at low density, mimicking reendothelization after vascular injury. Over 3 days of coculture, ECs transition from a network conformation to confluent 2D islands, as promoted by pulsatile flow, resulting in a "defected" EC monolayer. In defected regions, SMCs incorporated plasma fibronectin into fibers, increased proliferation, and formed multilayers, similarly to IH in vivo. These phenomena are inhibited under confluent EC layers, supporting therapeutic approaches that focus on endothelial regeneration rather than inhibiting proliferation, as illustrated in a proof-of-concept experiment with Paclitaxel. Thus, this in vitro system offers a new tool to study EC-SMC communication in IH pathophysiology, while providing an easy-to-use translational disease model platform for low-cost and high-content therapeutic development.

Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1).

Spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs, which has not yet been reported for spermatogonia-like cells generated in vitro. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including Sirtuin 1 (SIRT1) inhibitor Ex-527, DNA methyltransferase (DNMT) inhibitor RG-108 and electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of PGC to gonocyte differentiation. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3ß and MEK (2i) with leukemia inhibitory factor (LIF) (2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes by removal of 2iL and a specific schedule of twice daily partial medium replacement. Combination of this new protocol with the previously reported chemical intervention increased population-averaged gene expression of Lhx1 in the resulting CSMs. Furthermore, we detected single CSMs with strong nuclear LHX1/5 protein signal only in the chemical intervention group. We propose that further investigation of CSMs may provide new insights into male germline development.

Platelets drive fibronectin fibrillogenesis using integrin αIIbß3.

Platelets interact with multiple adhesion proteins during thrombogenesis, yet little is known about their ability to assemble fibronectin matrix. In vitro three-dimensional superresolution microscopy complemented by biophysical and biochemical methods revealed fundamental insights into how platelet contractility drives fibronectin fibrillogenesis. Platelets adhering to thrombus proteins (fibronectin and fibrin) versus basement membrane components (laminin and collagen IV) pull fibronectin fibrils along their apical membrane versus underneath their basal membrane, respectively. In contrast to other cell types, platelets assemble fibronectin nanofibrils using αIIbß3 rather than α5ß1 integrins. Apical fibrillogenesis correlated with a stronger activation of integrin-linked kinase, higher platelet traction forces, and a larger tension in fibrillar-like adhesions compared to basal fibrillogenesis. Our findings have potential implications for how mechanical thrombus integrity might be maintained during remodeling and vascular repair.