Cell-matrix Interactions Research Articles

A kidney proximal tubule model to evaluate effects of basement membrane stiffening on renal tubular epithelial cells.

The kidney tubule consists of a single layer of epithelial cells supported by the tubular basement membrane (TBM), a thin layer of specialized extracellular matrix (ECM). The mechanical properties of the ECM are important for regulating a wide range of cell functions including proliferation, differentiation and cell survival. Increased ECM stiffness plays a role in promoting multiple pathological conditions including cancer, fibrosis and heart disease. How changes in TBM mechanics regulate tubular epithelial cell behavior is not fully understood. Here we introduce a cell culture system that utilizes in vivo-derived TBM to investigate cell-matrix interactions in kidney proximal tubule cells. Basement membrane mechanics was controlled using genipin, a biocompatibility crosslinker. Genipin modification resulted in a dose-dependent increase in matrix stiffness. Crosslinking had a marginal but statistically significant impact on the diffusive molecular transport properties of the TBM, likely due to a reduction in pore size. Both native and genipin-modified TBM substrates supported tubular epithelial cell growth. Cells were able to attach and proliferate to form confluent monolayers. Tubular epithelial cells polarized and assembled organized cell-cell junctions. Genipin modification had minimal impact on cell viability and proliferation. Genipin stiffened TBM increased gene expression of pro-fibrotic cytokines and altered gene expression for N-cadherin, a proximal tubular epithelial specific cell-cell junction marker. This work introduces a new cell culture model for cell-basement membrane mechanobiology studies that utilizes in vivo-derived basement membrane. We also demonstrate that TBM stiffening affects tubular epithelial cell function through altered gene expression of cell-specific differentiation markers and induced increased expression of pro-fibrotic growth factors.

Glycosaminoglycan Mimetics Obtained by Microwave-Assisted Sulfation of Marine Bacterium Sourced Infernan Exopolysaccharide.

Sulfated glycosaminoglycans (GAGs) are fundamental constituents of both the cell surface and extracellular matrix. By playing a key role in cell-cell and cell-matrix interactions, GAGs are involved in many physiological and pathological processes. To design GAG mimetics with similar therapeutic potential as the natural ones, the specific structural features, among them sulfate content, sulfation pattern, and chain length, should be considered. In the present study, we describe a sulfation method based on microwave radiation to obtain highly sulfated derivatives as GAG mimetics. The starting low-molecular-weight (LMW) derivative was prepared from the infernan exopolysaccharide, a highly branched naturally slightly sulfated heteropolysaccharide synthesized by the deep-sea hydrothermal vent bacterium Alteromonas infernus. LMW highly sulfated infernan derivatives obtained by conventional heating sulfation have already been shown to display GAG-mimetic properties. Here, the potential of microwave-assisted sulfation versus that of the conventional method to obtain GAG mimetics was explored. Structural analysis by NMR revealed that highly sulfated derivatives from the two methods shared similar structural features, emphasizing that microwave-assisted sulfation with a 12-fold shorter reaction time is as efficient as the classical one.

Relative Importance of Different Elements of Mitochondrial Oxidative Phosphorylation in Maintaining the Barrier Integrity of Retinal Endothelial Cells: Implications for Vascular-Associated Retinal Diseases.

Mitochondrial dysfunction is central to breaking the barrier integrity of retinal endothelial cells (RECs) in various blinding eye diseases such as diabetic retinopathy and retinopathy of prematurity. Therefore, we aimed to investigate the role of different mitochondrial constituents, specifically those of oxidative phosphorylation (OxPhos), in maintaining the barrier function of RECs. Electric cell-substrate impedance sensing (ECIS) technology was used to assess in real time the role of different mitochondrial components in the total impedance (Z) of human RECs (HRECs) and its components: capacitance (C) and the total resistance (R). HRECs were treated with specific mitochondrial inhibitors that target different steps in OxPhos: rotenone for complex I, oligomycin for complex V (ATP synthase), and FCCP for uncoupling OxPhos. Furthermore, data were modeled to investigate the effects of these inhibitors on the three parameters that govern the total resistance of cells: Cell-cell interactions (Rb), cell-matrix interactions (α), and cell membrane permeability (Cm). Rotenone (1 µM) produced the greatest reduction in Z, followed by FCCP (1 µM), whereas no reduction in Z was observed after oligomycin (1 µM) treatment. We then further deconvoluted the effects of these inhibitors on the Rb, α, and Cm parameters. Rotenone (1 µM) completely abolished the resistance contribution of Rb, as the Rb became zero immediately after the treatment. Secondly, FCCP (1 µM) eliminated the resistance contribution of Rb only after 2.5 h and increased Cm without a significant effect on α. Lastly, of all the inhibitors used, oligomycin had the lowest impact on Rb, as evidenced by the fact that this value became similar to that of the control group at the end of the experiment without noticeable effects on Cm or α. Our study demonstrates the differential roles of complex I, complex V, and OxPhos coupling in maintaining the barrier functionality of HRECs. We specifically showed that complex I is the most important component in regulating HREC barrier integrity. These observed differences are significant since they could serve as the basis for future pharmacological and gene expression studies aiming to improve the activity of complex I and thereby provide avenues for therapeutic modalities in endothelial-associated retinal diseases.

Sensing Cells-Peptide Hydrogel Interaction In Situ via Scanning Ion Conductance Microscopy.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.

Heparin-mimicking polymer-based hydrogel matrix regulates macrophage polarization by controlling cell adhesion

The physicochemical properties of biomaterials influence cell adhesion, shape, and polarization of macrophages. In this study, we aimed to evaluate the polarization of macrophages in terms of the regulation of cell adhesion and how synthetic mimics for heparin and poly(sodium-4-styrenesulfonate) can regulate macrophage polarization by modulating cell shape, focal adhesion, cell traction force, and intracellular tension. Our initial findings showed that macrophages cultured with heparin-mimicking polymer-based hydrogel matrix showed reduced expression of cell adhesion markers such as integrins, vinculin, RhoA, and ROCK1/2 and reduced cell shape, elongation, cell-matrix traction force, and intracellular tension. Furthermore, we observed a significant decrease in cell adhesion in cells cultured on the hydrogel, resulting in the promotion of M1 polarization. These findings offer insights into the important roles of cell-matrix interactions in macrophage polarization and offer a platform for heparin-mimicking polymer-based hydrogel matrices to induce M1 polarization by inducing cell adhesion without classical activators.

Cell-Matrix Interactions in Renal Fibrosis.

Renal fibrosis is a hallmark of end-stage chronic kidney disease. It is characterized by increased accumulation of extracellular matrix (ECM), which disrupts cellular organization and function within the kidney. Here, we review the bi-directional interactions between cells and the ECM that drive renal fibrosis. We will discuss the cells involved in renal fibrosis, changes that occur in the ECM, the interactions between renal cells and the surrounding fibrotic microenvironment, and signal transduction pathways that are misregulated as fibrosis proceeds. Understanding the underlying mechanisms of cell-ECM crosstalk will identify novel targets to better identify and treat renal fibrosis and associated renal disease.

CCN2/CTGF promotes liver fibrosis through crosstalk with the Slit2/Robo signaling

Liver fibrosis is the common outcome of many chronic liver diseases, resulting from altered cell-cell and cell-matrix interactions that promote hepatic stellate cell (HSC) activation and excessive matrix production. This study aimed to investigate functions of cellular communication network factor 2 (CCN2)/Connective tissue growth factor (CTGF), an extracellular signaling modulator of the CYR61/CTGF/Nov (CCN) family, in liver fibrosis. Tamoxifen-inducible conditional knockouts in mice and hepatocyte-specific deletion of this gene in rats were generated using the Cre-lox system. These animals were subjected to peri-central hepatocyte damage caused by carbon tetrachloride. Potential crosstalk of this molecule with a new profibrotic pathway mediated by the Slit2 ligand and Roundabout (Robo) receptors was also examined. We found that Ccn2/Ctgf was highly upregulated in periportal hepatocytes during carbon tetrachloride-induced hepatocyte damage, liver fibrosis and cirrhosis in mice and rats. Overexpression of this molecule was observed in human hepatocellular carcinoma (HCC) that were surrounded with fibrotic cords. Deletion of the Ccn2/Ctgf gene significantly reduced expression of fibrosis-related genes including Slit2, a smooth muscle actin (SMA) and Collagen type I during carbon tetrachloride-induced liver fibrosis in mice and rats. In addition, Ccn2/Ctgf and its truncated mutant carrying the first three domains were able to interact with the 7th -9th epidermal growth factor (EGF) repeats and the C-terminal cysteine knot (CT) motif of Slit2 protein in cultured HSC and fibrotic murine livers. Ectopic expression of Ccn2/Ctgf protein upregulated Slit2, promoted HSC activation, and potentiated fibrotic responses following chronic intoxication by carbon tetrachloride. Moreover, Ccn2/Ctgf and Slit2 synergistically enhanced activation of phosphatidylinositol 3-kinase (PI3K) and AKT in primary HSC, whereas soluble Robo1-Fc chimera protein could inhibit these activities. These observations demonstrate conserved cross-species functions of Ccn2/Ctgf protein in rodent livers. This protein can be induced in hepatocytes and contribute to liver fibrosis. Its novel connection with the Slit2/Robo signaling may have therapeutic implications against fibrosis in chronic liver disease.

Transcriptome and proteome profiling of activated cardiac fibroblasts supports target prioritization in cardiac fibrosis.

Activated cardiac fibroblasts (CF) play a central role in cardiac fibrosis, a condition associated with most cardiovascular diseases. Conversion of quiescent into activated CF sustains heart integrity upon injury. However, permanence of CF in active state inflicts deleterious heart function effects. Mechanisms underlying this cell state conversion are still not fully disclosed, contributing to a limited target space and lack of effective anti-fibrotic therapies. To prioritize targets for drug development, we studied CF remodeling upon activation at transcriptomic and proteomic levels, using three different cell sources: primary adult CF (aHCF), primary fetal CF (fHCF), and induced pluripotent stem cells derived CF (hiPSC-CF). All cell sources showed a convergent response upon activation, with clear morphological and molecular remodeling associated with cell-cell and cell-matrix interactions. Quantitative proteomic analysis identified known cardiac fibrosis markers, such as FN1, CCN2, and Serpine1, but also revealed targets not previously associated with this condition, including MRC2, IGFBP7, and NT5DC2. Exploring such targets to modulate CF phenotype represents a valuable opportunity for development of anti-fibrotic therapies. Also, we demonstrate that hiPSC-CF is a suitable cell source for preclinical research, displaying significantly lower basal activation level relative to primary cells, while being able to elicit a convergent response upon stimuli.

Modeling ameloblast-matrix interactions using 3D cell culture.

The distinct morphology adopted by ameloblasts during amelogenesis is highly stage specific and involved intimately with the development of a hierarchical enamel microstructure. The molecular mechanisms that govern the development of an elongated and polarized secretory ameloblast morphology and the potential roles played by the enamel matrix proteins in this process are not fully understood. Thus far, the in vitro models that have been developed to mimic these early cell-matrix interactions have either been unable to demonstrate direct morphological change or have failed to adapt across ameloblast cell lines. Here, we use a recently established 3D cell culture model to examine the interactions between HAT-7 cells and the major enamel matrix proteins, amelogenin and ameloblastin. We demonstrate that HAT-7 cells selectively respond to functional EMPs in culture by forming clusters of tall cells. Aspect ratio measurements from three-dimensional reconstructions reveal that cell elongation is 5-times greater in the presence of EMPs when compared with controls. Using confocal laser scanning microscopy, we observe that these clusters are polarized with asymmetrical distributions of Par-3 and claudin-1 proteins. The behavior of HAT-7 cells in 3D culture with EMPs is comparable with that of ALC and LS-8 cells. The fact that the 3D model presented here is tunable with respect to gel substrate composition and ameloblast cell type highlights the overall usefulness of this model in studying ameloblast cell morphology in vitro.

Transcriptomic analysis of a 3D blood–brain barrier model exposed to disturbed fluid flow

Cerebral aneurysms are more likely to form at bifurcations in the vasculature, where disturbed fluid is prevalent due to flow separation at sufficiently high Reynolds numbers. While previous studies have demonstrated that altered shear stress exerted by disturbed flow disrupts endothelial tight junctions, less is known about how these flow regimes alter gene expression in endothelial cells lining the blood–brain barrier. Specifically, the effect of disturbed flow on expression of genes associated with cell–cell and cell–matrix interaction, which likely mediate aneurysm formation, remains unclear. RNA sequencing of immortalized cerebral endothelial cells isolated from the lumen of a 3D blood–brain barrier model reveals distinct transcriptional changes in vessels exposed to fully developed and disturbed flow profiles applied by both steady and physiological waveforms. Differential gene expression, validated by qRT-PCR and western blotting, reveals that lumican, a small leucine-rich proteoglycan, is the most significantly downregulated gene in endothelial cells exposed to steady, disturbed flow. Knocking down lumican expression reduces barrier function in the presence of steady, fully developed flow. Moreover, adding purified lumican into the hydrogel of the 3D blood–brain barrier model recovers barrier function in the region exposed to fully developed flow. Overall, these findings emphasize the importance of flow regimes exhibiting spatial and temporal heterogeneous shear stress profiles on cell–matrix interaction in endothelial cells lining the blood–brain barrier, while also identifying lumican as a contributor to the formation and maintenance of an intact barrier.

Characterization of TGF-β signaling in a human organotypic skin model reveals that loss of TGF-βRII induces invasive tissue growth.

Transforming growth factor-β (TGF-β) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-β signaling pathway, including the ligands TGF-β1, TGF-β2, and TGF-β3, the receptor TGF-βRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-β signaling, the loss of TGF-β1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-βRII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-βRII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-βRII. This study provides a framework for exploring TGF-β signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.

Evaluation of a Gene Signature Related to Thrombotic Manifestations in Antiphospholipid Syndrome

Introduction: Whether different manifestations of thrombotic antiphospholipid syndrome (APS) share pathological mechanisms has not been established. Transcriptome analysis may constitute a new approach to evaluate the mechanisms behind thrombotic manifestations in APS. Aim: To determine in patients with primary thrombotic APS (t-PAPS) and aPL asymptomatic (individuals carrying the antibodies but without clinical manifestation) the expression of genes already related to venous and arterial thrombosis in general population compared with control. Method: mRNA was obtained from total leucocyte and gene expression was measured by qPCR and the results were analyzed by QuantStudio™ Software. Results: 100 t-PAPS, 17 asymptomatic aPL carriers and 100 control individuals were included. The group of t-PAPS were 63.73% female, and the median age was 40 years (IQR 31-53). Asymptomatic aPL carriers were mostly female (82.36%) and the median age was 36 years (IQR 24-59). Controls were mostly female (67%) and their median was 39 years (IQR 29-49). No significant demographic differences between the groups was observed. Cardiovascular risk factors were more prevalent among t-PAPS than among asymptomatic aPL carriers and controls (37.25%, 5.88% and 9% respectively). ANXA3 (P=0.0292) and TNFAIP6 (P=0.0006) mRNA were up-regulated while TXK (P=< 0.0001), BACH2 (P=0.0085) and SERPINB2 (P=0.0491) were down-regulated in t-PAPS when compared to controls. In aPL carriers, the relative expression levels of TNFAIP6 were similar to t-PAPS while that of BACH was similar to controls; as shown in Figure 1. In a subgroup analysis, ANXA3 and TNFAIP6 mRNA expression was more pronounced in t-PAPS with multiple thrombosis (P= 0.0027 and P=< 0.0001 respectively), while TXK mRNA expression was lower in all t-PAPS subgroups in comparison with controls, being particularly lower in patients who had first thrombotic event and non-triple positivity (P=< 0.0001 and P= < 0.0001 respectively), BACH2 mRNA expression was lower in t-PAPS subgroup with first thrombotic event (P= 0.0019) compared to the control group and SERPINB2 mRNA expression was only significantly lower in t-PAPS with first thrombotic event (P= 0.0071). Table 1 demonstrates fold changes by different manifestations of t-PAPS. Discussion: We observed a dysregulation of genes related to hemostasis (ANXA3 and SERPINB2) and inflammation (TNFAIP6, TXK and BACH2) in t-PAPS, when compared to controls. ANXA3 plays a role in cell growth regulation and signal transduction pathways, and has previously been linked to stroke in animal studies. TNFAIP6 is involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis. Among TXK related pathways are the transmigration of immune cells and plays a role in regulating the adaptive immune response. BACH2 is responsible for immune regulation, mainly related to the adaptive immune response and has been implicated in B cell. SERPINB2 regulates the production of the plasminogen activator inhibitor-2 (PAI-2), being directly related to the hemostasis pathways. Such mechanisms were not evident among asymptomatic aPL carriers. In subgroup analysis, the relative expression of genes related to innate immunity has also been associated with signs of disease severity, such as multiple thrombosis and triple positivity. Conclusions: Our results showed that innate immunity and hemostasis pathways are associated with t-PAPS at a transcriptome level and innate immunity may play a role in disease severity. Acknowledgements: São Paulo Research Foundation FAPESP (2019/20136-0) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Silk Bone Marrow-like Niches for Red Cell Production Ex Vivo

Background Human adults produce up to 3 million red blood cells every second, equating to about 200 billion generated every day (Caulier and Sankaran Blood 2022). Leading studies point to the erythroblastic islands as the core of red blood cell production within the bone marrow microenvironment. Here, cellular, chemical, and physical elements define a specialized three-dimensional (3D) niche that orchestrates lineage commitment, cell maturation, and final enucleation. The extracorporeal generation of physiologically relevant models of the bone marrow is central to multiple healthcare challenges including studying the physio-pathological mechanisms of human erythropoiesis, personalized drug screening, and production of red blood cells on-demand. Different culture protocols have been developed, aiming at the mass production of erythrocytes in vitro, including bioengineering techniques for developing physiologically relevant models of the hematopoietic niche (Di Buduo et al. Haematologica 2021). Extrapolating the 3D structure and function of the erythroblastic islands is still challenging. Consequently, enucleation remains the main critical limiting step of efficient erythrocyte production ex vivo. Aim We have pioneered bone marrow tissue engineering by using silk fibroin, derived from Bombyx mori silkworm cocoons, to provide solutions for human platelet production (Di Buduo et al. Blood 2015). Here, we aimed at exploiting this technology by manufacturing innovative 3D silk-based bone marrow niches and flow chambers to direct the generation of compartmentalized erythroblastic islands and set effective protocols to produce fully mature human erythrocytes ex vivo. Methods We developed an all-natural tissue model consisting of silk fibroin and extracellular matrix proteins. The spongy scaffold was enclosed in a protective chamber providing a dynamic flow of nutrients and cytokines through an interconnected vascular-like network. The system was cultured for up to 3 weeks with hematopoietic stem and progenitor cells (HSPCs) from human mobilized peripheral blood, upon informed consent and in accordance with the Declaration of Helsinki. Results HSPCs could be efficiently differentiated into erythroblasts under the control of a bespoke cocktail of cytokines in combination with increasing concentrations of transferrin. After 1 week of culture, flow cytometry analysis demonstrated the gradual loss of stem-associated markers, such as CD34, CD105, and CD117, and the acquisition of the lineage-specific markers CD71 (transferrin receptor), CD36 (a transmembrane scavenger receptor), and CD235a (glycophorin-α). The analysis of the spatial distribution of cells within the silk scaffold, by real-time imaging, demonstrated the spontaneous development of cellular nests, resembling the physiological structure of the erythroblastic islands typically, but not exclusively, aggregating around and/or next to co-cultured CD68+ macrophages. After 2 weeks of differentiation, the population turned to a CD36-CD71lowCD235ahigh profile, indicative of terminal erythroblast maturation. Starting from week 3, fully mature CD36-C71-CD235ahigh erythrocytes were mainly localized at the periphery of the erythroblastic islands, in close contact with macrophages. Dynamic perfusion of the system at physiologically relevant shear stress allowed the collection of >90% enucleated CD235ahigh erythrocytes. Cells were fully hemoglobinized and expressed both α- and β-globin. May-Grunwald Giemsa staining revealed that the size, shape, and color of cultured erythrocytes were perfectively comparable to donor cells. The recovered erythrocytes showed significant downregulation of the expression of α4β1 integrin, which was retained by immature erythroblasts adhering to the silk scaffolds. Conclusion Our data demonstrated that the spatiotemporal control of the dynamics of cell-cell and cell-matrix interaction, together, with controllable shear forces, within a 3D physiologically relevant microenvironment, may be the key to guiding efficient erythropoiesis ex vivo. The silk bone marrow model represents a unique sustainable resource to gain insights into mechanisms of human erythropoiesis, and advance basic research to robust clinical translation.

The Roles of CD44 Membrane Ruffling and Signaling in Leukocyte Transmigration across Cryptococcus Neoformans-Coinfected Blood-Brain Barrier with HIV-1 gp41 in Vitro

Cryptococcus neoformans (Cn) is a critical human pathogen, mainly causes Cryptococcal meningitis with severe symptoms and high mortality. As an environmental yeast, Cn initiates infection after inhalation and spreads hematogenously to almost all organs. To cause Cryptococcal meningitis, Cn must invade the blood-brain barrier (BBB) that consists of brain microvascular endothelial cells (BMEC) and adhere to the host cell to resist the blood-flow. Previous studies have demonstrated that CD44, a cell adhesion molecule that mediates cell-cell and cell-matrix interactions, is involved in the invasion of Cn into human non-small cell lung cancer cells, providing a fundamental insight into the mechanism of Cn penetrating the blood-air barrier. Moreover, the binding between Cn and BMEC is enhanced by HIV-1 gp41 ectodomain (gp41-I90), which is related to its enhanced role in Cn-induced cytoskeletal changes. Both gp41-I90 and Cn are able to promote the CD44-mediated monocyte transmigration separately, and co-exposure to both would further accelerate the transmigration. The Cn CPS1 gene encodes a hyaluronic acid synthase, a product of which hyaluronic acid is the main receptor for CD44 in vivo. In addition, CD44 functions as the primary receptor during Cn infection. Although the mechanism how Cn crosses the BBB and causes CNS infection keeps mysterious, we suggest that CD44 plays a vital role in the interaction among Cn, leukocyte and BMEC. To investigate the effect of CD44 in Cn infection into BMEC, we explored several cellular and molecular events during Cn invasion. Results showed that CD44 molecules from lipid rafts can interact with hyaluronic acid. The adherence and invasion of Cn were abrogated while CD44 was perturbed by Bikunin, a protein which can disturb the oligomerization of CD44 on membrane lipid rafts. The lipid raft marker, ganglioside GM1, co-localizes with CD44 on the plasma membrane, allowing Cn cells to adhere to host cells in GM1-enriched sites, illustrating that CD44 anchors on the lipid rafts and serves as the host receptor for Cn. Upon Cn engagement, GM1 internalized through vesicular structures to the nuclear membrane. This endocytic redistribution process is abolished by cytochalasin D, nocodazole, or anti-DYRK3 siRNA. The knockdown of DYRK3 significantly reduces Cn invasion across the BMEC. Cav1, a characteristic protein of caveolae/lipid rafts, colocalized with CD44 in certain regions on the plasma membrane (10~15%). The knockdown of Cav1 significantly reduced Cn invasion and the crossing of the BMEC. Upon Cn engagement, Cav1 was phosphorylated in a CD44-dependent manner, which was diminished by filipin, a disrupter of lipid raft structure. These findings imply that CD44 anchors on lipid rafts and plays a vital role in Cn infection and the perturbation of CD44, Cav-1 or DYRK3 would block Cn adherence and invasion to BMEC. Furthermore, CD44 has an effect on leukocyte migration mediated by Cn. Cn infection into BMEC significantly enhanced the adhesion and migration of THP-1 cell (human leukemic monocytes) across the monolayers (P < 0.01), depending on the concentration and exposure time of Cn (P < 0.05). The addition of anti-CD44 and Bikunin to BMEC after Cn infection showed both adhesion and migration of THP-1 were significantly decreased (P < 0.01) in a dose dependent manner of the antibody (within 0-1 μg) and inhibitor (within 0-20 nmol/L). Both THP-1 adhesion and migration decreased in the BBB model infected with CPS1 gene-knockout Cn strain (P < 0.01, P < 0.05), but increased in complemented strain and wild-type strain. What's more, we also elaborated that gp41-I90 has an effect of enhancement in monocyte transmigration across Cn-infected BMEC, which may be induced by CD44. In conclusion, both CD44 and gp41-I90 play a vital role in THP-1 adhering to Cn-stimulated BMEC and migrating across BBB. In summary, we indicate the CD44 molecule plays a crucial role in leukocyte transmigration. CD44 functions in conjunction with DYRK3, Cav1 and gp41-I90 on lipid rafts. Anti-CD44 and Bikunin, especially the latter, can significantly reduce leukocytes migration, and have certain application prospects in reducing inflammatory responses. The specific mechanism, however, is beneath the surface and studies is urgently appreciated. (Acknowledgements: Corresponding author: Hong Cao, gzhcao@smu.edu.cn; Grant from School of Public Health of Southern Medical University, China, No. GW202222 to H.C.)

Establishing an Easy-to-Use 3D BM Niche Model for the Co-Culture of Primary Bone Marrow Mesenchymal Stromal Cells (BM-MSC) with Hematopoietic Stem and Progenitor Cells (HSPC)

Background BM-MSC are a collection of primitive cell populations, a major stromal component of the BM niche and are essential to maintain tissue homeostasis. 2D culture protocols are popular, though not standardized, and effective for BM-MSC expansion, but display limited niche function in co-culture systems. 3D co-culture leverages the importance of cell signaling networks activated by cell-cell and cell-matrix interactions in native tissues. Here, we establish a superior and easy-to-use 3D culture protocol and compare to conventional 2D approaches. Methods, Results and Discussion First, we compared commercially available expansion media to the most widely cited investigator-defined media for optimal 2D expansion and 3D culture of ex vivo murine BM-MSC. Based on this, we developed a five-marker flow cytometry panel to accurately quantify BM-MSC culture purity. To test BM-MSC niche function in 2D vs 3D cultures, we generated 3D spheroid hanging drop (HD) cultures from 2D expanded BM-MSC. In HD spheroids, cells are in direct contact with each other as well as extracellular matrix components and can be used to co-culture multiple different cell types. To assess the niche-like function of 3D HD cultures compared to 2D adherent cultures, we assessed Nestin protein expression since Nestin is a marker of HSC niches within mouse BM in vivo. BM-MSC were cultured in 2D or HD and Nestin protein expression assessed by immunofluorescence imaging, showing a significant increase in percent of Nestin-positive cells in HD-cultured BM-MSC, compared to matched 2D controls, in agreement with previous reports in human BM-MSC 3D culture. To assess 3D in vitro BM-MSC niche (IVBMN) function, we assayed stemness markers and survival of FACS isolated primary HSPC (Lineage-Sca1+cKit+, LSK) from mouse BM. LSK cells were cultured as standard (alone) or seeded as co-cultures with BM-MSC within HD with specific cytokine support, in low oxygen (6.5%) conditions (IVBMN) (Figure 1). LSK cells cultured within IVBMN exhibited superior survival as well as retention of stemness markers, demonstrating the benefit of BM-MSC support. In vivo, the BM niche also includes BM niche resident macrophages (BM-Mɸ), which were recently shown to be a limiting factor for engraftment and HSC reconstitution in irradiated mice, and are part of long term and short term HSC niches. To further extend our IVBMN, BM-Mɸ [F4/80+ Ly6G- VCAM1+ CD169+] were collected by FACS and added to IVBMN cultures (IVBMN-Mɸ). Notably, the IVBMN-Mɸ co-culture model further enhanced survival and stemness of primary LSK cells, when compared to LSK cultured alone or within IVBMN. Based on these preliminary data, we demonstrate that our IVBMN-Mɸ culture system is a unique tool which mimics niche properties in vitro and is easy-to-use for HSPC manipulation. Conclusions Here we describe a standardized flow panel for reliable murine BM-MSC immunophenotyping, and characterize a simple but effective 3D BM-MSC co-culture system which allows niche-like function in vitro. Fig. 1. In vitro BM niche (IVBMN) experimental workflow: A) Ex vivo expanded primary BM-MSC (20x) (i) -/+ FACS isolated BM niche resident Mɸ (ii) in co-culture with primary BM HPSC (LSK) (iii). B) Cells were cultured in a 3D mixed culture system with specific cytokine support, in 6.5% low oxygen. C) IVBMN-Mɸ [MSC+ BM-Mɸ +LSK] co-culture promotes maintenance of primary viable LSK compared to IVBMN [MSC+LSK] or LSK standard culture conditions alone, after 5 days culture. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nanoceria-GO-intercalated multicellular spheroids revascularize and salvage critical ischemic limbs through anti-apoptotic and pro-angiogenic functions

Critical limb ischemia (CLI) is a serious form of peripheral arterial disease that involves severe blockage of blood flow in lower extremities, often leading to foot necrosis and limb loss. Lack of blood flow and high pro-inflammation with overproduced reactive oxygen species (ROS) in CLI aggravate the degenerative events. Among other therapies, cell delivery is considered potential for restoring regenerative capacity, and preservation of cell survival under high oxidative stress has been challenging and prerequisite to harness cellular functions. Here, we introduce a multicellular delivery system that is intercalated with nanoceria-decorated graphene oxide (CeGO), which is considered to have high ROS scavenging ability while providing cell-matrix interaction signals. The CeGO nano-microsheets (8-nm-nanoceria/0.9-μm-GO) incorporated in HUVEC/MSC (7/3) could form cell-material hybrid spheroids mediated by cellular contraction. Under in vitro oxidative-stress-challenge with H2O2, the CeGO-intercalation enhanced the survival and anti-apoptotic capacity of cellular spheroids. Pro-angiogenic events of cellular spheroids, including cell sprouting and expression of angiogenic markers (HIF1α, VEGF, FGF2, eNOS) were significantly enhanced by the CeGO-intercalation. Proteomics analysis also confirmed substantial up-regulation of a series of angiogenesis-related secretome molecules. Such pro-angiogenic events with CeGO-intercalation were proven to be mediated by the APE/Ref-1 signaling pathway. When delivered to ischemic hindlimb in mice, the CeGO-cell spheroids could inhibit the accumulation of in vivo ROS rapidly, preserving high cell survival rate (cells were more proliferative and less apoptotic vs. those in cell-only spheroids), and up-regulated angiogenic molecular expressions. Monitoring over 28 days revealed significantly enhanced blood reperfusion and tissue recovery, and an ultimate limb salvage with the CeGO-cell delivery (∼60% salvaged vs. ∼29% in cell-only delivery vs. 0% in ischemia control). Together, the CeGO intercalated in HUVEC/MSC delivery is considered a potential nano-microplatform for CLI treatment, by scavenging excessive ROS and enhancing transplanted cell survival, while stimulating angiogenic events, which collectively help revascularization and tissue recovery, salvaging critical ischemic limbs.

Cell-surface contacts determine volume and mechanical properties of human embryonic kidney 293 T cells.

Alterations in the organization of the cytoskeleton precede the escape of adherent cells from the framework of cell-cell and cell-matrix interactions into suspension. With cytoskeletal dynamics being linked to cell mechanical properties, many studies elucidated this relationship under either native adherent or suspended conditions. In contrast, tethered cells that mimic the transition between both states have not been the focus of recent research. Using human embryonic kidney 293 T cells we investigated all three conditions in the light of alterations in cellular shape, volume, as well as mechanical properties and relate these findings to the level, structure, and intracellular localization of filamentous actin (F-actin). For cells adhered to a substrate, our data shows that seeding density affects cell size but does not alter their elastic properties. Removing surface contacts leads to cell stiffening that is accompanied by changes in cell shape, and a reduction in cellular volume but no alterations in F-actin density. Instead, we observe changes in the organization of F-actin indicated by the appearance of blebs in the semi-adherent state. In summary, our work reveals an interplay between molecular and mechanical alterations when cells detach from a surface that is mainly dominated by cell morphology.

Design of synthetic collagens that assemble into supramolecular banded fibers as a functional biomaterial testbed

Collagens are the most abundant proteins of the extracellular matrix, and the hierarchical folding and supramolecular assembly of collagens into banded fibers is essential for mediating cell-matrix interactions and tissue mechanics. Collagen extracted from animal tissues is a valuable commodity, but suffers from safety and purity issues, limiting its biomaterials applications. Synthetic collagen biomaterials could address these issues, but their construction requires molecular-level control of folding and supramolecular assembly into ordered banded fibers, comparable to those of natural collagens. Here, we show an innovative class of banded fiber-forming synthetic collagens that recapitulate the morphology and some biological properties of natural collagens. The synthetic collagens comprise a functional-driver module that is flanked by adhesive modules that effectively promote their supramolecular assembly. Multiscale simulations support a plausible molecular-level mechanism of supramolecular assembly, allowing precise design of banded fiber morphology. We also experimentally demonstrate that synthetic fibers stimulate osteoblast differentiation at levels comparable to natural collagen. This work thus deepens understanding of collagen biology and disease by providing a ready source of safe, functional biomaterials that bridge the current gap between the simplicity of peptide biophysical models and the complexity of in vivo animal systems.

Analysis of CRSsNP Proteome Using a Highly Multiplexed Approach in Nasal Mucus.

Chronic rhinosinusitis without nasal polyps (CRSsNP) represents a phenotype of CRS, whose immunological mechanisms are still unclear. So far there are neither suitable biomarkers to determine the course of the disease nor an individual therapy. The purpose of this study was to characterize the CRSsNP endotype by identifying and validating non-invasive proteomic biomarkers. A highly-multiplexed proteomic array consisting of antibodies against 2000 proteins was used to identify proteins that are differentially expressed in the nasal mucus of the CRSsNP and control groups (n = 7 per group). The proteins identified to be most differentially expressed were validated in matched nasal mucus samples using western blots and enzyme-linked immunosorbent assay (ELISA). Validation was also done in a second cohort using western blots (CRSsNP n = 25, control n = 23) and ELISA (n = 30 per group). Additionally, immunohistochemistry in CRSsNP and control tissue samples was performed to characterize the selected proteins further. Out of the 2000 proteins examined, 7 from the most differentially expressed proteins were chosen to be validated. The validation results showed that 4 proteins were significantly upregulated in CRSsNP mucus, including macrophage inflammatory protein-1beta (MIP-1β), resistin, high mobility group box 1 (HMGB1), and forkhead box protein 3 (FOXP3). Cartilage acidic protein 1 (CRTAC1) was not significantly upregulated. Two proteins were significantly downregulated including scavenger receptor class F member 2 (SCARF2) and P-selectin. All proteins selected are mainly associated with inflammation, cell proliferation/differentiation, apoptosis and cell-cell or cell-matrix interaction. Proteomic analysis of CRSsNP and control mucus has confirmed known and revealed novel disease-associated proteins that could potentially serve as a new biosignature for CRSsNP. Analysis of the associated pathways will specify endotypes of CRSsNP and will lead to an improved understanding of the pathophysiology of CRSsNP. Furthermore, our data contribute to the development of a reproducible, non-invasive, and quantitative "liquid biopsy" for rhinosinusitis.

A rapid high throughput bioprinted colorectal cancer spheroid platform for in vitro drug- and radiation-response

We describe the development of a high-throughput bioprinted colorectal cancer (CRC) spheroid platform with high levels of automation, information content, and low cell number requirement. This is achieved via the formulation of a hydrogel bioink with a compressive Young’s modulus that is commensurate with that of colonic tissue (1–3 kPa), which supports exponential growth of spheroids from a wide range of CRC cell lines. The resulting spheroids display tight cell–cell junctions, bioink matrix-cell interactions and necrotic hypoxic cores. By combining high content light microscopy imaging and processing with rapid multiwell plate bioprinting, dose-response profiles are generated from CRC spheroids challenged with oxaliplatin (OX) and fluorouracil (5FU), as well as radiotherapy. Bioprinted CRC spheroids are shown to exhibit high levels of chemoresistance relative to cell monolayers, and OX was found to be significantly less effective against tumour spheroids than in monolayer culture, when compared to 5FU.