Mesenchymal condensation is critical for organogenesis, yet little is known about how this process is controlled. Here we show that Fgf8 and Sema3f, produced by early dental epithelium, respectively, ...attract and repulse mesenchymal cells, which cause them to pack tightly together during mouse tooth development. Resulting mechanical compaction-induced changes in cell shape induce odontogenic transcription factors (Pax9, Msx1) and a chemical cue (BMP4), and mechanical compression of mesenchyme is sufficient to induce tooth-specific cell fate switching. The inductive effects of cell compaction are mediated by suppression of the mechanical signaling molecule RhoA, and its overexpression prevents odontogenic induction. Thus, the mesenchymal condensation that drives tooth formation is induced by antagonistic epithelial morphogens that manifest their pattern-generating actions mechanically via changes in mesenchymal cell shape and altered mechanotransduction.
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► Fgf8 and Sema3f induce mesenchymal condensation during tooth development ► Resulting physical cell compaction causes odontogenic induction ► Inhibition of RhoA mediates mechanical induction of odontogenesis ► Mechanical compression alone can trigger tooth organ formation
Increased vascular permeability contributes to many diseases, including acute respiratory distress syndrome, cancer and inflammation. Most past work on vascular barrier function has focused on ...soluble regulators, such as tumour-necrosis factor-α. Here we show that lung vascular permeability is controlled mechanically by changes in extracellular matrix structure. Our studies reveal that pulmonary vascular leakage can be increased by altering extracellular matrix compliance in vitro and by manipulating lysyl oxidase-mediated collagen crosslinking in vivo. Either decreasing or increasing extracellular matrix stiffness relative to normal levels disrupts junctional integrity and increases vascular leakage. Importantly, endotoxin-induced increases of vascular permeability are accompanied by concomitant increases in extracellular matrix rigidity and lysyl oxidase activity, which can be prevented by inhibiting lysyl oxidase activity. The identification of lysyl oxidase and the extracellular matrix as critical regulators of lung vascular leakage might lead to the development of new therapeutic approaches for the treatment of pulmonary oedema and other diseases caused by abnormal vascular permeability.
Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals ...integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP (also known as GRLF1), controls capillary network formation in vitro in human microvascular endothelial cells and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors, TFII-I (also known as GTF2I) and GATA2, that govern gene expression of the VEGF receptor VEGFR2 (also known as KDR). Moreover, this new angiogenesis signalling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is, to our knowledge, the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.
Dendritic cells (DCs)--immunomodulatory cells that initiate adaptive immune responses--have recently been shown to exert proangiogenic effects when infiltrating the tumor microenvironment. As tumors ...that escape immune surveillance inhibit DC maturation, we explored whether maturation status determines their ability to promote angiogenesis and whether angiogenesis depends on the presence of DCs. Using mouse xenograft models of human tumors, we show that fast-growing "angiogenic" tumors are infiltrated by a more immature DC population than respective dormant avascular tumors. Accordingly, supplementation of immature DCs, but not mature DCs, enhanced tumor growth. When DCs were mixed with Matrigel and injected subcutaneously into mice, only immature DCs promoted the ingrowth of patent blood vessels. Notably, depletion of DCs in a transgenic mouse model that allows for their conditional ablation completely abrogated basic fibroblast growth factor-induced angiogenesis in Matrigel plugs, and significantly inhibited tumor growth in these mice. Because immature DCs actively promote angiogenesis and tumor growth, whereas DC maturation or ablation suppresses this response, we conclude that angiogenesis is dependent on the presence of immature DCs. Thus, cancer immunotherapies that promote DC maturation may act by both augmenting the host immune response to the tumor and by suppressing tumor angiogenesis.--Fainaru, O., Almog, N., Yung, C. W., Nakai, K., Montoya-Zavala, M., Abollahi, A., D'Amato, R., Ingber, D. E. Tumor growth and angiogenesis are dependent on the presence of immature dendritic cells.
Abstract
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly ...matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
Here we describe a blood-cleansing device for sepsis therapy inspired by the spleen, which can continuously remove pathogens and toxins from blood without first identifying the infectious agent. ...Blood flowing from an infected individual is mixed with magnetic nanobeads coated with an engineered human opsonin--mannose-binding lectin (MBL)--that captures a broad range of pathogens and toxins without activating complement factors or coagulation. Magnets pull the opsonin-bound pathogens and toxins from the blood; the cleansed blood is then returned back to the individual. The biospleen efficiently removes multiple Gram-negative and Gram-positive bacteria, fungi and endotoxins from whole human blood flowing through a single biospleen unit at up to 1.25 liters per h in vitro. In rats infected with Staphylococcus aureus or Escherichia coli, the biospleen cleared >90% of bacteria from blood, reduced pathogen and immune cell infiltration in multiple organs and decreased inflammatory cytokine levels. In a model of endotoxemic shock, the biospleen increased survival rates after a 5-h treatment.
Sepsis is a lethal disease caused by a systemic microbial infection that spreads via the bloodstream to overwhelm the body's defenses. Current therapeutic approaches are often suboptimal, in part, ...because they do not fully eliminate the pathogen, and hence the source of deadly toxins. Here we describe an extracorporeal blood cleansing device to selectively remove pathogens from contaminated blood and thereby enhance the patient's response to antibiotic therapy. Immunomagnetic microbeads were modified to create magnetic opsonins that were used to cleanse flowing human whole blood of Candida albicans fungi, a leading cause of sepsis-related deaths. The micromagnetic-microfluidic blood cleansing device generates magnetic field gradients across vertically stacked channels to enable continuous and high throughput separation of fungi from flowing whole blood. A multiplexed version of the device containing four parallel channels achieved over 80% clearance of fungi from contaminated blood at a flow rate of 20 mL/h in a single pass, a rate 1000 times faster than a previously described prototype micromagnetic-microfluidic cell separation system. These results provide the first proof-of-principle that a multiplexed micromagnetic-microfluidic separation system can be used to cleanse pathogens from flowing human blood at a rate and separation efficiency that is relevant for clinical applications.
In the encapsulated environment of biohybrid artificial organs, cells often encounter a deficiency in oxygen, which lead to apoptosis, necrosis, and lost of productivity. Two vectors with ...constitutive CMV promoters were constructed to examine the ability of Bcl-2Δ to help C2C12 mouse myoblasts maintain exogenous protein production under hypoxia. Two additional vectors with hypoxia-inducible promoters (5HRE) that switched on Bcl-2Δ expression based on low oxygen levels (0.0%, 0.5%, 1.0%, 2.0%, 5.0%, or 21.0%) were tested for protein productivity and protection against hypoxic stresses. A yellow fluorescent protein was used as a model protein in all vector constructs. C2C12 cells with Bcl-2Δ consistently produced more protein regardless of the oxygen level or promoter used. Cells utilizing the 5HRE rather than the CMV promoter showed an increased level of protein production as the oxygen was decreased. Among the cells with 5HRE promoters, the presence of Bcl-2Δ also increased viability and decreased apoptosis.
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