Development of N-Terminally Modified Variants of the CXCR4-Antagonistic Peptide EPI-X4 for Enhanced Plasma Stability.

EPI-X4, a natural peptide CXCR4 antagonist, shows potential for treating inflammation and cancer, but its short plasma stability limits its clinical application. We aimed to improve the plasma stability of EPI-X4 analogues without compromising CXCR4 antagonism. Our findings revealed that only the peptide N-terminus is prone to degradation. Consequently, incorporating d-amino acids or acetyl groups in this region enhanced peptide stability in plasma. Notably, EPI-X4 leads 5, 27, and 28 not only retained their CXCR4 binding and antagonism but also remained stable in plasma for over 8 h. Molecular dynamic simulations showed that these modified analogues bind similarly to CXCR4 as the original peptide. To further increase their systemic half-lives, we conjugated these stabilized analogues with large polymers and albumin binders. These advances highlight the potential of the optimized EPI-X4 analogues as promising CXCR4-targeted therapeutics and set the stage for more detailed preclinical assessments.

The fin roundabout: Slit-Robo and S1P signaling coordinate fin morphogenesis.

Development of vertebrate limbs and fins requires that tissue growth is directed outwards, away from the body. How such directed growth is achieved is a fascinating biological problem. For limb/fin formation and outgrowth, signaling between mesenchymal cells and the overlying epithelium is essential. In particular, the epithelium at the distal margin of the growing limb/fin bud, termed the apical ectodermal ridge (AER), promotes directed outgrowth of the underlying mesenchyme, e.g., by providing polarization cues for mesenchymal cell migration. Several classical signaling pathways, such as fibroblast growth factor (Fgf), hedgehog, and Wnt signaling, are involved in the regulation of the cellular events that shape the limb/fin bud (Iovine, 2007). In this issue of EMBO Reports, Carney and colleagues surprisingly find that the Slit-Robo pathway, which is best known for its function in axon guidance, regulates the polarity of developing zebrafish fins (Mahabaleshwar et al, 2007). Intriguingly, they identify an intricate back and forth of signals between the mesenchyme and the AER. Slit ligands derived from mesenchyme act on Robo receptors in the AER to stimulate the production of sphingosine-1-phosphate, which then acts back on the mesenchyme to regulate cell polarity and orientation.

Zebrafish fin regeneration involves generic and regeneration-specific osteoblast injury responses.

Successful regeneration requires the coordinated execution of multiple cellular responses to injury. In amputated zebrafish fins, mature osteoblasts dedifferentiate, migrate towards the injury, and form proliferative osteogenic blastema cells. We show that osteoblast migration is preceded by cell elongation and alignment along the proximodistal axis, which require actomyosin, but not microtubule (MT) turnover. Surprisingly, osteoblast dedifferentiation and migration can be uncoupled. Using pharmacological and genetic interventions, we found that NF-ĸB and retinoic acid signalling regulate dedifferentiation without affecting migration, while the complement system and actomyosin dynamics affect migration but not dedifferentiation. Furthermore, by removing bone at two locations within a fin ray, we established an injury model containing two injury sites. We found that osteoblasts dedifferentiate at and migrate towards both sites, while accumulation of osteogenic progenitor cells and regenerative bone formation only occur at the distal-facing injury. Together, these data indicate that osteoblast dedifferentiation and migration represent generic injury responses that are differentially regulated and can occur independently of each other and of regenerative growth. We conclude that successful fin bone regeneration appears to involve the coordinated execution of generic and regeneration-specific responses of osteoblasts to injury.

Human α-Defensin-6 Neutralizes Clostridioides difficile Toxins TcdA and TcdB by Direct Binding.

Rising incidences and mortalities have drawn attention to Clostridioides difficile infections (CDIs) in recent years. The main virulence factors of this bacterium are the exotoxins TcdA and TcdB, which glucosylate Rho-GTPases and thereby inhibit Rho/actin-mediated processes in cells. This results in cell rounding, gut barrier disruption and characteristic clinical symptoms. So far, treatment of CDIs is limited and mainly restricted to some antibiotics, often leading to a vicious circle of antibiotic-induced disease recurrence. Here, we demonstrate the protective effect of the human antimicrobial peptide α-defensin-6 against TcdA, TcdB and the combination of both toxins in vitro and in vivo and unravel the underlying molecular mechanism. The defensin prevented toxin-mediated glucosylation of Rho-GTPases in cells and protected human cells, model epithelial barriers as well as zebrafish embryos from toxic effects. In vitro analyses revealed direct binding to TcdB in an SPR approach and the rapid formation of TcdB/α-defensin-6 complexes, as analyzed with fluorescent TcdB by time-lapse microscopy. In conclusion, the results imply that α-defensin-6 rapidly sequesters the toxin into complexes, which prevents its cytotoxic activity. These findings extend the understanding of how human peptides neutralize bacterial protein toxins and might be a starting point for the development of novel therapeutic options against CDIs.

Wnt/ß-catenin signaling acts cell-autonomously to promote cardiomyocyte regeneration in the zebrafish heart.

Zebrafish can achieve scar-free healing of heart injuries, and robustly replace all cardiomyocytes lost to injury via dedifferentiation and proliferation of mature cardiomyocytes. Previous studies suggested that Wnt/ß-catenin signaling is active in the injured zebrafish heart, where it induces fibrosis and prevents cardiomyocyte cell cycling. Here, via targeting the destruction complex of the Wnt/ß-catenin pathway with pharmacological and genetic tools, we demonstrate that Wnt/ß-catenin activity is required for cardiomyocyte proliferation and dedifferentiation, as well as for maturation of the scar during regeneration. Using cardiomyocyte-specific conditional inhibition of the pathway, we show that Wnt/ß-catenin signaling acts cell-autonomously to promote cardiomyocyte proliferation. Our results stand in contrast to previous reports and rather support a model in which Wnt/ß-catenin signaling plays a positive role during heart regeneration in zebrafish.

Zebrafish Fin: Complex Molecular Interactions and Cellular Mechanisms Guiding Regeneration.

The zebrafish caudal fin has become a popular model to study cellular and molecular mechanisms of regeneration due to its high regenerative capacity, accessibility for experimental manipulations, and relatively simple anatomy. The formation of a regenerative epidermis and blastema are crucial initial events and tightly regulated. Both the regenerative epidermis and the blastema are highly organized structures containing distinct domains, and several signaling pathways regulate the formation and interaction of these domains. Bone is the major tissue regenerated from the progenitor cells of the blastema. Several cellular mechanisms can provide source cells for blastemal (pre-)osteoblasts, including dedifferentiation of differentiated osteoblasts and de novo formation from other cell types, providing intriguing examples of cellular plasticity. In recent years, omics analyses and single-cell approaches have elucidated genetic and epigenetic regulation, increasing our knowledge of the surprisingly complex coordination of various mechanisms to achieve successful restoration of a seemingly simple structure.

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish.

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.

An optimized derivative of an endogenous CXCR4 antagonist prevents atopic dermatitis and airway inflammation.

Aberrant CXCR4/CXCL12 signaling is involved in many pathophysiological processes such as cancer and inflammatory diseases. A natural fragment of serum albumin, named EPI-X4, has previously been identified as endogenous peptide antagonist and inverse agonist of CXCR4 and is a promising compound for the development of improved analogues for the therapy of CXCR4-associated diseases. To generate optimized EPI-X4 derivatives we here performed molecular docking analysis to identify key interaction motifs of EPI-X4/CXCR4. Subsequent rational drug design allowed to increase the anti-CXCR4 activity of EPI-X4. The EPI-X4 derivative JM#21 bound CXCR4 and suppressed CXCR4-tropic HIV-1 infection more efficiently than the clinically approved small molecule CXCR4 antagonist AMD3100. EPI-X4 JM#21 did not exert toxic effects in zebrafish embryos and suppressed allergen-induced infiltration of eosinophils and other immune cells into the airways of animals in an asthma mouse model. Moreover, topical administration of the optimized EPI-X4 derivative efficiently prevented inflammation of the skin in a mouse model of atopic dermatitis. Thus, rationally designed EPI-X4 JM#21 is a novel potent antagonist of CXCR4 and the first CXCR4 inhibitor with therapeutic efficacy in atopic dermatitis. Further clinical development of this new class of CXCR4 antagonists for the therapy of atopic dermatitis, asthma and other CXCR4-associated diseases is highly warranted.

Computational modeling and experimental validation of the EPI-X4/CXCR4 complex allows rational design of small peptide antagonists.

EPI-X4, a 16-mer fragment of albumin, is a specific endogenous antagonist and inverse agonist of the CXC-motif-chemokine receptor 4 (CXCR4) and thus a key regulator of CXCR4 function. Accordingly, activity-optimized synthetic derivatives of EPI-X4 are promising leads for the therapy of CXCR4-linked disorders such as cancer or inflammatory diseases. We investigated the binding of EPI-X4 to CXCR4, which so far remained unclear, by means of biomolecular simulations combined with experimental mutagenesis and activity studies. We found that EPI-X4 interacts through its N-terminal residues with CXCR4 and identified its key interaction motifs, explaining receptor antagonization. Using this model, we developed shortened EPI-X4 derivatives (7-mers) with optimized receptor antagonizing properties as new leads for the development of CXCR4 inhibitors. Our work reveals the molecular details and mechanism by which the first endogenous peptide antagonist of CXCR4 interacts with its receptor and provides a foundation for the rational design of improved EPI-X4 derivatives.

Gran1: A Granulysin-Derived Peptide with Potent Activity against Intracellular Mycobacterium tuberculosis.

Granulysin is an antimicrobial peptide (AMP) expressed by human T-lymphocytes and natural killer cells. Despite a remarkably broad antimicrobial spectrum, its implementation into clinical practice has been hampered by its large size and off-target effects. To circumvent these limitations, we synthesized a 29 amino acid fragment within the putative cytolytic site of Granulysin (termed "Gran1"). We evaluated the antimicrobial activity of Gran1 against the major human pathogen Mycobacterium tuberculosis (Mtb) and a panel of clinically relevant non-tuberculous mycobacteria which are notoriously difficult to treat. Gran1 efficiently inhibited the mycobacterial proliferation in the low micro molar range. Super-resolution fluorescence microscopy and scanning electron microscopy indicated that Gran1 interacts with the surface of Mtb, causing lethal distortions of the cell wall. Importantly, Gran1 showed no off-target effects (cytokine release, chemotaxis, cell death) in primary human cells or zebrafish embryos (cytotoxicity, developmental toxicity, neurotoxicity, cardiotoxicity). Gran1 was selectively internalized by macrophages, the major host cell of Mtb, and restricted the proliferation of the pathogen. Our results demonstrate that the hypothesis-driven design of AMPs is a powerful approach for the identification of small bioactive compounds with specific antimicrobial activity. Gran1 is a promising component for the design of AMP-containing nanoparticles with selective activity and favorable pharmacokinetics to be pushed forward into experimental in vivo models of infectious diseases, most notably tuberculosis.

Delivery by Dendritic Mesoporous Silica Nanoparticles Enhances the Antimicrobial Activity of a Napsin-Derived Peptide Against Intracellular Mycobacterium tuberculosis.

Tuberculosis remains a serious global health problem causing 1.3 million deaths annually. The causative pathogen Mycobacterium tuberculosis (Mtb) has developed several mechanisms to evade the immune system and resistances to many conventional antibiotics, so that alternative treatment strategies are urgently needed. By isolation from bronchoalveolar lavage and peptide optimization, a new antimicrobial peptide named NapFab is discovered. While showing robust activity against extracellular Mtb, the activity of NapFab against intracellular bacteria is limited due to low intracellular availability. By loading NapFab onto dendritic mesoporous silica nanoparticles (DMSN) as a carrier system, cellular uptake, and consequently antimycobacterial activity against intracellular Mtb is significantly enhanced. Furthermore, using lattice light-sheet fluorescence microscopy, it can be shown that the peptide is gradually released from the DMSN inside living macrophages over time. By electron microscopy and tomography, it is demonstrated that peptide loaded DMSN are stored in vesicular structures in proximity to mycobacterial phagosomes inside the cells, but the nanoparticles are typically not in direct contact with the bacteria. Based on the combination of functional and live-cell imaging analyses, it is hypothesized that after being released from the DMSN NapFab is able to enter the bacterial phagosome and gain access to the bacilli.

Is zebrafish heart regeneration "complete"? Lineage-restricted cardiomyocytes proliferate to pre-injury numbers but some fail to differentiate in fibrotic hearts.

Adult zebrafish are frequently described to be able to "completely" regenerate the heart. Yet, the extent to which cardiomyocytes lost to injury are replaced is unknown, since existing evidence for cardiomyocyte proliferation is indirect or non-quantitative. We established stereological methods to quantify the number of cardiomyocytes at several time-points post cryoinjury. Intriguingly, after cryoinjuries that killed about 1/3 of the ventricular cardiomyocytes, pre-injury cardiomyocyte numbers were restored already within 30 days. Yet, many hearts retained small residual scars, and a subset of cardiomyocytes bordering these fibrotic areas remained smaller, lacked differentiated sarcomeric structures, and displayed defective calcium signaling. Thus, a subset of regenerated cardiomyocytes failed to fully mature. While lineage-tracing experiments have shown that regenerating cardiomyocytes are derived from differentiated cardiomyocytes, technical limitations have previously made it impossible to test whether cardiomyocyte trans-differentiation contributes to regeneration of non-myocyte cell lineages. Using Cre responder lines that are expressed in all major cell types of the heart, we found no evidence for cardiomyocyte transdifferentiation into endothelial, epicardial, fibroblast or immune cell lineages. Overall, our results imply a refined answer to the question whether zebrafish can completely regenerate the heart: in response to cryoinjury, preinjury cardiomyocyte numbers are indeed completely regenerated by proliferation of lineage-restricted cardiomyocytes, while restoration of cardiomyocyte differentiation and function, as well as resorption of scar tissue, is less robustly achieved.

Recent advancements in understanding fin regeneration in zebrafish.

Zebrafish have the remarkable ability to fully regenerate a lost appendage, faithfully restoring its size, shape and tissue patterning. Studies over the past decades have identified mechanisms underlying the formation, spatial organization, and regenerative growth of the blastema, a pool of proliferative progenitor cells. The patterning of newly forming tissue is tightly regulated to ensure proper rebuilding of anatomy. Precise niche regulation of retinoic acid and sonic hedgehog signaling ensures adherence to ray-interray boundaries. The molecular underpinnings of systems underlying re-establishment of pre-amputation size and shape (positional information) are also slowly starting to emerge. Osteoblasts play an important role as a cellular source of regenerating skeletal elements, and in zebrafish both osteoblast dedifferentiation as well as de novo osteoblast formation occurs. Both dedifferentiation and proliferation are tightly controlled, which makes it interesting to compare it to tumorigenesis, and to identify potential players involved in these processes. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

Unbiased Identification of Angiogenin as an Endogenous Antimicrobial Protein With Activity Against Virulent Mycobacterium tuberculosis.

Tuberculosis is a highly prevalent infectious disease with more than 1.5 million fatalities each year. Antibiotic treatment is available, but intolerable side effects and an increasing rate of drug-resistant strains of Mycobacterium tuberculosis (Mtb) may hamper successful outcomes. Antimicrobial peptides (AMPs) offer an alternative strategy for treatment of infectious diseases in which conventional antibiotic treatment fails. Human serum is a rich resource for endogenous AMPs. Therefore, we screened a library generated from hemofiltrate for activity against Mtb. Taking this unbiased approach, we identified Angiogenin as the single compound in an active fraction. The antimicrobial activity of endogenous Angiogenin against extracellular Mtb could be reproduced by synthetic Angiogenin. Using computational analysis, we identified the hypothetical active site and optimized the lytic activity by amino acid exchanges. The resulting peptide-Angie1-limited the growth of extra- and intracellular Mtb and the fast-growing pathogens Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Toward our long-term goal of evaluating Angie1 for therapeutic efficacy in vivo, we demonstrate that the peptide can be efficiently delivered into human macrophages via liposomes and is not toxic for zebrafish embryos. Taken together, we define Angiogenin as a novel endogenous AMP and derive the small, bioactive fragment Angie1, which is ready to be tested for therapeutic activity in animal models of tuberculosis and infections with fast-growing bacterial pathogens.

NF-κB Signaling Negatively Regulates Osteoblast Dedifferentiation during Zebrafish Bone Regeneration.

Dedifferentiation of mature cells is an intriguing cellular process associated with regeneration of several organs. During zebrafish fin regeneration, osteoblasts dedifferentiate to osteogenic progenitors that provide source cells for bone restoration. We performed a high-content in vivo chemical screen for regulators of osteoblast dedifferentiation and fin regenerative growth. NF-κB signaling emerged as a specific regulator of dedifferentiation. The pathway is active in mature osteoblasts and downregulated prior to dedifferentiation. Pathway activation blocked osteoblast dedifferentiation, while NF-κB signaling inhibition enhanced dedifferentiation. Conditional Cre-lox-mediated NF-κB signaling manipulation specifically in osteoblasts showed that the pathway acts cell autonomously to interfere with osteoblast dedifferentiation. NF-κB signaling acts upstream of retinoic acid (RA) signaling, which also needs to be downregulated for dedifferentiation to occur, via suppression of the RA-degrading enzyme cyp26b1. Our findings shed light on the molecular regulation of regenerative cellular plasticity.

Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart.

While the heart regenerates poorly in mammals, efficient heart regeneration occurs in zebrafish. Studies in zebrafish have resulted in a model in which preexisting cardiomyocytes dedifferentiate and reinitiate proliferation to replace the lost myocardium. To identify which processes occur in proliferating cardiomyocytes we have used a single-cell RNA-sequencing approach. We uncovered that proliferating border zone cardiomyocytes have very distinct transcriptomes compared to the nonproliferating remote cardiomyocytes and that they resemble embryonic cardiomyocytes. Moreover, these cells have reduced expression of mitochondrial genes and reduced mitochondrial activity, while glycolysis gene expression and glucose uptake are increased, indicative for metabolic reprogramming. Furthermore, we find that the metabolic reprogramming of border zone cardiomyocytes is induced by Nrg1/ErbB2 signaling and is important for their proliferation. This mechanism is conserved in murine hearts in which cardiomyocyte proliferation is induced by activating ErbB2 signaling. Together these results demonstrate that glycolysis regulates cardiomyocyte proliferation during heart regeneration.

Single-molecule imaging correlates decreasing nuclear volume with increasing TF-chromatin associations during zebrafish development.

Zygotic genome activation (ZGA), the onset of transcription after initial quiescence, is a major developmental step in many species, which occurs after ten cell divisions in zebrafish embryos. How transcription factor (TF)-chromatin interactions evolve during early development to support ZGA is largely unknown. We establish single molecule tracking in live developing zebrafish embryos using reflected light-sheet microscopy to visualize two fluorescently labeled TF species, mEos2-TBP and mEos2-Sox19b. We further develop a data acquisition and analysis scheme to extract quantitative information on binding kinetics and bound fractions during fast cell cycles. The chromatin-bound fraction of both TFs increases during early development, as expected from a physical model of TF-chromatin interactions including a decreasing nuclear volume and increasing DNA accessibility. For Sox19b, data suggests the increase is mainly due to the shrinking nucleus. Our single molecule approach provides quantitative insight into changes of TF-chromatin associations during the developmental period embracing ZGA.

Molecular mechanisms of glucocorticoids on skeleton and bone regeneration after fracture.

Glucocorticoid hormones (GCs) have profound effects on bone metabolism. Via their nuclear hormone receptor - the GR - they act locally within bone cells and modulate their proliferation, differentiation, and cell death. Consequently, high glucocorticoid levels - as present during steroid therapy or stress - impair bone growth and integrity, leading to retarded growth and glucocorticoid-induced osteoporosis, respectively. Because of their profound impact on the immune system and bone cell differentiation, GCs also affect bone regeneration and fracture healing. The use of conditional-mutant mouse strains in recent research provided insights into the cell-type-specific actions of the GR. However, despite recent advances in system biology approaches addressing GR genomics in general, little is still known about the molecular mechanisms of GCs and GR in bone cells. Here, we review the most recent findings on the molecular mechanisms of the GR in general and the known cell-type-specific actions of the GR in mesenchymal cells and their derivatives as well as in osteoclasts during bone homeostasis, GC excess, bone regeneration and fracture healing.

Regulatory T Cells Know What Is Needed to Regenerate.

Adaptive immunity has been suggested to limit regeneration in mammals. However, in this issue of Developmental Cell, Hui et al. (2017) report that regulatory T cells are required for regeneration of heart, spinal cord, and retina in the zebrafish. Intriguingly, in each organ system, Treg cells secrete organ-specific regeneration factors.

Generic wound signals initiate regeneration in missing-tissue contexts.

Despite the identification of numerous regulators of regeneration in different animal models, a fundamental question remains: why do some wounds trigger the full regeneration of lost body parts, whereas others resolve by mere healing? By selectively inhibiting regeneration initiation, but not the formation of a wound epidermis, here we create headless planarians and finless zebrafish. Strikingly, in both missing-tissue contexts, injuries that normally do not trigger regeneration activate complete restoration of heads and fin rays. Our results demonstrate that generic wound signals have regeneration-inducing power. However, they are interpreted as regeneration triggers only in a permissive tissue context: when body parts are missing, or when tissue-resident polarity signals, such as Wnt activity in planarians, are modified. Hence, the ability to decode generic wound-induced signals as regeneration-initiating cues may be the crucial difference that distinguishes animals that regenerate from those that cannot.