Allogenic tissue-specific decellularized scaffolds promote long-term muscle innervation and functional recovery in a surgical diaphragmatic hernia model

Congenital diaphragmatic hernia (CDH) is a neonatal defect in which the diaphragm muscle does not develop properly, thereby raising abdominal organs into the thoracic cavity and impeding lung development and function. Large diaphragmatic defects require correction with prosthetic patches to close the malformation. This treatment leads to a consequent generation of unwelcomed mechanical stress in the repaired diaphragm and hernia recurrences, thereby resulting in high morbidity and significant mortality rates. We proposed a specific diaphragm-derived extracellular matrix (ECM) as a scaffold for the treatment of CDH. To address this strategy, we developed a new surgical CDH mouse model to test the ability of our tissue-specific patch to regenerate damaged diaphragms. Implantation of decellularized diaphragmatic ECM-derived patches demonstrated absence of rejection or hernia recurrence, in contrast to the performance of a commercially available synthetic material. Diaphragm-derived ECM was able to promote the generation of new blood vessels, boost long-term muscle regeneration, and recover host diaphragmatic function. In addition, using a GFP\u202f+\u202fSchwann cell mouse model, we identified re-innervation of implanted patches. These results demonstrated for the first time that implantation of a tissue-specific biologic scaffold is able to promote a regenerating diaphragm muscle and overcome issues commonly related to the standard use of prosthetic materials

Customized bioreactor enables the production of 3D diaphragmatic constructs influencing matrix remodeling and fibroblast overgrowth

The production of skeletal muscle constructs useful for replacing large defects in vivo, such as in congenital diaphragmatic hernia (CDH), is still considered a challenge. The standard application of prosthetic material presents major limitations, such as hernia recurrences in a remarkable number of CDH patients. With this work, we developed a tissue engineering approach based on decellularized diaphragmatic muscle and human cells for the in vitro generation of diaphragmatic-like tissues as a proof-of-concept of a new option for the surgical treatment of large diaphragm defects. A customized bioreactor for diaphragmatic muscle was designed to control mechanical stimulation and promote radial stretching during the construct engineering. In vitro tests demonstrated that both ECM remodeling and fibroblast overgrowth were positively influenced by the bioreactor culture. Mechanically stimulated constructs also increased tissue maturation, with the formation of new oriented and aligned muscle fibers. Moreover, after in vivo orthotopic implantation in a surgical CDH mouse model, mechanically stimulated muscles maintained the presence of human cells within myofibers and hernia recurrence did not occur, suggesting the value of this approach for treating diaphragm defects

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape

Envenomations by Bothrops and Crotalus Snakes Induce the Release of Mitochondrial Alarmins

Skeletal muscle necrosis is a common manifestation of viperid snakebite envenomations. Venoms from snakes of the genus Bothrops, such as that of B. asper, induce muscle tissue damage at the site of venom injection, provoking severe local pathology which often results in permanent sequelae. In contrast, the venom of the South American rattlesnake Crotalus durissus terrificus, induces a clinical picture of systemic myotoxicity, i.e., rhabdomyolysis, together with neurotoxicity. It is known that molecules released from damaged muscle might act as ‘danger’ signals. These are known as ‘alarmins’, and contribute to the inflammatory reaction by activating the innate immune system. Here we show that the venoms of B. asper and C. d. terrificus release the mitochondrial markers mtDNA (from the matrix) and cytochrome c (Cyt c) from the intermembrane space, from ex vivo mouse tibialis anterior muscles. Cyt c was released to a similar extent by the two venoms whereas B. asper venom induced the release of higher amounts of mtDNA, thus reflecting hitherto some differences in their pathological action on muscle mitochondria. At variance, injection of these venoms in mice resulted in a different time-course of mtDNA release, with B. asper venom inducing an early onset increment in plasma levels and C. d. terrificus venom provoking a delayed release. We suggest that the release of mitochondrial ‘alarmins’ might contribute to the local and systemic inflammatory events characteristic of snakebite envenomations

Effects of snake neurotoxins and lipids on neurons

The venom of many snakes contains presynaptic neurotoxins (SPAN, snake presynaptic PLA2 neurotoxin), which are able to block neurotransmission and induce muscular paralysis. Structural motives are conserved across toxins, but quaternary structures vary considerably; we have therefore considered four toxins characterised by different structural complexity: Notexin (monomer), ?-bungarotoxin (heterodimer), taipoxin (trimer) e textilotoxin (pentamer). These toxins are endowed with PLA2 enzymatic activity; they hydrolyse the sn-2 ester bond of glycerophospholipids, thereby producing lysophospholipids (lysoPLs) and fatty acids. It has been shown, by using equimolar mixtures of such lipids on isolated neuromuscular junction and on cultivated neurons, that they are able to reproduce the effects of toxins: the block of neurotransmission with alteration of the morphology of nerve terminals (depletion of synaptic vesicles (SVs), swelling, alteration of mitochondria) and the formation of bulges on neurites, characterised by accumulation of specific marker of SVs. These findings have led to interpreting the toxin mechanism of action as driven by an imbalance between exocytosis and endocytosis, due to the different structural characteristics of the lipids being produced: the lysophosphatidylcholine (lysoPC) has an inverted-cone shape and remains in the external leaflet of the membrane; on the contrary, fatty acids have a cone shape and move fast to both leaflets of the membrane. Such alteration of the lipid composition of the membrane causes its deformation, which facilitates its fusion with the SVs (ie, it facilitates the transition between the hemifusion intermediate and the open pore) and at the same time inhibits endocytosis, which is necessary to recover empty vesicles and to maintain the functionality of the synapses. Similar results, with regard to the block of neurotransmission, have been obtained when testing lysoPLs with different polar heads; in particular, lysoPLs with a negative charge are able, in presence of high magnesium concentrations, to induce a triphasic pattern of paralysis (initial depression followed by increment of neurotransmitter release, and finally a progressive lowering of the muscular contraction), which is considered specific to SPANs. The phenomenon can be reversed by washing with albumin, which complexes lipids and restores the equilibrium. SPANs and their lipid products have been tested also on the neuromuscular junction of Drosophila melanogaster. In this model, however, the toxins resulted inactive. This suggests that the specific receptors, which are necessary to their action, are missing. On the contrary, the mixture of lysoPC and oleic acid (OA) causes an initial increase of their excitatory postsynaptic potential, followed by the block of neurotransmission, and the bulging of synaptic buttons. Similar to what observed on the mouse neuromuscular junction, lysoPC is the active component; its effect on the fusion of SVs is more general than that of the toxins. Significant amounts of lysoPC were detected in the saliva of hematophagous arthropods, which are at an earlier stage of the evolution process than snakes. When used in laboratory experiments, this is sufficient to cause a range of effects which are similar to those induced by the synthetic lipids. It is therefore conceivable that such lipids represent a very simple poison obtained from digestive phospholipases, from which neurotoxins could then have evolved. The effect on the probability of SV fusion is not sufficient to explain the extensive depletion which is observed after intoxication. The prolonged production of lysoPLs and fatty acids causes an alteration of the membrane permeability to calcium, which enters into the cell mainly from the external medium. This effect induces a massive fusion of SVs, also involving the reserve pool, and the alteration of cell metabolism, notably for what concern the mitochondria. Toxins conjugated with fluorophores are visible inside the neurons a few minutes after intoxication, and they colocalise with mitochondria. When used on preparations of isolated mitochondria, such toxins are able to promote the opening of the permeability transition pore with an efficiency which is directly proportional to their phospholipasic activity; similar results were obtained with mixtures of lysoPC and fatty acids. Other amphipathic molecules, with different chemical structure than lysoPC, but a broadly similar inverted-cone shape, are also able to induce a reversible block of neurotransmission, the formation of bulges in the neurons and an increase in the intracellular calcium levels, with similar kinetics to lysoPC. In particular, miltefosine and perifosine (used in anti-tumoral therapies) as well as the lysolipid derived from PAF (Platelet Activated Factor) have already been tested. The neurotoxicity of the snake phospholipases derives therefore from a high level of specificity and localisation of enzymatic activity; however, their mechanism of action is more general, as it is related to the general role of lipids in the biological membrane fusion process .Il veleno di molti serpenti contiene neurotossine ad azione presinaptica (SPAN, snake presynaptic PLA2 neurotoxin) che sono in grado di provocare blocco della neurotrasmissione e paralisi muscolare. I motivi strutturali sono molto conservati, ma la struttura quaternaria è molto variabile; si sono perciò prese in considerazione quattro tossine di diversa complessità strutturale: notexin (monomero), ?-bungarotoxin (eterodimero), taipoxin (trimero) e textilotoxin (pentamero). Queste tossine sono dotate di attività enzimatica di tipo PLA2, cioè idrolizzano il legame estereo in posizione 2 dei glicerofosfolipidi, producendo lisofosfolipidi e acidi grassi. Utilizzando miscele equimolari di questi lipidi su giunzione neuromuscolare isolata e su neuroni in coltura si è dimostrato che essi sono in grado di riprodurre gli effetti delle tossine: blocco della neurotrasmissione con alterazione della morfologia dei terminali (deplezione delle vescicole sinaptiche, rigonfiamento, alterazione dei mitocondri) e formazione di rigonfiamenti (“bulges”) sui neuriti, caratterizzati da accumulo di proteine specifiche delle vescicole sinaptiche. Questi dati hanno portato a interpretare il meccanismo d’azione delle tossine come uno sbilanciamento tra eso ed endocitosi, dovuto alle caratteristiche strutturali dei lipidi che vengono prodotti: la lisofosfatidilcolina (lisoPC) ha forma di cono invertito e rimane confinata nel foglietto esterno della membrana, gli acidi grassi hanno forma a cono, ma si ripartiscono velocemente su entrambe i foglietti. Questa alterazione della composizione lipidica della membrana plasmatica ne provoca una deformazione che rende più facile il processo di fusione con le vescicole sinaptiche, cioè facilita la transizione tra intermedio di emifusione e poro aperto; per le stesse ragioni invece inibisce l’endocitosi, necessaria per il recupero delle vescicole stesse e il mantenimento della funzionalità della sinapsi. Analoghi risultati di blocco della neurotrasmissione sono stati ottenuti testando lisofosfolipidi con diverse teste polari; in particolare, lisolipidi dotati di carica negativa, in presenza di alte concentrazioni di magnesio, sono in grado di provocare un andamento trifasico della paralisi (iniziale depressione, facilitazione, diminuzione progressiva della contrazione muscolare) ritenuto tipico delle SPANs. Il fenomeno è reversibile mediante lavaggio con albumina, capace di complessare i lipidi e riequilibrare la situazione. Le SPANs e i loro prodotti lipidici sono stati testati anche sulla giunzione neuromuscolare di Drosophila melanogaster: le tossine sono risultate inattive in questo modello, suggerendo la mancanza di recettori specifici, necessari alla loro azione. La miscela di lisoPC e acido oleico provoca invece un iniziale aumento del potenziale post-sinaptico eccitatorio, seguito dal blocco della neurotrasmissione, e un rigonfiamento dei bottoni sinaptici. Analogamente a quanto osservato sulla giunzione neuromuscolare di topo, il componente attivo è la lisoPC e il suo effetto sulla fusione delle vescicole sinaptiche sembra essere di carattere generale. Anche animali meno evoluti dei serpenti, come alcuni insetti ematofagi, contengono nella loro saliva concentrazioni significative di lisoPC, sufficienti a provocare nei modelli sperimentali effetti analoghi a quelli osservati con i lipidi sintetici. E’ possibile che questi lipidi rappresentino una forma molto semplice di veleno ottenuto da fosfolipasi digestive, da cui si sono in seguito evolute le neurotossine. L’effetto sulla fusione delle vescicole non è sufficiente a spiegare l’estensiva deplezione che si osserva dopo l’intossicazione. La prolungata produzione di lisolipidi e acidi grassi provoca un’alterazione della permeabilità della membrana allo ione calcio, che entra nella cellula principalmente dal mezzo esterno. Questo comporta una massiccia fusione delle vescicole sinaptiche, che coinvolge anche il pool di riserva, e alterazioni del metabolismo cellulare, soprattutto a carico dei mitocondri. Tossine coniugate con fluorofori sono visibili all’interno dei neuroni dopo tempi brevi di intossicazione e colocalizzano con i mitocondri. Su preparazioni di mitocondri isolati sono in grado di promuovere l’apertura del poro di transizione della permeabilità mitocondriale con un’efficienza proporzionale alla loro attività fosfolipasica; analoghi risultati si sono ottenuti con miscele di lisoPC e acidi grassi. Blocco reversibile della neurotrasmissione, formazione di bulges nei neuroni e aumento dei livelli intracellulari di calcio sono prodotti, con cinetiche simili a quelle della lisoPC, anche da altre molecole anfipatiche di diversa struttura chimica, ma aventi una forma complessiva di cono invertito. Tra queste sono state testate miltefosina e perifosina, usate in terapia come agenti anti-tumorali, e il lisolipide derivato dal PAF (Platelet Activated Factor). La neurotossicità delle fosfolipasi di serpente deriva quindi da una elevata specificità e localizzazione dell’attività enzimatica; accanto a ciò è da considerare che la loro azione si esplica su un meccanismo che ha carattere più generale, cioè il ruolo dei lipidi nei processi di fusione delle membrane biologiche

Neurotoxicity of inverted-cone shaped lipids

Many amphipatic molecules are characterized by an inverted-cone shape capable of altering the curvature and other properties of the plasma membrane of cells. We have recently shown that several lysophospholipids which have this shape impair nerve terminals by promoting neuroexocytosis and inhibiting endocytosis. This results in a bulging of neurites and nerve terminals and block of neurotransmission with paralysis of the neuromuscular junction. Here, we have determined the neurotoxicity of four inverted-cone shaped molecules of great interest because of their biological and pharmacological activities: miltefosine, perifosine, lysoPAF and lysophosphatidylcholine. These compounds were found to cause a complete, but reversible, paralysis of the nerve-hemidiaphragm preparation and to induce bulging of neurons in culture with entry of calcium from the external medium

Reversible skeletal neuromuscular paralysis induced by different lysophospholipids

Lysophosphatidylcholine rapidly paralyses the neuromuscular junction (NMJ), similarly to snake phospholipase A2 neurotoxins, implicating a lipid hemifusion-pore transition in neuroexocytosis. The mode and kinetics of NMJ paralysis of different lysophospholipids (lysoPLs) in high or low [Mg2+] was investigated. The following order of potency was found: lysophosphatidylcholine>lysophosphatidylethanolamine>lysophosphatidic acid>lysophosphatidylserine>lysophosphatidylglycerol. The latter two lysoPLs closely mimic the profile of paralysis caused by the toxins in high [Mg2+]. This paralysis is fully reversed by albumin washing. These findings provide novel insights on the mode of action of snake neurotoxins and qualify lysoPLs as novel agents to study neuroexocytosis

Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins.

Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca(2+) which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of beta-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca(2+) influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca(2+) overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins

The synaptotagmin juxtamembrane domain is involved in neuroexocytosis

AbstractSynaptotagmin is a synaptic vesicle membrane protein which changes conformation upon Ca2+ binding and triggers the fast neuroexocytosis that takes place at synapses. We have synthesized a series of peptides corresponding to the sequence of the cytosolic juxtamembrane domain of synaptotagmin, which is highly conserved among different isoforms and animal species, with or without either a hexyl hydrophobic chain or the hexyl group plus a fluorescein moiety. We show that these peptides inhibit neurotransmitter release, that they localize on the presynaptic membrane of the motor axon terminal at the neuromuscular junction and that they bind monophosphoinositides in a Ca2+-independent manner. Based on these findings, we propose that the juxtamembrane cytosolic domain of synaptotagmin binds the cytosolic layer of the presynaptic membrane at rest. This binding brings synaptic vesicles and plasma membrane in a very close apposition, favouring the formation of hemifusion intermediates that enable rapid vesicle fusion