The blue shark (Prionace glauca) exhibits a striking dorsoventral color gradient, transitioning from vibrant blue dorsally to silver and white ventrally, a pattern widely interpreted as pelagic countershading. Despite its ecological significance, the physical basis of this coloration remains unresolved. Here we show that this color system does not arise from dermal chromatophores, as in most vertebrates, but from a previously unrecognised photonic architecture housed within the pulp cavity of individual dermal denticles that cover the skin. Optical imaging reveals discrete color domains within denticle crowns, while external denticle morphology remains similar across color zones. Using spectroscopy, micro-computed tomography, histology, and correlative electron microscopy, we demonstrate that color variation is organized across coupled micro- and nanoscale architectures. In blue denticles, iridophores and melanophores form a densely packed tessellated reflector-absorber system within an expanded crown-restricted pulp cavity. Transition-zone denticles exhibit partial cellular layering, whereas white denticles lack melanophores and contain only reflective cells. At the nanoscale, ordered purine-crystal stacks generate narrowband blue reflection, whereas disordered assemblies produce broadband white scattering. Together, these results reveal denticles as mechanically protected optical "pixels" whose hierarchical cellular and nanocrystal organization generates the shark's countershaded coloration.

Structural blue colors are common in animals, with the tissue nanostructures and material systems that produce them-especially bright blues-typically based on highly ordered nano-architectures. In this study, we describe an unusually bright and angle-independent structural blue from the skin of ribbontail stingray, arising from a more disordered array of scattering elements with a previously undescribed core-shell ultrastructure, involving nano-vesicles enclosing guanine nano-platelets. We show that this skin architecture functions as an intracellular photonic glass, coherently scattering blue, while broadband absorption from closely associated melanophores obviates the low color saturation typical for photonic glasses. Our characterization of skin ultrastructure and color in a stingray demonstrates how disordered systems can be harnessed to produce brilliant hues while illustrating that the capacity for guanine-based colors likely arose extremely early in vertebrate evolution. Moreover, the material-structure-function associations underlying ribbontail stingray coloration, employing two distinct photonic phenomena, illustrate how the evolution of nanoscale architectures can have profound effects at much larger size scales (e.g., in visual ecology and communication), and provide fundamental guidelines for color-saturated manmade photonic glasses. Combining multiscale materials, spectroscopy, microscopy, and optical modeling approaches, we show that the electric blue color of ribbontail stingray skin arises from a unique natural core-shell photonic glass. The color-producing tissue is composed of nanoscale crystals within quasi-ordered vesicles, coherently scattering blue light. Additionally, tissue melanin granules cause broadband absorption, preventing the low color-saturation typical for manmade photonic glasses. image

In animals, pigments but also nanostructures determine skin coloration, and many shades are produced by combining both mechanisms. Recently, we discovered a new mechanism for blue coloration in the ribbontail stingray Taeniura lymma, a species with electric blue spots on its yellow-brown skin. Here, we characterize finescale differences in cell composition and architecture distinguishing blue from non-blue regions, the first description of elasmobranch chromatophores and the nanostructures responsible for the stingray’s novel structural blue, contrasting with other known mechanisms for making nature’s rarest color. In blue regions, the upper dermis comprised a layer of chromatophore units —iridophores and melanophores entwined in compact clusters framed by collagen bundles— this structural stability perhaps the root of the skin color’s robustness. Stingray iridophores were notably different from other vertebrate light-reflecting cells in having numerous fingerlike processes, which surrounded nearby melanophores like fists clenching a black stone. Iridophores contained spherical iridosomes enclosing guanine nanocrystals, suspended in a 3D quasi-order, linked by a cytoskeleton of intermediate filaments. We argue that intermediate filaments form a structural scaffold with a distinct optical role, providing the iridosome spacing critical to produce the blue color. In contrast, black-pigmented melanosomes within melanophores showed space-efficient packing, consistent with their hypothesized role as broadband-absorbers for enhancing blue color saturation. The chromatophore layer’s ultrastructure was similar in juvenile and adult animals, indicating that skin color and perhaps its ecological role are likely consistent through ontogeny. In non-blue areas, iridophores were replaced by pale cells, resembling iridophores in some morphological and nanoscale features, but lacking guanine crystals, suggesting that the cell types arise from a common progenitor cell. The particular cellular associations and structural interactions we demonstrate in stingray skin suggest that pigment cells induce differentiation in the progenitor cells of iridophores, and that some features driving color production may be shared with bony fishes, although the lineages diverged hundreds of millions of years ago and the iridophores themselves differ drastically.

Many fish use a set of pharyngeal jaws in their throat to aid in prey capture and processing, particularly of large or complex prey. In this study-combining dissection, CT scanning, histology, and performance testing-we demonstrate a novel use of pharyngeal teeth in the ocean sunfish (Mola mola), a species for which pharyngeal jaw anatomy had not been described. We show that sunfish possesses only dorsal pharyngeal jaws where, in contrast to their beaklike oral teeth, teeth are recurved spikes, arranged in three loosely connected rows. Fang-like pharyngeal teeth were tightly socketed in the skeletal tissue, with shorter, incompletely-formed teeth erupting between, suggesting tooth replacement. Trichrome staining revealed teeth anchored into their sockets via a combination of collagen bundles originating from the jaw connective tissue and mineralized trabeculae extending from the teeth bases. In resting position, teeth are nearly covered by soft tissue; however, manipulation of a straplike muscle, running transversely on the dorsal jaw face, everted teeth like a cat's claws. Adult sunfish suction feed almost exclusively on gelatinous prey (e.g., jellyfish) and have been observed to jet water during feeding and other activities; flume experiments simulating jetting behavior demonstrated adult teeth caught simulated gelatinous prey with 70%-100% success, with the teeth immobile in their sockets, even at 50x the jetting force, demonstrating high safety factor. We propose that sunfish pharyngeal teeth function as an efficient retention cage for mechanically challenging prey, a curious evolutionary convergence with the throat spikes of divergent taxa that employ spitting and jetting.

Abstract Blue structural colors, produced by diverse tissue nanostructures, are known from all major vertebrate clades except cartilaginous fishes (e.g. sharks, rays). We describe a bright angle-independent structural blue from ribbontail stingray skin, arising from a novel cell type with unique quasi-ordered arrays of nano-vesicles enclosing guanine nanoplatelets. This natural architecture —an intracellular photonic glass— coherently scatters blue, while broadband absorption from closely-associated melanophores obviates the low color-saturation typical for photonic glasses. This first demonstration of structural color in elasmobranchs (the oldest extant clade of jawed vertebrates) illustrates that the capacity for guanine-based colors likely arose extremely early in vertebrate evolution. The structure-function mechanisms underlying ribbontail stingray coloration point to selective pressures driving elasmobranch visual ecology and communication, but also strategies for biomimetic color production.

Background:Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. Methods:Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-alpha/beta receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. Results:Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn(-/-), Tlr3(-/-), and Ifnar1(-/-) mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-alpha/beta receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. Conclusions:This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

In humans, the incidence of congenital defects of the intraembryonic celom and its associated structures has increased over recent decades. Surgical treatment of abdominal and diaphragmatic malformations resulting in congenital hernia requires deep knowledge of ventral body closure and the separation of the primary body cavities during embryogenesis. The correct development of both structures requires the coordinated and fine-tuned synergy of different anlagen, including a set of molecules governing those processes. They have mainly been investigated in a range of vertebrate species (e.g., mouse, birds, and fish), but studies of embryogenesis in humans are rather rare because samples are seldom available. Therefore, we have to deal with a large body of conflicting data concerning the formation of the abdominal wall and the etiology of diaphragmatic defects. This review summarizes the current state of knowledge and focuses on the histological and molecular events leading to the establishment of the abdominal and thoracic cavities in several vertebrate species. In chronological order, we start with the onset of gastrulation, continue with the establishment of the three-dimensional body shape, and end with the partition of body cavities. We also discuss well-known human etiologies.

The liver has been proposed as an important “immune organ” of the body, as it is critically involved in a variety of specific and unique immune tasks. It contains a huge resident immune cell repertoire, which determines the balance between tolerance and inflammation in the hepatic microenvironment. Liver-resident immune cells, populating the sinusoids and the space of Disse, include professional antigen-presenting cells, myeloid cells, as well as innate and adaptive lymphoid cell populations. Machine perfusion (MP) has emerged as an innovative technology to preserve organsex vivowhile testing for organ quality and function prior to transplantation. As for the liver, hypothermic and normothermic MP techniques have successfully been implemented in clinically routine, especially for the use of marginal donor livers. Although there is evidence that ischemia reperfusion injury-associated inflammation is reduced in machine-perfused livers, little is known whether MP impacts the quantity, activation state and function of the hepatic immune-cell repertoire, and how this affects the inflammatory milieu during MP. At this point, it remains even speculative if liver-resident immune cells primarily exert a pro-inflammatory and hence destructive effect on machine-perfused organs, or in part may be essential to induce liver regeneration and counteract liver damage. This review discusses the role of hepatic immune cell subtypes during inflammatory conditions and ischemia reperfusion injury in the context of liver transplantation. We further highlight the possible impact of MP on the modification of the immune cell repertoire and its potential for future applications and immune modulation of the liver.

BackgroundMuscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration.MethodsA clamping model of murine (C57b/6J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 hours (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1x10 6 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration.Results 2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI.ConclusionWIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

The bones are of mesenchymal or ectomesenchymal origin, form the skeleton of most vertebrates, and are essential for locomotion and organ protection. As a living tissue they are highly vascularized and remodelled throughout life to maintain intact. Bones consist of osteocytes entrapped in a mineralized extracellular matrix, and via their elaborated network of cytoplasmic processes they do not only communicate with each other but also with the cells on the bone surface (bone lining cells). Bone tissue develops through a series of fine-tuned processes, and there are two modes of bone formation, referred to either as intramembranous or endochondral ossification. In intramembranous ossification, bones develop directly from condensations of mesenchymal cells, and the flat bones of the skull, the clavicles and the perichondral bone cuff develop via this process. The bones of the axial (ribs and vertebrae) and the appendicular skeleton (e.g. upper and lower limbs) form through endochondral ossification where mesenchyme turns into a cartilaginous intermediate with the shape of the future skeletal element that is gradually replaced by bone. Endochondral ossification occurs in all vertebrate taxa and its onset involves differentiation of the chondrocytes, mineralization of the extracellular cartilage matrix and vascularization of the intermediate, followed by disintegration and resorption of the cartilage, bone formation, and finally - after complete ossification of the cartilage model - the establishment of an avascular articular cartilage. The epiphyseal growth plate regulates the longitudinal growth of the bones, achieved by a balanced proliferation and elimination of chondrocytes, and the question whether the late hypertrophic chondrocytes die or transform into osteogenic cells is still being hotly debated. The complex processes leading to endochondral ossification have been studied for over a century, and this review aims to give an overview of the histological and molecular events, arising from the long bones' (e.g. femur, tibia) development. The fate of the hypertrophic chondrocytes will be discussed in the light of new findings obtained from cell tracking studies.

Tessellated cartilage is a distinctive composite tissue forming the bulk of the skeleton of cartilaginous fishes (e.g. sharks and rays), built from unmineralized cartilage covered at the surface by a thin layer of mineralized tiles called tesserae. The finescale structure and composition of elasmobranch tessellated cartilage has largely been investigated with electron microscopy, micro-computed tomography and histology, but many aspects of tissue structure and composition remain uncharacterized. In our study, we demonstrate that the tessellated cartilage of a stingray exhibits a strong and diverse autofluorescence, a native property of the tissue which can be harnessed as an effective label-free imaging technique. The autofluorescence signal was excited using a broad range of wavelengths in confocal and light sheet microscopy, comparing several sample preparations (fresh; demineralized and paraffin-embedded; non-demineralized and plastic-embedded) and imaging the tissue at different scales. Autofluorescence varied with sample preparation with the signal in both plastic- and paraffin-embedded samples strong enough to allow visualization of finescale (≥ 1 μm) cellular and matrix structures, such as cell nuclei and current and former mineralization fronts, identifiable by globular mineralized tissue. A defined pericellular matrix (PCM) surrounding chondrocytes was also discernible, described here for the first time in elasmobranchs. The presence of a PCM suggests similarities with mammalian cartilage regarding how chondrocytes interact with their environment, the PCM in mammals acting as a transducer for biomechanical and biochemical signals. A posterior analysis of hyperspectral images by an MCR-ALS unmixing algorithm allowed identification of several distinct fluorescence signatures associated to specific regions in the tissue. Some fluorescence signatures identified could be correlated with collagen type II, the most abundant structural molecule of cartilage. Other fluorescence signatures, however, remained unidentified, spotlighting tissue regions that deserve deeper characterization and suggesting the presence of molecules still unidentified in elasmobranch skeletal cartilage. Our results show that autofluorescence can be a powerful exploratory imaging tool for characterizing less-studied skeletal tissues, such as tessellated cartilage. The images obtained are largely comparable with more commonly used techniques, but without the need for complicated sample preparations or external staining reagents standard in histology and electron microscopy (TEM, SEM).

An accepted uniting character of modern cartilaginous fishes (sharks, rays, chimaera) is the presence of a mineralized, skeletal crust, tiled by numerous minute plates called tesserae. Tesserae have, however, never been demonstrated in modern chimaera and it is debated whether the skeleton mineralizes at all. We show for the first time that tessellated cartilage was not lost in chimaera, as has been previously postulated, and is in many ways similar to that of sharks and rays. Tesserae in Chimaera monstrosa are less regular in shape and size in comparison to the general scheme of polygonal tesserae in sharks and rays, yet share several features with them. For example, Chimaera tesserae, like those of elasmobranchs, possess both intertesseral joints (unmineralized regions, where fibrous tissue links adjacent tesserae) and recurring patterns of local mineral density variation (e.g. Liesegang lines, hypermineralized ‘spokes’), reflecting periodic accretion of mineral at tesseral edges as tesserae grow. Chimaera monstrosa's tesserae, however, appear to lack the internal cell networks that characterize tesserae in elasmobranchs, indicating fundamental differences among chondrichthyan groups in how calcification is controlled. By compiling and comparing recent ultrastructure data on tesserae, we also provide a synthesized, up-to-date and comparative glossary on tessellated cartilage, as well as a perspective on the current state of research into the topic, offering benchmark context for future research into modern and extinct vertebrate skeletal tissues.

Cleavage of amyloid precursor protein (APP) by β-secretase BACE1 initiates the production and accumulation of neurotoxic amyloid-β peptides, which is widely considered an essential pathogenic mechanism in Alzheimer's disease (AD). Here, we report that BACE1 is essential for normal auditory function. Compared with wild-type littermates, BACE1-/- mice of either sex exhibit significant hearing deficits, as indicated by increased thresholds and reduced amplitudes in auditory brainstem responses (ABRs) and decreased distortion product otoacoustic emissions (DPOAEs). Immunohistochemistry revealed aberrant synaptic organization in the cochlea and hypomyelination of auditory nerve fibers as predominant neuropathological substrates of hearing loss in BACE1-/- mice. In particular, we found that fibers of spiral ganglion neurons (SGN) close to the organ of Corti are disorganized and abnormally swollen. BACE1 deficiency also engenders organization defects in the postsynaptic compartment of SGN fibers with ectopic overexpression of PSD95 far outside the synaptic region. During postnatal development, auditory fiber myelination in BACE1-/- mice lags behind dramatically and remains incomplete into adulthood. We relate the marked hypomyelination to the impaired processing of Neuregulin-1 when BACE1 is absent. To determine whether the cochlea of adult wild-type mice is susceptible to AD treatment-like suppression of BACE1, we administered the established BACE1 inhibitor NB-360 for 6 weeks. The drug suppressed BACE1 activity in the brain, but did not impair hearing performance and, upon neuropathological examination, did not produce the characteristic cochlear abnormalities of BACE1-/- mice. Together, these data strongly suggest that the hearing loss of BACE1 knock-out mice represents a developmental phenotype.SIGNIFICANCE STATEMENT Given its crucial role in the pathogenesis of Alzheimer's disease (AD), BACE1 is a prime pharmacological target for AD prevention and therapy. However, the safe and long-term administration of BACE1-inhibitors as envisioned in AD requires a comprehensive understanding of the various physiological functions of BACE1. Here, we report that BACE1 is essential for the processing of auditory signals in the inner ear, as BACE1-deficient mice exhibit significant hearing loss. We relate this deficit to impaired myelination and aberrant synapse formation in the cochlea, which manifest during postnatal development. By contrast, prolonged pharmacological suppression of BACE1 activity in adult wild-type mice did not reproduce the hearing deficit or the cochlear abnormalities of BACE1 null mice.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The production of blood cells (haematopoiesis) occurs in the limb bones of most tetrapods but is absent in the fin bones of ray-finned fish. When did long bones start producing blood cells? Recent hypotheses suggested that haematopoiesis migrated into long bones prior to the water-to-land transition and protected newly-produced blood cells from harsher environmental conditions. However, little fossil evidence to support these hypotheses has been provided so far. Observations of the humeral microarchitecture of stem-tetrapods, batrachians, and amniotes were performed using classical sectioning and three-dimensional synchrotron virtual histology. They show that Permian tetrapods seem to be among the first to exhibit a centralised marrow organisation, which allows haematopoiesis as in extant amniotes. Not only does our study demonstrate that long-bone haematopoiesis was probably not an exaptation to the water-to-land transition but it sheds light on the early evolution of limb-bone development and the sequence of bone-marrow functional acquisitions. eLife digest For many aquatic creatures, the red blood cells that rush through their bodies are created in organs such as the liver or the kidney. In most land vertebrates however, blood-cell production occurs in the bone marrow. There, the process is shielded from the ultraviolet light or starker temperature changes experienced out of the water. It is possible that this difference evolved long before the first animal with a backbone crawled out of the aquatic environment and faced new, harsher conditions: yet very little fossil evidence exists to support this idea. A definitive answer demands a close examination of fossils from the water-to-land transition including lobe-finned fish and early limbed vertebrates. To support the production of red blood cells, their fin and limb bones would have needed an internal cavity that can house a specific niche that opens onto a complex network of blood vessels. To investigate this question, Estefa et al. harnessed the powerful x-ray beam produced by the European Synchrotron Radiation Facility and imaged the fin and limb bones from fossil lobe-finned fish and early limbed vertebrates. The resulting three-dimensional structures revealed spongy long bones with closed internal cavities where the bone marrow cells were probably entrapped. These could not have housed the blood vessels needed to create an environment that produces red blood cells. In fact, the earliest four-legged land animals Estefa et al. found with an open marrow cavity lived 60 million years after vertebrates had first emerged from the aquatic environment, suggesting that blood cells only began to be created in bone marrow after the water-to-land transition. Future work could help to pinpoint exactly when the change in blood cell production occurred, helping researchers to identify the environmental and biological factors that drove this change. Introduction Tetrapod long bones are among the most studied skeletal elements in the field of bone biology as they constitute a unit of reference for understanding the development and biomechanics of the appendicular skeleton (e.g. Duboule, 1994; Fröbisch, 2008; Hall, 2008; Shubin et al., 1997). The recent discovery of fossil tetrapod trackways (Ahlberg, 2018; Niedźwiedzki et al., 2010) suggested that limbs supported weight and engaged substrate locomotion earlier than previously thought in early tetrapod evolution. Not only crucial for their biomechanical properties, long bones also host bone marrow including stem-cell niches for the production of blood cells, that is haematopoiesis (Orkin and Zon, 2008). After birth, bone marrow is the definitive haematopoietic system in mostly terrestrial mammals and many other tetrapods (Akiyoshi and Inoue, 2012; Kapp et al., 2018; Orkin and Zon, 2008) but not in fish or some aquatic tetrapods (Akiyoshi and Inoue, 2012; Avagyan and Zon, 2016; Kapp et al., 2018). Indeed, red blood cells are produced in the supraspinal organ in the lamprey, the kidney and liver in actinopterygians (ray-finned fish) and some amphibians (tadpoles and aquatic adults, Akiyoshi and Inoue, 2012), and the kidney in lungfish (Amemiya et al., 2007; Kapp et al., 2018). Several studies proposed that the skeleton would have played a major role in hosting blood-cell production over the water-to-land transition and (1) protecting it against temperature changes (Weiss and Wislocki, 1956), (2) protecting it against potential DNA mutations induced by UV exposure on land (Horton, 1980; Kapp et al., 2018) or (3) providing a better efficiency in red-blood-cell production necessary for metabolically-demanding terrestrial locomotion and aerial respiration (Tanaka, 1976). Our study focusses on characterising the early evolution of the bone marrow and long-bone growth in fossils to contextualise these hypotheses. Tetrapod long bones are regionalised in three parts mirrored from midshaft (Figure 1): (1) the middle of the shaft is called diaphysis; (2) the metaphyses are located at each extremity of the shaft and (3) the epiphyses start above the ossification notch (i.e. where the cortical bone stops forming), extend beyond the metaphyses, and comprise one or more condyles in the case of concave articulations (Francillon-Vieillot et al., 1990). Long bones elongate from the growth plate, which is located in the metaphysis (Figure 1). In this region, the cartilage is progressively substituted with bone: this process is called endochondral ossification (Francillon-Vieillot et al., 1990; Hall, 2005). Figure 1 Download asset Open asset Schematic drawing of the long-bone epiphyses of extant amniotes (A) and amphibians (B). Four conditions are figured here. They are separated by yellow dashed lines: A1, condition in crocodiles (interpreted from Haines, 1938); A2, condition in mammals at an early developmental stage before the appearance of the secondary ossification centre (Anderson and Shapiro, 2010; Tanaka, 1976); B1, condition in Triturus (Cynops) pyrrhogaster (Quilhac et al., 2014; Tanaka, 1976); B2, condition in Rana catesbeiana (Francillon, 1981; Tanaka, 1976). Abbreviations: c., cortex; Dia., diaphysis; e., endosteal bone; Epi., epiphysis; h.c., hypertrophied chondrocytes; Meta., metaphysis; m.p., marrow process; s., sinusoids; sept., septum; trab., trabeculae. In extant amniotes, long-bone elongation results from the proliferation of longitudinal columns of hypertrophic cartilage cells, called hypertrophic chondrocytes (Francillon-Vieillot et al., 1990; Haines, 1942; Xie et al., 2020; Figure 1A). The latter express collagen type X which facilitates the calcification of the surrounding matrix (Gudmann and Karsdal, 2016; Lüllmann-Rauch, 2015). To do so, the hypertrophic chondrocytes secrete matrix vesicles containing calcium phosphate crystals (Amizuka, 2012; Anderson and Shapiro, 2010). The vesicles align longitudinally along the septa. The crystals penetrate the vesicle membranes to form stellate clusters of needle-shaped apatite in the extra cellular matrix (Amizuka, 2012). The mineralisation thus propagates following the longitudinal organisation of the septa (Amizuka, 2012; Anderson and Shapiro, 2010; Figure 1A). Blood vessels and marrow processes invade the growth plate along these columns of hypertrophic cartilage (Lüllmann-Rauch, 2015; Figure 1A). Lytic enzymes secreted by the bone-marrow cells degrade the cartilage matrix, which is progressively substituted by bone deposition (Lüllmann-Rauch, 2015; Suzuki et al., 1981). Growth factors, such as the vascular endothelial growth factor (VEGF), trigger cartilage calcification and regulate endochondral ossification through stimulation of blood-vessel ingrowth into the diaphysis (Gerber et al., 1999). The lines of calcifying stellate clusters of crystals therefore form a scaffold for future trabecular bone deposition (Amizuka, 2012). This results in the formation of a bony mesh of longitudinal trabeculae (Figure 1A), which is progressively incorporated into the metaphysis where haematopoietic stem cell (HSC) niches (Figure 1A) are located (Calvi et al., 2003; Zhang et al., 2003) in the close vicinity of trabecular/endothelial surfaces (Gong, 1978; Nilsson et al., 2001; Wilson and Trumpp, 2006). HSC form localised niches whose environment is greatly controlled and regulated (Orkin and Zon, 2008; Sipkins et al., 2005; Zhang et al., 2003). Often in mature animals the growth plate disappears, causing the senescence of long-bone elongation (Kilborn et al., 2002). In most amniotes, the trabecular mesh in the metaphysis can be vastly remodelled (Haines, 1975). HSC can thereafter be observed adjacent to epiphyseal trabeculae (Askenasy and Farkas, 2002). In extant urodeles (e.g. Pleurodeles waltl, De Ricqlès, 1964; De Ricqlès, 1965), the elongation of limb bones differs from the process in amniotes (Francillon-Vieillot et al., 1990; Haines, 1938; Figure 1B). Unlike mammals, endochondral ossification starts at a later stage in urodeles (De Ricqlès, 1964). The diaphyseal cartilaginous matrix is first hollowed by the formation of lacunae that are subsequently filled in with bone marrow, far before endochondral ossification starts (De Ricqlès, 1964). In mammals and birds (e.g. mouse, Zelzer et al., 2002; chicken, Carlevaro et al., 2000), VEGF initiates vascular ingrowth before endochondral ossification starts. In the amphibian Bufo gargarizan, a peak of VEGF expression is present in the hindlimb at metamorphic climax (Gao et al., 2018) paralleling an increase of endochondral ossification activity (Bo et al., 2018). VEGF would therefore seem to play a major role in amphibian long-bone endochondral ossification as well, but this role still needs to be characterised. The growth plate in the metaphysis of urodeles exhibits no aligned columns of hypertrophic cartilage cells or drastically reduced alignment of a few cells at most (De Ricqlès, 1965; Dickson, 1982; Felisbino and Carvalho, 1999; Felisbino and Carvalho, 2001; Figure 1B). Contrary to amniotes, when present, these aligned columns of hypertrophic cartilage do not constitute the location where the ossification takes place (Figure 1B). Instead, the mineralisation front is located in the underlying areas of the growth plate (i.e. in a layer of non-oriented hypertrophic cartilage or stratified non-oriented hypertrophic cartilage, De Ricqlès, 1965). There, after erosion of the cartilage, mineralisation occurs in urodeles via the formation of globular structures called globuli ossei (Figure 1B; De Ricqlès, 1965; Quilhac et al., 2014) and spherical mineralisation around them (forming Liesegang's rings, Francillon-Vieillot et al., 1990). Globuli ossei are either (1) opened hypertrophic cartilaginous cells which died and were subsequently invaded by a cell from the blood/marrow system to initiate mineralisation or (2) uneroded hypertrophic cartilaginous cells modified into active cells which synthesise bone-like collagen fibrils (of intermediate size between type II of the cartilage and type I of the bone, Quilhac et al., 2014). The endochondral ossification therefore does not produce a longitudinally-oriented trabecular network (Figure 1B), but forms instead a light reticular mesh (De Ricqlès, 1965; Quilhac et al., 2014; Sanchez et al., 2008), rich in globuli ossei (De Ricqlès, 1964; Haines, 1938; Quilhac et al., 2014; Sanchez et al., 2010a). The epiphyses of urodeles remain cartilaginous while they often ossify in anurans (Castanet et al., 2003; Francillon, 1981; Sanchez et al., 2008). This is probably an adaptation to a demanding jumping locomotion and/or heterochronic mechanisms relevant to this clade (Francillon, 1981). In the medullary cavity of their long bones however, the cartilaginous cells hypertrophy with no preferential orientation as in urodeles (Dickson, 1982; Felisbino and Carvalho, 1999; Felisbino and Carvalho, 2001; Miura et al., 2008; Rozenblut and Ogielska, 2005). The resulting spongiosa is largely reduced (even quite often absent, Francillon, 1981). The epiphyseal cartilage hangs over the ossification notch and the shaft (Francillon, 1981) to ossify straight after the metamorphosis (Miura et al., 2008; Rozenblut and Ogielska, 2005). The function of bone marrow in amphibian long bones also differs from the function in extant amniotes. Indeed, in amphibians, the sites for haematopoiesis almost exclusively comprise the thymus, spleen and liver (Akiyoshi and Inoue, 2012; Hightower and Pierre, 1971). Bone marrow only plays a role of haematopoiesis in a few amphibian species (e.g. Xenopus laevis, Rana catesbeiana, Tanaka, 1976; Phillobates terribilis, Dendrobates tinctorius, Kapp et al., 2018). In these cases, haematopoiesis occurs in endosteal regions of the diaphysis between sinusoids (i.e. fenestrated capillaries; Figure 1B2) and endosteum (Tanaka, 1976). No HSC has been observed so far in the epiphysis of frogs. Is the urodele model the plesiomorphic or the derived condition for tetrapod long-bone elongation and bone-marrow function? Very little attention has been given to these aspects of limb-bone evolution. On the one hand, some authors suggest that the amniote-like elongation process may have been the primitive state (Haines, 1942) but no fossil evidence was provided. On the other hand, early tetrapods had cartilaginous epiphyses like extant urodeles. Could that be an indication for a urodele-like primitive condition (e.g. Sanchez et al., 2008; Sanchez et al., 2010a)? This debate relied on the absence of evidence from stem-tetrapod data. Recently, palaeohistological studies revealed a fan-like longitudinal trabecular arrangement in the long-bone metaphysis of the 380-million-year-old lobe-finned fish Eusthenopteron (Sanchez et al., 2014), and in the 365-million-year-old limbed stem-tetrapod Acanthostega (Sanchez et al., 2016). These patterns would result from the same elongation process as in amniotes and would rather suggest that amphibians exhibit a derived condition. Using three-dimensional (3D) virtual histology based on propagation phase-contrast X-ray synchrotron radiation micro-computed tomography (PPC-SRµCT), as well as classical thin-section histology, we herein investigate several stem amphibians and stem amniotes to provide the first glimpses for characterising the early evolution of long-bone elongation and bone-marrow roles. Results The diaphyseal and metaphyseal microarchitectures of the stem tetrapods Eusthenopteron and Hyneria were described by Sanchez et al., 2014 and Kamska et al., 2018, respectively. The diaphysis of Discosauriscus (Sanchez et al., 2008), Apateon (Sanchez et al., 2010a; Sanchez et al., 2010b), Metoposaurus (Konietzko-Meier and Sander, 2013) and Seymouria (Estefa et al., 2020) were thoroughly described, but the metaphyseal organisation of their humeri was only succinctly mentioned in the cited articles. Here, we provide a detailed description of them (Table 1) in 3D when possible. Table 1 Table summarising the material used. Skull length measurements and ontogenetic stages determined by Berman et al., 1987b; Sanchez et al., 2008; Sanchez et al., 2010a and Klembara et al., 2006. SpeciesCollection numberSkull length (cm)Ontogenetic stageBoneApateon caducusGPIM-N 12971.52JuvenileHumerusRadiusUlnaGPIM-N 1572Estimated to 1.60AdultRadiusUlnaApateon pedestrisSMNS 549810.86AdultHumerusRadiusUlnaSMNS 549881.06AdultHumerusRadiusUlnaSeymouria sanjuanensisMNG 77475.6JuvenileHumerusCM 285978.8AdultHumerusDiscosauriscus austriacusSNM Z 155686.2SubadultHumerusMetoposaurus sp.MUZ PGI OS-220/171-Subadult or adultHumerus Apateon caducus, juvenile specimen GPIM-N 1297, humerus As the humerus was crushed (Figure 2A), only a small region of the metaphysis could be sectioned and visualised (Figure 2B). Nevertheless, a relatively complete sequence of calcification (extending over 600 µm) can be described here. The upper part of the section reflects the irregular surface of the calcification front (separating the unpreserved eroded non-calcified cartilage from the preserved calcified cartilage) (Figure 2B). Under this region, obvious figures of globuli ossei are entrapped in Liesegang's rings (g.o. and l.r., Figure 2Bb1-2). They are numerous and unevenly arranged. Their sizes (ranging from 9 to 15 µm in diameter) seem as well unevenly distributed. The trabeculae are very few in this thin section (t., Figure 2Bb2). Figure 2 Download asset Open asset Juvenile specimen of Apateon caducus, GPIM-N 1297. (A) Skeleton. (a) Right limb. (B) Epiphyseal and metaphyseal histology of the proximal end of the humerus. (C) Epiphyseal and metaphyseal histology of the proximal end of the radius (c2-3) and ulna (c1). Abbreviations: c.b., cortical bone; c.c., cluster of chondrocytes; c.f., calcification front; c.m., cartilage matrix; dia., diaphysis; e.b., erosion bay; e.l., erosion lacunae; g.o., globuli ossei; H., humerus; l.r., Liesegang's rings; meta., metaphysis; m.f., mineralisation front; o.n., ossification notch; Prox., proximal end; R. and U., radius and ulna; t., trabeculae. A. caducus, juvenile specimen GPIM-N 1297, radius and ulna Both bones exhibit large sequences of cartilage calcification which spread over more than a third of the total bone length on each side of the long bone (Figure 2C). The mineralisation front (m.f., Figure 2C) is located relatively far under the ossification notch (400 µm) (o.n., Figure 2C). Numerous globuli ossei can be visualised in the metaphysis (g.o., Figure 2Cc1,3). They are unevenly distributed and their size ranges between 8 and 25 µm. Clusters of chondrocytes can be observed (c.c., Figure 2Cc3). The top of the epiphysis probably exhibited a uniform matrix of uncalcified cartilage before the fossilisation that was not preserved afterwards. The mesh of ossified trabeculae is very scattered and shows no preferential orientation (t., Figure 2Cc1). A. caducus, adult specimen GPIM-N 1572, radius and ulna The epiphysis and metaphysis of the radius and ulna of this individual (Figure 3A) are more hollowed than those of the specimen GPIM-N 1297, with less cartilaginous matrix between the mineralised trabeculae (Figure 3B). Fewer globuli ossei are visible (g.o., Figure 3Bb1). Instead, large empty lacunae can be observed (75 µm) (e.l., Figure 3Bb1). Large bays of erosion open as well between these lacunae (e.b., Figure 3Bb2). The process of mineralisation therefore seems more advanced but no obvious trabecular organisation can be observed. Figure 3 Download asset Open asset Adult specimen of Apateon caducus, GPIM-N 1572. (A) Skeleton. (a) Right limb. (B) Epiphyseal and metaphyseal histology of the proximal end of the radius (b1) and ulna (b2). Abbreviations: c.b., cortical bone; c.m., cartilage matrix; dia., diaphysis; e.b., erosion bay; e.l., erosion lacunae; g.o., globuli ossei; H., humerus; l.r., Liesegang's rings; meta., metaphysis; Prox., proximal end; R. and U., radius and ulna. Apateon pedestris, adult specimen SMNS 54981, humerus In the humerus of SMNS 54981 (Figure 4A), the process of mineralisation seems relatively advanced as the globuli ossei only remain along a few mineralised trabeculae (g.o., Figure 4B). They are 16 µm large. In the metaphysis, the cartilage has been removed by erosional process (e.b., Figure 4B). The uncalcified cartilage in the epiphysis has not been preserved during the fossilisation (at least in this slide). Figure 4 Download asset Open asset Adult specimen of Apateon pedestris, SMNS 54981. (A) Skeleton. (a) Right limb. (B) Epiphyseal and metaphyseal histology of the distal end of the humerus. (C) Epiphyseal and metaphyseal histology of the proximal end of the radius (c2) and ulna (c1). Abbreviations: c.b., cortical bone; c.c.t., calcified-cartilage trabecula; Dist., distal end; e.b., erosion bay; g.o., globuli ossei; H., humerus; l.r., Liesegang's rings; meta., metaphysis; m.f., mineralisation front; m.t., mineralised trabecula; Prox., proximal end; R. and U., radius and ulna. A. pedestris, adult specimen SMNS 54981, radius and ulna The quantity of calcified cartilage is higher in the zeugopod (i.e. radius and ulna) than in the stylopod (i.e. humerus) (Figure 4Cc1-2). Most of the uncalcified cartilage has been eroded. The calcified cartilage is hollowed, thereby forming multiple bays of erosion (e.b., Figure 4Cc2). Nevertheless, the globuli ossei remain connected to each other by calcified-cartilage trabeculae (c.c.t., Figure 4Cc2) or mineralised trabeculae (m.t., Figure 4C) present in the metaphysis. A. pedestris, adult specimen SMNS 54988, humerus This thin section in the humerus of SMNS 54988 (Figure 5A) shows a very remodelled bone with large bays of erosion in the cartilaginous matrix (e.b., Figure 5Bb) and only a few remaining globuli ossei at the surface of the bone trabeculae (g.o., Figure 5Bb). Most of the cartilaginous matrix has been eroded. There is no preserved cartilage in the epiphysis. The bony trabeculae have no preferential orientation. Figure 5 Download asset Open asset Adult specimen of Apateon pedestris, SMNS 54988. (A) Skeleton. (a) Right limb. (B) Epiphyseal and metaphyseal histology of the distal end of the humerus. (C) Epiphyseal and metaphyseal histology of the proximal end of the radius and ulna. Abbreviations: c.b., cortical bone; c.m., cartilage matrix; dia., diaphysis; Dist., distal end; e.b., erosion bay; e.l., erosion lacunae; g.o., globuli ossei; H., humerus; meta., metaphysis; Prox., proximal end; R. and U., radius and ulna. A. pedestris, adult specimen SMNS 54988, radius and ulna As for the zeugopod of the specimen SMNS 54988 (Figure 5Aa), the globuli ossei seem to be replaced by large empty lacunae (30 µm, e.l., Figure 5C). A certain amount of uncalcified cartilage has been eroded in the distal epiphyses and metaphyses. Nevertheless, a large amount of cartilage is still present in the proximal metaphyses of both long bones (c.m., Figure 5C). No or very few trabeculae can be observed. Metoposaurus sp., (sub-)adult specimen MUZ PGI OS-220/171, humerus Transverse thin sections were made in the metaphysis of the femur (Konietzko-Meier and Sander, 2013) of Metoposaurus diagnosticus krasiejowensis (Sulej, 2002) recently re-diagnosed as Metoposaurus krasiejowensis (Brusatte et al., 2015). They revealed a dense trabecular mesh. The longitudinal virtual thin sections, made with PPC-SRµCT and presented here, were made in the proximal and distal metaphyses of a humerus of Metoposaurus sp. and confirm the presence of a dense trabecular mesh in the overall humerus (Figure 6Aa1-2). Additionally, a directional coloured light effect (cf. Materials and method section, Sanchez et al., 2014) shows that this mesh is oriented longitudinally and exhibits a fan-like shape in the metaphyses (purple trabeculae, Figure 6Aa2). The trabecular mesh covers the entire volume of the metaphysis and spreads into the diaphysis (Figure 6Aa1-2). The mineralisation front (m.f., Figure 6Aa1) contacts the sediment in which the bone is embedded (Figure 6Ba). The surface of the mineralisation front is irregular. No ossified epiphysis was found, thereby suggesting that a cartilaginous cap was probably covering the bone. This cap did not preserve over the fossilisation. In the metaphysis, the trabeculae are homogeneously distributed (t., Figure 6Bb). Some remnants of calcified cartilage are visible through Liesegang's rings forming within the cartilage remaining between the metaphyseal trabeculae (Figure 6Bc). The mean thickness of the trabeculae is 117 µm (Table 2). Tubular structures can be observed (m.p., Figure 6Ba). They end blindly at the location of the mineralisation front. They are well defined tubes (248 µm in diameter, Table 2), although anastomosed. They ossified through endochondral ossification. These tubes are locally slightly eroded (Figures 6Ba and 10A). The size of these tubes, their intimate connection to each other and their location strongly support their identification as marrow processes (Haines, 1938). Figure 6 Download asset Open asset Left humerus of a (sub-)adult specimen of Metoposaurus sp., MUZ PGI OS-220/171 imaged using PPC-SRµCT. (A) Frontal view. (a1) Longitudinal virtual thin section (40 µm thick) and (a2) longitudinal virtual thin section of the segmented model of the bone (50 µm thick). The longitudinally-oriented trabeculae are highlighted in purple (white arrows), while the transversally-oriented trabeculae appear in green. (B) Ventral view. (a) Longitudinal virtual thin section of the proximal metaphysis (40 µm thick), (b) transverse virtual thin section made in the metaphysis and (c) longitudinal thin section made in the distal metaphysis. Abbreviations: c.b., cortical bone; dia., diaphysis; Dist., distal end; l.r., Liesegang's rings; meta., metaphysis; m.f., mineralisation front; m.p., marrow process; Prox., proximal end; s., sediment; t., trabeculae. Table 2 Microanatomical measurements made on the samples using VGStudio MAX (version 3.2, Volume Graphics Inc, Germany). The protocol details are provided by Estefa et al., 2020. SpeciesThickness of the trabeculae (µm)Diameter of the marrow processes (µm)DiaphysisMetaphysisMetaphysisMetoposaurus sp. (Subadult or Adult, MUZ PGI OS-220/171)131117248Seymouria sanjuanensis (Juvenile, MNG 7747)9425100S. sanjuanensis (Adult, CM 28597)7930100Discosauriscus austriacus (Subadult, SNM Z 15568)8054111 Seymouria sanjuanensis, juvenile specimen MNG 7747, humerus This specimen was investigated using PPC-SRµCT. The spongiosa occupies the entire bone area (Figure 7A). The metaphyseal trabeculae are about four times thinner (25 µm on average) than the diaphyseal trabeculae (94 µm on average, Table 2 and Estefa et al., 2020). A longitudinal section reveals that the trabecular mesh becomes denser towards the distal and proximal ends of the bone (Figure 7Aa1-2). As the shape of the bone widens and flattens from midshaft towards the metaphyseal surfaces, the longitudinal trabeculae tilt, thereby forming a fan-like configuration (Figure 7Aa2). In the metaphysis, the trabecular mesh is mostly arranged longitudinally (obviously appearing purple and progressively shifting to green as the deltopectoral crest tilts to 90 degrees, Figure 7Aa2) although a few anastomoses (highlighted in green in most of the metaphysis apart from the tilted region of the deltopectoral crest, Figure 7Aa2) run radially. A few remnants of calcified cartilage (Francillon-Vieillot et al., 1990) are very rarely visible (Estefa et al., 2020). Marrow processes form an intricate network, while anastomosing to each other, and connecting to cavities of irregular shapes and sizes (Figure 7Ba,c and 10B). These tubular structures are around 100 µm in diameter under the mineralisation front (Table 2). They contact each other when they reach the mineralisation front (Figure 10B). No ossified epiphysis was found. The humeral epiphysis was probably not preserved due to being unmineralised cartilage. Figure 7 Download asset Open asset Left humerus of a juvenile specimen of Seymouria sanjuanensis, MNG 7747 imaged using PPC-SRµCT. (A) Frontal view. (a1) Longitudinal virtual thin section (40 µm thick), the darker part is an artefact in the original data due to electron reinjection in the synchrotron storage ring (refilling) during the scan and (a2) longitudinal virtual thin section of the segmented model of the bone (250 µm thick). The longitudinally-oriented trabeculae (pointed by horizontal arrows) are highlighted in purple, while the transversally-oriented trabeculae appear in green. Note that, due to the shape of the metaphysis, the trabeculae exhibit an overall fan-like configuration which progressively tilts to 90 degrees at the location of the deltopectoral crest (Asterisk). For that reason, the longitudinal trabeculae appear green and the transverse trabeculae appear purple at this location. (B) Ventral view. (a) Longitudinal virtual thin section in the proximal metaphysis (40 µm thick), (b) transverse virtual thin section in the metaphysis, the large ring artefact results from the synchrotron electron refilling visible in a1, (c) detail of (b) showing marrow processes and cavities in transverse section. Abbreviations: c., cavity; dia., diaphysis; Dist., distal end; meta., metaphysis; m.f., mineralisation front; m.p., marrow process; Prox., proximal end; s., sediment; t., trabeculae. S. sanjuanensis, adult specimen CM 28597, humerus A longitudinal virtual thin section from the PPC-SRµCT data shows that the trabecular network remains relatively dense in the metaphyses (Figure 8A) at the adult stage. The trabecular mesh is longitudinally and radially oriented like a fan although slightly less organised than in the juvenile specimen (purple trabeculae, Figure 8Aa2). The trabeculae appear to be more remodelled, leaving large cavities resulting from an intense erosional process (Figure 8Aa2). The cortex is almost inexistent in the metaphysis (Figure 8Bb). The thickness of the trabeculae averages 30 µm (Table 2, Estefa et al., 2020), which is equivalent to the thickness of the trabeculae in the juvenile metaphysis (MNG 7747). There is no endosteal bone on the surface of the medullary cavity. Very few remnants of calcified cartilage were found in the metaphysis of the adult humerus, that is in much lower frequency than in the juvenile specimen (Estefa et al., 2020). The spongiosa contains a few longitudinal interconnected marrow processes (100 µm in diameter, Figure 8Bb

AIMS:As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia.METHODS AND RESULTS:Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography.CONCLUSION:The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium.

Cytokine-expression profiles revealed IL-1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL-1β antibody in limb transplantation. Following allogenic hind-limb transplantation, Lewis rats were either left untreated or treated with anti-lymphocyte serum + tacrolimus (baseline); baseline immunosuppression + anti-IL-1β (1 mg/kg once/week, 6-8 subcutaneous injections) into the transplanted or contralateral limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti-IL-1β injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL-1β, IL-4, IL-13, IP-10, MCP-1, and MCP-3 in long-term-survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti-IL-1β. Inhibition of IL-1β with short-term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.