The long Filamin-A isoform is required for intestinal development and motility: implications for chronic intestinal pseudo-obstruction.

Filamin A (FLNA) is a cytoplasmic actin binding protein, recently shown to be expressed as a long and short isoform. Mutations in FLNA are associated with a wide spectrum of disorders, including an X-linked form of chronic intestinal pseudo-obstruction (CIPO). However, the role of FLNA in intestinal development and function is largely unknown. In this study, we show that FLNA is expressed in the muscle layer of the small intestine from early human fetal stages. Expression of FLNA variants associated with CIPO, blocked expression of the long flna isoform and led to an overall reduction of RNA and protein levels. As a consequence, contractility of human intestinal smooth muscle cells was affected. Lastly, our transgenic zebrafish line showed that the flna long isoform is required for intestinal elongation and peristalsis. Histological analysis revealed structural and architectural changes in the intestinal smooth muscle of homozygous fish, likely triggered by the abnormal expression of intestinal smooth muscle markers. No defect in the localization or numbers of enteric neurons was observed. Taken together, our study demonstrates that the long FLNA isoform contributes to intestinal development and function. Since loss of the long FLNA isoform does not seem to affect the enteric nervous system, it likely results in a myopathic form of CIPO, bringing new insights to disease pathogenesis.

Direct Interaction of Sox10 With Cadherin-19 Mediates Early Sacral Neural Crest Cell Migration: Implications for Enteric Nervous System Development Defects.

BACKGROUND AND AIMS: The enteric nervous system, which regulates many gastrointestinal functions, is derived from neural crest cells (NCCs). Defective NCC migration during embryonic development may lead to enteric neuropathies such as Hirschsprung's disease (hindgut aganglionosis). Sox10 is known to be essential for cell migration but downstream molecular events regulating early NCC migration have not been fully elucidated. This study aimed to determine how Sox10 regulates migration of sacral NCCs toward the hindgut using Dominant megacolon mice, an animal model of Hirschsprung's disease with a Sox10 mutation. METHODS: We used the following: time-lapse live cell imaging to determine the migration defects of mutant sacral NCCs; genome-wide microarrays, site-directed mutagenesis, and whole embryo culture to identify Sox10 targets; and liquid chromatography and tandem mass spectrometry to ascertain downstream effectors of Sox10. RESULTS: Sacral NCCs exhibited retarded migration to the distal hindgut in Sox10-null embryos with simultaneous down-regulated expression of cadherin-19 (Cdh19). Sox10 was found to bind directly to the Cdh19 promoter. Cdh19 knockdown resulted in retarded sacral NCC migration in vitro and ex vivo, whereas re-expression of Cdh19 partially rescued the retarded migration of mutant sacral NCCs in vitro. Cdh19 formed cadherin-catenin complexes, which then bound to filamentous actin of the cytoskeleton during cell migration. CONCLUSIONS: Cdh19 is a direct target of Sox10 during early sacral NCC migration toward the hindgut and forms cadherin-catenin complexes which interact with the cytoskeleton in migrating cells. Elucidation of this novel molecular pathway helps to provide insights into the pathogenesis of enteric nervous system developmental defects.

Development of a porcine acellular bladder matrix for tissue-engineered bladder reconstruction.

PURPOSE: Enterocystoplasty is adopted for patients requiring bladder augmentation, but significant long-term complications highlight need for alternatives. We established a protocol for creating a natural-derived bladder extracellular matrix (BEM) for developing tissue-engineered bladder, and investigated its structural and functional characteristics. METHODS: Porcine bladders were de-cellularised with a dynamic detergent-enzymatic treatment using peristaltic infusion. Samples and fresh controls were evaluated using histological staining, ultrastructure (electron microscopy), collagen, glycosaminoglycans and DNA quantification and biomechanical testing. Compliance and angiogenic properties (Chicken chorioallantoic membrane [CAM] assay) were evaluated. T test compared stiffness and glycosaminoglycans, collagen and DNA quantity. p value of < 0.05 was regarded as significant. RESULTS: Histological evaluation demonstrated absence of cells with preservation of tissue matrix architecture (collagen and elastin). DNA was 0.01 µg/mg, significantly reduced compared to fresh tissue 0.13 µg/mg (p < 0.01). BEM had increased tensile strength (0.259 ± 0.022 vs 0.116 ± 0.006, respectively, p < 0.0001) and stiffness (0.00075 ± 0.00016 vs 0.00726 ± 0.00216, p = 0.011). CAM assay showed significantly increased number of convergent allantoic vessels after 6 days compared to day 1 (p < 0.01). Urodynamic studies showed that BEM maintains or increases capacity and compliance. CONCLUSION: Dynamic detergent-enzymatic treatment produces a BEM which retains structural characteristics, increases strength and stiffness and is more compliant than native tissue. Furthermore, BEM shows angiogenic potential. These data suggest the use of BEM for development of tissue-engineered bladder for patients requiring bladder augmentation.

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia.

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease.

The Somatic Mutation Paradigm in Congenital Malformations: Hirschsprung Disease as a Model.

Patients with Hirschsprung disease (HSCR) do not always receive a genetic diagnosis after routine screening in clinical practice. One of the reasons for this could be that the causal mutation is not present in the cell types that are usually tested-whole blood, dermal fibroblasts or saliva-but is only in the affected tissue. Such mutations are called somatic, and can occur in a given cell at any stage of development after conception. They will then be present in all subsequent daughter cells. Here, we investigated the presence of somatic mutations in HSCR patients. For this, whole-exome sequencing and copy number analysis were performed in DNA isolated from purified enteric neural crest cells (ENCCs) and blood or fibroblasts of the same patient. Variants identified were subsequently validated by Sanger sequencing. Several somatic variants were identified in all patients, but causative mutations for HSCR were not specifically identified in the ENCCs of these patients. Larger copy number variants were also not found to be specific to ENCCs. Therefore, we believe that somatic mutations are unlikely to be identified, if causative for HSCR. Here, we postulate various modes of development following the occurrence of a somatic mutation, to describe the challenges in detecting such mutations, and hypothesize how somatic mutations may contribute to 'missing heritability' in developmental defects.

Loss-of-Function Variants in MYLK Cause Recessive Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital disorder characterized by loss of smooth muscle contraction in the bladder and intestine. To date, three genes are known to be involved in MMIHS pathogenesis: ACTG2, MYH11, and LMOD1. However, for approximately 10% of affected individuals, the genetic cause of the disease is unknown, suggesting that other loci are most likely involved. Here, we report on three MMIHS-affected subjects from two consanguineous families with no variants in the known MMIHS-associated genes. By performing homozygosity mapping and whole-exome sequencing, we found homozygous variants in myosin light chain kinase (MYLK) in both families. We identified a 7 bp duplication (c.3838_3844dupGAAAGCG [p.Glu1282_Glyfs∗51]) in one family and a putative splice-site variant (c.3985+5C>A) in the other. Expression studies and splicing assays indicated that both variants affect normal MYLK expression. Because MYLK encodes an important kinase required for myosin activation and subsequent interaction with actin filaments, it is likely that in its absence, contraction of smooth muscle cells is impaired. The existence of a conditional-Mylk-knockout mouse model with severe gut dysmotility and abnormal function of the bladder supports the involvement of this gene in MMIHS pathogenesis. In aggregate, our findings implicate MYLK as a gene involved in the recessive form of MMIHS, confirming that this disease of the visceral organs is heterogeneous with a myopathic origin.

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System.

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest branch of the peripheral nervous system, comprising complex networks of neurons and glia, which are present throughout the gastrointestinal tract. Although development of a fully functional ENS is required for gastrointestinal motility, little is known about the ontogeny of ENS function in humans. We studied the development of neuronal subtypes and the emergence of evoked electrical activity in the developing human ENS. METHODS: Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterized by immunohistochemistry, calcium imaging, RNA sequencing, and quantitative real-time polymerase chain reaction analyses. RESULTS: Human fetal colon samples have dense neuronal networks at the level of the myenteric plexus by embryonic week (EW) 12, with expression of excitatory neurotransmitter and synaptic markers. By contrast, markers of inhibitory neurotransmitters were not observed until EW14. Electrical train stimulation of internodal strands did not evoke activity in the ENS of EW12 or EW14 tissues. However, compound calcium activation was observed at EW16, which was blocked by the addition of 1 µmol/L tetrodotoxin. Expression analyses showed that this activity was coincident with increases in expression of genes encoding proteins involved in neurotransmission and action potential generation. CONCLUSIONS: In analyses of human fetal intestinal samples, we followed development of neuronal diversity, electrical excitability, and network formation in the ENS. These processes are required to establish the functional enteric circuitry. Further studies could increase our understanding of the pathogenesis of a range of congenital enteric neuropathies.

Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

Dual origin of enteric neurons in vagal Schwann cell precursors and the sympathetic neural crest.

Most of the enteric nervous system derives from the "vagal" neural crest, lying at the level of somites 1-7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the "vagal crest" subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations. Crest cells adjacent to somites 1 and 2 produce Schwann cell precursors that colonize the vagus nerve, which in turn guides them into the esophagus and stomach. Crest cells adjacent to somites 3-7 belong to the crest streams contributing to sympathetic chains: they migrate ventrally, seed the sympathetic chains, and colonize the entire digestive tract thence. Accordingly, enteric ganglia, like sympathetic ones, are atrophic when deprived of signaling through the tyrosine kinase receptor ErbB3, while half of the esophageal ganglia require, like parasympathetic ones, the nerve-associated form of the ErbB3 ligand, Neuregulin-1. These dependencies might bear relevance to Hirschsprung disease, with which alleles of Neuregulin-1 are associated.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.

Identification of Variants in RET and IHH Pathway Members in a Large Family With History of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is an inherited congenital disorder characterized by absence of enteric ganglia in the distal part of the gut. Variants in ret proto-oncogene (RET) have been associated with up to 50% of familial and 35% of sporadic cases. We searched for variants that affect disease risk in a large, multigenerational family with history of HSCR in a linkage region previously associated with the disease (4q31.3-q32.3) and exome wide. METHODS: We performed exome sequencing analyses of a family in the Netherlands with 5 members diagnosed with HSCR and 2 members diagnosed with functional constipation. We initially focused on variants in genes located in 4q31.3-q32.3; however, we also performed an exome-wide analysis in which known HSCR or HSCR-associated gene variants predicted to be deleterious were prioritized for further analysis. Candidate genes were expressed in HEK293, COS-7, and Neuro-2a cells and analyzed by luciferase and immunoblot assays. Morpholinos were designed to target exons of candidate genes and injected into 1-cell stage zebrafish embryos. Embryos were allowed to develop and stained for enteric neurons. RESULTS: Within the linkage region, we identified 1 putative splice variant in the lipopolysaccharide responsive beige-like anchor protein gene (LRBA). Functional assays could not confirm its predicted effect on messenger RNA splicing or on expression of the mab-21 like 2 gene (MAB21L2), which is embedded in LRBA. Zebrafish that developed following injection of the lrba morpholino had a shortened body axis and subtle gut morphological defects, but no significant reduction in number of enteric neurons compared with controls. Outside the linkage region, members of 1 branch of the family carried a previously unidentified RET variant or an in-frame deletion in the glial cell line derived neurotrophic factor gene (GDNF), which encodes a ligand of RET. This deletion was located 6 base pairs before the last codon. We also found variants in the Indian hedgehog gene (IHH) and its mediator, the transcription factor GLI family zinc finger 3 (GLI3). When expressed in cells, the RET-P399L variant disrupted protein glycosylation and had altered phosphorylation following activation by GDNF. The deletion in GDNF prevented secretion of its gene product, reducing RET activation, and the IHH-Q51K variant reduced expression of the transcription factor GLI1. Injection of morpholinos that target ihh reduced the number of enteric neurons to 13% ± 1.4% of control zebrafish. CONCLUSIONS: In a study of a large family with history of HSCR, we identified variants in LRBA, RET, the gene encoding the RET ligand (GDNF), IHH, and a gene encoding a mediator of IHH signaling (GLI3). These variants altered functions of the gene products when expressed in cells and knockout of ihh reduced the number of enteric neurons in the zebrafish gut.

ACTG2 variants impair actin polymerization in sporadic Megacystis Microcolon Intestinal Hypoperistalsis Syndrome.

Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a rare congenital disorder, in which heterozygous missense variants in the Enteric Smooth Muscle actin γ-2 (ACTG2) gene have been recently identified. To investigate the mechanism by which ACTG2 variants lead to MMIHS, we screened a cohort of eleven MMIHS patients, eight sporadic and three familial cases, and performed immunohistochemistry, molecular modeling and molecular dynamics (MD) simulations, and in vitro assays. In all sporadic cases, a heterozygous missense variant in ACTG2 was identified. ACTG2 expression was detected in all intestinal layers where smooth muscle cells are present in different stages of human development. No histopathological abnormalities were found in the patients. Using molecular modeling and MD simulations, we predicted that ACTG2 variants lead to significant changes to the protein function. This was confirmed by in vitro studies, which showed that the identified variants not only impair ACTG2 polymerization, but also contribute to reduced cell contractility. Taken together, our results confirm the involvement of ACTG2 in sporadic MMIHS, and bring new insights to MMIHS pathogenesis.

Transplanted enteric neural stem cells integrate within the developing chick spinal cord: implications for spinal cord repair.

Spinal cord injury (SCI) causes paralysis, multisystem impairment and reduced life expectancy, as yet with no cure. Stem cell therapy can potentially replace lost neurons, promote axonal regeneration and limit scar formation, but an optimal stem cell source has yet to be found. Enteric neural stem cells (ENSC) isolated from the enteric nervous system (ENS) of the gastrointestinal (GI) tract are an attractive source. Here, we used the chick embryo to assess the potential of ENSC to integrate within the developing spinal cord. In vitro, isolated ENSC formed extensive cell connections when co-cultured with spinal cord (SC)-derived cells. Further, qRT-PCR analysis revealed the presence of TuJ1+ neurons, S100+ glia and Sox10+ stem cells within ENSC neurospheres, as well as expression of key neuronal subtype genes, at levels comparable to SC tissue. Following ENSC transplantation to an ablated region of chick embryo SC, donor neurons were found up to 12 days later. These neurons formed bridging connections within the SC injury zone, aligned along the anterior/posterior axis, and were immunopositive for TuJ1. These data provide early proof of principle support for the use of ENSCs for SCI, and encourage further research into their potential for repair.

Decellularized Diaphragmatic Muscle Drives a Constructive Angiogenic Response In Vivo.

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31⁺, αSMA⁺, and vWF⁺ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF⁺ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration.

Enteric nervous system development in avian and zebrafish models.

Our current understanding of the developmental biology of the enteric nervous system (ENS) and the genesis of ENS diseases is founded almost entirely on studies using model systems. Although genetic studies in the mouse have been at the forefront of this field over the last 20 years or so, historically it was the easy accessibility of the chick embryo for experimental manipulations that allowed the first descriptions of the neural crest origins of the ENS in the 1950s. More recently, studies in the chick and other non-mammalian model systems, notably zebrafish, have continued to advance our understanding of the basic biology of ENS development, with each animal model providing unique experimental advantages. Here we review the basic biology of ENS development in chick and zebrafish, highlighting conserved and unique features, and emphasising novel contributions to our general understanding of ENS development due to technical or biological features.

Regulators of gene expression in Enteric Neural Crest Cells are putative Hirschsprung disease genes.

The enteric nervous system (ENS) is required for peristalsis of the gut and is derived from Enteric Neural Crest Cells (ENCCs). During ENS development, the RET receptor tyrosine kinase plays a critical role in the proliferation and survival of ENCCs, their migration along the developing gut, and differentiation into enteric neurons. Mutations in RET and its ligand GDNF cause Hirschsprung disease (HSCR), a complex genetic disorder in which ENCCs fail to colonize variable lengths of the distal bowel. To identify key regulators of ENCCs and the pathways underlying RET signaling, gene expression profiles of untreated and GDNF-treated ENCCs from E14.5 mouse embryos were generated. ENCCs express genes that are involved in both early and late neuronal development, whereas GDNF treatment induced neuronal maturation. Predicted regulators of gene expression in ENCCs include the known HSCR genes Ret and Sox10, as well as Bdnf, App and Mapk10. The regulatory overlap and functional interactions between these genes were used to construct a regulatory network that is underlying ENS development and connects to known HSCR genes. In addition, the adenosine receptor A2a (Adora2a) and neuropeptide Y receptor Y2 (Npy2r) were identified as possible regulators of terminal neuronal differentiation in GDNF-treated ENCCs. The human orthologue of Npy2r maps to the HSCR susceptibility locus 4q31.3-q32.3, suggesting a role for NPY2R both in ENS development and in HSCR.

Genetics of enteric neuropathies.

Abnormal development or disturbed functioning of the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, is associated with the development of neuropathic gastrointestinal motility disorders. Here, we review the underlying molecular basis of these disorders and hypothesize that many of them have a common defective biological mechanism. Genetic burden and environmental components affecting this common mechanism are ultimately responsible for disease severity and symptom heterogeneity. We believe that they act together as the fulcrum in a seesaw balanced with harmful and protective factors, and are responsible for a continuum of symptoms ranging from neuronal hyperplasia to absence of neurons.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies.

Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.

FKBPL is a critical antiangiogenic regulator of developmental and pathological angiogenesis.

OBJECTIVE: The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models. APPROACH AND RESULTS: FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL's critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl(+/-) mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish. CONCLUSIONS: FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes.

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model.

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.