Anti-apoptotic effect of adipose tissue-derived stromal vascular fraction in denervated rat muscle.

BACKGROUND: Recovery of muscle function after peripheral nerve injury is often poor, and this can be attributed to muscle fiber atrophy and cell death. In the current study, we have investigated the effects of stromal vascular fraction (SVF) on muscle cell apoptosis and its potential to preserve muscle tissue following denervation. METHODS: Rat gastrocnemius muscle was denervated by sciatic nerve transection. At 2 and 4 weeks after injury, muscles were examined histologically and apoptosis was measured using TUNEL assay and PCR array for a range of apoptotic genes. Additionally, an in vitro TNF-α apoptosis model was established using SVF cells co-cultured indirectly with primary rat myoblasts. Annexin V and TUNEL were used together with Western blotting to investigate the signaling pathways. RESULTS: Denervated muscles showed significantly higher TUNEL reactivity at 2 and 4 weeks following nerve injury, and an increased expression of caspase family genes, mitochondria-related apoptotic genes, and tumor necrosis factor family genes. In cultured rat primary myoblasts, Annexin V labeling was significantly increased at 12 h after TNF-α treatment, and this was followed by a significant increase in TUNEL reactivity at 48 h. Western blotting showed that caspase-7 was activated/cleaved as well as the downstream substrate, poly (ADP-ribose) polymerase (PARP). Co-culture of myoblasts with SVF significantly reduced all these measures of apoptosis. Bax and Bcl-2 levels were not changed suggesting that the TNF-α-induced apoptosis occurred via mitochondria-independent pathways. The protective effect of SVF was also shown in vivo; injections of SVF cells into denervated muscle significantly improved the mean fiber area and diameter, as well as reduced the levels of TUNEL reactivity. CONCLUSIONS: This study provides new insights into how adipose tissue-derived cells might provide therapeutic benefits by preserving muscle tissue.

Combining in silico and in vitro models to inform cell seeding strategies in tissue engineering.

The seeding density of therapeutic cells in engineered tissue impacts both cell survival and vascularization. Excessively high seeded cell densities can result in increased death and thus waste of valuable cells, whereas lower seeded cell densities may not provide sufficient support for the tissue in vivo, reducing efficacy. Additionally, the production of growth factors by therapeutic cells in low oxygen environments offers a way of generating growth factor gradients, which are important for vascularization, but hypoxia can also induce unwanted levels of cell death. This is a complex problem that lends itself to a combination of computational modelling and experimentation. Here, we present a spatio-temporal mathematical model parametrized using in vitro data capable of simulating the interactions between a therapeutic cell population, oxygen concentrations and vascular endothelial growth factor (VEGF) concentrations in engineered tissues. Simulations of collagen nerve repair constructs suggest that specific seeded cell densities and non-uniform spatial distributions of seeded cells could enhance cell survival and the generation of VEGF gradients. These predictions can now be tested using targeted experiments.

The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts.

BACKGROUND: Adipose tissue is an excellent source for isolation of stem cells for treating various clinical conditions including injuries to the neuromuscular system. Many previous studies have focused on differentiating these adipose stem cells (ASCs) towards a Schwann cell-like phenotype (dASCs), which can enhance axon regeneration and reduce muscle atrophy. However, the stromal vascular fraction (SVF), from which the ASCs are derived, also exerts broad regenerative potential and might provide a faster route to clinical translation of the cell therapies for treatment of neuromuscular disorders. METHODS: The aim of this study was to establish the effects of SVF cells on the proliferation and differentiation of myoblasts using indirect co-culture experiments. A Growth Factor PCR Array was used to compare the secretomes of SVF and dASCs, and the downstream signaling pathways were investigated. RESULTS: SVF cells, unlike culture-expanded dASCs, expressed and secreted hepatocyte growth factor (HGF) at concentrations sufficient to enhance the proliferation of myoblasts. Pharmacological inhibitor studies revealed that the signal is mediated via ERK1/2 phosphorylation and that the effect is significantly reduced by the addition of 100 pM Norleual, a specific HGF inhibitor. When myoblasts were differentiated into multinucleated myotubes, the SVF cells reduced the expression levels of fast-type myosin heavy chain (MyHC2) suggesting an inhibition of the differentiation process. CONCLUSIONS: In summary, this study shows the importance of HGF as a mediator of the SVF effects on myoblasts and provides further evidence for the importance of the secretome in cell therapy and regenerative medicine applications.

The effects of N-acetyl-cysteine and acetyl-L-carnitine on neural survival, neuroinflammation and regeneration following spinal cord injury.

Traumatic spinal cord injury induces a long-standing inflammatory response in the spinal cord tissue, leading to a progressive apoptotic death of spinal cord neurons and glial cells. We have recently demonstrated that immediate treatment with the antioxidants N-acetyl-cysteine (NAC) and acetyl-l-carnitine (ALC) attenuates neuroinflammation, induces axonal sprouting, and reduces the death of motoneurons in the vicinity of the trauma zone 4weeks after initial trauma. The objective of the current study was to investigate the effects of long-term antioxidant treatment on the survival of descending rubrospinal neurons after spinal cord injury in rats. It also examines the short- and long-term effects of treatment on apoptosis, inflammation, and regeneration in the spinal cord trauma zone. Spinal cord hemisection performed at the level C3 induced a significant loss of rubrospinal neurons 8 weeks after injury. At 2 weeks, an increase in the expression of the apoptosis-associated markers BCL-2-associated X protein (BAX) and caspase 3, as well as the microglial cell markers OX42 and ectodermal dysplasia 1 (ED1), was seen in the trauma zone. After 8 weeks, an increase in immunostaining for OX42 and the serotonin marker 5HT was detected in the same area. Antioxidant therapy reduced the loss of rubrospinal neurons by approximately 50%. Treatment also decreased the expression of BAX, caspase 3, OX42 and ED1 after 2 weeks. After 8 weeks, treatment decreased immunoreactivity for OX42, whereas it was increased for 5HT. In conclusion, this study provides further insight in the effects of treatment with NAC and ALC on descending pathways, as well as short- and long-term effects on the spinal cord trauma zone.

Nerve repair with adipose-derived stem cells protects dorsal root ganglia neurons from apoptosis.

Novel approaches are required in the clinical management of peripheral nerve injuries because current surgical techniques result in deficient sensory recovery. Microsurgery alone fails to address extensive cell death in the dorsal root ganglia (DRG), in addition to poor axonal regeneration. Incorporation of cultured cells into nerve conduits may offer a novel approach in which to combine nerve repair and enhance axonal regeneration with neuroprotective therapies. We examined apoptotic mediator expression in rat DRG neurons following repair of a 10-mm sciatic nerve gap using a novel synthetic conduit made of poly ε-caprolactone (PCL) and primed with adipose-derived stem cells (ADSC) differentiated towards a Schwann cell phenotype or with primary adult Schwann cells. Differentiated ADSC expressed a range of neurotrophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and neurotrophin-4 (NT4). Incorporation of either differentiated ADSC or Schwann cells significantly increased anti-apoptotic Bcl-2 mRNA expression (P<0.001) in the DRG, while significantly decreasing pro-apoptotic Bax (P<0.001) and caspase-3 mRNA (P<0.01) expression. Cleaved caspase-3 protein was observed in the DRG following nerve injury which was attenuated when nerve repair was performed using conduits seeded with cells. Cell incorporation into conduit repair of peripheral nerves demonstrates experimental promise as a novel intervention to prevent DRG neuronal loss.

Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts.

Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Laryngeal transplantation in minipigs: vascular, myologic and functional outcomes.

There is no effective way of replacing all the functions of the larynx in those requiring laryngectomy. Regenerative medicine offers promise, but cannot presently deliver implants with functioning neuromuscular units. A single well-documented laryngeal transplant in man was a qualified success, but more information is required before clinical trials may be proposed. We studied the early response of the larynx to laryngeal transplantation between 17 pairs of NIH minipigs full matched at the MHC2 locus. Following iterative technical improvements, pigs had good swallowing and a patent airway at 1 week. No significant changes in mucosal blood flux were observed compared with pre-operative measurements. Changes in muscle morphology and fibre phenotype were observed in transplant muscles retrieved after 7 days: the levels of fast and slow myosin heavy chain (MyHC) protein were reduced and embryonic MyHC was up regulated consistent with denervation induced atrophy. At 1 week laryngeal transplantation can result in good swallowing, and is not associated with clinical evidence of ischemia-reperfusion injury in MHC-matched pigs.

Regeneration potential and survival of transplanted undifferentiated adipose tissue-derived stem cells in peripheral nerve conduits.

Adipose tissue-derived stem cells (ADSCs) have shown potential for the treatment of nerve injuries. Most previous efforts have aimed at stimulating regeneration by using neural-differentiation protocols, but the potential of undifferentiated ADSCs to enhance axonal growth as well as their ability to transdifferentiate in situ have been poorly investigated. In this study, using a rat sciatic nerve model we show that ADSCs, transplanted in an artificial nerve conduit, stimulate axonal outgrowth from the proximal nerve stump and evoke greater Schwann cell (SC) proliferation/intrusion in the distal stump. To track the fate of the transplanted cells, we used green fluorescent protein (GFP)-labelling and polymerase chain reaction (PCR) for the detection of the sex determining region Y (SRY) gene in the donor male cells. Both methods indicated a lack of significant quantities of viable cells 14 days after transplantation. These results suggest that any regenerative effect of transplanted ADSCs is more likely to be mediated by an initial boost of released growth factors and/or by an indirect effect on endogenous SCs activity. Future studies need to address long-term cell survival in tissue-engineered nerve conduits to improve the neuroregenerative potential of ADSCs.

In vitro and in vivo testing of novel ultrathin PCL and PCL/PLA blend films as peripheral nerve conduit.

In an attempt to obviate the drawbacks of nerve autograft, ultrathin microporous biodegradable PCL and PCL/PLA films were tested for their compatibility with motor neuron-like NG108-15 cells and primary Schwann cells. Data obtained from MTS colorimetric and DNA fluorimetric assays showed that both cell lines readily attached and proliferated on these materials. Images taken using scanning electron microscope and fluorescence microscope confirmed these observations. Enhanced cell-surface interaction was achieved by pretreating the films in NaOH solution. Importantly, NG108-15 cells could be induced into differentiated phenotype with long, un-branched neurites growing across the surface of the materials. The bipolar spindle-shaped phenotype of Schwann cells was also retained on these scaffolds. Positive immunochemical staining using antibodies against neurofilament for NG108-15 cells and S100 for Schwann cells indicated the expression of these marker proteins. In a small-scaled pilot testing, the performance of PCL conduits in bridging up a 10 mm gap in rat sciatic nerve model was assessed. Immunohistochemical staining showed that regenerated nerve tissue and penetrated Schwann cells have the potential to span the whole length of the conduit in 2 weeks.

Adipose-derived stem cells enhance peripheral nerve regeneration.

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

[Fibrin matrix enhances adherence of peripheral nerve regenerative cells]. / Fibrinmatrix erhöht adhärenz von regenerativen peripheren nervenzellen.

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.

Fibrin matrix for suspension of regenerative cells in an artificial nerve conduit.

Peripheral nerve injury presents with specific problems of neuronal reconstructions, and from a clinical viewpoint a tissue engineering approach would facilitate the process of repair and regeneration. We have previously used artificial nerve conduits made from bioresorbable poly-3-hydroxybutyrate (PHB) in order to refine the ways in which peripheral nerves are repaired and reconnected to the target muscles and skin. The addition of Schwann cells (SC) or differentiated mesenchymal stem cells (dMSC) to the conduits enhances regeneration. In this study, we have used a matrix based on fibrin (Tisseel) to fill optimally the nerve-conduits with cells. In vitro analysis showed that both SC and MSC adhered significantly better to PHB in the presence of fibrin and cells continued to maintain their differentiated state. Cells were more optimally distributed throughout the conduit when seeded in fibrin than by delivery in growth medium alone. Transplantation of the nerve conduits in vivo showed that cells in combination with fibrin matrix significantly increased nerve regeneration distance (using PGP9.5 and S100 distal and proximal immunohistochemistry) when compared with empty PHB conduits. This study shows the beneficial combinatory effect of an optimised matrix, cells and conduit material as a step towards bridging nerve gaps which should ultimately lead to improved functional recovery following nerve injury.

Eosinophil and airway nerve interactions.

In vivo, eosinophils localise to airway nerves in patients with asthma as well as in animal models of hyperreactivity. In both, in vivo and in vitro studies, we have shown that this localisation changes both cholinergic nerve and eosinophil function. In particular, it leads to an increase in acetylcholine release due to loss of function of a neuronal autoreceptor, the M(2) muscarinic receptor. This loss of M(2) receptor function occurs because eosinophils become activated and degranulate as a result of interactions that occur via specific adhesion molecules expressed on nerves that are recognised by counter ligands on eosinophils.

Microglial secreted cathepsin B induces neuronal apoptosis.

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z-FA-fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA-activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.

Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release.

Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia or the murine N9 microglial cell line with chromogranin A resulted in nitric oxide production, which triggered microglial apoptosis. Exposure of microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity.

Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation.

Senile plaques of Alzheimer's brain are characterized by activated microglia and immunoreactivity for the peptide chromogranin A. We have investigated the mechanisms by which chromogranin A activates microglia, producing modulators of neuronal survival. Primary cultures of rat brain-derived microglia display a reactive phenotype within 24 h of exposure to 10 nM chromogranin A, culminating in microglial death via apoptotic mechanisms mediated by interleukin-1beta converting enzyme. The signalling cascade initiated by chromogranin A triggers nitric oxide production followed by enhanced microglial glutamate release, inhibition of which prevents microglial death. The plasma membrane carrier inhibitor aminoadipate and the type II/III metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-sulphonophenylglycine are equally protective. A significant amount of the released glutamate occurs from bafilomycin-sensitive stores, suggesting a vesicular mode of release. Inhibition of this component of release affords significant microglial protection. Conditioned medium from activated microglia kills cerebellar granule cells by inducing caspase-3-dependent neuronal apoptosis. Brain-derived neurotrophic factor is partially neuroprotective, as are ionotropic glutamate receptor antagonists, and, when combined with boiling of conditioned medium, full protection is achieved; nitric oxide synthase inhibitors are ineffective.