Mesenchymal stromal cell-derived exosomes ameliorate peripheral neuropathy in a mouse model of diabetes.

AIMS/HYPOTHESIS: Diabetic peripheral neuropathy (DPN) is one of the major complications of diabetes, which contributes greatly to morbidity and mortality. There is currently no effective treatment for this disease. Exosomes are cell-derived nanovesicles and play an important role in intercellular communications. The present study investigated whether mesenchymal stromal cell (MSC)-derived exosomes improve neurological outcomes of DPN. METHODS: Exosomes were isolated from the medium of cultured mouse MSCs by ultracentrifugation. Diabetic mice (BKS.Cg-m+/+Leprdb/J, db/db) at the age of 20 weeks were used as DPN models. Heterozygous mice (db/m) of the same age were used as the control. MSC-exosomes were administered weekly via the tail vein for 8 weeks. Neurological function was evaluated by testing motor and sensory nerve conduction velocities, and thermal and mechanical sensitivity. Morphometric analysis was performed by myelin sheath staining and immunohistochemistry. Macrophage markers and circulating cytokines were measured by western blot and ELISA. MicroRNA (miRNA) array and bioinformatics analyses were performed to examine the exosomal miRNA profile and miRNA putative target genes involved in DPN. RESULTS: Treatment of DPN with MSC-exosomes markedly decreased the threshold for thermal and mechanical stimuli and increased nerve conduction velocity in diabetic mice. Histopathological analysis showed that MSC-exosomes markedly augmented the density of FITC-dextran perfused blood vessels and increased the number of intraepidermal nerve fibres (IENFs), myelin thickness and axonal diameters of sciatic nerves. Western blot analysis revealed that MSC-exosome treatment decreased and increased M1 and M2 macrophage phenotype markers, respectively. Moreover, MSC-exosomes substantially suppressed proinflammatory cytokines. Bioinformatics analysis revealed that MSC-exosomes contained abundant miRNAs that target the Toll-like receptor (TLR)4/NF-κB signalling pathway. CONCLUSIONS/INTERPRETATION: MSC-derived exosomes alleviate neurovascular dysfunction and improve functional recovery in mice with DPN by suppression of proinflammatory genes.

Pathophysiology of diabetic erectile dysfunction: potential contribution of vasa nervorum and advanced glycation endproducts.

Erectile dysfunction (ED) due to diabetes mellitus remains difficult to treat medically despite advances in pharmacotherapeutic approaches in the field. This unmet need has resulted in a recent re-focus on the pathophysiology, in order to understand the cellular and molecular mechanisms leading to ED in diabetes. Diabetes-induced ED is often resistant to PDE5 inhibitor treatment, thus there is a need to discover targets that may lead to novel approaches for a successful treatment. The aim of this brief review is to update the reader in some of the latest development on that front, with a particular focus on the role of impaired neuronal blood flow and the formation of advanced glycation endproducts.

Bone marrow mononuclear cells have neurovascular tropism and improve diabetic neuropathy.

Bone marrow-derived mononuclear cells (BMNCs) have been shown to effectively treat ischemic cardiovascular diseases. Because diabetic neuropathy (DN) is causally associated with impaired angiogenesis and deficiency of angiogenic and neurotrophic factors in the nerves, we investigated whether DN can be ameliorated by local injection of BMNCs. Severe peripheral neuropathy, characterized by a significant decrease in the motor and sensory nerve conduction velocities (NCVs), developed 12 weeks after the induction of diabetes with streptozotocin in rats. The injection of BMNCs restored motor and sensory NCVs to normal levels and significantly improved vascular density and blood flow in diabetic nerves over 4 weeks. Fluorescent microscopic observation revealed that DiI-labeled BMNCs preferentially engrafted in sciatic nerves. Whole-mount fluorescent imaging and confocal microscopic evaluation demonstrated that many of the BMNCs localized following the course of the vasa nervorum in close proximity to blood vessels without incorporation into vasa nervorum as endothelial cells at a detectable level. Real-time reverse transcription-polymerase chain reaction analysis showed that the levels of angiogenic and neurotrophic factors were significantly increased in the nerves by BMNC injection. Local transplantation of BMNCs improved experimental DN by augmenting angiogenesis and increasing angiogenic and neurotrophic factors in peripheral nerves. These findings suggest that BMNC transplantation may represent a novel therapeutic option for treating DN.

Differential vulnerability of neuropeptides in nerves of the vasa nervorum to streptozotocin-induced diabetes.

Neuropeptides in perivascular nerves of vasa nervorum supplying blood to rat optic, sciatic, vagus and sympathetic chain nerve trunks are differentially vulnerable to streptozotocin (STZ)-induced diabetes. Immunohistochemical analysis of epineurial/perineurial nerve sheaths showed that 8 weeks after induction of diabetes, the density of neuropeptide Y (NPY)-immunoreactive nerve fibres in optic nerve sheaths was increased, while it was decreased in sciatic, vagus and sympathetic nerve sheaths. Vasoactive intestinal polypeptide (VIP)-immunoreactivity was increased in vasa and nervi nervorum of optic, sciatic, vagus and sympathetic chain nerve sheaths. Immunoassay of NPY confirmed increased levels in optic nerve sheaths and showed that substance P and calcitonin gene-related peptide levels increased in sciatic but not optic nerve sheaths. Neuropeptide levels in the intrafascicular nerve fibres were unaffected. This provides further evidence for a disturbance in the autonomic control of blood flow to peripheral and cranial nerve trunks via vasa nervorum in STZ-induced diabetes, which may lead to ischaemic changes, alter local axon reflexes and contribute to the pathogenesis of the disease.

Decreased density of beta-adrenergic and muscarinic cholinergic receptor sites in the vasa nervorum of aged rats.

The pharmacological profile and the anatomical localization of beta-adrenergic and muscarinic cholinergic receptors of the vasa nervorum were studied in sections of sciatic nerve using radioreceptor binding and light microscope autoradiography techniques. Sprague-Dawley rats of 4 and 24 months of age were used. [3H]Dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB) were used to label beta-adrenergic and muscarinic cholinergic receptors, respectively. The ligands were bound to sections of rat sciatic nerve in a manner consistent with the labelling of beta-adrenergic or muscarinic cholinergic receptors in the 2 age groups investigated. The dissociation constant (Kd) values (about 1.37 nM for [3H]DHA and 0.75 nM for [3H]QNB) did not significantly change between 4- and 24-month-old rats. The maximum concentration of binding sites (Bmax) for [3H]DHA was decreased by about 35% in 24 in comparison with 4-month-old rats. The Bmax value for [3H]QNB was reduced by about 50% in the aged rats. Light microscope autoradiography revealed the development of specific silver grains in the medial layer of epineurial and perineurial arteries in sections of sciatic nerve exposed either to [3H]DHA or [3H]QNB. The number of silver grains developed in epineurial and perineurial arteries of rats of 24 months is significantly lower than in animals of 4 months. The above results suggest the occurrence of an age-dependent loss in the density of beta-adrenergic and muscarinic cholinergic receptors of vasa nervorum. Vasa nervorum are the blood vessels which supply peripheral nerve trunks. They are constituted by outer (epineurial) and inner (perineurial) arteries and veins as well as by a capillary (endoneurial) plexus. Vasa nervorum are innervated by both sympathetic and non-sympathetic nerves which probably play a role in the pathogenesis of some neuropathies. Several different neurotransmitter containing nerve fibres have been identified in the vasa nervorum perivascular plexuses. However, no information is so far available concerning the neurotransmitter receptors of vasa nervorum. Moreover, although the occurrence of age-dependent changes in peripheral nerve morphology and function is well documented, very few reports were centered on the age-dependent changes of the vasa nervorum. The aims of the present study were to characterize pharmacologically and to localize anatomically the beta-adrenergic and muscarinic cholinergic receptors in the rat vasa nervorum. Moreover, the effect of ageing on the density and pattern of these receptors was investigated.

Glucose transporters at the blood-nerve barrier are associated with perineurial cells and endoneurial microvessels.

The blood-nerve barrier consists of continuous layers of cells linked by tight junctions and includes the endothelial cells which line the endoneurial capillaries and the perineurial cells which surround fascicles of nerve fibers. A facilitated transport carrier protein allows D-glucose to penetrate the barrier. To determine the specific cellular location of the transport system, an antiserum to a synthetic peptide corresponding to the carboxyl-terminus of the glucose transporter protein was used for light and electron immunocytochemical analyses. Glucose transporters were abundant both in endoneurial capillaries and the perineurial sheath. In perineurium, transporters were located in the plasma membranes and cytoplasm of the perineurial cells. Approximately two-thirds of the transporters associated with perineurial cells were localized in the plasma membranes. Perineurial cells are thus similar to cerebral endothelial cells in that they lack a large intracellular pool of transporters which might be sensitive to hormonal regulation. The presence of hexose carriers in perineurium suggests that glucose transport from epineurium to endoneurium may play a significant role in the metabolism of peripheral nerve fibers. These results support the concept that the blood-nerve barrier serves as an important nutrient delivery system.

The noradrenergic innervation of the vasa nervorum in old rats: a fluorescence histochemical study.

The effect of ageing on the density and pattern of noradrenergic nerves in the perivascular nerve plexus supplying the vasa nervorum of the rat sciatic nerve was studied using combined catecholamine histofluorescence and quantitative image analysis techniques. The density of noradrenergic fibres around arteries and arterioles of the rat sciatic nerve vasa nervorum increased in the old animals. In contrast, no changes in perivascular nerve fibres supplying the veins and venules were found in the vasa nervorum of old rats. The increase in old age of noradrenergic innervation of arteries and arterioles of the vasa nervorum may be related to the pathogenesis of some peripheral nerve diseases.

Tangier disease. A case with sensorimotor distal polyneuropathy and lipid accumulation in striated muscle and vasa nervorum.

A 65-year-old man with Tangier disease (analphalipoproteinemia) had had a progressive sensorimotor distal neuropathy with sensory ataxia for 1 year. Muscle biopsy demonstrated excess lipid vacuoles on histochemical and electron-microscopic techniques. Sural nerve biopsy showed a marked loss of large fibers and an increase in small myelinated fibers, with presence of remyelinating fibers and clusters of regeneration; a few aspects of active demyelination and some onion-like formations were also present. Lipid accumulation chiefly affected the Schwann cells of unmyelinated fibers and, to a lesser degree, of myelinated fibers, endoneurial fibroblast, and vasa nervorum. Teased fibers showed prevalent aspects of de-/remyelination and, often in association, marked myelin wrinkling suggesting axonal atrophy. This Tangier patient differs from known cases for the presence of a distal symmetrical sensorimotor polyneuropathy (not previously reported in Tangier disease) and because of the morphological findings of de-/remyelination coexisting with aspects of axonal atrophy and previous degeneration, and of lipid accumulation within striated muscle and vasa nervorum. This latter finding contrasts with the assumption that in Tangier disease vessel walls are not a site of lipid storage: probably the vasa nervorum are different, in this respect, from other vessels, because of the intense lipid metabolism of the nervous tissue. Thus we suggest that involvement of vasa nervorum in Tangier disease may be more important than previously suspected, possibly playing a role in the causation of neuropathy.