Nerve growth factor gene therapy improves bone marrow sensory innervation and nociceptor-mediated stem cell release in a mouse model of type 1 diabetes with limb ischaemia

Aims/hypothesis Sensory neuropathy is common in people with diabetes; neuropathy can also affect the bone marrow of individuals with type 2 diabetes. However, no information exists on the state of bone marrow sensory innervation in type 1 diabetes. Sensory neurons are trophically dependent on nerve growth factor (NGF) for their survival. The aim of this investigation was twofold: (1) to determine if sensory neuropathy affects the bone marrow in a mouse model of type 1 diabetes, with consequences for stem cell liberation after tissue injury; and (2) to verify if a single systemic injection of the NGF gene exerts long-term beneficial effects on these phenomena. Methods A mouse model of type 1 diabetes was generated in CD1 mice by administration of streptozotocin; vehicle was administered to non-diabetic control animals. Diabetic animals were randomised to receive systemic gene therapy with either human NGF or β-galactosidase. After 13 weeks, limb ischaemia was induced in both groups to study the recovery post injury. When the animals were killed, samples of tissue and peripheral blood were taken to assess stem cell mobilisation and homing, levels of substance P and muscle vascularisation. An in vitro cellular model was adopted to verify signalling downstream to human NGF and related neurotrophic or pro-apoptotic effects. Normally distributed variables were compared between groups using the unpaired Student’s t test and non-normally distributed variables were assessed by the Wilcoxon–Mann–Whitney test. The Fisher’s exact test was employed for categorical variables. Results Immunohistochemistry indicated a 3.3-fold reduction in the number of substance P-positive nociceptive fibres in the bone marrow of type 1 diabetic mice (p < 0.001 vs non-diabetic). Moreover, diabetes abrogated the creation of a neurokinin gradient which, in non-diabetic mice, favoured the mobilisation and homing of bone-marrow-derived stem cells expressing the substance P receptor neurokinin 1 receptor (NK1R). Pre-emptive gene therapy with NGF prevented bone marrow denervation, contrasting with the inhibitory effect of diabetes on the mobilisation of NK1R-expressing stem cells, and restored blood flow recovery from limb ischaemia. In vitro hNGF induced neurite outgrowth and exerted anti-apoptotic actions on rat PC12 cells exposed to high glucose via activation of the canonical neurotrophic tyrosine kinase receptor type 1 (TrkA) signalling pathway. Conclusions/interpretation This study shows, for the first time, the occurrence of sensory neuropathy in the bone marrow of type 1 diabetic mice, which translates into an altered modulation of substance P and depressed release of substance P-responsive stem cells following ischaemia. NGF therapy improves bone marrow sensory innervation, with benefits for healing on the occurrence of peripheral ischaemia. Nociceptors may represent a new target for the treatment of ischaemic complications in diabetes. Electronic supplementary material The online version of this article (10.1007/s00125-019-4860-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

ELISA assays ELISA assays for hNGF (Cloud-Clone Corp., Katy, TX, USA) and pro-hNGF (CUSABIO, Houston, TX, USA) were performed according to manufacturer instructions on plasma collected from animals 3 days post-gene transfer to assess the levels of the circulating proteins. All samples were assayed in duplicates. This was used also to confirm that the mature hNGF was the only form of the recombinant protein.
ELISA for SP (Cayman Chemical Company, Ann Arbor, Michigan, USA) was employed to detect SP levels in mice BM, plasma, and ischaemic adductor muscles. Briefly, samples were diluted in 2M acetic acid, fractionated with C-18 reverse-phase single-use columns (Cayman Chemical Company), and concentrated under vacuum. Quantification of SP was performed by measuring the changes in the absorbance of nitrobenzoic acid at 412 nm.

Competitive assay to check specificity of the anti-NGF antibody
A peptide competition assay was performed to rule out non-specific binding of the anti-NGF antibody to other proteins in the mouse tissues. The NGF peptide was purchased from Abcam (Cambridge, UK) (Ab154297). This peptide was recommended by the vendor to block the anti-NGF antibody Ab52918. According to manufacture instructions, the antibody was incubated with five times excess blocking peptide by weight in blocking solution (PBS + 1% w/v BSA), for 30 min at RT, with gentle agitation. A tube with the antibody but without peptide served as control. The staining of the BM tissue was performed following the normal protocol using the blocked and control antibody on two different slices of the same samples.
Culture and characterisation of PC12 cells PC12 cells were cultured in DMEM containing 1g/l Glucose + Glutamine, 10% v/v FBS and pen/strep and passaged at confluence every 3 days. For phenotypic characterisation, cells were fixed with 4% w/v PFA for 15 min at 15-25°C and permeabilised using 0.1% TritonX for 5 min as required. After blocking with 10% v/v normal goat serum (Cell Signalling Technology), antibodies were incubated for 16 h at 4°C (as reported in ESM Table 1). Alexa488-conjugated secondary antibodies raised in goat were used to visualise the staining, and the nuclei counter-stained using DAPI. Cells were analysed at a x200 magnification.

Rat fibroblasts transduction and collection of conditioned media
Primary rat fibroblasts were cultured in DMEM containing 1g/l glucose with glutamine, 10% v/v FBS and pen/strep, and passaged at confluency (̴ 3 days). Cultures that were 80% confluent were transduced with either Ad.hNGF or Ad.βGal at a total dose of 5 × 10 7 viral particles/ml. Cells not transduced served as control (NV, no virus). After 48h, the conditioned media (CM) was centrifuged for 10 min at 1000 x g at 4°C to remove floating cells/debris and frozen for subsequent studies with PC12 cells. An aliquot of medium was used to check the expression of the human NGF using western blotting.
A further aliquot was used to measure the amount of NGF and proNGF using ELISA assays.
Biological assays on PC12 cells exposed to the CM from NGF-transduced fibroblasts PC12 cells were maintained under basal (BG, 5 mmol/l) or high (HG, 30mmol/l) D-Glucose. Mannitol (25mmol/l) was used as osmotic control when required.
For signalling experiments, PC12 pre-conditioned for 48h with BG or HG media were exposed to the fibroblasts CM (NV, hNGF, βGAL) for 30 min and total cell extracts were collected for Western Blotting. For Caspase 3/7 apoptosis assay, PC12 were incubated for 48h with the fibroblasts CM in the presence of either BG or HG or Mannitol. At the end of this incubation time, the Caspase Glo 3/7 Assay (Promega, Madison, Wisconsin, USA) was used to measure caspase activity. This was quantified as relative luminescence units (RLU) and is expressed as fold change compared with the BG-NV group, using 4 replicates per group. For neuronal differentiation and neurites outgrowth experiments, low density PC12 were incubated for 72h with the fibroblasts CM in the presence of either BG or HG. At the end of the protocol, images were snapped using a x 200 magnification. The total neurites length per cell was measured as an index of neurites outgrowth (N=50 cells randomly measured in 3-4 different optical fields), while cells presenting at least one axon longer than the cell body were considered as differentiated "neuron-like" cells and expressed as a percentage of the total number of cell (per each group, N=250 to 300 cells analysed in 4 different optical fields). For all experiments, fibroblasts CM was diluted 1:10 with fresh culture media, with appropriate adjustment for the final level of glucose or mannitol. The final hNGF concentration given to the PC12 cells was 540 pg/ml, as measured by ELISA assay.

Proteins extraction and western blotting
Total proteins from cells were obtained using RIPA lysis buffer supplemented with protease and phosphatase inhibitors cocktails. In addition, CM from cells was collected for measuring secreted proteins. This was centrifuged for 10 min at 1000 x g at 4°C to remove floating cells/debris and supplemented with Laemmli loading buffer. The protein concentration in cell extracts was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein samples (10-15μg) were prepared in Laemmli loading buffer, incubated for 8 min at 98°C, resolved on 8-12% SDS-PAGE according to the proteins MW and transferred onto 0.2 μm PVDF membranes (Bio-Rad, Hercules, California, USA). Membranes were blocked using 5% w/v non-fat dried milk in Tris-buffered saline (TBS) supplemented with 0.05% v/v Tween-20 (TBS-T) for 2 h at 15-25°C. Primary antibodies -listed in ESM Table 1 were incubated for 16h at 4°C using 5% w/v BSA in TBS-T. Secondary antibodieslisted in ESM Table 1 -were incubated for 1h at 15-25C using 5% w/v non-fat dried milk in TBS-T. β-tubulin was used as loading control. Membrane development was performed by an enhanced chemiluminescence-based detection method (ECL™ Prime Western Blotting Detection Reagent, GE Healthcare, Chicago, Illinois, USA) in a ChemiDoc-MP system (Bio-Rad). Proteins with similar molecular weight were assessed on different gels. When necessary, no more than 2 stripping procedures were performed in the same membrane (RestoreTM Plus Western Blot Stripping Buffer, Thermo Scientific). All samples to be compared were loaded on the same gel for densitometry analysis. Data were analysed using the free ImageJ software to determine optical density (OD) of the bands. The OD readings of phosphorylated proteins were expressed as a ratio relative to non-phosphorylated protein (N=1 replicate). Final data are showed as the fold change of the BG-NV group (considered to be the basal control).

Flow cytometry
Analyses of progenitor cells in PB, BM and adductor muscles of mice were conducted using multicolour flow cytometry. Cell collection. Freshly harvested cells were washed with ice-cold Hank's balanced salt solution containing 0.5% w/v BSA and 0.02% w/v sodium azide. Adductor muscles were harvested, minced with fine scissors and placed into a digest solution of Collagenase A 100 µg/µl (Roche, Basel, Switzerland), Dispase II (1X) 2.4 U/ml (Roche), DNase I 10 mg/ml (Roche), BSA 10% w/v, CaCl2 50 mmol/l, MgCl2 1 mol/l in PBS at 37 o C for 1 h, triturated through 40μm diameter nylon mesh and washed with 0.2% w/v BSA + 0.1% w/v DNAse I in PBS. Analysis: Cells were fixed and permeabilised using a fix/perm solution (BD, Franklin Lakes, New Jersey, USA) followed by blocking with anti-mouse CD16/32 (eBioscience, Thermo Scientific, Waltham, Massachusetts, USA) in PBS with 1% w/v BSA for 15 min at 4˚C. Cells were then incubated with lineage and stem cells/progenitor cell antibodies: anti-Lineage Mixture (mouse CD3e, CD11b, CD45R, Ly-6C/G, TER119, Invitrogen, Carlsbad, California, USA) and anti-Ly6-A/E (Sca-1), anti-Cd117 (c-Kit) (both from eBioscience), and rabbit anti-Neurokinin 1 Receptor (NK1R) (Novus Biologicals, Centennial, Colorado, USA) for 30 min at 4˚C. The concentration of primary antibodies is reported in ESM Table 1. Cells were then washed, treated with Goat Anti-Rabbit PE secondary antibodies for 30 min at 4˚C, and finally analysed using a FACS Canto II equipped with FACS Diva software (BD).

BM cells migration
To verify the effect of type 1 diabetes on the sensitivity of BM cells to SP, we performed in vitro migration assays using 6 well-plates transwell cell culture inserts equipped with 3-5μm pore size filters (Corning, New York, USA). Freshly-isolated BM cells were plated onto the upper compartment (2.5x10 6 , cells/well) and 100 mmol/l SP was added to the lower compartment in DMEM and 0.5% w/v foetal bovine serum. Cells were allowed to migrate overnight in a humidified atmosphere (37°C, 5% CO2). Cells from the upper (non-migrated cells) and lower compartments (migrated cells) were collected and processed for flow cytometry analysis with the use of AccuCheck counting beads (Invitrogen) for absolute and reproducible quantification of cell numbers. Enrichment of antigenically-defined cell populations was expressed as the ratio of cells in lower and upper chambers and then normalised to control (cells treated with vehicle).

Statistical analysis
The D'Agostino & Pearson omnibus normality test was applied to check for normal distribution of data. Normally-distributed continuous variables were expressed as mean ± SEM and compared between groups using the unpaired Student t-test. When continuous variables did not follow a normal distribution, values were expressed as median (interquartile range IQR) and compared with the Wilcoxon-Mann-Whitney test. Categorical variables were compared using the chi-squared or the Fisher's exact test. All reported p-values are two-sided. A p-value <0.05 was considered statistically significant. Statistical analyses were performed with STATA 11 software (StataCorp. 2009. Stata Statistical Software: Release 11. College Station, TX: StataCorp LP) or GraphPad Prism software, Version 6.01. Antibodies validation: whenever possible, antibodies were previously validated by our team using positive or negative control tissues/cells. All antibodies used in this study have been previously used by other Investigators, and references are available either from the company or in online databases such as PubMed. The companies are often providing information on how antibodies specificity was tested.

Sensory fibres in mouse bone marrow express neurotrophins receptors. (a-c)
Sensory fibres in mouse bone marrow (BM) were identified by staining for substance P (SP, red fluorescence). Co-staining for TrkA (a), p75 NTR (b), and RET (c) receptors, all in green fluorescence, demonstrated sensory fibres innervating the BM are potentially responsive to neurotrophins stimuli. DAPI (blue) identifies nuclei. Scale bar, 25 μm. Bone is identified by a dashed line.
Dorsal root ganglia express substance P. Immunofluorescence staining for substance P (green) in the spinal cord. Dorsal root ganglia (DRG) and dorsal horns (DH) are positive to SP. Scale bar, 10 μm.
Expression of NGF in PGP9.5 neuronal fibres. Immunohistochemistry staining for NGF (a) and PGP9.5 (b) on two consecutive slices of bone marrow indicates fibres co-express the 2 markers. Scale bar, 50 μm.