p75NTR-dependent activation of NF-κB regulates microRNA-503 transcription and pericyte–endothelial crosstalk in diabetes after limb ischaemia

The communication between vascular endothelial cells (ECs) and pericytes in the microvasculature is fundamental for vascular growth and homeostasis; however, these processes are disrupted by diabetes. Here we show that modulation of p75NTR expression in ECs exposed to high glucose activates transcription of miR-503, which negatively affects pericyte function. p75NTR activates NF-κB to bind the miR-503 promoter and upregulate miR-503 expression in ECs. NF-κB further induces activation of Rho kinase and shedding of endothelial microparticles carrying miR-503, which transfer miR-503 from ECs to vascular pericytes. The integrin-mediated uptake of miR-503 in the recipient pericytes reduces expression of EFNB2 and VEGFA, resulting in impaired migration and proliferation. We confirm operation of the above mechanisms in mouse models of diabetes, in which EC-derived miR-503 reduces pericyte coverage of capillaries, increased permeability and impaired post-ischaemic angiogenesis in limb muscles. Collectively, our data demonstrate that miR-503 regulates pericyte–endothelial crosstalk in microvascular diabetic complications.


Supplementary Figure 2: miR-503 expression in microvascular ECs and miRNA array analysis
A, HMVECs were exposed to high-glucose (HG), cultured in normal glucose (NG) or osmotic control (Cont; L-Glucose) conditions for 24hrs and expression of p75 NTR was analyzed by qPCR. **p<0.01 vs NG or Cont (n=3), unpaired two-tailed Student's t-test; B, Representative Western blot for p75 NTR and tubulin and protein quantification *p<0.05 vs NG or Cont (n=3), unpaired two-tailed Student's t-test C, Expression of p75 NTR in HUVECs after transduction with Ad.p75. Gene expression was normalized to 18S expression. **p<0.01 vs. Ad.Null (n=3), unpaired two-tailed Student's t-test; All values are mean±SEM of three independent experiments. D, Ranked list of miRNA differentially expressed in HUVECs transduced with Ad.p75 or Ad.Null (as control). miRNAs were ranked by differential expression and statistical significance. The default table of Limma is ranked by B-statistics (B), closely related to the adjusted p-value. The B-statistic used for this analysis is the logodds that that gene is differentially expressed and it is automatically adjusted for multiple testing by assuming that 1% of the genes are expected to be differentially expressed. Figure 3: The p75 NTR receptor regulates EC function A, p75 NTR expression in HUVECs transfected with siRNA for p75 NTR or scramble oligos (scr oligos) and treated with HG or L-Glucose (Cont) for 24hrs; **p<0.01; two-way ANOVA (n=5); B, miR-183* expression in the conditions described above; E, Proliferation assay in HUVECs transduced with Ad.Null, Ad.p75 or Ad.decoy503 adenovirus; F, Matrigel assay in the same conditions described above. Results are described as total tube length. For E and F: *p<0.05 vs. Ad.Null, # p<0.05 vs. Ad.p75 (n=3), unpaired two-tailed Student's t-test. All values are mean±SEM of three independent experiments. Figure 4: Clinical outcomes and target gene expression after p75 NTR gene transfer in limb muscles of ischemic mice A, Analysis of necrotic toes of non-diabetic mice injected with Ad.p75, Ad.Null or Ad.p75 and Ad.decoy503 together in ischemic limb muscles (n=12/group). *p<0.05 vs. Ad.Null, #p<0.05 vs. Ad.p75, necrotic toes endopoint was analyzed using Cochran-Armitage trend test B, Relative expression of CDC25A and CCNE1 in ischemic adductors of non-diabetic mice given Ad.p75, Ad.Null or Ad.p75 and Ad.decoy503 together (n=6/group). *p<0.05 vs. Ad.Null, # p<0.05 vs. Ad.p75, Mann-Whitney nonparametric test was applied. All values are mean±SEM of three independent experiments.

Supplementary Figure 5: miR-503 regulation by NF-kB and clinical outcomes after dnIKK2 gene transfer in limb muscles of diabetic and ischemic mice
A, HUVECs were transduced with Ad.Null or Ad.dnIKK2 and treated with HG or osmotic control (L-Glucose). qPCR was carried out to measure the expression of pri-miR-503 and mature miR-503; *p<0.05 vs Cont+Ad.Null; # p<0.05 vs HG+Ad.Null (n=3), unpaired twotailed Student's t-test All values are mean±SEM of three independent experiments. B, Representative color laser Doppler images are taken at 21 days post-ischemia. C, Analysis of necrotic toes of non-diabetic mice and diabetic mice injected with Ad.dnIKK2, Ad.miR-503 or Ad.Null in ischemic limb muscles (n=12/group). **p<0.01 vs. Non Diab+Ad.Null; # p<0.05 vs. Diab+Ad.Null, necrotic toes endopoint was analysed using Cochran-Armitage trend test. Data show individual data points, where a designation of "6" indicates foot necrosis or impaired foot locomotion. Figure 6: Endothelial microparticles analysis A, Size distribution of isolated MPs from p75 NTR -transduced HUVECs (n=500 cells). B, Flow cytometric analysis of MPs from p75 NTR -transduced HUVECs. p75 NTR increased the release of HUVEC-derived MPs expressing AnnexinV (AnnV Pos ) on the MP-surface; C, Expression of miR-126, miR-143 and miR-145 in HUVECs and in D, isolated MPs from medium of p75 NTR -transduced HUVECs. E, HUVECs were cultured in HG and osmotic control medium (Cont) and treated with Rho kinase inhibitors Y27632 (10µM) or HA-1077 (10µM) and MPs were collected from the medium by centrifugation and analyzed for Annexin V by flow cytometry. **p<0.01 vs Cont; #p<0.05 vs HG (n=3), unpaired two-tailed Student's t-test. F, HUVECs were cultured in same conditions described above and transduced with the Ad.Null or Ad.dnIKK2. G, Caspase-3 activity in HUVECs treated as described above, For F and E: *p<0.05 vs Cont/Ad.Null; # p<0.05 vs HG/Ad.Null (n=3), unpaired two-tailed Student's t-test. All values are mean±SEM of three independent experiments.

Supplementary Figure 8: Overxpression of 3'UTR-mutated target gene restores proliferation and migration in pericytes overexpressing miR-503
HUVECs expressing either ∆EFNB2 or ∆VEGFA or vector alone (pcDNA3.1) were transfected with mature miR-503 or a scramble sequence and further cultivated for 24 hrs. Then, proliferation (A and B) or migration (C and D) were assayed. ∆EFNB2 or ∆VEGFA partially restored the proliferation (A and B, respectively) and migration (C and D, respectively) in HUVEC transfected with pre-miR-503. **p<0.01 or *p<0.05 vs. scr oligos+pcDNA3.1; # p<0.05 vs. miR-503+ pcDNA3.1 (n=5), unpaired two-tailed Student's ttest. All values are mean±SEM of three independent experiments. Figure 9: The uptake of MPs by pericytes A, pericytes incubated for 24h with MPs-miR-503 increased significantly the intracellular levels of miR-503 in dose-dependent manner. *p<0.05, **p<0.01 vs. vehicle` (n=5), unpaired two-tailed Student's t-test. All values are mean±SEM of three independent experiments. B, An in vitro co-culture system of HUVECs (top compartment) with pericytes (bottom compartment) in which the cells are separated by a membrane to prevent direct cell contact has been setup HUVECs were labeled with DiO (green fluorescence) and transduced with p75 NTR -overexpressing adenovirus or Null-control adenovirus and fluorescence was analyzed in pericytes after 48hrs of co-culture by fluorescent microscopy; Scale bar 50 m.

Supplementary Figure 10: Flow cytometry analysis of CD31 and NG2 sorted cells from limb muscle
Cells were isolated using magnetic beads conjugated with CD31 and NG2 antibodies. Sorted cells were stained using A, CD31-FITC or B, NG2-PE conjugated antibody. Boxed: percentage of CD31 and NG2 in sorted cells (n=3); All values are mean±SEM of three independent experiments.

Supplementary Figure 11: miR-503 in situ hybridization
Localization of miR-503 (green fluorescence) by in situ hybridization in endothelial cells (Isolectin-B4 blue fluorescence) and pericytes (NG2, red fluorescence) in the ischemic limb muscles of diabetic and non-diabetic mice. A, Ischemic adductor muscle of diabetic mice hybridized with scrambled sequence probes as negative control (n=6). B, Non diabetic adductor muscle hybridized with miR-503 probe (n=6). C, Ischemic adductor muscle of diabetic mice hybridized with miR-503 probe (n=6). Scale bars 50 μm. Insert: Higher magnification image of small vessel in ischemic adductor muscles of diabetic mice. Scale bars 10μm.