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Neurotrophin p75 Receptor (p75NTR) Promotes Endothelial Cell Apoptosis and Inhibits Angiogenesis

Implications for Diabetes-Induced Impaired Neovascularization in Ischemic Limb Muscles

Andrea Caporali, Elisabetta Pani, Anton J.G. Horrevoets, Nicolle Kraenkel, Atsuhiko Oikawa, Graciela B. Sala-Newby, Marco Meloni, Brunella Cristofaro, Gallia Graiani, Aurelie S. Leroyer, Chantal M. Boulanger, Gaia Spinetti, Sung Ok Yoon, Paolo Madeddu, Costanza Emanueli
https://doi.org/10.1161/CIRCRESAHA.108.177386
Circulation Research. 2008;103:e15-e26
Originally published July 17, 2008

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Abstract

Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75NTR), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we show that gene transfer-induced p75NTR expression impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells (EPCs) and inhibits angiogenesis in vitro. Moreover, intramuscular p75NTR gene delivery impairs neovascularization and blood flow recovery in a mouse model of limb ischemia. These disturbed functions are associated with suppression of signaling mechanisms implicated in EC survival and angiogenesis. In fact, p75NTR depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels of ITGB1 [beta (1) integrin], BIRC5 (survivin), PTTG1 (securin) and VEZF1. Diabetic mice, which typically show impaired postischemic muscular neovascularization and blood perfusion recovery, have these defects corrected by intramuscular gene transfer of a dominant negative mutant form of p75NTR. Collectively, our data newly demonstrate the antiangiogenic action of p75NTR and open new avenues for the therapeutic use of p75NTR inhibition to combat diabetes-induced microvascular liabilities.

Diabetes impairs endothelial function and induces apoptosis of endothelial cells (ECs) and endothelial progenitor cells (EPCs). As a result, postischemic reparative angiogenesis and vasculogenesis are both impaired in diabetic subjects.1–12 The consequences are harmful: in the leg, severe microangiopathy aggravates atherosclerosis-induced muscular ischemia, thus contributing to gangrene and cutaneous ulcers with a severely impaired healing potential, which makes amputation of the diabetic foot an all-too-frequent necessity.13,14 A remedy to prevent and treat diabetic microvascular complications is urgently needed.

Neurotrophins (NTs) have been extensively studied for their actions on the nervous system. However, it is becoming increasingly evident that the expression and function of NTs is also important in the cardiovascular system. Both ECs and EPCs express tropomyosin kinase receptors (trk), which are tyrosine-kinases binding NTs with high affinity. The NT nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), acting on trkA and trkB, respectively, promote EC survival and angiogenesis.3,15–19 Moreover, NGF, via trkA, is an autocrine survival factor for cardiomyocytes.20 NTs have another receptor of 75 kDa MW (p75NTR). The p75NTR contains a death domain and belongs to the tumor necrosis factor α (TNF-α) receptor superfamily, but it does not bind TNF-α. In neural cells, p75NTR mediates apoptosis and cell cycle arrest.21–23 It was proposed that p75NTR has a low affinity for mature NTs, as it preferentially binds proforms of NTs.24–26 However, the mechanisms underpinning p75NTR activation and downstream molecular signaling have not been fully elucidated. Notably, no investigation of the role of p75NTR on EC and EPC survival and function or on angiogenesis has been attempted to date.

We previously reported that p75NTR is scarcely represented in capillary ECs of healthy murine limb muscles, but that expression strikingly increases after induction of type-1 diabetes and hindlimb ischemia, 2 increasingly prevalent conditions that synergistically activate EC apoptosis.3,10,27

This study provides novel evidence that p75NTR impairs survival and functions of cultured ECs and EPCs and hampers reparative neovascularization in ischemic limb muscles. We attribute the antiangiogenic effect of p75NTR to inhibition of the vascular endothelial growth factor A (VEGF-A)/Akt kinase axis. Finally, we show that p75NTR inhibition benefits postischemic healing in diabetes.

Materials and Methods

A detailed description of material and methods used is provided as online supplement (available online at http://circres.ahajournals.org). The experiments involving mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and with prior approval of the British Home Office and the University of Bristol.

Cell Cultures

Human umbilical vein ECs (HUVECs) and human microvascular ECs (MVECs) were grown in EGM-2 medium. EPCs were enriched from peripheral blood mononuclear cells (PB-MNC) and cultured on fibronectin in the EPC medium EGM-2MV.

Adenoviruses (Ad) and In Vitro Gene Transfer

We prepared Ad.p75NTR, containing the complete coding sequence of human p75NTR with or without a V5 tag. The Ad.DN-p75NTR, consisting of extracellular and transmembrane domains of rat p75NTR linked to a kinase-dead form the human EGF (cytoplasmic domain), has been previously shown to inhibit apoptotic responses mediated by ligand-mediated activation of the endogenous p75NTR receptor.28 Ad.Null and Ad.GFP served as controls. Ad.VEGF-A and Ad.Myr-Akt (expressing a constitutively active mutant form of Akt) have been previously described.12,27 Cells were infected with Ads at 10 to 500 multiplicity of infection.

Western Blot Analyses

Western blot analyses for: mouse p75NTR, phosphorylated and total eNOS, phosphorylated and total Akt, VEGF-A, phospho-Rb, phospho-FAK, cleaved-caspase-3, and GAPDH (loading control) were performed.

FACS Analysis of HUVECs for p75NTR, Annexin-V, and Propidium Iodide

Surface antigen expression by HUVECs was analyzed by flow cytometry using a combination of a phycoerythrin-conjugated mouse antihuman p75NTR antibody and FITC-conjugated Annexin-V. Annexin-V binds to phosphatidyl serine exposed on the extracellular membrane of apoptotic cells. Cells were also stained with propidium iodide (PI).

In Vitro Apoptosis Assays

Caspase-3 activity was measured with a luminescent cell death detection kit. TUNEL assay was performed according to the manufacturer’s instructions.

Analysis of Endothelial Apoptotic Microparticles

Endothelial apoptotic microparticles (EMPs) were collected from the HUVEC-conditioned medium by centrifugation. EMPs were numbered by flow cytometry analysis as AnnexinV-binding microparticles. Analyses were performed using an antihuman p75NTR fluorescent antibody and its isotype control. EMPs were analyzed in the presence of Flowcount calibrator beads. EMP gate was defined as events with a 0.1 to 1 μm diameter and examined on a fluorescence/forward light scatter plot.

Cell Cycle Analysis

Transduced HUVECs were synchronized by serum deprivation before being released in complete medium, and harvested at different time points. After fixation and RNase treatment, cells were stained with PI. DNA content was analyzed by flow cytometric analysis.

BrdU Incorporation

Transduced HUVECs and EPCs were incubated with BrdU for 24 hours and 72 hours, respectively. BrdU incorporation was measured using a BrdU immunofluorescence assay kit (HUVECs) or by FACS (EPCs).

Migration Assay

Migration of transduced HUVECs and EPCs toward stromal cell–derived factor-1 (SDF-1) or 0.1% BSA (negative control indicative of spontaneous migration) was analyzed by using fibronectin-coated 24-well plate transwell migration inserts with a polycarbonate membrane of 8-μm (HUVECs) or 5-μm (EPCs) pore size.

Adhesion Assay

Adhesion of transduced HUVECs and EPCs to fibronectin was evaluated.

Matrigel Assay

Transduced HUVECs were seeded in 24-well plates coated with Matrigel supplemented with growth factors. Tube formation was quantified in randomly captured microscopic fields by counting the number of intersection points and by measuring the % of area covered by connected tubular structures.

NO Generation Assay

NO generation by transduced HUVECs was assessed in a FACS Calibur flow cytometer by using DAF-2DA in the presence or absence of the eNOS preferential inhibitor L-N5-(1-Iminoethyl) ornithine (L-NIO).

Gene Transfer to Murine Muscles

Adenoviruses were delivered to the normoperfused or ischemic left adductor muscles of anesthetized mice.

Illumina Beadarray Gene Expression Profiling

Analyses are described in supplementary methods. Microarray expression data are available at the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/project/geo) under accession number GSE9910.

GEArray

Human GEArray kit, composed of 113 genes connected to the p53 signaling pathway and 2 housekeeping genes (actin and GAPDH), was used to analyze the RNA of transduced HUVECs. The results were validated by real-time quantitative PCR.

Real-Time Quantitative RT-PCR

Quantitative RT-PCR for mouse and human VEGF-A, ITGB1 and VEZF-1; human BIRC5, PTTG1, and p75NTR; and 18s rRNA was performed in a LightCycler.

Limb Ischemia and Diabetes Models

Left limb ischemia was induced in anesthetized CD1 male mice. Type-1 diabetes was induced by streptozotocin.

Clinical Outcome From Ischemia and BF Measurements

The number of autoamputated or necrotic fingers in the ischemic foot was calculated. Superficial BF of the ischemic and contralateral foot was analyzed by using the Lisca color laser Doppler. Intramuscular BF was measured by Oxylite/Oxyflow.

Histology and Immunohistochemistry

Capillary density was determined on muscular sections stained with H&E or for isolectin B4 (to identify ECs) and DAPI (to identify the nuclei). Cell apoptosis was recognized by TUNEL and proliferation by staining for the proliferation cell nuclear antigen (PCNA) or the mini-chromosome maintenance protein 2 (MCM2). Transgene expression by capillary ECs was determined by costaining sections for V5 and with isolectinB4. Endogenous mouse p75NTR expression by ECs was identified by costaining for mouse p75NTR and with isolectinB4.

FACS Analyses of Bone Marrow Cells

BM-MNC cells were obtained from mouse legs. Surface antigen expression of MNC was analyzed by flow cytometry using combinations of fluorescent antibodies for Sca-1, lineage markers, and mouse p75NTR plus fluorescent Annexin-V.

Statistical Analysis

Results are expressed as the mean±SEM. Statistical analysis was performed via a t test when 2 groups were analyzed, or via an ANOVA, followed by a Bonferroni post hoc test for multiple comparisons. A probability value of <0.05 was taken as statistically significant. The statistical analysis used for Illumina data are described apart.

Results

p75NTR Transduction Impairs the Survival and Proangiogenic Capacity of ECs

p75NTR expression in ECs is increased by diabetes and tissue injury.3,29 To evaluate the functional relevance of p75NTR in ECs, we induced p75NTR expression in HUVECs by gene transfer. The successful p75NTR transduction was demonstrated by both Western blot (Figure 1a) and FACS (Figure 1b) analyses. FACS analysis at 12, 24, and 48 hours from transduction showed that the appearance of the p75NTR protein (which was already present at 12 hours; Figure 1b) precedes apoptosis in p75NTR-transduced HUVECs (p75NTR-HUVECs; Figure 1c). In fact, early apoptosis (cells positive for Annexin-V binding and negative for PI) peaked at 24 hours, followed, at 48 hours, by late apoptosis (Annexin-V+PI+ cells). We confirmed the proapoptotic effect of p75NTR in HUVECs by TUNEL apoptosis assay (Figure 1d), Western blot for the apoptosis marker cleaved (activated) caspase-3 (Figure 1a), and caspase-3 activity assay (Figure 1e). Additionally, we measured the release of EMP30 from p75NTR- and Null-transduced HUVECs (Null-HUVECs) with or without exposure to the apoptosis inducer staurosporin. Under both circumstances, p75NTR enhanced EMP release and p75NTR was present in EMP (Figure 1g). Taken together, these results provide evidence that p75NTR promotes HUVEC apoptosis, via caspase-3 activation. The proapoptotic effect of p75NTR was also present in MVECs (Figure 1f).

Figure1

Figure 1. Adenovirus-mediated p75NTR transduction promotes EC apoptosis. a, HUVECs were infected with 100, 250, and 500 multiplicity of infection (MOI) of Ad.Null or Ad.p75NTR. After 48 hours, cell lysates were collected and subjected to Western blotting with antibodies to p75NTR, cleaved caspase-3, and GAPDH (used as loading control). Bands are representative of n=3 experiments. b, FACS analysis of HUVECs infected with Ad.p75NTR (250 MOI) showed abundant receptor expression (95.8±3.0% of total cells) at 12 hours from gene transfer as compared with low p75NTR expression in Ad.Null-infected cells (6.7±0.7%). c, To detect apoptosis, p75NTR-expressing HUVECs were gated and studied at 12 hours, 24 hours, and 48 hours for Annexin-V binding and uptake of propidium iodide (PI). This analysis revealed that, at 12 hours from Ad.p75NTR, less than 9% of p75NTR-carrying cells binds Annexin-V. Early apoptosis (cells positive for Annexin-V and negative for PI, lower right squares) peaked at 24 hours (39.1±3.0% of p75NTR-expressing HUVECs), followed by late apoptosis (cells positive for both Annexin-V and PI, upper right squares) at 48 hours from Ad.p75NTR (17.1±2.0% of p75NTR-expressing HUVECs). d, Apoptotic nuclei of transduced HUVECs were detected by TUNEL assay. Fluorescent images are representative of apoptosis rate in Null-HUVECs and p75NTR-HUVECs (250 MOI). Original magnification, 400×; scale bar, 40 μm. Green fluorescence, TUNEL-positive nuclei; Blue fluorescence, all the nuclei. Arrows point to TUNEL-positive nuclei. Bar graphs quantify apoptosis, which is expressed as percentage of TUNEL-positive nuclei to total nuclei. Data are presented as means±SEM *P<0.05 and **P<0.001 vs Ad.Null. e and f, Caspase-3 activity assay in transduced HUVECs (e) and MVECs (f). The apoptosis inducer staurosporin (stauro) was used as reference in e. Values are means±SEM *P<0.05 and **P<0.001 vs Ad.Null. g, Upper panel: Concentration of endothelial apoptotic microparticles (EMP) released by Null-HUVECs or p75NTR-HUVECs under unstimulated conditions or following incubation with staurosporin. Values are means±SEM **P<0.01 vs Ad.Null. Lower panel: p75NTR is present in EMP released by HUVECs. The shadow peak corresponds to fluorescence background obtained with the isotypic control of p75NTR antibody. The second peak represents the specific labeling of EMPs with fluorescent p75NTR antibody.

In neural cells, p75NTR promotes cell cycle arrest.21 Because EC replication is intrinsic to the angiogenesis process, we studied the impact of p75NTR on HUVEC cycle progression. HUVECs were synchronized at G1/S boundary by serum starvation and the progression of cell cycle on release from the G1-block was analyzed by flow cytometry. As shown in Figure 2a, the control Null-HUVECs entered the cell cycle and progressed through S-phase and G2. p75NTR-HUVECs were delayed in cycling, as shown by the reduced percentage of cells in S-phase (P<0.01 versus Null-HUVECs at 21 hours). Correspondingly, the level of phosphorylated tumor suppressor retinoblastoma protein (Rb), a marker of cell cycle progression,31 was less in p75NTR-HUVECs (Figure 2b). The antiproliferative effect of p75NTR on ECs was confirmed by using a BrdU incorporation assay (Figure 2c).

Figure2

Figure 2. p75NTR inhibits EC cell cycle progression and impairs EC ability to proliferate, migrate, adhere, and form capillary-like tubular structures in vitro. a, HUVECs were infected with either Ad.Null or Ad.p75NTR (250 multiplicity of infection [MOI]). Cells were synchronized by overnight serum starvation. After release of cell cycle arrest by restoring normal culture medium, cell cycle progression was assessed by flow cytometric analysis, at the indicated time points. The percentage of cells in G1, S, and G2 phases is indicated in figures. b, Western blot analysis of phosphorylated tumor suppressor retinoblastoma protein (p-Rb) (marker of cell cycle progression) on HUVEC extracts. GAPDH was used as loading control. c, Proliferation (measured by BrdU incorporation) of HUVECs infected with Ad.Null or Ad.p75NTR (250 MOI) or uninfected (PBS). **P<0.01 vs Ad.Null. d, Migration toward SDF-1 (100 ng/mL) or PBS (vehicle) of p75NTR-HUVECs and Null-HUVECs. In bar graph, values are means±SEM #P<0.05 vs Ad.Null combined with PBS, *P<0.05 vs Ad.Null combined with SDF-1. Upper panels show representative microscopic fields (original magnification 100×; scale bar: 100 μm) e, Adhesion to fibronectin of p75NTR-HUVECs and Null-HUVECs. Cell adhesion was quantified by counting adherent cells following washing of nonadherent cells and DAPI staining of nuclei of adherent cells. In bar graph, values are means±SEM **P<0.01 vs Ad.Null. Upper panels show representative photomicrographs (magnification 200×, scale bar: 100 μm) f, Time-course (up to 24 hours from cell seeding) of tubular structure formation in matrigel. Twelve hours before being seeded on Matrigel-coated dishes, HUVECs were infected with Ad.Null or Ad.p75NTR. In bar graphs, the quantification of EC tube network formation at 24 hours from seeding is expressed as the number of intersecting points of tubular structures for microscopic field (left) as well as the % of microscopic field area covered by connected tubular structures (right). Values are means±SEM **P<0.01 versus Ad.Null. Upper panels show representative photomicrographs (original magnification 100×; scale bar: 100 μm).

EC migration is essential for neovessel formation. We tested the effect of p75NTR on EC migratory capacity toward SDF-1, which is a classic chemotactic stimulus for cells of the endothelial lineage.32 At both 12 hours and 48 hours from gene transfer, the migratory capacity of p75NTR-HUVECs to SDF-1 was severely impaired compared to Null-HUVECs (Figure 2d for 48 hours; 12-hour data not shown).

Cell-matrix adhesion is also fundamental for vessel growth and maintenance. At both 12 hours and 48 hours from infection, p75NTR-HUVECs showed reduced adhesion capacity to fibronectin (Figure 2e for 48 hours; 12-hour data not shown).

Finally, we tested the angiogenic potential of p75NTR-HUVECs in a matrigel assay, in which ECs stimulated by growth factors give rise to vessel-like tubular structures in vitro, thus mimicking angiogenesis. In this assay, which was initiated at 12-hour postgene transfer, the antiangiogenic effect of p75NTR was evident (Figure 2f).

p75NTR Induces a Proapoptosis and Antiangiogenesis Signaling Pathway in ECs

p75NTR-HUVECs showed reduced levels of VEGF-A and Ser473-phospho-Akt (Figure 3a). Phospho-Akt positively controls EC survival and angiogenesis by several downstream mediators, including endothelial nitric oxide synthase (eNOS).33 Correspondingly, p75NTR transduction impaired Ser1177-phosphorylation of eNOS (Figure 3a) and eNOS-derived NO production (Figure 3b). VEGF-A gene transfer improved Ser473-phospho-Akt level in p75NTR-HUVECs, whereas Myr-Akt did not rescue VEGF-A expression (data not shown), thus confirming that VEGF-A lies upstream of Akt phosphorylation.34 Phospho-eNOS content was increased by either VEGF-A or a Myr-Akt gene transfer in p75NTR-HUVECs. Focal adhesion kinase (FAK), upstream of Akt, controls EC migration, proliferation, and survival.35 Phospho (active)-FAK was less in p75NTR-HUVECs and, as expected, Myr-Akt did not restore phospho-FAK to normal levels. By contrast, VEGF-A increased phospho-FAK in p75NTR-HUVECs (data not shown). To investigate the role of depressed VEGF-A and phospho-Akt in the antiangiogenic action of p75NTR, we performed a rescue experiment in the matrigel assay model. The capacity of p75NTR-HUVECs to form tubular structures was restored by either VEGF-A or Myr-Akt (Figure 3c), thus providing evidence that the antiangiogenic action of p75NTR is caused by inhibition of the VEGF-A/Akt pathway.

Figure3

Figure 3. p75NTR inhibits proangiogenesis and prosurvival pathways in ECs. a, Western blot bands of Null-HUVECs and p75-HUVECs show protein expression of VEGF-A, phospho-Akt, total Akt, phospho-eNOS, and total eNOS. GAPDH was used as loading control. HUVECs were infected with Ad.Null or Ad.p75NTR (100, 250, and 500 multiplicity of infection [MOI]). Bands are representative of 3 independent experiments. b, HUVECs were infected with Ad.Null or Ad.p75NTR (250 MOI). The release of NO was analyzed using the fluorescent probe DAF-2DA in the presence or absence of the eNOS preferential inhibitor L-NIO. FL1 fluorescence, emitted by DAF-2DA after reaction with NO, was determined by FACS. The bar graph shows the NOS-mediated FL1 mean fluorescence intensity after subtraction of eNOS-independent fluorescence emitted in presence of L-NIO. Representative histographs are shown on the right. Full curves are representative of NO release in the absence of L-NIO. Empty curves show NO release in the presence of L-NIO. Values in the bar graph are means±SEM **P<0.01 vs Ad.Null; c, Both Ad.VEGF-A and Ad.Myr-Akt rescued tube formation capacity of p75NTR-transduced HUVECs. Twelve hours before being seeded in matrigel, HUVECs were infected with Ad.Null or Ad.p75NTR (each at 250 MOI) plus Ad.VEGF-A or its control Ad.Null (each at 10 MOI) or Ad.Myr-Akt or its control Ad.Null (each at 100 MOI). Images were taken at 24 hours from seeding. (Original magnification, 100×; scale bar, 100 μm). Values in the bar graph are means±SEM (average of 3 independent experiments performed in triplicate). *P<0.05 vs Ad.Null plus Ad.Null, +P<0.05 vs Ad.p75NTR plus Ad.Null. d, Relative mRNA expression (determined by real-time RT-PCR, using RNA 18S as reference) of BIRC5 (survivin) and PTTG1 (securin) in p75NTR-HUVECs and Null-HUVECs. Coinfection with Myr-Akt enhanced BIRC5 and PTTG1 content of p75NTR-HUVECs. Values are means±SEM *P<0.05 vs Ad.Null, +P<0.05 vs Ad.p75NTR alone.

To further understand the p75NTR-induced molecular program contributing to EC apoptosis and antiangiogenesis, we performed a GeArray for apoptosis and cell cycle-related genes on RNA extracted from p75NTR-HUVECs and Null-HUVECs. Results (not shown) were validated by real-time RT-PCR. As shown in Figure 3d, p75NTR reduced the mRNA expression of BIRC5 (survivin) and PTTG1 (pituitary tumor-derived transforming gene, also named securin), which both mediate EC survival and angiogenesis and are transcriptionally controlled by phospho-Akt.36 Accordingly, Myr-Akt increased BIRC5 and PTTG1 in p75NTR-HUVECs (Figure 3d).

p75NTR Transduction Impairs EPC Survival and Functions

The growth of new blood vessels needs the participation of both angiogenesis, developed by preexisting vessels, and vasculogenesis, which involves EPCs. To study the impact of p75NTR in EPCs derived from the human blood, we performed p75NTR gene transfer (Figure 4a). We found that p75NTR negatively impacted on EPC survival (caspase-3 activity assay, Figure 4b), proliferation (FACS analysis of BrdU incorporation, Figure 4c), migration toward SDF-1 (Figure 4d), and adhesion (data not shown). Moreover, p75NTR transduction reduced the levels of both phospho-Akt and phospho-eNOS in EPCs (Figure 4e).

Figure4

Figure 4. p75NTR negatively impacts on EPC survival, proliferation, and migration. a, Western blot analysis of p75NTR expression in EPCs infected with Ad.p75NTR or Ad.Null (100 to 500 multiplicity of infection [MOI]). GAPDH expression was used as a loading control. b, Caspase-3 activity assay tested on EPCs infected with Ad.p75NTR or Ad.Null (250 and 500 MOI). Values are means±SEM *P<0.05 and **P<0.01 vs Ad.Null. c, FACS analysis of BrdU incorporation by EPCs. EPCs were infected with Ad.Null or Ad.p75 and cultured in the presence of BrdU. Aliquots were stained with anti-BrdU followed by secondary FITC-conjugated antibody or with secondary antibody only (negative control). The percentage of BrdU incorporating EPCs (indicated by marker bar) was lower in p75NTR-HUVECs; d, EPC migration (after infection with Ad.Null or Ad.p75NTR, 500 MOI) toward SDF-1 (100 ng/mL). In bar graph, values are means±SEM. #P<0.05 vs Ad.Null plus PBS, **P<0.01 vs Ad.Null and SDF-1. Upper panels show representative microscopic fields (original magnification, 100×; scale bar, 100 μm). e, Western blot analyses for phospho-Akt, total Akt, phospho-eNOS, total eNOS, and GAPDH (as loading control) of EPCs infected with Ad.Null or Ad.p75NTR (500 MOI).

p75NTR Transduction Impairs Postischemic Neovascularization and Blood Flow Recovery in a Mouse Model of Limb Ischemia

To test the impact of p75NTR on blood vessel growth in vivo, Ad.p75NTR (V5-tagged) or Ad.Null was injected into the ischemic adductor of normoglycemic mice. We verified successful gene transfer by Western blot analysis for p75NTR (supplemental Figure Ia). In addition, by immunohistochemistry for V5 (red fluorescence), we observed localization of the transgene in capillary ECs (recognized by FITC-isolectin staining -green fluorescence-) of the ischemic muscles (supplemental Figure Ib). Limb blood flow (BF) was measured by color laser Doppler at 7 and 14 days postischemia (n=12 mice per group). Reduce BF to the ischemic foot was observed in the p75NTR group. In Figure 5a, the color laser Doppler images are representative of the BF to the ischemic foot at 14 days postischemia, and the graph shows results at the same time (data at 7 days are not shown). Correspondingly, capillary density was reduced in p75NTR-transduced muscles (Figure 5b). In additional mice euthanized at 5 days postischemia, which, in comparison to 14 days, is a time point more informative on ischemia-induced EC proliferation and EC apoptosis responses, we observed increased EC apoptosis (Figure 5c) and reduced EC proliferation (Figure 5d) in p75NTR-transduced muscles.

Figure5

Figure 5. Effects of p75NTR transduction of ischemic limb muscles. a, Recovery of the blood flow (BF) to the ischemic foot (expressed as the ratio between the BF in the ischemic foot to the BF in the contralateral foot) at 14 days after ischemia induction and intramuscular delivery of Ad.p75NTR or Ad.Null. Values are means±SEM. *P<0.05 vs Ad.Null. Representative laser Doppler images taken at 14 days postischemia induction are also shown. Squares include the ischemic feet. Color from blue to brown indicates progressive increases in BF. b, Capillary density of ischemic adductor muscles of mice described in a. Values are means±SEM. **P<0.01 vs Ad.Null. Representative microphotographs show muscle sections stained with isolectin B4 (red fluorescence) to recognize ECs and counterstained with DAPI (blue fluorescence) to recognize nuclei. (Original magnification, 400×; scale bar, 500 μm). Apoptosis (c; revealed by TUNEL assay) and proliferation (d; revealed by immunhistochemistry for the proliferating cell antigen [PCNA]) of ECs in limb muscles at 5 days postischemia induction and gene transfer. Values are means±SEM **P<0.01 vs Ad.Null.

Expressional Changes Induced by p75NTR Transduction or Diabetes in Limb Muscles

We have used full genome expression profiling using Illumina bead-arrays interrogating 46 600 murine genome transcripts to study the global impact of p75NTR transduction on the gene expression program of normoperfused mouse muscle. The overall changes in the transcriptome were analyzed at the levels of specific functional pathways using Panther-annotated protein group analysis.37 This analysis identified, in addition to a number of inflammatory or muscle-specific pathways, a specific, statistically significant reduction in gene-sets involved in endothelial survival and angiogenesis processes, ie, VEGF-signaling and integrin-signaling (Figure 6a). Analysis of the corresponding genes in more details identified 3 pivotal genes (VEGF-A, ITGB1-[beta (1) integrin]-, VEZF1) that were suppressed by p75NTR, relative to a number of well-established endothelial-specific genes (data not shown). By real-time RT-PCR, we confirmed that p75NTR alters the expression of VEGF-A, ITGB1, and VEZF1 in murine muscles (Figure 6b). To further confirm that these molecular changes are relative to the effect of p75NTR on ECs, we analyzed VEGF-A, ITGB1 and VEZF1 mRNA levels in p75- HUVECs and found all of them reduced in comparisons to Null-HUVECs (Figure 6c). VEGF-A transfer corrected ITGB1, but not VEZF1 deficit in p75NTR-HUVECs (Figure 6c). Notably, muscular expression of VEGF-A, ITGB1, and VEZF1 is also dramatically downregulated by diabetes (Figure 6b), thus suggesting that this program may be involved in the diabetes-induced apoptosis of ECs. The scheme in Figure 6d illustrates our interpretation of the molecular changes induced by p75NTR in ECs.

Figure6

Figure 6. p75NTR transduction, similarly to diabetes, inhibits proangiogenesis and prosurvival pathways in mouse limb muscles. a, Changes at the levels of specific functional pathways in the transcriptome of p75NTR-transduced muscles. Gene sets linked to VEGF signaling and integrin signaling, which are both involved in endothelial survival and angiogenesis processes were found affected. b, Relative mRNA expression of VEGF-A, ITGB1, and VEZF1 in adductor muscles of normoglycemic mice following infection with Ad.Null (Null) or Ad.p75NTR (p75NTR) and in Null-transduced limb muscles of diabetic mice (diabetes). Values are means±SEM. *P<0.05 and **P<0.01 vs Ad.Null in normoglycemic muscles. c, Relative mRNA expression of VEGF-A, ITGB1, and VEZF1 in p75NTR-HUVECs and Null-HUVECs. Coinfection with VEGF-A rescued the mRNA level of ITGB1 but not of VEZF1 in p75NTR-HUVECs. Values are means±SEM. **P<0.01 versus Ad.Null, +P<0.05 vs Ad.p75NTR alone. d, Schematic representation of the molecular signaling emanating from p75NTR in ECs.

Diabetes Increases p75NTR Expression and Apoptosis of BM-Resident Progenitor Cells

In the murine bone marrow, putative progenitor cell populations able to stimulate blood vessel growth have been described based on their antigenic profile. One of these populations is represented by MNC cells which are Sca-1+ and lineage negative (Sca-1+Lin−). In previous studies, Sca-1+Lin− cells were recognized to be hematopoietic progenitor cells able to promote neovascularization of ischemic muscles.38,39 Consequently, to understand whether endogenous p75NTR may influence the survival of progenitor cells with neovascularization potential, we initially assessed p75NTR expression in bone marrow–resident Sca-1+Lin− cells. Thus, extending our previous findings of an association between diabetes, p75NTR expression, and apoptosis in ECs,3 here we provide new evidence that—even in the absence of superimposed ischemia—diabetes increases the abundance of both Sca-1+p75NTR+ cells (Figure 7a) and Sca-1+Lin−p75NTR+ cells (Figure 7c) in limb BM. By contrast, the abundance of Lin− p75NTR+ cells is not changed by diabetes (Figure 7b). Diabetic Sca-1+p75NTR+ cells bind the apoptosis marker Annexin-V (Figure 7d). These data suggest a role for p75NTR in diabetes-induced progenitor cell apoptosis.

Figure7

Figure 7. Diabetes increases p75NTR and apoptosis in BM progenitor cells. a and b, The percentage of BM-MNC coexpressing Sca-1 and p75NTR (a) was increased in diabetic mice, whereas the percentage of BM-MNC Lin− cell expressing p75NTR (b) did not change. c, The percentage of Sca-1+Lin−p75NTR+ was higher in diabetic BM-MNC. d, Left: The percentage of BM Sca-1+p75NTR+ cells binding the apoptosis marker Annexin-V was enhanced by diabetes. Values are means±SEM. *P<0.05 vs healthy mice. Right: Identification of apoptotic cells (gate R4) among Sca-1+p75NTR+ (gate R3) BM-MNC. After selection of monocytic cells according to size and granularity (not shown) and further identification of Sca-1+p75NTR+ cells, the relative abundance of Annexin-V-binding cells was determined.

Inhibition of p75NTR Action Reestablishes Proper Postischemic Neovascularization and BF Recovery in Diabetic Mice

Supplemental Figure II confirms our previous findings of increased p75NTR expression by capillary ECs in ischemic muscles of diabetic mice in comparison to normoglycemic controls.3 We finally tested the possibility that the above association reflects a functional relevance of p75NTR in diabetes-induced impairment of reparative neovascularization. This was accomplished using an adenovirus carrying a dominant negative mutant form of p75NTR (Ad.DN-p75NTR) in diabetic mice subjected to unilateral limb ischemia. This dominant negative mutant was designed to inhibit the function of the constitutive p75NTR protein, without affecting its expression.28 In fact, it did not change the mRNA content of murine p75NTR in diabetic muscles (data not shown). As shown in Figure 8a, foot BF recovery overtime was impaired in diabetic mice receiving the control virus Ad.GFP (in comparison to normoglycemic mice treated with Ad.GFP). Ad.DN-p75NTR had no effect in normoglycemic mice, whereas it improved BF recovery in diabetic mice, making it comparable to that of normoglycemic controls. Representative Doppler images of hindlimbs at 15 days postischemia are presented (Figure 8a). We observed a similar trend when measuring deep muscular BF (Figure 8b). Additionally, postischemic clinical outcome (calculated as the number of autoamputated or necrotic fingers in the ischemic foot) was worsened by diabetes. Notably, Ad.DN-p75NTR improved the clinical outcome of diabetic mice (number of necrotic/amputated fingers at 15 days: 1.6±0.9 in diabetic with Ad.DN-p75NTR versus 3.7±0.9 in diabetic with Ad.GFP, P<0.05 and versus 1.4±0.7 in healthy with Ad.GFP, P=N.S.). Doppler measurements and clinical analyses were performed on groups of at least n=12 mice. As shown in Figure 8c, the capillarization response to ischemia was not altered by Ad.DN-p75NTR in normoglycemic mice. Diabetes impaired postischemic neovascularization, with this effect being prevented by Ad.DN-p75NTR. Furthermore, Ad.DN-p75NTR inhibited EC apoptosis (TUNEL assay; Figure 8d and supplemental Figure III) and improved EC proliferation (assessed by MCM-2 staining; Figure 8e and supplemental Figure III) in diabetic ischemic muscles. Notably, intramuscular Ad.DN-p75NTR reduced apoptosis (recognized by Annexin-V binding) of Sca-1+Lin− cells in the diabetic BM (Figure 8f). To provide an interpretation of the latter result, we further investigated whether gene delivery to ischemic muscles can infect BM-resident cells. To this aim, we injected muscles with Ad.GFP and, after 3 days, we performed FACS analysis for GFP fluorescence of total BM cells of both legs. We did not detect any GFP fluorescence in BM (data not shown). These results suggest that limb BM cells cannot be transduced by adenovirus-mediated gene delivery to ischemic limb muscles.

Figure8

Figure 8. Inhibition of p75NTR signaling in diabetic limb muscles restores proper neovascularization and BF recovery after limb ischemia. Unilateral limb ischemia was induced in diabetic and normoglycemic mice before an adenovirus carrying a dominant negative mutant of p75NTR (Ad.DN-p75NTR) or Ad.GFP (control) was delivered to the ischemic adductor. a, Left: Time-course of postischemic BF recovery (calculated by laser color Doppler flowmetry) in diabetic and normoglycemic mice treated with Ad.DN-p75NTR or Ad.GFP; Right: Representative laser Doppler images taken at 14 days after induction of ischemia are shown. Squares include the ischemic feet; (b) Recovery of BF (measured by Oxford Optronic) is expressed as the ratio between the BF to ischemic muscle and the BF to the contralateral muscle at 14 days after ischemia induction. Values are mean±SEM. *P<0.05 vs normoglycemic mice with Ad.GFP, §P<0.05 vs diabetic with Ad.GFP. c, Capillary density in the ischemic adductor at 14 days postischemia. Values are means±SEM **P<0.01 vs normoglycemic mice with Ad.GFP, §§P<0.05 vs diabetic with Ad.GFP. d, Apoptosis (revealed by TUNEL assay) and (e) proliferation (revealed by immunhistochemistry for the proliferation antigen MCM-2) of capillary ECs in diabetic limb muscles at 14 days postischemia. Values are means±SEM *P<0.05 versus Ad.GFP. f, Left: The percentage of BM-resident Sca-1+Lin− progenitor cells binding the apoptosis marker Annexin-V is significantly reduced by intramuscular delivery of Ad.DN-p75NTR in diabetic mice. Values are means±SEM. +P<0.05 vs normoglycemic mice with Ad.Null.*P<0.05 vs diabetic with Ad.Null). Right: Representative examples of FACS analyses of diabetic BM are shown. First, monocytic cells were identified by size/granularity, followed by selection of Sca-1+Lin− cells (R2). Finally, the percentage of cells binding Annexin-V was calculated (R4).

Discussion

We previously showed and confirm here that diabetes induces p75NTR expression in capillary ECs belonging to ischemic limb muscles.3 With this study, we provide evidence that p75NTR severely impairs the survival and phenotype of cultured ECs and EPCs and inhibits postischemic neovascularization. The apoptotic and cell cycle inhibitor effects of p75NTR on neural cells were already known.21–23 In neural cells, p75NTR plays an ambiguous “Jekyll-and-Hyde” role, being able to either kill or stimulate cell survival and differentiation.22,23 By contrast, according to our findings, p75NTR has only detrimental actions on cells belonging to the endothelial lineage.

We have showed the potent antineovascularization effect of p75NTR in a mouse model of limb ischemia. In normoglycemic mice, p75NTR transduction of ischemic muscles also enhanced apoptosis and reduced proliferation of capillary ECs, thus ultimately depressing postischemic neovascularization and BF recovery. Conclusive proof that p75NTR plays a key role in diabetes-induced impairment of reparative neovascularization is provided by the experiment in which p75NTR activity in ischemic limb muscles was blocked by a dominant negative p75NTR mutant form. Whereas the p75NTR dominant negative had no effect in normoglycemic mice, it inhibited EC apoptosis, normalized EC proliferation, and restored proper muscular capillarization and BF recovery in diabetic mice. Notably, p75NTR inhibition in diabetic limb muscles additionally resulted in survival of BM-resident Sca-1+Lin− progenitor cells. Several antigenic definitions have been proposed to identify BM progenitor cells with the capacity to promote neovascularization of ischemic hindlimb muscles. In this study, we have analyzed the effect of diabetes on p75NTR expression in mouse BM Sca-1+Lin− cells, which were previously shown to be able to promote neovascularization of ischemic muscle.38,39 We recognize as a possible limitation of our study not to have investigated whether diabetes upregulates p75NTR expression in progenitor cells characterized by different antigenic profiles, including Sca-1+flk+ cells.40,41

The finding that limb BM cells were not transduced by adenovirus-mediated intramuscular gene transfer suggests that the reduced apoptosis of bone marrow–resident cells may be the consequence of improved global perfusion of the ischemic limb attributable to p75NTR signaling inhibition, rather than a direct effect on the BM. However, this possibility needs to be further investigated before reaching reliable conclusions.

Overall, our in vivo findings have important clinical implications and may help to design innovative therapeutic approaches able to promote angiogenesis and vasculogenesis in a diabetic context.

A possible limitation of our study is that we have not elucidated the mechanisms by which diabetes leads to expression of p75NTR in ECs and bone marrow progenitor cells. Preliminary data suggest that endothelial cells increase their p75NTR content after culture in high glucose and reduced serum, 2 situations mimicking diabetes and ischemia in vitro. (Caporali, Emanueli, unpublished data, 2007) The focus of the present work was dissecting the mechanisms triggered by p75NTR in ECs and their implications for blood vessel growth and maintenance. The diabetes- and ischemia-dependent mechanisms modulating p75NTR induction will be investigated in a specifically designed study.

Notably, the molecular signaling pathways that we identified to be downstream of p75NTR in ECs totally differ from those previously described in neural cells. In fact, here we newly report that p75NTR inhibits the VEGF-A/Akt/eNOS/NO pathway. This is probably the most relevant molecular signaling for angiogenesis and EC survival,33,34 and its deficit may per se justify the impairment of EC function. In line with this hypothesis, cotransduction with either VEGF-A or Myr-Akt rescued the capacity of p75NTR-HUVECs to form vascular-like tubular structures in matrigel. Moreover, Myr-Akt improved survival of p75NTR-HUVECs (Caporali, Emanueli, unpublished data, 2007). Reportedly, phospho-Akt positively regulates the transcription of BIRC536 and PTTG1,42 which both resulted being downregulated by p75NTR. Notably, Myr-Akt totally or partially rescued BIRC5 and PTTG1 mRNA expression levels in p75NTR-HUVECs. BIRC5, which was named survivin for its prosurvival action, was recently discovered to be a positive mediator of angiogenesis.36,43,44 Interestingly, BIRC5 also modulates cell cycle entry by interaction with Cdk4 and activation of Cdk2/Cyclin E complex leading to Rb phosphorylation.45 This may be reconciled with the impaired cell cycling capacity and reduced phospho-Rb levels observed in p75NTR-HUVECs. PTTG1 was also reported to positively modulate angiogenesis.42,46

Illumina technology-based analysis of p75NTR- and Null-transduced murine muscles followed by real-time RT-PCR of the same muscles and of p75- and Null-HUVECs identified that p75NTR downregulates the mRNA expression of ITGB1 [beta(1) integrin] and of the endothelial specific VEZF1 transcription factor in ECs, in vitro and in vivo. Notably, we also found downregulated VEGF-A, ITGB1, and VEZF-1 in diabetic muscles, which confirms previous reports of VEGF-A reduction as the cause of diabetes-induced impaired neovascularization in skeletal muscles and myocardium.1,4,47,48 Reduction in ITGB1 content may be explained with the p75NTR-induced decrease in VEGF-A. In fact, in microvascular ECs, VEGF-A upregulates the mRNA expression of alpha(6)beta(1) integrin. Interestingly, VEGF-A also modulates the activity of alpha(6)beta(1) and alpha(9)beta(1) integrins, which in turn mediate VEGF-A driven angiogenesis.49,50 In line, VEGF-A rescued ITGB1 level in p75NTR-HUVECs, once again suggesting that VEGF-A downregulation plays an important role in orchestrating the molecular changes triggered by p75NTR in ECs. The only outsider to this VEGF-A–based molecular network seems to be VEZF1, whose impaired mRNA content was not corrected by VEGF-A transfer in p75NTR-HUVECs. VEZF1 is a relatively scarcely explored transcription factor that was implicated in in utero vasculogenesis, postnatal angiogenesis, and EC survival.51–53 Our expressional data discount the control of VEZF1 by VEGF-A. It is also improbable that VEZF1 lies upstream of VEGF-A, because the antiangiogenic effect caused by knocking down VEZF1 expression was reportedly not corrected in the presence of VEGF-A53. Further studies are necessary to define the role of decreased VEZF1 in diabetes-induced microvascular complications.

Our interpretation of the coordinated changes induced by p75NTR in the molecular program of ECs is summarized in Figure 6d. Future studies are necessary to elucidate the molecular mechanisms by which p75NTR modulates the expressional level of VEGF-A in HUVECs and murine limb muscles. In addition, considering the importance of VEGF-A for the survival of neurons,54 we speculate that VEGF-A downregulation might be also involved in the well established proapoptotic effect of p75NTR on neural cells.55 This hypothesis should be validated by future studies.

We finally conclude that p75NTR overexpression is sufficient and necessary for altering angiogenesis in diabetes and thus becomes an obligatory target for therapy.

Acknowledgments

We thank Dr Mark Bond (Bristol Heart Institute) for important suggestions and gift of reagents, Dr Andrew Herman (Molecular and Cellular Medicine, University of Bristol) for help in setting the conditions for FACS analyses, Mauro Siragusa (Bristol Heart Institute) for help in preparing fluorescent images, and Dr Elinor Griffiths (Bristol Heart Institute) for editing the manuscript.

Sources of Funding

This study was supported by the British Heart Foundation (BHF) (RJ4769, RJ4430, and SM6266 to C.E.) and by the European community FP6 through the European Vascular Genomic Network of Excellence (EVGN) (to A.J.G.H., C.M.B., P.M., and C.E.).

Disclosures

None.

Footnotes

  • ↵*These authors contributed equally to this study.

  • Original received April 11, 2008; revision received June 2, 2008; accepted June 11, 2008.

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  • Neurotrophin p75 Receptor (p75NTR) Promotes Endothelial Cell Apoptosis and Inhibits Angiogenesis
    Andrea Caporali, Elisabetta Pani, Anton J.G. Horrevoets, Nicolle Kraenkel, Atsuhiko Oikawa, Graciela B. Sala-Newby, Marco Meloni, Brunella Cristofaro, Gallia Graiani, Aurelie S. Leroyer, Chantal M. Boulanger, Gaia Spinetti, Sung Ok Yoon, Paolo Madeddu and Costanza Emanueli
    Circulation Research. 2008;103:e15-e26, originally published July 17, 2008
    https://doi.org/10.1161/CIRCRESAHA.108.177386
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