VEGF vascularization pathway in human intervertebral disc does not change during the disc degeneration process
Capossela et al. BMC Res Notes
VEGF vascularization pathway in human intervertebral disc does not change during the disc degeneration process
Simona Capossela 0
Alessandro Bertolo 0
Kapila Gunasekera 0
Tobias Pötzel 2
Martin Baur 1 2
Jivko V. Stoyanov 0
0 Biomedical Laboratories, Swiss Paraplegic Research , 6207 Nottwil , Switzerland
1 Cantonal Hospital of Lucerne , Lucerne , Switzerland
2 Swiss Paraplegic Centre , Nottwil , Switzerland
Objective: During degeneration of the intervertebral disc ingrowth of blood vessels and nerves into the disc are associated with back pain. Vascular endothelial growth factors promote vasculogenesis by binding to the membrane vascular endothelial growth factor receptor 1, while shorter soluble forms of this receptor can inhibit vascularization. We hypothesized that membrane and soluble receptor forms might change between stages of intervertebral disc degeneration. Results: Expression of soluble and membrane forms of vascular endothelial growth factor receptor 1 in human degenerated intervertebral discs and healthy bovine caudal discs was assessed by qRT-PCR and immunoblot. Comparative microarray meta-analysis across disc degeneration grades showed that membrane and soluble forms of this receptor, together with other components of classic vascularization pathways, are constitutively expressed across human disc degeneration stages. Contrary to our hypothesis, we observed that expression of the classic vascularization pathway is stable across degeneration stages and we assume that soluble vascular endothelial growth factor receptor 1 does not contribute to prevent disc degeneration. However, we observed increased expression levels of genes involved in alternative vascularization signalling pathways in severely degenerated discs, suggesting that abnormal vascularization is part of the pathological progression of disc degeneration.
VEGFR1; FLT1; Vascularization; Intervertebral disc
The intervertebral disc (IVD) is a complex avascular
structure composed of outer annulus fibrosus (AF) and
central nucleus pulposus (NP), sandwiched by
cartilage endplates [
]. Essential nutrients for cell activity
are supplied by diffusion through the dense
extracellular matrix, from the blood vessels at the endplates and
the disc margins [
]. Aggrecan, the major proteoglycan
in human IVD, has been demonstrated to inhibit nerve
] and blood vessels migration into the disc [
Loss of proteoglycans and water content in degenerated
disc tissues [
] is associated with disc vascularization
and innervation [
]. Vascular and neural ingrowth into
IVD has been correlated with the presence of vascular
endothelial growth factors (VEGF) and their receptors
]. VEGF regulate blood and lymphatic vessel
development by binding to cell membrane receptors and
activating specific signalling pathways [
]. VEGF receptor
1 (VEGFR1; also known as FLT1) is a transmembrane
receptor for VEGFA, which promotes angiogenesis and
vascular permeability. While VEGFR1 gene encodes the
full-length membrane receptor (mVEGFR1), an
alternative pre-mRNA splicing encodes for a negative regulator
soluble receptor (sVEGFR1), which binds VEGFA and
decreases its bio-availability [
]. sVEGFR1 has shorter
and distinct C-terminus and lacks the transmembrane
]. In such way the expression of mVEGFR1 and
sVEGFR1 can be utilised to regulate the local response
to VEGF [
]. Interestingly, sVEGFR1 has an essential
role in conferring corneal avascularity in diverse
] and reduced levels of this protein were found
in people with age-related macular degeneration [
Recently, it has been shown that inhibitory factors,
present in conditioned media generated from porcine
notochordal-enriched NP, can inhibit in vitro angiogenesis by
suppressing VEGF signalling pathway [
]. In this study
we tested the hypothesis that human IVD expresses
sVEGFR1 as an additional way to maintain its
avascularity and that sVEGFR1 expression may change with disc
Materials and methods
Isolation and culture of human and bovine disc cells
Human degenerated IVD fragments were obtained from
patients undergoing surgical intervention (Additional
file 1: Table S1), after informed consent and approval
by ethics committee of North and Central Switzerland
(EKNZ). Degeneration grade was determined by
magnetic resonance imaging (MRI) and based on surgeons
expertise, according to the Pfirrmann’s modification of
the Thompson classification. Five-score grades from
healthy (grade I) to the most advanced degenerated
(grade V) disc are defined in terms of sequential changes
to MRI features: disc height, distinction between AF and
NP, brightness and uniformity of the NP [
Human degenerated IVD fragments were digested with
0.05% Collagenase Type-2 (Worthington—Bioconcept,
Allschwil, Switzerland) in DMEM/F12 + GlutaMAX with
5% fetal bovine serum (FBS) (Gibco—Paisley, UK) for 6 h
at 37 °C. As healthy human IVD (control) were
inaccessible for ethical reason, we isolated discs from bovine tail
obtained from a local slaughterhouse. Healthy bovine
disc fragments were digested with 0.3% pronase (Sigma,
Buchs, Switzerland) in DMEM/F12 + GlutaMAX with
5% FBS, for 1 h at 37 °C, then further digested with
Collagenase overnight at 37 °C. Disc cells were expanded in
monolayer culture in DMEM/F12 + GlutaMAX
supplemented with 10% FBS (Gibco) and 5 ng/ml recombinant
bFGF (Peprotech—LuBioScience, Lucerne, Switzerland)
at 37 °C in a humid atmosphere containing 5% CO2.
RNA extraction and gene expression
IVD tissues stored at − 80 °C were disintegrated
mechanically while still frozen and lysed in RNA Lysis Buffer
(Aurum Total Mini Kit, Bio-Rad—Cressier, Switzerland).
Disc cells in monolayer were trypsinized and the
pellet was dissolved in RNA Lysis Buffer. Total RNA was
extracted (Aurum Total Mini Kit, Bio-Rad) and used for
synthesis of cDNA (SuperScript VILO cDNA Synthesis
Kit, Invitrogen—LuBioScience). Template cDNA was
mixed with PCR reaction solution (IQ SYBR Green
Supermix, Bio-Rad) containing 0.25 μM specific
primers (Additional file 2: Table S2). Quantitative PCR
(qRTPCR) reactions were carried out in duplicate in 96-well
plates (Bio Rad) for 40 amplification cycles, followed by
melting curve analysis. Relative quantification was
calculated based on the 2−ΔΔCt method and normalized to
Flow-through washes from the RNA extraction
procedure were stored overnight at − 20 °C to allow protein
precipitation, and then centrifuged at 10,000g for 15 min
at 4 °C. Protein pellets were washed three times with 70%
ethanol and resuspended in CelLytic M Buffer (Sigma)
with Protease Inhibitor Cocktail (Sigma). In Western
blot assays, proteins were separated by SDS-PAGE using
Tris–Glycine 5–15% gradient gels (Bio-Rad) with a
Protein Standard 10–250 kDa (Bio-Rad) and transferred onto
a nitrocellulose membrane using the semi-dry Trans-Blot
Turbo system (Bio-Rad). After blocking of non-specific
sites with 5% milk (Rapilait, Migros, Switzerland) in PBS,
membranes were incubated overnight at 4 °C with
primary antibodies diluted in 5% milk: anti-VEGFR1 1:1000
(AF321 R&D System—Abingdon, UK); anti-soluble
VEGFR1 1:50 (36–1100 Thermo
Scientific—LuBioScience); anti β-Actin 1:10,000 (AC-15
Novus—LuBioScience). Secondary antibody HRP-conjugated (anti-mouse
and anti-rabbit 1:10,000, anti-goat 1:20,000—Bethyl,
LuBioScience) was incubated 1 h at room temperature in
5% milk-PBS and detected with chemiluminescence
substrate (LumiGlo Reserve, KPL—Bio Concept, Allschwil,
Switzerland). Acquisition was performed with digital SLR
camera (Nikon D600—Nikon, Zürich, Switzerland) [
Western blot quantification was performed with
Microarray data analysis
Two microarray data sets (GSE15227 and GSE23130) [
] were downloaded from the Gene Expression
Omnibus (GEO) using GEOquery, an R bioconductor package.
These datasets have been generated from AF tissues of
human degenerated IVD classified according to
Thompson grading system. A quality check was performed using
ArrayQualityMetrics to identify the arrays with poor
quality. It was found that there were no notable deviations
amongst 26 chips out of 38 chips. The intensities were
re-calculated and normalized using Robust Multichip
Average (RMA), Guanine Cytosine Robust Multi-Array
(GCRMA) and Variance Stabilizing (VSN) methods.
Having observed that the normalization could bring the
intensity distribution of the selected 26 chips to similar
characteristics, we decided to use these chips in our
analysis (Additional file 3: Table S3). We then filtered out
unwanted information (e.g. genes without entrez
information, duplicated entrez gene identifiers etc.) together
with genes having low variance, which would not pass the
statistical tests for differential expression. Subsequently
the filtered data sets were processed using limma R
package to identify deferentially expressed genes. The results
presented are based on VSN normalization.
For statistical analysis, we used non-parametric Mann–
Whitney–Wilcoxon U test for independent variables.
Data analysis was performed with SPSS version 24.0
for Windows (SPSS Inc.). Significance was indicated as
*p < 0.05. After publication the data will be shared on
Human degenerated IVD express membrane and soluble
Immunoblotting assay with anti-VEGFR1 antibody
against the N-terminus region of the protein showed
the expression of the full-length membrane form
(mVEGFR1 ~ 200 kDa) in human degenerated (Fig. 1a)
and healthy bovine (Fig. 1c) disc cells. An antibody
against the C-terminal region of VEGFR1 soluble form
showed a band of ~ 130 kDa (sVEGFR1) in human
degenerated (Fig. 1a) and healthy bovine (Fig. 1c) disc cells.
Both antibodies showed a band at ~ 60 kDa. β-Actin was
used as a control. Western blot quantification showed no
significant differences between mVEGFR1 and sVEGFR1
in human degenerated IVD (n = 3—Fig. 1b) and healthy
bovine discs (n = 2—Fig. 1d). Relative protein levels
represent the average of the area (square pixels)
normalized on β-Actin. qRT-PCR analysis with specific primers
(Additional file 2: Table S2) designed in the unique
regions of VEGFR1 gene to discriminate between
different isoforms, showed that mVEGFR1 and sVEGFR1 were
comparably expressed in human degenerated IVD tissues
(n = 11) and disc cells cultures (n = 11) (Additional file 4:
Unchanged VEGF pathways and abnormal vascularization in IVD degeneration process
Comparative analysis of AF tissues (n = 26) expression
profiles from two microarray data sets [
that sVEGFR1 and mVEGFR1, along with other
members of the classic signalling vascularization pathway
 (Fig. 2a), were unchanged at the level of transcript
abundance through the degeneration grades (Fig. 2b).
An exception was VEGFA, which exhibited a fluctuating
expression between degeneration grades (Fig. 2b).
On the other hand, the expression levels of genes
regulating abnormal alternative vascularization, such as
hypoxia-inducible factor-1A (HIF1A) and
High-Temperature Requirement A Serine Peptidase 1 (HTRA1) were
significantly increased in degenerated AF grade IV and
V (Fig. 3a). There were also increased expression levels
of some of the components of the interactome of HIF1A
and HTRA1 proposed by STRING database (Fig. 3b).
Meta-analysis gene expression data are given in
Additional file 5: Table S4 and Additional file 6: Table S5.
The qRT-PCR validation of six selected genes
regulating abnormal vascularization (Fig. 3c) showed
significant increased expression levels of Ubiquitin C (UBC)
in severe degenerated AF tissues (grade IV and V; n = 5),
compared to grade II and III (n = 5). HIF1A, HTRA1, 40S
Ribosomal Protein S27A (RPS27A) and Ubiquitin A-52
(UBA52) showed higher expression levels in
degeneration grade IV and V, but the results were not significant.
In this study, we observed that gene expression in the
classic vascular endothelial growth factor (VEGF)
vascularization pathway is preserved across human
intervertebral disc (IVD) degeneration stages and we propose that
alternative vascularization pathways may be involved
with the pathological progression of disc degeneration.
The degeneration of IVD is associated with
vascularization and innervation [
]. VEGF promotes
vasculogenesis binding to the full-length transmembrane vascular
endothelial growth factor receptor 1 (mVEGFR1), while
shorter soluble forms of this receptor (sVEGFR1)
behave as competitive inhibitors of vascularization.
Since sVEGFR1 has the essential role to preserve
corneal avascularity in diverse mammals [
hypothesized that human IVD expresses the decoy sVEGFR1
as an additional way to maintain its avascularity and
the expression of sVEGFR1 and mVEGFR1 may change
during degeneration process. We expected that healthy
avascular discs prevalently express the inhibitor soluble
form, while the vascularized degenerated discs express
more the membrane form. Contrary to our expectations,
we showed that both forms were similarly expressed in
human degenerated IVD tissues and disc cell cultures. By
immunoblot, we observed the expression of mVEGFR1
(~ 200 kDa) and sVEGFR1 (~ 130 kDa), both isoforms
well described in literature [
]. It has been
demonstrated that VEGFR1 can undergo proteolytic
fragmentation, resulting in the formation of soluble form and
cytoplasmic fragment of ~ 60 kDa . We observed, by
microarray meta-analysis, unchanged expression levels
of sVEGFR1 and mVEGFR1 through the IVD
degeneration grades, as well as of the other components of classic
vascularization pathways [
], except for the fluctuating
transcript levels expressed by VEGFA. VEGFR1 together
with VEGFA are the imperative upstream components
in a highly studied signalling pathway that regulates
]. The mechanism of
neovascularization mediated by VEGF and its receptors has been
closely correlated with inflammation, chronic back pain
and accelerated IVD degeneration [
]. However, our
meta-analysis rejected the initial hypothesis and showed
that the classic vascularization signalling pathway is
constitutively expressed across disc degeneration stages.
This leaves the option that other vascularization
pathways may be involved in the pathological vascularization
progression during IVD degeneration. Interestingly, the
meta-analysis and our qRT-PCR validation experiments
revealed number of genes-either known or proposed
to regulate abnormal vascularization—with increased
expression levels in severely degenerated discs. Above
all, hypoxia-inducible factor-1A (HIF1A), which has
been shown to regulate VEGF [
] and induce
angiogenesis , High-Temperature Requirement A Serine
Peptidase 1 (HTRA1), which has a potential role in IVD
] and abnormal vascularization [
], and Ubiquitin C (UBC), which was hypothesized to
have a crucial role in inhibiting cell proliferation of
annulus fibrosus in IVD degeneration [
In conclusion, this study showed that the classic VEGF
vascularization pathway is unchanged across disc
degeneration, advancing that decoy sVEGFR1 does not have a
major role in protecting from disc degeneration process.
Our results allow us to speculate that IVD cells response
to the degenerative processes may trigger an alternative
vascularization signalling pathway activated by some
of the components of the interactome of HIF1A and
HTRA1. However, this is a new hypothesis, which needs
to be tested.
In this study we used as healthy control caudal bovine
discs, because healthy human IVD were inaccessible for
ethical reason. Bovine discs have been proposed as a
suitable biological and biomechanical model for studying
human disc disorders, since they are very similar to the
human IVD in terms of cell distribution, cell phenotype,
disc composition, disc size and mechanical loading [
Additional file 1: Table S1. Demographic details of Intervertebral disc
Additional file 2: Table S2. Human primers used in qRT-PCR.
Additional file 3: Table S3. Microarray samples information.
Additional file 4: Figure S1. (a) Schematic representation of transcript
isoforms of VEGFR1. (UniProtKB-P17948). In red are marked identical
N-terminus coding sequences, while in yellow the C-terminus unique
sequences belonging to splicing isoforms. Specific primers (depicted in
dotted lines) were designed in the unique regions to discriminate the
different isoforms by qRT-PCR. (b) Agarose gel shows PCR products of
VEGFR1 isoforms analysed in HUVEC; GAPDH was used as housekeeping
gene. PCR product sizes were expressed in base pair (bp). (c) In human
degenerated IVD tissues (n = 11) and (d) monolayer disc cell cultures
(n = 11) mVEGFR1 and sVEGFR1 were similarly expressed. Soluble
variants isoform 3 and isoform 4 were not detected by qRT-PCR. Box plots
represent relative mRNA expression normalized on GAPDH in annulus
fibrosus (AF) and nucleus pulposus (NP). The line across the box indicates
the median, bubbles indicate outliers, stars indicate extreme values. No
statistical significance with Non-parametric Mann–Whitney–Wilcoxon U
test for independent variables.
Additional file 5: Table S4. Expression profiles of genes of classic
angiogenesis signalling pathway.
Additional file 6: Table S5. Expression profiles of genes of abnormal
angiogenesis signalling pathway.
IVD: intervertebral disc; mVEGFR1: membrane vascular endothelial growth
factor receptor 1; sVEGFR1: soluble vascular endothelial growth factor receptor 1;
AF: annulus fibrosus; NP: nucleus pulposus; VEGF: vascular endothelial growth
factors; VEGFA: vascular endothelial growth factor A; HIF1A: hypoxia-inducible
factor-1A; HTRA1: High-Temperature Requirement A Serine Peptidase 1; UBC:
ubiquitin C; RPS27A: 40S Ribosomal Protein S27A; UBA52: ubiquitin A-52;
BIRC2: baculoviral IAP repeat-containing protein 2.
SC performed, analysed and interpreted the data regarding the presence of
the receptors in intervertebral discs and was a major contributor in writing
the manuscript. AB contributed in data interpretation, figures preparations. KG
analysed microarray data sets. TP and MB provided intervertebral disc samples,
participated in MRI data analysis and established the grade of disc
degeneration. JS helped coordinating the study, discussing results and edited the
manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The two microarray data sets analysed (GSE15227 and GSE23130) were
downloaded from the Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.
Consent for publication
Ethics approval and consent to participate
Human samples were obtained after written informed consent and approval
by ethics committee of Northwest and Central Switzerland (Ethikkommission
Nordwest und Zentralschweiz—EKNZ).
This work was supported by the Swiss Paraplegic Foundation and Swiss
National Foundation (Grant CR2313_159744 to JS).
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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