Activation of Low-Oxygen Response Pathway May Protect Against Spine Disc Degeneration

To assess the effect of HIF-1α on disc regeneration, NP cells were cultured in either DMSO or hypoxia-mimetic agents desferrioxamine (DFO) for 48 h, and the mRNA level was quantified using qRT-PCR. NP cells cultured under hypoxia showed more Aggrecan and Collagen II mRNA expression than those cultured in normoxia (Figure 1).

To determine whether pHIF-1α Mut A and B were degraded under normoxic conditions, nuclear and cytoplasmic fractions were obtained, and the HIF-1α protein levels of each group were analyzed by Western blotting. HIF-1α protein levels in both pHIF-1α Mut A and B were significantly higher than those of pHIF-1α WT in the nuclear fractions (p < 0.01) (Figure 2A). This suggests that HIF-1α mutated cells have increased stability and nuclear localization even under normoxic conditions, as HIF-1α is not degraded by the ubiquitin–proteasome system.

Next, to evaluate the transcriptional activity of pHIF-1α Mut A and B, a luciferase reporter assay was performed. In the luciferase reporter assay performed with pGL2-EPO-HRE reporter plasmids, pHIF-1α Mut A and B showed 4.7- and 10.3-fold higher transcriptional activity, respectively, compared with pHIF-1α WT with statistical significance, while no significant difference between the transcriptional activity of pHIF-1α Mut A and B and that of pHIF-1α WT was observed in the assay performed with pGL2-EPO-mHRE (Figure 2B). Therefore, the mutants of HIF-1α showed increased transcriptional activity in a specific manner in the EPO-HRE sequence. pHIF-1α Mut B was selected for further study because pHIF-1α Mut B was more active than pHIF-1α Mut A. A pHIF-1α Mut B will be referred to as CA HIF-1α from this point.

Then, to investigate the effects of CA HIF-1α (pHIF-1α Mut B) on VEGF and glucose transporter (Glut-1 and Glut-3) expression, NP cells from patient tissues were transfected with pcDNA3, pHIF-1α WT, and CA HIF-1α. After 48 h, qRT-PCR analysis was performed to evaluate VEGF, Glut-1, and Glut-3 expression. CA HIF-1α induced significant upregulation of VEGF, Glut-1, and Glut-3 expression in NP cells. To validate the effect of CA HIF-1α on the disc matrix, Sox9, aggrecan, and type II collagen expression were also analyzed, and the results indicated that CA HIF-1α increased Sox9, aggrecan, and type II collagen expression in NP cells (Figure 2C).

To examine the role of CA HIF-1α in NP cell apoptosis, NP cells were transfected with pHIF-1α WT or CA HIF-1α, and apoptosis was induced by treatment with 50 ng/mL FasL. After 24 h, apoptosis was measured by enumerating annexin V-positive cells. FasL induced apoptosis in 34.7% of NP cells, although CA HIF-1α reduced the proportion of apoptotic cells to 12.5% (Figure 3). This result implies that CA HIF-1α protects NP cells from apoptosis during IVD degeneration.

The T2 signal intensity was decreased in the PBS- and pcDNA3-alone segments at 4 and 8 weeks following needle puncture. However, the signal intensity in the pHIF-1α WT and pHIF-1α Mut B segments was increased at 4 and 8 weeks following injection compared with that of the PBS and pcDNA3 alone segments, and the difference was statistically significant (p < 0.05; Figure 4A). The degree of disc height index (DHI) reduction comparing control in the pHIF-1α WT and pHIF-1α Mut B segments was smaller than that in the PBS and pcDNA3 segments at 4 and 8 weeks after injection; this difference was also statistically significant (p < 0.05; Figure 4B).

Representative images of safranin O staining are shown in Figure 4C. The NP area in the disc stained strongly for safranin O, although the stained levels were not homogenous in the PBS and pcDNA3 segments. Histological analysis of IVD tissues was performed using H&E and toluidine blue staining. In the PBS and pcDNA3 segments, ruptured and serpentine-patterned fibers were observed in the AF. Furthermore, the border between the AF and NP was interrupted and unclear and exhibited features of severe degeneration. However, the AF and the border between the AF and NP in the pHIF-1α WT and pHIF-1α Mut B segments demonstrated an intact circumferential AF, and the border between the AF and NF was clear compared with those observed in the PBS and pcDNA3 segments. Immunohistochemical staining revealed that the staining for HIF-1α and collagen 2 were stronger in the pHIF-1α Mut B transfected segments compared with pHIF-1α WT segments. HIF-1α and collagen 2 expression levels were negligible in the PBS segments. Conversely, the HIF-1α and collagen 2 expression levels were notably increased in the NP cells of the pHIF-1α Mut B transfected segments. A histopathology scoring system was applied according to Lai et al. [22], and pHIF-1α Mut B had a significantly lower score than the other segments.

pcDNA3 plasmid and pcDNA3 HIF-1α wild-type (pHIF-1α WT, plasmid#18949) were purchased from Invitrogen and Addgene, Inc., respectively. The HIF-1α wild-type (HIF-1α WT) gene was mutated using a polymerase chain reaction-based (PCR-based) mutagenesis method (Stratagene, Lajolla, CA, USA) according to the manufacturer’s instructions. Two types of HIF-1α mutants (HIF-1α Mut) were created by point mutation as follows: (1) pHIF-1α Mut A was created by replacing proline 402 (Pro402) and Pro564 with alanines, and (2) pHIF-1α Mut B was created by replacing Pro402, Pro564, and asparagine 803 (Asn803) with alanines. These three sites were selected because Pro402 and Pro564 were involved in oxygen-dependent degradation, and mutations at Asn803 are known to prevent the interaction with p300 for transcriptional activation [40].

To compare the activity of HIF-1α WT proteins between mutants A and B and the control under normoxic conditions (20% O₂), the following five groups were transfected into NP cells using Lipofectamine (Invitrogen, Waltham, MA, USA): (1) Desferrioxamine (DFO) only, (2) pcDNA3 only, (3) pHIF-1α WT, (4) pHIF-1α Mut A, and (5) pHIF-1α Mut B. DFO was used as a positive control, because it inhibits both PHD enzyme and FIH-1 enzyme activities [41]. The transfected cells were lysed using a cell fractionation kit (Cell Signaling Technology, Danvers, MA, USA). The cells were separated into nuclear and cytoplasmic fractions in Nupage 4–12% Bis-Tris gels (Invitrogen, Waltham, MA, USA) and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk and washed with phosphate-buffered saline and 0.2% Tween 20 buffer. The membranes were blotted with mouse anti-HIF-1α (Cell Signaling Technology, Danvers, MA, USA), mouse anti-nuclear lamin A/C, or anti-α-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. The blots were treated with goat anti-mouse conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA) and visualized using an enhanced chemiluminescence system (Uppsala, Sweden).

To determine the transcriptional activity of pHIF-1α Mut A and B, a luciferase reporter assay was performed. The following two different types of luciferase reporter plasmids were used to determine whether pHIF-1α Mut A and B had increased transcriptional activity specifically related to the erythropoietin (EPO)-HRE sequence: (1) pGL2-EPO-HRE, containing the wild-type HRE sequence in the EPO gene, and (2) pGL2-EPO-mHRE, containing the mutant HRE sequence in the EPO gene. DFO only, pcDNA3 only, pHIF-1α WT, and pHIF-1α Mut A and B were co-transfected into HEK NP cells with pGL2-EPO-HRE or pGL2-EPO-mHRE using Lipofectamine (Invitrogen, Waltham, MA, USA). The cells were lysed, and luciferase assays (Promega, Madison, WI, USA) were performed using a luminometer (Titertek Berthold FB14, Bad Wildbad, Germany). Firefly luciferase activity was normalized to that of Renilla luciferase, which was used as an internal control.

The Institutional Review Board of the CHA Bundang Medical Center approved this study. Patients diagnosed with degenerative lumbar spinal stenosis were recruited after obtaining informed consent. Before sample collection, the degree of IVD degeneration was evaluated according to the Pfirrman grading system [42], and degenerative discs of grade IV or higher were collected in this study.

Human NP cells donated by patients were obtained within 2 h after surgery. NP tissues were identified and separated by a stereotaxic microscope. The NP tissues were washed with phosphate-buffered saline (PBS) and digested for 40 min in 0.2% pronase (Gibco BRL, Carlsbad, CA, USA). Following being washed, the tissues were incubated in 0.25% type II collagenase (Gibco BRL, Carlsbad, CA, USA) at 37 °C under gentle agitation for 4 h. The cell suspension was centrifuged at 300× g for 10 min, and the pellet was washed with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Waltham, MA, USA). The cells were plated in culture dishes and maintained in DMEM supplemented with 10% fetal bovine serum and 50 µg/mL gentamicin (Invitrogen, Waltham, MA, USA) at 37 °C in 5% CO₂. First-passage cells maintained in a monolayer were obtained, and second-passage cells were used in the experiments. NP cells were transfected with empty pcDNA3 only or pHIF-1α (WT, Mut A and Mut B).

Total RNA was isolated from NP cells using TRIzol reagent (Invitrogen, Waltham, MA, USA). First-strand cDNA was synthesized using the SuperScript system (Invitrogen, Waltham, MA, USA). PCR amplification conditions were as follows: denaturation at 94 °C for 20 s, annealing at 55 °C for 30 s, and extension at 72 °C for all genes. PCR reactions were performed using the QuantiTech SYBR Green PCR kit (Qiagen, Valencia, CA, USA) and an Exicycler 96 system (Bioneer, Daejeon, Korea). Specific primers for the genes examined were based on their PrimerBank sequences and are listed in Table 1.

NP cells were cultured and fractionated into cytosolic and nuclear fractions using a cell fractionation kit (Abcam, Inc., Cambridge, UK) according to the manufacturer’s instructions. Trypsinized NP cells were collected and centrifuged for 5 min at 300× g. The collected cells were washed with buffer A (wash buffer). Then, the cells were resuspended in buffer A. An equal volume of buffer B (lysis buffer for Cyt extraction) was added to the cell suspensions, mixed via pipetting, and then incubated for 7 min at room temperature (RT). The cell lysates were centrifuged at 5000× g for 1 min at 4 °C, and the cytosol fractions (cell lysate supernatants) were collected in new tubes. The remaining pellets containing the nuclear protein fraction were resuspended in buffer A.

Transfected NP cell lysates were boiled in an SDS sample buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. After the transfer, the membranes were blocked in 5% skim milk in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 40 min and incubated with the specific primary antibodies in the blocking solution for overnight at 4 °C. Antibodies against HIF-1α (sc-13515, 1:1000) and α-Tubulin (sc-8035, 1:1000) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against Lamin A/C (#4777, 1:1000) was purchased from Cell Signaling Technology (Danvers, MA, USA). The membranes were then washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody followed by detection using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

NP cells were transfected with pHIF-1α WT or CA-HIF-1α and then treated with 50 ng/mL of Fas ligand (FasL) (R&D Systems, Minneapolis, MN, USA). After 24 h, apoptosis was measured using an apoptosis detection kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Briefly, cells were collected and incubated with fluorescein isothiocyanate-labeled annexin (V annexin V-FITC) in Annexin V Binding Buffer. Annexin V-positive cells were enumerated by flow cytometry (FACScan™ Calibur, Becton Dickinson, San Jose, CA, USA).

Ten male Sprague Dawley rats (each weighing 220–250 g and 2 months old) were used in this study. The animals were housed with free access to commercial rodent chow and water. The temperature was maintained at 24 °C, and the light schedule comprised 12 h of daylight starting at 8:00 a.m. and 12 h of darkness starting at 8:00 p.m. Humidity was maintained at 50%. All experiments were performed according to the guidelines for the ethical treatment of animals approved by the Laboratory Animal Ethics Committee of CHA Bundang Medical Center (Seongnam, Korea).

The rat tail segments were divided into four groups: (1) phosphate-buffered saline (PBS), (2) pcDNA3 only, (3) pHIF-1α WT, and (4) pHIF-1α Mut B. Ten rat tails were prepared for the drug treatment. The animals were anesthetized by inhalation of anesthetic isoflurane, and the operative field was prepared in a sterile fashion. After identifying the level of the caudal segments by fluoroscopy, a puncture was made parallel to the endplates through the annulus fibrosus (AF) into the NP using a syringe needle (21-gauge) (Figure 5) [43]. The needle was then rotated by 180° and held for 5 s. During needle puncture, fluoroscopy was performed to ensure that the needle reached the center of the NP [20]. For the MRI assessment, the caudal segments 4/5 and 5/6 from the same rat tail were used for the PBS- and pcDNA3-only treatment, and the caudal segments 6/7 and 7/8 for the pHIF-1α WT and pHIF-1α Mut B (Figure 5).

Images were obtained using a 3.0T MRI machine (Philips Healthcare, Amsterdam, The Netherlands) using a dedicated coil for small animals. The animals were serially followed by MRI immediately and 4 and 8 weeks following injection. At each time point, the rats were anesthetized with a 2% isoflurane/oxygen mixture throughout the MRI examination. The animals were placed in a prone position, and their tails were straightened in the MR scanner. Serial T2-weighted sagittal and transverse images covering the entire experimental disc area were obtained, and T2-weighted signal intensity and disc height were measured using Image J software (National Institutes of Health, Bethesda, MD, USA). Additionally, intervertebral disc height index (DHI) under X-ray immediately before and after injection was calculated using modification of previous methods [44,45,46]. Image assessments were conducted by two independent, blinded, and experienced observers. The data are presented as the means of the two evaluations.

At 8 weeks after injection, all rats were killed, and the discs were harvested for histological examination. Each disc was fixed, decalcified, using 10% ethylenediaminetetraacetic acid (EDTA; pH = 7.4) for 1 month. The EDTA solution was replaced every 2 days. The decalcified discs were washed, dehydrated and then processed for paraffin embedding and sectioning into sagittal sections (5 μm thickness) using a microtome. Sagittal sections were stained with hematoxylin and eosin (H&E), safranin O, and toluidine blue. Histological images were analyzed qualitatively under a light microscope (BX51, Olympus, Inc., Tokyo, Japan) at magnifications ranging from 4 to 100× to investigate changes in the NP, AF, and endplates. The extent of disc degeneration was graded using a height index as described previously by Masuda et al. [47].

Immunohistochemistry (IHC) was performed using a Ready-to-use IHC/ICC kit (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer’s protocol. Briefly, paraffin-embedded sections were deparaffinized, rehydrated, immersed in a retrieval solution (10 mmol/L citrate, pH 6.0), and then placed in a microwave for 10 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 10 min. The slides were incubated in 3% H2O2 at room temperature for 30 min to quench endogenous peroxidase activity and then blocked in blocking buffer (BioVision, Inc., Milpitas, CA, USA) at room temperature for 10 min, followed by incubation with anti- HIF-1α (sc-13515; 1:100) or anti-collagen II (1:100) antibodies at room temperature for 30 min. After incubation with HRP-anti-mouse or -rabbit IgG polymer at room temperature for 20 min and washing with PBS, the tissue sections were treated with 3,3′-diaminobenzidine at room temperature for 10 min, followed by counterstaining with hematoxylin at room temperature for 1 min.

All data were analyzed at least three times for each group. The means of the groups were compared using the Mann–Whitney U test or Kruskal–Wallis test with post hoc Bonferroni tests. Means and standard deviations were calculated from numerical data, as presented in the text, figures, and figure legends. In the figures, bar graphs represent the means, whereas error bars represent one standard deviation. SPSS version 18.0 (SPSS, Chicago, IL, USA) was used for all statistical analyses. The Kruskal–Wallis test was used to analyze the differences in T2 signal intensities, and one-way analysis of variance was used to analyze the differences in disc height. Statistical significance was set at p < 0.05.

Not applicable.