Interaction Between HIF-1α and Notch Signaling Pathways in Nucleus Pulposus Cells of Patients with Modic Changes

Background The HIF-1α/Notch signaling pathway has been shown to regulate proliferation, apoptosis, and metabolism in the intervertebral disc (IVD). The NP is an important structure adjacent to the disc. However, the roles of HIF1α and Notch signaling pathways in NP cells of patients with different Modic changes (MCs) are unclear. The purpose of this research was to assess the expression and association of HIF-1α and components of the Notch pathway in nucleus pulposus (NP) tissue of patients with various MCs. Methods Eighty-five surgical NP tissue samples were obtained from patients undergoing microdiscectomy procedures for the treatment of low back and root pain caused by prolapse of the IVD. The NP tissues were divided into four groups based on the adjacent endplate degeneration: MC I, II, III, and negative MC groups. The expression of HIF-1α and Notch-related components were measured and compared. Results The expression of HIF-1α, Notch1, and Notch2 were gradually increased in the MC I and MC II groups compared with that of the negative MC group. Meanwhile, HIF-1α and Notch-related components were rarely detected in MC III group. between HIF-1α/Notch signaling pathway be promising target diagnosis treatment disc degeneration MC patients. there is a correlation between and hypoxia (through HIF-1α) in the patients with IVD herniation. the expression of in the NP cells of the patients with IVD protrusion with HIF-1α. speculate that in the NP cells the disc herniation


Background
Modic changes (MCs) are changes of vertebral body marrow and endplate lesions that appear are visual signals on magnetic resonance images (MRI). In 1988, this phenomenon was first described and validated by Modic et al [1,2]. In their study, these changes were classified into 3 general types. The Previous studies of MCs have focused mainly on their pathogenesis and the clinical significance of changes between different types of MCs, as well as their relationship with low back pain (LBP) [3,4].
The main cause of MCs has been attributed to minor trauma of the endplate as a result of repetitive loading [5] or to recurrent disc injury which causes an inflammatory reaction within the nucleus pulposus (NP). This initiates endplate changes and intervertebral disc (IVD) degeneration [6]. Therefore, investigating whether or not there is an association between IVD degeneration and MCs was desired. In order to clarify the correlation between IVD degeneration and MCs, we have focused on their change of hypoxic biomarkers.
Hypoxia-inducible factor (HIF) is a master transcription factor induced upon hypoxia and directs coordinated cellular responses to hypoxic environments. The HIF family of proteins comprises several distinct HIF proteins: HIF-1, HIF-2, and HIF-3. Each of these consist of an α-subunit and a constitutively expressed β-subunit known as aryl hydrocarbon receptor nuclear translocator [7,8]. Risbud et al. [9] examined the expression of HIF-1α in rat, human, and sheep NP cells under both hypoxia and normoxia (2% and 21% oxygen); they found that NP cells consistently expressed functionally active HIF-1α protein under hypoxia. Thus, we apply HIF-1α as an index of the ischemia and anoxia of NP cells of the IVD. Therefore, it is reasonable to predict that HIF1-α may be a potential target for the prevention and treatment of IVD degeneration.
Notch is a hypoxia-sensitive receptor protein that can widely regulate proliferation of progenitor cells.
Several studies have investigated the relationship between HIF-1 and Notch in physiological and pathological conditions [10][11][12]. In short, hypoxia activates the Notch signaling pathway to maintain IVD cell proliferation, accelerating catabolism. Therefore, HIF-1 and Notch may play an important role in the pathological process of disc degeneration. We predict that HIF-1/Notch might be an important signaling pathway in the maintenance of disc cell proliferation, and thus offers a therapeutic target for the restoration of cell numbers during degenerative disc disease.
In this study, we investigated the expression of HIF-1α and Notch in the bulgy discs adjacent to end plates with MCs and discuss the relationship between MCs and disc degeneration through imaging, biochemical, immunohistochemical methods to determine whether the expression of HIF-1α and

Materials And Methods Human Tissue Collection
The Ethics Committee of the Hospital and Medical College approved this study and waived the requirement for informed consent. Eighty-five surgical IVD tissue samples were obtained from patients undergoing micro discectomy procedures for the treatment of LBP and root pain caused by prolapse of the IVD from January 2013 to January 2016. Each sample was obtained from the protrusive region of the IVD. The average LBP intensity was reported by the patient on a 0-10 numerical rating scale (NRS): 0 = no pain, 10 = worst possible pain. (Table 1)

Immunohistochemical Analysis
Immunohistochemistry was used to confirm and localize production of HIF-1α and Notch1 in IVDs.
Tissue sections were dewaxed and rehydrated, endogenous peroxidases were quenched, and,

Statistical Analysis
All measurements were performed in triplicate. Data were analyzed using the Graphpad Prism statistical software (version 7.00). Unpaired t-test was used for comparison between different groups.
Differences between groups were assessed by analysis of variance. P values less than 0.05 were considered statistically significant.

Results
Histological Analysis degenerated IVDs. The results showed that the NP tissues from MC I and MC II groups expressed higher level of HIF-1α protein compared with that of the control group. The expression of HIF-1α protein did not show any significant change in the MC III group (Fig. 2a). Additional statistical analysis showed that the percentage of cells immunopositive for HIF-1α in the MC I and MC II groups was significantly higher than that in the control group (MC I:33.96% vs. 9.8%, MC II: 41.52% vs. 9.8%, P < 0.01; Fig. 2b). In accordance with the immunoactivity for HIF-1α, the Notch1 intracellular domain (NICD) expression was correlated to elevated HIF-1α level in MC I and MC II groups (MC I:29.75% vs. 18.32%, MC II: 41.96% vs. 18.32%, p < 0.01; Fig. 2c).

Gene Expression
An increase in Notch1 and Notch2 mRNA levels were seen in MC I and MC II groups, which positively correlated with HIF-1α upregulation. However, the activities of those genes in patients with MC III showed no statistically significant difference compared with that of the control group (Fig. 3a-c).
Additionally, there was no significant difference in the expression of Notch3 in patients with MC I and MC III compared with that of the control group (Fig. 3d). The transcriptional level of Notch3 receptor in the MC II group increased moderately compared to that of the control group. The transcriptional activities of Notch4 in MC groups displayed no statistically significant difference compared with that of the control group (Fig. 3e). Moreover, a significantly increased expression of HES1 (Fig. 3f), the target gene of Notch signaling, was detected in MC I and MC II groups when compared with that in the control group.

Correlation Analysis
The expression of HIF-1α and Notch1 ( Fig. 3g; p = 0.0001), HIF-1α and Notch2 ( Fig. 3h; p = 0.0077), and HIF-1α and Notch3 ( Fig. 8i; p = 0.0011), and HIF-1α and Notch4 ( Fig. 8j; p = 0.0077) in patients with MC II were significantly correlated with one another (Table 3). No significant correlations were observed between NRS back pain and HIF-1α or Notch receptors in any MC groups (Table 4). Table 3 Correlation of mRNA expression between HIF-1α and Notch receptors in different groups.  Notch2. The expression levels of NICD and HES1 were prominently increased in MC I and MC II groups compared with those in the control group, which was consistent with the trend observed for HIF-1α.
These results indicated that the expression of HIF-1α correlated with the Notch signaling pathway to a great extent in MC tissues.

Discussion
In this research, the expression of HIF-1α was elevated in the NP cells of patients with MC I and MC II compared with that of the cells of patients with pure disc herniation. First of all, HIF-1α is an indicator of anaerobic condition. In response to hypoxic conditions, cells up-regulate the synthesis of HIF proteins [14]. HIF-1α plays an important role in the regulation of the biological behaviors of NP cells [7,15]. Therefore, the increased expression of HIF-1α indicates conditions of degeneration or ischemia and hypoxia. Furthermore, other studies found that the HIF-1α/Notch signaling pathway plays an important role in the anoxic pathologic process such as tumor and neural degeneration disease. Based on the above results, we hypothesize that there is a correlation between Notch and hypoxia (through HIF-1α) in the patients with IVD herniation. Our study shows that the expression of NICD in the NP cells of the patients with IVD protrusion was higher, and it was positively correlated with HIF-1α. Therefore, we can speculate that in the NP cells of the disc herniation patients HIF-1α may work through Notch signaling and change the downstream products.
Based on the interesting observation in our immunohistochemical results above, we assessed the mRNA expression of major components of the Notch signaling pathway in lumbar disc cells of different MC groups. There was an increased expression of Notch1, NICD, and HES1 in MC I and MC II groups, which was consistent with the histological results obtained. The evident correlation between HIF-1α and the Notch signaling pathway strengthens the point that HIF-1α regulates disc regeneration through activation of the Notch-HES1 pathway. Specifically, HIF-1α may promote recruitment of the NICD to the CSL-binding motifs in the HES1 promoter and maintain the homeostasis of the ECM in NP [16,17].
Furthermore, to illustrate the association between MCs and lumbar disc degeneration, a Spearman's rank correlation analysis between NRS score and gene expression of HIF-1α/Notch receptors was conducted. Contrary to the imaging methods to evaluate the degree of IVD degeneration [18], neither of the biochemical markers above are positively corelated with the clinical symptoms of LBP in different MC groups. This inconsistent result is likely because the Notch-HES1 pathway is not involved in the initiation of LBP. A large study showed that there were significant correlations between LBP and inflammatory factors, such as IL-6, IL-8, PGE2, TNFα, etc [19,20]. Most of them have been successfully used to activate Notch signaling in NP [21,22]. In addition, HIF-1α expression was significantly increased in the IL-1β-stimulated NP cells under hypoxic condition [23]. We therefore speculate that the overexpressed inflammatory factors participate in consistent activation of the Notch and HIF-1α pathways and subsequent initiation of IVD degeneration in MCs patients, especially MC I and MC II on MRI [24].
Most studies have demonstrated crosstalk between HIF-1α and Notch signaling pathways in IVD [17].