Increased HIF-2α activity in the nucleus pulposus causes intervertebral disc degeneration in the aging mouse spine.

Hypoxia-inducible factors (HIFs) are essential to the homeostasis of hypoxic tissues. Although HIF-2α, is expressed in nucleus pulposus (NP) cells, consequences of elevated HIF-2 activity on disc health remains unknown. We expressed HIF-2α with proline to alanine substitutions (P405A; P531A) in the Oxygen-dependent degradation domain (HIF-2αdPA) in the NP tissue using an inducible, nucleus pulposus-specific K19CreERT allele to study HIF-2α function in the adult intervertebral disc. Expression of HIF-2α in NP impacted disc morphology, as evident from small but significantly higher scores of degeneration in NP of 24-month-old K19CreERT; HIF-2αdPA (K19-dPA) mice. Noteworthy, comparisons of grades within each genotype between 14 months and 24 months indicated that HIF-2α overexpression contributed to more pronounced changes than aging alone. The annulus fibrosus (AF) compartment in the 14-month-old K19-dPA mice exhibited lower collagen turnover and Fourier transform-infrared (FTIR) spectroscopic imaging analyses showed changes in the biochemical composition of the 14- and 24-month-old K19-dPA mice. Moreover, there were changes in aggrecan, chondroitin sulfate, and COMP abundance without alterations in NP phenotypic marker CA3, suggesting the overexpression of HIF-2α had some impact on matrix composition but not the cell phenotype. Mechanistically, the global transcriptomic analysis showed enrichment of differentially expressed genes in themes closely related to NP cell function such as cilia, SLIT/ROBO pathway, and HIF/Hypoxia signaling at both 14- and 24-month. Together, these findings underscore the role of HIF-2α in the pathogenesis of disc degeneration in the aged spine.

GLUT1 is redundant in hypoxic and glycolytic nucleus pulposus cells of the intervertebral disc.

Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose transporter, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and postnatal maintenance of the disc. Surprisingly, primary NP cells treated with 2 well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We showed in vitro that NP cells mitigated GLUT1 loss by rewiring glucose import through GLUT3. Of note, we demonstrated that substrates, such as glutamine and palmitate, did not compensate for glucose restriction resulting from dual inhibition of GLUT1/3, and inhibition compromised long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated 2 NP-specific knockout mice: Krt19CreERT Glut1fl/fl and Foxa2Cre Glut1fl/fl. There were no apparent defects in postnatal disc health or development and maturation in mutant mice. Microarray analysis verified that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence to our knowledge of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells.

Increased HIF-2α Activity in the Nucleus Pulposus Causes Intervertebral Disc Degeneration in the Aging Mouse Spine.

Hypoxia-inducible factors (HIFs) are essential to the homeostasis of hypoxic tissues. Although HIF-2α, is expressed in nucleus pulposus (NP) cells, consequences of elevated HIF-2 activity on disc health remains unknown. We expressed HIF-2α with proline to alanine substitutions (P405A;P531A) in the Oxygen-dependent degradation domain (HIF-2αdPA) in the NP tissue using an inducible, nucleus pulposus-specific K19 CreERT allele to study HIF-2α function in the adult intervertebral disc. Expression of HIF-2α in NP impacted disc morphology, as evident from small but significantly higher scores of degeneration in NP of 24-month-old K19 CreERT ; HIF-2α dPA (K19-dPA) mice. Noteworthy, comparisons of grades within each genotype between 14 months and 24 months indicated that HIF-2α overexpression contributed to more pronounced changes than aging alone. The annulus fibrosus (AF) compartment in the 14-month-old K19-dPA mice exhibited lower collagen turnover and Fourier transform-infrared (FTIR) spectroscopic imaging analyses showed changes in the biochemical composition of the 14-and 24-month-old K19-dPA mice. Moreover, there were changes in aggrecan, chondroitin sulfate, and COMP abundance without alterations in NP phenotypic marker CA3, suggesting the overexpression of HIF-2α had some impact on matrix composition but not the cell phenotype. Mechanistically, the global transcriptomic analysis showed enrichment of differentially expressed genes in themes closely related to NP cell function such as cilia, SLIT/ROBO pathway, and HIF/Hypoxia signaling at both 14- and 24-months. Together, these findings underscore the role of HIF-2α in the pathogenesis of disc degeneration in the aged spine.

Conditional Deletion of HIF-2α in Mouse Nucleus Pulposus Reduces Fibrosis and Provides Mild and Transient Protection From Age-Dependent Structural Changes in Intervertebral Disc.

Hypoxia-inducible factors (HIFs) are critical to the development and homeostasis of hypoxic tissues. Although HIF-2α, one of the main HIF-α isoforms, is expressed in nucleus pulposus (NP) cells, its functions remain unknown. We deleted HIF-2α in the NP tissue using a notochord-specific FoxA2Cre allele to study HIF-2α function in the adult intervertebral disc. Unlike observations in HIF-1αcKO mice, fate mapping studies using Rosa26-mTmG reporter showed that HIF-2α loss in NP did not negatively impact cell survival or affect compartment development. Rather, loss of HIF-2α resulted in slightly better attributes of NP morphology in 14-month-old HIF-2αcKO mice as evident from lower scores of degeneration. These 14-month-old HIF-2αcKO mice also exhibited significant reduction in NP tissue fibrosis and lower collagen turnover in the annulus fibrosis (AF) compartment. Imaging-Fourier transform-infrared (FTIR) analyses showed decreased collagen and protein content in the NP and maintained chondroitin sulfate levels in 14-month-old HIF-2αcKO . Mechanistically, global transcriptomic analysis showed enrichment of differentially expressed genes with Gene Ontology (GO) terms related to metabolic processes and cell development, molecular functions concerned with histone and protein binding, and associated pathways, including oxidative stress. Noteworthy, these morphological differences were not apparent in 24-month-old HIF-2αcKO , indicating that aging is the dominant factor in governing disc health. Together these data suggest that loss of HIF-2α in the NP compartment is not detrimental to the intervertebral disc development but rather mitigates NP tissue fibrosis and offers mild but transient protection from age-dependent early degenerative changes. © 2022 American Society for Bone and Mineral Research (ASBMR).

Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice.

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16INK4a, p19ARF, and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration.