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Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus.

Merceron C, Mangiavini L, Robling A, Wilson TL, Giaccia AJ, Shapiro IM, Schipani E, Risbud MV - PLoS ONE (2014)

Bottom Line: This structure is covered superior and inferior side by cartilaginous endplates (CEP).The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome.Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Medical School, University of Michigan, Ann Arbor, Michigan, United States of America; Inserm, UMRS 791-LIOAD, Centre for Osteoarticular and Dental Tissue Engineering, Group STEP 'Skeletal Tissue Engineering and Physiopathology', Nantes, France; LUNAM, Nantes University, Faculty of Dental Surgery, Nantes, France.

ABSTRACT
The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.

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Altered biomechanical properties of the mutant IVD lacking the NP.A. Load damping capacity in control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) IVD at 4 months. B. Phase shift angle (a) and energy dissipation (b) based on the load damping test in control (HIF-1αf/f) and mutant (Foxa2iCre;HIF-1αf/f) IVD at 4 months. Statistical analysis performed between control and mutant groups: rmANOVA p<0.001 (a) and p<0.05 (b).
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pone-0110768-g007: Altered biomechanical properties of the mutant IVD lacking the NP.A. Load damping capacity in control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) IVD at 4 months. B. Phase shift angle (a) and energy dissipation (b) based on the load damping test in control (HIF-1αf/f) and mutant (Foxa2iCre;HIF-1αf/f) IVD at 4 months. Statistical analysis performed between control and mutant groups: rmANOVA p<0.001 (a) and p<0.05 (b).

Mentions: To evaluate the impact of the NP loss on the IVD function, biomechanical analyses were performed on spinal motion segments of 4 month-old mice. Dynamic loading of the motion segments isolated from 4 months old mice at ±1.5 N resulted in ∼120–200 µm displacement in each direction (Figure 7A). Calculation of the mean phase shift angle across genotypes at each of the 19 frequencies tested revealed no significant frequency effect on phase shift angle (Figure 7B a). However, comparison between genotypes revealed a ∼40% reduction in phase shift angle among the mutant mice, compared with the controls (rmANOVA, p<0.001). The reduction in phase shift angle in mutant mice suggests that the load damping capacity of the spine was significantly reduced by the HIF-1α deletion. Energy dissipation, calculated from the area within the force-displacement loops, followed a similar but less-pronounced trend as was observed for phase shift angle (Figure 7B b). Again, no frequency effect was detected across genotypes, but there was a significant reduction in energy dissipation among the mutant mice, compared to the HIF-1αf/f mice (rmANOVA, p<0.05). The reduction in energy dissipation in the mice for which HIF-1α has been deleted in the notochordal lineage suggests that the mutant disc has impaired ability to absorb axial energy transmitted across it.


Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus.

Merceron C, Mangiavini L, Robling A, Wilson TL, Giaccia AJ, Shapiro IM, Schipani E, Risbud MV - PLoS ONE (2014)

Altered biomechanical properties of the mutant IVD lacking the NP.A. Load damping capacity in control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) IVD at 4 months. B. Phase shift angle (a) and energy dissipation (b) based on the load damping test in control (HIF-1αf/f) and mutant (Foxa2iCre;HIF-1αf/f) IVD at 4 months. Statistical analysis performed between control and mutant groups: rmANOVA p<0.001 (a) and p<0.05 (b).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206488&req=5

pone-0110768-g007: Altered biomechanical properties of the mutant IVD lacking the NP.A. Load damping capacity in control (HIF-1αf/f) (a) and mutant (Foxa2iCre;HIF-1αf/f) (b) IVD at 4 months. B. Phase shift angle (a) and energy dissipation (b) based on the load damping test in control (HIF-1αf/f) and mutant (Foxa2iCre;HIF-1αf/f) IVD at 4 months. Statistical analysis performed between control and mutant groups: rmANOVA p<0.001 (a) and p<0.05 (b).
Mentions: To evaluate the impact of the NP loss on the IVD function, biomechanical analyses were performed on spinal motion segments of 4 month-old mice. Dynamic loading of the motion segments isolated from 4 months old mice at ±1.5 N resulted in ∼120–200 µm displacement in each direction (Figure 7A). Calculation of the mean phase shift angle across genotypes at each of the 19 frequencies tested revealed no significant frequency effect on phase shift angle (Figure 7B a). However, comparison between genotypes revealed a ∼40% reduction in phase shift angle among the mutant mice, compared with the controls (rmANOVA, p<0.001). The reduction in phase shift angle in mutant mice suggests that the load damping capacity of the spine was significantly reduced by the HIF-1α deletion. Energy dissipation, calculated from the area within the force-displacement loops, followed a similar but less-pronounced trend as was observed for phase shift angle (Figure 7B b). Again, no frequency effect was detected across genotypes, but there was a significant reduction in energy dissipation among the mutant mice, compared to the HIF-1αf/f mice (rmANOVA, p<0.05). The reduction in energy dissipation in the mice for which HIF-1α has been deleted in the notochordal lineage suggests that the mutant disc has impaired ability to absorb axial energy transmitted across it.

Bottom Line: This structure is covered superior and inferior side by cartilaginous endplates (CEP).The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome.Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Medical School, University of Michigan, Ann Arbor, Michigan, United States of America; Inserm, UMRS 791-LIOAD, Centre for Osteoarticular and Dental Tissue Engineering, Group STEP 'Skeletal Tissue Engineering and Physiopathology', Nantes, France; LUNAM, Nantes University, Faculty of Dental Surgery, Nantes, France.

ABSTRACT
The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.

Show MeSH
Related in: MedlinePlus