Part 1 A histocytological and radiological overview of the natural history of intervertebral disc: From embryonic formation to age-related degeneration
Objective：To characterize the histocytological change during intervertebral disc (IVD) formation, maturation, and age-related degeneration.
Methods：Coronal sections of embryonic (E13、E14、E18、Neonatal) and postnatal (4W、12W、30W、60W) rat IVD were stained by a series of histological stainings: with HE staining to evaluate the change in cellular contents, Alcian blue staining to assess the accumulation of cartilagenous matrix, Picrosirius red staining for fibrotic matrix, Masson staining for collagenous fibers and myofibers, and PAS staining to identify the expression of glycoproteins and glycosaminoglycans. Growth kinemics within the different compartments of embryonic IVD were evaluated by immunohistochemical staining of Ki67 and PCNA. Postnatal maturation (Neonatal、2W、6W) and age-related degeneration (12W、60W) of rat IVD were visualized on radiology by X-ray, CT, and MR imaging.
Results：Embryonically, non-condensed sclerotome developed into vertebrae and cartilaginous endplate (CEP), condensed sclerotome generated annulus fibrosis (AF), and axial notochord formed nucleus pulposus (NP). During the formation of IVD, notochordal cells increased and enlarged the cytoplasmic vacuoles, the paired vertebrae anlagen fused and compressed the vacuolating notochord into the forming NP, which was enwrapped by the outward-expanding AF anlagen. Noteworthy, inner AF shared similar histocytological contents and growth kinetics as the CEP, drawing distinct boundaries between the inner and the outer AF of neonatal IVD.
At early development stages (E13.5–14.5) the axial notochord was surrounded by a thick notochord sheath that accumulated both cartilagenous and fibrotic matrix, in which the expression of glycoproteins and glycosaminoglycans and the accumulaton of more collagenous fibers than myofibers were also evident. While the notochord sheath was thick at E13.5, it became thinner and vanished as the axial notochord was compressed into the prospective NP.
Postnatally, vacuolated notochord cells were reduced by devacuolation while chondrocytic NP cells increased; cartilaginous layers of CEP were narrowed by vertebrae growth and secondary ossification; fibrotic portion of AF decreased as cartilagenous matrix accumulated and infiltrated outwards. In aged and degenerated IVD, large longitudinal fissures were detected near the boundaries between inner and outer AF, whereas both reduced cellularity and accumulated cell clusters were evident within the dehydrated NP.
Conclusion：From embryonic formation to age-related degeneration, the natural history of IVD is sophisticatedly orchestrated by a dynamic regulation of the histocytological components. NP is significantly different from AF in terms of embryonic origin and histocytological change.
Part 2 Hypoxic regulation of sumoylation pathways in intervertebral disc cells: implications for Hypoxia-Inducible Factor-1α stability and hypoxic adaptations
Objective：To identify the expression of sumoylation pathways within IVD. To explore the hypoxic regulation of sumoylation pathways and cell viability in NP and AF cells.
Methods:Expression of SUMO molecules SUMO1 and SUMO2/3, SUMO E1 activating enzymes SAE1 and SAE2, SUMO E2 conjugating enzyme UBC9, and SUMO specific proteases (de-Sumoylation enzyme) SENP1 was immunolocalized in vivo within the IVD of neonatal and 10 weeks-old (W) rat, and in vitro in the rat IVD cells cultured in monolayers. NP and AF cells were maintained in hypoxia (1% O2) for 0-24 hours (h) and cell viability was evaluated by quantifying cell proliferation with CCK-8 analysis, cellular senescence with SA-β-gal staining, apoptosis with Annexin-V/PI staining and flow cytometry analysis, and cell cycle distribution with PI staining and flow cytometry analysis. Hypoxic regulation of sumoylation pathways was studied by analyzing the transcription and expression of SUMO molecules and sumoylation enzymes with Realtime-PCR and Western-blot.
SENP-1 specific interfering RNA was used to silence the expression of SENP1 in NP cells. The transcription and stabilization of HIF-1α was evaluated by Realtime-PCR and Western-blot in the NP cells under hypoxia, while the transactivation of HIF-1α was evaluated by analyzing the expression of its downstream targets VEGF, Glut-1, and PDK1. SA-β-gal staining was used to quantify the hypoxia-induced senescence change, while Hoechst33342/PI staining and flow cytometry analysis were performed to evaluate the apoptosis in the NP cells with lowered SENP1 expression.
Results：SUMO1, SUMO2/3, SAE1, SAE2, UBC9, and SENP1 were expressed in the NP, AF, and CEP of neonatal and 10W rat IVD. On immunocytofluocent staining, sumoylation pathways were also expressed in the cultured IVD cells and localized predominantly in nuclei, with SUMO-1 expressed exclusively in the nuclei. On western blotting, free SUMO1 (~12kD) was detected only in the NP cells cultured under hypoxia for 4h, whereas conjugated SUMO1 (~80kD) was promoted by hypoxia in both NP and AF cells. By contrast, free SUMO2/3 (~11kD) was detectable under both normoxia and hypoxia. RanGAP1 was detected in IVD cells at ~80kD under both normoxia and hypoxia.
In NP cells, hypoxia for 4h significantly increased the transcription of SUMO1, SUMO2, and SUMO3, which returned to the normoxic level by 12h. In AF cells, the mRNA level of SUMO-1 was increased in hypoxia at 12h, while the transcription of SUMO2 and SUMO3 was decreased in hypoxic cultures. When NP cells were maintained in hypoxia for 4-12h, the transcription of SAE1 was decreased, followed by an increase to the nomoxic level by 24h; the mRNA expression of SAE2 and UBC9 was increased after hypoxic cultures for 4 h, followed by a return to the normoxic level by 12h. In contrast, AF cells reduced the transcription of SAE1, SAE2, and UBC9 after hypoxic cultures for 12-24h. In both NP and AF cells, the transcription and expression of SENP1 showed steady increase under hypoxic cultures.HIF-1α was constitutively expressed within rat NP cells under both normoxia and hypoxia. After SENP1 was silenced in NP cells, both the transcription and expression of HIF-1α were decreased under hypoxia. The expression of HIF-1α downstream targets GLUT-1, VEGF, and PDK1 was also decreased in the NP cells with lowered SENP1.
CCK-8 analysis showed that oxygen deprivation had no significant effects on cell proliferation of NP and AF cells. Under hypoxia, no significant increase of SA-β-gal staining, elevation of apoptosis, or attenuation of cell cycle transition was induced in NP or AF cells. After SENP1 expression was silenced in rat NP cells, hypoxia induced no significant elevation of cellular senescence or apoptosis, although both the expression and transactivation of HIF-1α were downregulated therein.
Conclusion：Sumoylation pathways are expressed in IVD cells and response to hypoxic microenvironment. NP and AF cells equally tolerate oxygen deficiency, but differently regulate the sumoylation pathways under hypoxia, with SENP1 involved in the regulation of HIF-1α expression and transactivation in NP cells. The constitutive expression and transactivation of HIF-1α is not the only factor essential for the survival of NP cells under hypoxia.