Angiogenesis Promoted Intervertebral Disc Degeneration Via Inhibiting The Autophagic Degradation Of HIF-1α

Part 1:Vascular Invasion mediated HIF-1α Levels abnormally Elevation in nucleus pulposus by disrupt hypoxia EnvironmentBackground:Intervertebral disc degeneration(IDD)is the pathological basis of many spinal diseases.Currently,there are no ideal treatment methods for this type of disease.The fundamental reason for this point is that the mechanism of IDD occurrence and development is still not fully understood.For a long time,many scholars have reported the phenomenon of vascular invasion observed in the process of IDD,but the experimental evidence of the impact of vascular invasion on IDD is very limited.Blood vessels increase the local oxygen concentration,and mediated HIF-1α degradation by the proteasome pathway.However,Recent studies have reported vascular invasion is companied with an increase in HIF-1αlevels during IDD.In addition,HIF-1α is necessary to adapt to this physiological hypoxia environment,but it is also the most important factor inducing angiogenesis.This suggests there is complex relationship between vascular invasion and HIF-1α during IDD.Therefore,exploring the role of vascular invasion in IDD,and elucidating the interaction between vascular invasion and HIF-1α is of great significance for understanding the pathogenesis of IDD.Objective:1.Demonstrate the role of vascular invasion in IDD;2.Elucidate the mutual regulation relationship between vascular invasion and HIF-1αin nucleus pulposus.Methods:1.Construct a mouse lumbar instability model and collect the intervertebral disc samples at five-time points after surgery:1 week postoperative(0m),3 months postoperative(3m),6 months postoperative(6m),9 months postoperative(9m),and 12 months postoperative(12m).Validate the induction effect of LSI on IDD by tissue pathological staining and observe the regularity of vascular invasion and HIF-1α level changes in IDD natural course through immunofluorescence experiments.2.Construct Vegfr2EC-mice or use pharmacological intervention to construct a mouse model that inhibits angiogenesis.Compare the difference in IDD severity between the mice with angiogenesis inhibited and the control mice at various time points through tissue pathological staining.Compare the changes in HIF-1α levels at various time points between the two groups through immunofluorescence staining.3.Construct a mouse model that promotes angiogenesis through AAV-VEGF,evaluate the difference in IDD severity between the accelerated vascular invasion group and the control group at various time points through tissue pathological staining,and compare the changes in HIF-1α levels at various time points between the two groups through immunofluorescence staining.4.Construct Phd2IVD-mice,Phd2AF+EP-mice,Phd2NP-mice,and HIF-1αIVD-mice respectively,and explore the effects of HIF-1α levels on vascular invasion and IDD through tissue pathological staining and immunofluorescence staining.5.Construct a mouse model that gradually increases the oxygen content in the intervertebral disc by hyperbaric oxygen,and evaluate the effects of vascular invasion on the hypoxia environment of the nucleus pulposus and HIF-1α through immunofluorescence experiments.Results:1.After LSI modeling of mice,there was no significant difference in IDD degree between the LSI group and Sham group at 0m and 3m,but the IDD degree of LSI group progressively increased at 6m,9m,and 12m and was significantly higher than that of Sham group.The vascular invasion of the intervertebral disc in LSI group gradually increased from 0m to 6m and reached its maximum at 6m,and then gradually decreased from 9m to 12m.In addition,the level of HIF-1α in LSI group showed no significant difference at 0m and 3m,significantly increased at 6m,and gradually disappeared at 9m-12m with fluctuations as the cell number decreased.2.Compared with control group,there was no significant difference in IDD degree between Vegfr2EC-or Anti-VEGF group at 0m and 3m,while the IDD degree of Vegfr2ECor Anti-VEGF group was significantly lower at 6m-12m.More importantly,inhibiting angiogenesis had no significant effect on the HIF-1α levels of the intervertebral disc in mice at 0m-6m,but it was slightly reduced at 9m-12m.3.Compared with control group,the vascular invasion degree of AAV-VEGF group significantly increased,and the IDD degree was significantly higher at 3m-9m.The HIF-1αfluctuation of the AAV-VEGF group advanced with vascular invasion and reached its highest level at 3m and gradually disappeared from 6m to 12m as the cell number decreased.4.Compared with control group,the IDD degree of Phd2IVD-,Phd2AF+EP-and Phd2NPwere higher,but the IDD degree of Phd2NP-and Phd2IVD-was similar and higher than that of Phd2NP-.In addition,the vascular invasion degree of Phd2NP-and Phd2IVD-was also significantly higher than that of Phd2AF+EP" and control.5.Compared with control group,the nucleus pulposus area of Vegfr2EC-always maintained a hypoxia environment.However,the abnormal increase in HIF-1α levels occurred after hyperbaric oxygen treatment.Conclusion:Vascular invasion of intervertebral disc disrupted the hypoxia environment in the nucleus pulposus during the early stages of IDD,and mediating the abnormal increase of HIF-1α.The increased HIF-1α levels in turn promote the angiogenesis of intervertebral disc,thereby forming a vicious cycle and promoting IDD.Part 2:Autophagic Degradation of HIF-1α in nucleus pulposus Cells under HypoxiaBackground:HIF-1α is stabilized under hypoxia,while it is degraded by proteasomes when the oxygen concentration is greater than 6%.However,the regulatory mechanism of HIF-1α in nucleus pulposus cells may be different.HIF-1α in nucleus pulposus cells were stable both under normoxia and hypoxia.In addition,the treatment of nucleus pulposus cells with autophagy inhibitors leads to an increase in HIF-1α levels.In the first part of the study,we also found that the destruction of the hypoxic environment in the nucleus pulposus during the IDD abnormally leads to an increase in HIF-1α levels.Given the critical role of HIF-1αin pathology and physiology of IDD,it is of great significance to further clarify the regulatory mechanism of HIF-1α in understanding the molecular mechanism of IDD.Objectives:1.To clarify that blood vessel invasion increases HIF-1α levels by inhibiting hypoxiadependent autophagy in nucleus pulposus cells.2.To elucidate the detailed mechanism of autophagic degradation of HIF-1α in nucleus pulposus cells under physiological hypoxia.Methods:1.After intervening with different oxygen concentrations in nucleus pulposus cells with or without inhibition of proteasome/lysosome function,changes in HIF-1α levels were detected by Western blotting.2.After knocking out autophagy-related genes under physiological hypoxia,changes in HIF-1α levels were detected by Western blotting.3.Constructed an Atg7NP-mice,and changes in HIF-1α levels were detected by immunofluorescence after transfection with AAV-VEGF or exposure to hyperbaric oxygen treatment.4.The autophagy receptor OPTN was screened by proximity ligation assay.After knocking out OPTN using shRNA,changes in HIF-1α levels were detected by Western blotting.OptnNP-mice were also constructed,and changes in HIF-1α levels were detected by immunofluorescence after transfection with AAV-VEGF or exposure to hyperbaric oxygen treatment.5.After co-transfecting nucleus pulposus cells with ubiquitin plasmids and HIF-1αoverexpression plasmids,changes in HIF-1α polyubiquitination at 1%oxygen concentration were detected by IP experiments.After knocking out PHD2 under physiological hypoxia,changes in HIF-1α hydroxylation and ubiquitination were detected by IP and Western blotting.Results:1.After intervention with 3%and 5%oxygen concentrations,the HIF-1α protein level in nucleus pulposus cells was significantly higher than that at 1%,7%,and 21%oxygen concentrations,while the HIF-1α mRNA level showed no significant difference between different oxygen concentrations.Proteasome inhibitor MG132 treatment of nucleus pulposus cells only inhibited HIF-1α degradation at 21%oxygen concentration,but not at 1%and 5%.Lysosome inhibitor E64 intervention increased HIF-1α level at 1%oxygen concentration,but not at 5%and 21%.After intervention with 1%oxygen concentration,the HIF-1α level in the lysosomal protein of nucleus pulposus cells significantly increased.2.Knockout of FIP200 and Atg7 significantly increased HIF-1α level at 1%oxygen concentration,while knockout of HSC70 and RUBICON did not.3.Compared to the control,Atg7NP-had increased HIF-1α levels in nucleus pulposus cells.Moreover,Atg7NP-abolished the induction of HIF-1α elevation by AAV-VEGF or hyperbaric oxygen.4.At 1%oxygen concentration,autophagy receptor OPTN was biotinylated in the proximity protein affinity experiment.Knockout of OPTN resulted in increased HIF-1αlevels in nucleus pulposus cells under hypoxia.In addition,compared to control,OptnNPhad increased HIF-1α levels in nucleus pulposus cells.Moreover,OptnNP-abolished the induction of HIF-1α elevation by AAV-VEGF or hyperbaric oxygen.5.At 1%oxygen concentration,HIF-1α was mainly ubiquitinated by K63.Furthermore,HIF-1α did not undergo K63 ubiquitination in at 5%and 21%oxygen concentrations.Knockout of PHD2 resulted in the disappearance of K63 ubiquitination of HIF-1α at 1%oxygen concentration,and PHD2 did not have hydroxylase activity at both 1%and 5%oxygen concentrations.Conclusion:The K63-ubiquitination of HIF-1α in nucleus pulposus cell during physiological hypoxia was abolished by increased oxygen concentration due to vascular invasion,and resulting in inhibition of OPTN-dependent autophagy degradation in the early stages of IDD.Part 3:Disrupting the Vicious Cycle between Blood Vessels and HIF-1α in the Nucleus Pulposus to Alleviate IDDBackground:Anti-VEGF has been widely used in the clinical treatment of tumors and age-related macular degeneration.In addition,preclinical studies have also shown the potential therapeutic effects of Anti-VEGF in diseases such as rheumatoid arthritis and osteoarthritis.In the previous part of our study,we found that Anti-VEGF can significantly alleviate IDD.However,due to the long duration of treatment,some mice in our study died from gastrointestinal perforation.Given that we found that vascular invasion in IDD mainly occurs in the early stage,we attempted to administer Anti-VEGF treatment in a staged manner during the early stage to treat IDD while reducing adverse reactions.Objectives:To evaluate the effectiveness of administering Anti-VEGF treatment in a staged manner during the early stage of IDD.Methods:1.Mice treated with Anti-VEGF at different periods were divided into 4 groups:early treatment group(Anti-VEGF treatment only during the 3m-6m periods),late treatment group(Anti-VEGF treatment only during the 6m-9m periods),Whole treatment group(Anti-VEGF treatment throughout the 0m-12m periods),and control group(Vehicle treatment throughout the 0m-12m periods).2.The levels of VEGF in the circulation of mice in each group at different time points were detected by ELISA.3.The changes in vascular invasion and HIF-1α levels at different time points in the four groups were compared by immunofluorescence staining.4.The degree of IDD at different time points in the four groups was compared by histological staining.Results:1.The levels of VEGF in the circulation of each group only decreased during the period of Anti-VEGF treatment and returned to normal levels after cessation of treatment.2.Neither the early treatment group nor the Whole treatment group showed significant vascular invasion throughout the observation period,while the degree of vascular invasion in the late treatment group was almost indistinguishable from that in the control group.In addition,the levels of HIF-1α in the early treatment group and Whole treatment group were relatively stable and gradually decreasing slightly with age.3.The degree of IDD in the early treatment group and whole treatment group was significantly lower than that in the control group and late treatment group.Conclusion:Targeted vascular invasion with VEGF antibody during the early stage blocked the vascular invasion and significantly delay IDD.