# Influence of Angiopoietin Treatment with Hypoxia and
## 4 Discussion
### Ang-1/2 and Its Effect on the ECM Gene Expression in Normoxia & Hypoxia  
Looking at the gene expression levels, we did not observe significant results between normoxia and hypoxia for the majority of the samples. The effect of hypoxia on gene expression associated with chondrogenic lineage and ECM production in the IVD is already well documented in the literature [38; 39]. At least, in the NP cell Tie2\({}^{+}\) population for ACAN, we detected a trend of upregulation in hypoxia, whereas for _COL2_, it was not the case. If Ang-1 and/or Ang-2 had a stimulatory effect, this would be the case for the NP cells Tie2\({}^{+}\) population, as this population has the Tie2 receptor for the binding of the Angl ligands. It is, therefore, astonishing and unexpected to find the highest upregulates for ACAN in normoxia in the NP cells mixed and Tie2\({}^{-}\) populations for Ang-1/2 at 10 ng/mL and Ang-1 at 100 ng/mL. A possible explanation for these particular increases could be related to any cross-reactions of the ligands Ang-1 and Ang-2 with another receptor. This hypothesis is difficult to defend in the sense that there are only two or three out of the six treated samples in normoxia that show an effect upon stimulation. However, the main explanation for the stimulation of Ang-1 and Ang-2 not having any effect is the outcome for the NP cells Tie2\({}^{+}\) population. The stimulation did not show any differences upon Ang-1 or Ang-2 protein stimulation, including their different concentrations. In the case of the NP cells Tie2\({}^{+}\) population, several explanations can be considered. First, there could have been a cross-reaction of the Tie2 receptor with other ligands, which subsequently blocked the binding of Ang-1/2 to the receptor and thus the downstream pathways. Second, it would be possible that the Tie2 receptor was not expressed after the expansion culture phase. Briefly, at the time of incubation with Ang-1 and Ang-2, the cells were not Tie2\({}^{+}\) progenitor cells anymore. To confirm the progenitor state of the cell right before the start of the experiment, another FACS analysis could have been performed. Third, the first FACS analysis itself could already have stressed the cells and favored the loss of their progenitor attributes. The hypothesis for the lack of effect of Ang stimulation is encouraged by the examination of the TEK gene. It is eminently important to see that, similar to _ACAN_ and _COL2_, we did miss out on any specific reaction in the relative gene expression of _TEK_ to either Ang-1, Ang-2, including their three different doses in the Tie2\({}^{+}\) cells population. This is unexpected for the same reasons as already mentioned for _ACAN_ and _COL2_. In the NP mixed and NP Tie2\({}^{-}\) cells populations, the relative gene expression in _TEK_ and _HIF1A_ do show the same patterns as _COL2_ and _ACAN_. The peak values in normoxia deserve special mention, such as Ang-1/2 at 10 ng/mL and Ang 1 at 100 ng/mL. However, again, it was unexpected to find such upregulation in normoxia of the genes within the NP mixed and NP cells Tie2\({}^{-}\) populations. As we find the same pattern several times, there must be attractive or favorable attributes for these doses in normoxia. Up to that point, explanations remain elusive. As already mentioned before, the data must be interpreted with caution, due to the high donor variation. The downregulation of _HIF1A_ in hypoxia in the NP cells mixed, NP cells Tie2\({}^{-}\) and Tie2\({}^{+}\) populations can be explained, as hypoxia progressively decreases _HIF1A_ expression at the mRNA level, due to the mRNA-destabilizing protein tristetraprolin [42]. However, [PERSON] et al. [32] detected that the level of relative gene expression of _HIF1A_ for DPCs of the NP remained constant under hypoxia, compared to normoxia. They suggest that incompetency of the DPCs of the NP to adapt to oxygen tension may be in part due to the inability to upregulate HIF expression.