# Influence of Angiopoietin Treatment with Hypoxia and
## 4 Discussion
### Ang-1/2 and Its Effect on DNA Measurement in Normoxic and Hypoxic Conditions  
Evidence has arisen in recent years suggesting that a tissue renin-angiotensin system (tRAS) is involved in the progression of various human diseases [28], including IVD degeneration [29]. Numerous reports and studies showed the positive effects of pathologic tRAS pathway inhibition and protective tRAS pathway stimulation on the treatment of cardiovascular, inflammatory, and autoimmune disease and the progression of neuropathic pain. Cell proliferation, senescence, and aging are known to be related to RAS pathways. In this context, the DNA concentration measured in our study in all the NP cells mixed and Tie2+ population samples cultured within normoxic conditions were statistically different from the controls at Day 0. None of the samples in the NP cells Tie2- population showed statistically significant differences. The amount of DNA in the NP cells mixed population decreased, whereas the amount was increased in the NP cells Tie2+ population on Day 7 for the control and the stimulated samples in normoxia, compared to their control Day 0 equivalents. In hypoxia, no effect on the proliferation was detected, except for the NP cells Tie2+ population treated with Ang-1 at 10 ng/mL. In the NP cells Tie2- population, a slight upregulation in the amount of DNA was seen in every sample, in normoxia and hypoxia, compared to the Day 0 sample. The reason for this discrepancy between the NP cells mixed and NP Tie2- cells populations is not clear. As the NP cells mixed population consists of 95 to 99% of the exact cell types as the Tie2- cells population, the 1 to 5% difference in cell type should not provoke the observed difference [1]. Furthermore, both samples underwent the same schedule of incubation and medium change, which should result in a more similar outcome. However, a possible cell-to-cell interaction or co-culture between the Tie2+ and Tie2- cells within the mixed population could influence the proliferation as shown in other tissue and cell types [30; 31]. All samples treated in the NP cells Tie2+ population showed an increased amount of DNA, compared to the Day 0 sample and a trend of higher amounts of DNA in normoxia, compared to hypoxia. A possible explanation for the lower amount of DNA detected within the cells population incubated under hypoxic conditions, compared to the normoxic condition, could be the potential differentiation within the chondrogenic lineage of the cells in hypoxia, reducing, at the same time, cell proliferation. As described by [PERSON] et al. [12], an explanation could be that hypoxia can exert either positive or negative effects on IVD progenitor cells. In particular, hypoxia can inhibit proliferation and induce apoptosis but might also promote the chondrogenic differentiation of IVD progenitor cells. [PERSON] et al. [32] identified tissue-specific intervertebral disc progenitor cells (DPCs) from healthy Rhesus monkeys; in particular, they found that they are sensitive to low oxygen tension and undergo apoptosis under hypoxic conditions due to their inability to induce/stabilize hypoxia-inducible factors (HIF). These results confirm our findings. Our findings and the findings of the previously mentioned studies contradict the publication of [PERSON] et al. [13], who found a better cell survival of Tie2+ cells cultured in hypoxia (2%), which were based on young-aged bovine cells. We identified in our results a trend for an increased amount of DNA in normoxia for the NP Tie2+ population. These are contradictory findings to several studies, in which it was shown that an oxemic shift would be expected to lead to a failure in progenitor cell activation [13; 33]. The variation in oxygen tension is possibly mediated by alterations in the vascular supply to the endplate cartilage or even the annulus fibrosus. In turn, the cells no longer reside in their native associated microenvironment and are associated with a reduced cell proliferation capacity [34; 35; 36].