# Influence of Angiopoietin Treatment with Hypoxia and
## 3 Results
### Effect of Oxygen Tension and Angiopoietin-1/2 on NP Single Cell's Metabolism  
Further, we examined the metabolic activity/DNA ratio at Day 7, which is the most relevant parameter, as it refers to single-cell metabolic activity (Figure 2).  
In the NP mixed population under normoxic conditions, the stimulated samples showed a 2- to 3-fold increase in the metabolism/DNA ratio, compared with the Day 7 control sample. In summary, for normoxia, a stimulation effect was detectable, but not specifically in concentration- or protein-dependency (Figure 2a). Furthermore, in hypoxic conditions, for the NP mixed population, the stimulated samples all presented an increase in their ratio metabolism/DNA, as they were 4 to 8 times higher than in normoxic conditions. Hence, no clear trend in concentration- or protein-dependency was observable. Concerning the Ang-1 stimulated samples, the metabolism/DNA ratio increased from 10 to 50 ng/mL concentrations and then decreased for the 100 ng/mL sample, with a significant increase for Ang-1 at 50 and 100 ng/mL. Regarding the Ang-2 treated samples, the metabolism/DNA ratio decreased from 10 to 50 ng/mL and then increased again for the Ang-2 for the 100 ng/mL treated sample, with a significant increase for Ang-2 at 10 and 100 ng/mL.  
For the NP Tie2\({}^{-}\) cells population under normoxic condition, the ratios of the Ang-2 at 10 ng/mL and the Ang-1 at 50 ng/mL samples were 3- to 3.5-fold increased, compared to the Day 7 control sample (Figure 3b). For the other Ang-1 or Ang-2 treated samples, the ratios stayed at an equal level to the Day 7 control sample, or they even decreased. In hypoxia, all the stimulated samples showed a decrease in their metabolism/DNA ratio, except for the Ang-2 at the 100 ng/mL sample, whose ratio stayed at a similar level to the Day 7 control sample. as Additionally, for the mixed population, no trend in the effect of the concentration, nor the protein, was assessable. The Ang-1 treated samples presented an increase in their metabolism from the 10 to 50 ng/mL samples and then a decrease to the 100 ng/mL sample. The Ang-2 treated samples showed a decrease from the 10 ng/mL sample to the 50 ng/mL sample and then an increase to the 100 ng/mL sample. However, no significant increase or decrease was detected.  
Figure 2: Metabolic activity on single-cell level of NP cells populations in normoxia and hypoxia on Day 7. Ang-1 and Ang-2 were administrated in three concentrations: 10, 50 and 100 ng/mL. The control Day 7 samples were used as reference. (**a**) Metabolic activity on single-cell level of NP cells mixed population; (**b**) metabolic activity on single-cell level of NP cells Tie2\({}^{-}\) population; (**c**) metabolic activity on single-cell level of NP cells Tie2\({}^{+}\) population. Bars represent mean \(\pm\) SD. A \(p\)-value < 0.05 was considered significant. Asterisks within figures denote the degree of statistical relevance observed: * < 0.05; ** < 0.01; *** < 0.001; *** < 0.0001.  
In the NP Tie2\({}^{+}\) cells population, we observed a similar ratio for all the samples in normoxia, either stimulated with Ang-1/2 or not (Figure 2c). The same picture was present in hypoxia. All samples showed similar or slightly elevated ratios, compared to the day 7 control sample, except for the Ang-2 at the 10 ng/mL sample, which significantly decreased in its ratio, compared to the control sample. Therefore, a significant increase in stimulation with Ang-1 at 50 ng/mL and Ang-2 at 10, 50 and 100 ng/mL was detected.