Characterization of the angiogenic potential of human monocytes and programmable cells of monocytic origin (PCMO) under hypoxic conditions

Zitta K, Berndt R, Hummitzsch L, Faendrich F, Gross J, Albrecht M

After reprogramming in-vitro, human blood monocytes can acquire a state of plasticity. These so called programmable cells of monocytic origin (PCMOs) hold great promise in diseases in which damaged or non-functional cells need to be replaced1-4. A pilot study of our group showed that PCMO cells release proteins involved in angiogenesis and suggested that PCMO inoculation may present a treatment strategy for ischemia-associated illnesses. As reprogramming of monocytes to PCMO requires prolonged in-vitro cultivation and stimulation with growth factors, the aim of the present study was to investigate whether hypoxia-stimulated naïve human monocytes are also able to release angiogenic factors.
Mononuclear cells were isolated from peripheral blood by density gradient centrifugation and PCMO cells were obtained as described earlier5. Enzymatic hypoxia6,7 was applied on monocytes or PCMO for 3h (pO2 <10 mmHg) and 24h after the end of hypoxia the respective conditioned culture medium (CCM) was collected. Assays for lactate dehydrogenase (LDH) release and angiogenesis proteome profiling arrays were used to characterize the cells. 
3h of hypoxia reduced the release of LDH in cultures of human naïve monocytes (n=11; p<0.001), while no significant differences in LDH release were observed in PCMO cultures (n=5; p>0.05). 24h after the end of hypoxia period, 28 of 55 (51%) angiogenic proteins were detectable in monocyte-CCM. Of these, 17/28 (61%) were up regulated, 3/28 (11%) were down-regulated and 8/28 (28%) did not show any regulation. In PCMO-CCM, 26 of 55 angiogenesis proteins (47%) were detectable. 12/26 (46%) proteins were up regulated, 6/26 (23%) were down-regulated, while 8/26 (31%) proteins did not reveal regulation by hypoxia (Figure 1). 
 
Figure 1: Regulation of protein secretion by hypoxia.
Proteins with the strongest up regulation by hypoxia were: MCP-1 (92-fold in monocyte-CCM and 53-fold in PCMO-CCM), MIP-1α (35-fold in monocyte-CCM and 16-fold in PCMO-CCM) and GM-CSF (40-fold in monocyte-CCM and 15-fold in PCMO-CCM, Figure 2). 
 
Figure 2: Relative protein secretion of selected angiogenic factors after hypoxia.
Naïve monocytes and PCMO show similarities in the release of several hypoxia-induced angiogenic factors. However, we also detected cell type specific responses to the hypoxic stimulus (e.g. LDH release; protein secretion, Figure 2), which might influence the potential of both cell types in therapies aiming at the reduction of ischemia/reperfusion injury and/or induction of angiogenesis in different organs.

1Ungefroren et al. Stem cells int 2016.2Ungefroren et al. PloS one 2015.3Zachos et al. J orthopaedic res 2014.4Ungefroren H, Fandrich F Adv Exp Med Biol 2010. 5Ungefroren et al. Stem cells dev 2010. 6Zitta et al. Eur j pharmacol 2016. 7Hummitzsch et al. Exp cell res 2014.

11 / Characterization of the angiogenic potential of human monocytes and programmable cells of monocytic origin (PCMO) under hypoxic conditions