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Additional file 1: Figure S1. Red dots indicate the spatial representation of the mean endpoint after the evaluation of cell movements of at least 20 cells per experiment (N = 4–5). A: oxygen gradient (hypoxia); B: no oxygen gradient (normoxia). P-values were calculated using circular statistics (Rayleigh test). Black lines inside the quadrants show the linear correlation of the center of mass in each experimental group. Figure S2. Detailed description of the array proteins. Figure S3. Red dots indicate the spatial representation of the mean endpoint after the evaluation of cell movements of at least 20 cells per experiment (N = 3). A: no oxygen gradient (normoxia) in combination with a gradient of CCL26; B: oxygen gradient (hypoxia) with homogeneously distributed CCL26. P-values were calculated using circular statistics (Rayleigh test). Black lines inside the quadrants show the linear correlation of the center of mass in each experimental group. Figure S4. Upper panel: Detection of RAGE by Western blotting in cell lysates of monocytes subjected to hypoxic and normoxic conditions (3 representative samples out of N = 7 are shown). Columns show the mean of N = 7 experiments, bars denote SD. Exp, experiment; H, hypoxia, N, normoxia. Lower panel: Colocalization of RAGE and CCL26 in monocytes (3 representative samples out of N = 7 are shown). Yellow color is indicative of cellular RAGE/CCL26 colocalization. Scale bars represent 3 µm. Exp, experiment.

Objectives The sequence of initial tissue ischaemia and consecutive blood flow restoration leads to ischaemia/reperfusion (I/R) injury, which is typically characterized by a specific inflammatory response. Migrating monocytes seem to mediate the immune response in ischaemic tissues and influence detrimental as well as regenerative effects during I/R injury. Materials and Methods To clarify the role of classical monocytes in I/R injury, isolated human monocytes were subjected to I/R in vitro (3 hours ischaemia followed by 24 hours of reperfusion). Cellular resilience, monocyte differentiation, cytokine secretion, as well as influence on endothelial tube formation, migration and cell recovery were investigated. Results We show that I/R supported an enhanced resilience of monocytes and induced intracellular phosphorylation of the prosurvival molecules Erk1/2 and Akt. FACS analysis showed no major alteration in monocyte subtype differentiation and surface marker expression under I/R. Further, our experiments revealed that I/R changes the cytokine secretion pattern, release of angiogenesis associated proteins and MMP‐9 activity in supernatants of monocytes exposed to I/R. Supernatants from monocytes subjected to I/R attenuated endothelial tube formation as indicator for angiogenesis as well as endothelial cell migration and recovery. Conclusion In summary, monocytes showed no significant change in cellular integrity and monocyte subtype after I/R. Functionally, monocytes might have a rather detrimental influence during the initial phase of I/R, suppressing endothelial cell migration and neoangiogenesis.

Perinatal asphyxia is characterized by oxygen deprivation and lack of perfusion in the perinatal period, leading to hypoxic-ischemic encephalopathy and sequelae such as cerebral palsy, mental retardation, cerebral visual impairment, epilepsy and learning disabilities. On cellular level PA is associated with a decrease in oxygen and glucose leading to ATP depletion and a compromised mitochondrial function. Upon reoxygenation and reperfusion, the renewed availability of oxygen gives rise to not only restoration of cell function, but also to the activation of multiple detrimental biochemical pathways, leading to secondary energy failure and ultimately, cell death. The formation of reactive oxygen species, nitric oxide and peroxynitrite plays a central role in the development of subsequent neurological damage. In this review we give insight into the pathophysiology of perinatal asphyxia, discuss its clinical relevance and summarize current neuroprotective strategies related to therapeutic hypothermia, ischemic postconditioning and pharmacological interventions. The review will also focus on the possible neuroprotective actions and molecular mechanisms of the selective neuronal and inducible nitric oxide synthase inhibitor 2-iminobiotin that may represent a novel therapeutic agent for the treatment of hypoxic-ischemic encephalopathy, both in combination with therapeutic hypothermia in middle- and high-income countries, as well as stand-alone treatment in low-income countries.

Additional file 1. Effects of I/R and RIPC on LDH release.

Table S2. Presenting patient characteristics prior to treatment with PCMO. (DOCX 18Â kb)

Figure S2. Quantification of cell damage by measuring LDH after 1 h, 2 h, 3 h and 4 h and representative images of PCMO cell culture under normoxia and hypoxia. (PDF 674 kb)

Table S1. Presenting primers relate to hypoxia-induced gene expression in monocytes/macrophages. (DOCX 308Â kb)

Table S3. Presenting human angiogenesis and tissue recovery-related proteins analyzed in PCMO cell culture. (DOCX 813Â kb)

Figure S1. Showing the evaluation of hypoxia-induced gene expression in PCMO. (PDF 828Â kb)

Characterization of the angiogenic potential of human monocytes and programmable cells of monocytic origin (PCMO) under hypoxic conditionsZitta K, Berndt R, Hummitzsch L, Faendrich F, Gross J, Albrecht MAfter 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 nau00efve 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 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-1u03b1 (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.Nau00efve 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.

Combining 2-iminobiotin and hypothermia for reducing hypoxia-induced neuronal damage: Implications from a human cell culture model of hypoxic-ischemic brain injuryKarina Zitta1, Cacha Peeters-Scholte2, Lena Sommer1, Kerstin Parczany1, Markus Steinfath1, Martin Albrecht1Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Kiel, Germany 1, Neurophyxia B.V.,u2019s-Hertogenbosch, The Netherlands2Background and Goal of Study: Perinatal asphyxia represents one of the major causes of neonatal morbidity and mortality. Hypothermia is currently the only established treatment for hypoxic-ischemic encephalopathy, and no specific pharmacological therapy exists so far. We have shown that the biotin analogue 2-iminobiotin (2-IB) is able to reduce neuronal cell damage both in vitro as well as in vivo. Here we evaluated whether 2-IB has the potential to attenuate hypoxia-induced injury of neuronal cells also under hypothermic conditions. Materials and Methods: In-vitro hypoxia was induced in cultures of IMR-32 cells. After the hypoxic injury, cells were incubated with vehicle or 2-IB (10, 30, 50, 100 and 300ng/ml) under hypothermic conditions (33.5u00b0C). Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS) was measured using fluorometric assays. Westernblotting for PARP and phosphorylation of akt and erk1/2 was performed. Results: Hypoxia led to morphological signs of cell damage. Under hypothermia, LDH measurements revealed a significant LDH increase earliest at 25h after the hypoxic insult (p

Background: Aspirin and its major metabolite salicylic acid (SA), confer protection against glutamate neurotoxicity via COX dependent and independent mechanisms. There is also evidence that SA may protect against hypoxia-induced neuronal cell damage which is associated with increased levels of reactive oxygen species (ROS). Here we investigated the effects of chronically increased levels of SA on the ability of neuronal cells to survive hypoxia-induced neurotoxicity in-vitro and evaluated the underlying mechanisms.Materials and methods: Human neuronal IMR-32 cells were cultured with or without SA (0.1, 1 and 10u00b5M) for a total of 7 months. In-vitro hypoxia was induced for 3 hours using our recently described system. Cell morphology was evaluated by brightfield microscopy, cell damage was measured by lactate dehydrogenase (LDH) assays and cell metabolism was quantified by colorimetric MTS assays. Production of ROS was analyzed using fluorometric assays. Westernblotting for catalase (CAT) and glutathione peroxidase (GPX) was performedResults: Long term incubation of neuronal IMR-32 cells with clinically relevant concentrations of SA (0.1, 1 and 10u00b5M) did not change morphology or proliferative potential of the cultures. However, neuronal cells that were exposed to chronic SA treatment showed statistically significant protection against hypoxia/reoxygenation injury (P

Additional file 2: Figure 2. Effects of different colloid containing solutions on Claudin-3 protein expression. Ratio of Claudin-3 and Actin protein expression in intestinal tissue of the Albumin, HES and HES/Alb group after perfusion. No changes in the protein expression levels of Claudin-3 are evident in the different treatment groups. Bars denote the mean ± SD. Albumin (N = 6), HES (N = 6), HES/Alb (N = 6).