Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications.

BACKGROUND: Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ. AIM OF THE STUDY: To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. METHODS: Differentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. RESULTS: Mreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased. CONCLUSION: Exposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

Large extracellular vesicles derived from human regulatory macrophages (L-EVMreg) attenuate CD3/CD28-induced T-cell activation in vitro.

Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EVMreg) with an average size of 7.5 µm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EVMreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EVMreg were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EVMreg. Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EVMreg was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B-positive cells (P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 106 Mreg/ml) led to a reduction of T-cell activation (number of granzyme B-positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EVMreg (P < 0.05 for 3.2 × 106 L-EVMreg/ml). A differential analysis of the effects of Mreg and L-EVMreg on CD4+ and CD8+ T-cells showed an inhibition of CD4+ T-cells by Mreg (P < 0.01) and L-EVMreg (P < 0.05 for 1.6 × 106 L-EVMreg/ml; P < 0.01 for 3.2 × 106 L-EVMreg/ml). A moderate inhibition of CD8+ T-cells was observed by Mreg (P < 0.05) and by L-EVMreg (P < 0.01 for 1.6 × 106 L-EVMreg/ml and 3.2 × 106 L-EVMreg/ml). PS was restricted to confined regions of the Mreg surface, while L-EVMreg showed strong signals for PS in the exoplasmic leaflet. L-EVMreg attenuate CD3/CD28-mediated activation of CD4+ and CD8+ T-cells. L-EVMreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity. KEY MESSAGES: Mreg release large extracellular vesicles (L-EVMreg) with an average size of 7.5 µm L-EVMreg exhibit phosphatidylserine positivity L-EVMreg suppress CD4+ and CD8+ T-cells L-EVMreg hold clinical potential in T-cell-related diseases.

Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis?

BACKGROUND: Large extracellular vesicles (L-EV) with a diameter between 1 and 10 µm are released by various cell types. L-EV contain and transport active molecules which are crucially involved in cell to cell communication. We have shown that secretory products of human regulatory macrophages (Mreg) bear pro-angiogenic potential in-vitro and our recent findings show that Mreg cultures also contain numerous large vesicular structures similar to L-EV with so far unknown characteristics and function. AIM OF THIS STUDY: To characterize the nature of Mreg-derived L-EV (L-EVMreg) and to gain insights into their role in wound healing and angiogenesis. METHODS: Mreg were differentiated using blood monocytes from healthy donors (N = 9) and L-EVMreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EVMreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EVMreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays. RESULTS: Mreg and L-EVMreg show an average diameter of 13.73 ± 1.33 µm (volume: 1.45 ± 0.44 pl) and 7.47 ± 0.75 µm (volume: 0.22 ± 0.06 pl) respectively. Flow cytometry analyses revealed similarities between Mreg and L-EVMreg regarding their surface marker composition. However, compared to Mreg fewer L-EVMreg were positive for CD31 (P < 0.01), CD206 (P < 0.05), CD103 (P < 0.01) and CD45 (P < 0.05). Proteome profiling suggested that L-EVMreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EVMreg positively influenced in-vitro wound healing (P < 0.05) and several pro-angiogenic parameters in tube formation assays (all segment associated parameters, P < 0.05; number of meshes, P < 0.05). CONCLUSION: L-EVMreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EVMreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Effects of remote ischemic preconditioning (RIPC) and chronic remote ischemic preconditioning (cRIPC) on levels of plasma cytokines, cell surface characteristics of monocytes and in-vitro angiogenesis: a pilot study.

Remote ischemic preconditioning (RIPC) protects the heart against myocardial ischemia/reperfusion (I/R) injury and recent work also suggested chronic remote ischemic conditioning (cRIPC) for cardiovascular protection. Based on current knowledge that systemic immunomodulatory effects of RIPC and the anti-inflammatory capacity of monocytes might be involved in cardiovascular protection, the aim of our study was to evaluate whether RIPC/cRIPC blood plasma is able to induce in-vitro angiogenesis, identify responsible factors and evaluate the effects of RIPC/cRIPC on cell surface characteristics of circulating monocytes. Eleven healthy volunteers were subjected to RIPC/cRIPC using a blood pressure cuff inflated to > 200 mmHg for 3 × 5 min on the upper arm. Plasma and peripheral blood monocytes were isolated before RIPC (Control), after 1 × RIPC (RIPC) and at the end of 1 week of daily RIPC (cRIPC) treatment. Plasma concentrations of potentially pro-angiogenic humoral factors (CXCL5, Growth hormone, IGFBP3, IL-1α, IL-6, Angiopoietin 2, VEGF, PECAM-1, sTie-2, IL-8, MCSF) were measured using custom made multiplex ELISA systems. Tube formation assays for evaluation of in-vitro angiogenesis were performed with donor plasma, monocyte conditioned culture media as well as IL-1α, CXCL5 and Growth hormone. The presence of CD14, CD16, Tie-2 and CCR2 was analyzed on monocytes by flow cytometry. Employing in-vitro tube formation assays, several parameters of angiogenesis were significantly increased by cRIPC plasma (number of nodes, P < 0.05; number of master junctions, P < 0.05; number of segments, P < 0.05) but were not influenced by culture medium from RIPC/cRIPC treated monocytes. While RIPC/cRIPC treatment did not lead to significant changes of the median plasma concentrations of any of the selected potentially pro-angiogenic humoral factors, in-depth analysis of the individual subjects revealed differences in plasma levels of IL-1α, CXCL5 and Growth hormone after RIPC/cRIPC treatment in some of the volunteers. Nevertheless, the positive effects of RIPC/cRIPC plasma on in-vitro angiogenesis could not be mimicked by the addition of the respective humoral factors alone or in combination. While monocyte conditioned culture media did not affect in-vitro tube formation, flow cytometry analyses of circulating monocytes revealed a significant increase in the number of Tie-2 positive and a decrease of CCR2 positive monocytes after RIPC/cRIPC (Tie-2: cRIPC, P < 0.05; CCR2: RIPC P < 0.01). Cardiovascular protection may be mediated by RIPC and cRIPC via a regulation of plasma cytokines as well as changes in cell surface characteristics of monocytes (e.g. Tie-2). Our results suggest that a combination of humoral and cellular factors could be responsible for the RIPC/cRIPC mediated effects and that interindividual variations seem to play a considerable part in the RIPC/cRIPC associated mechanisms.

Effects of different ischemic preconditioning strategies on physiological and cellular mechanisms of intestinal ischemia/reperfusion injury: Implication from an isolated perfused rat small intestine model.

BACKGROUND: Intestinal ischemia/reperfusion (I/R)-injury often results in sepsis and organ failure and is of major importance in the clinic. A potential strategy to reduce I/R-injury is the application of ischemic preconditioning (IPC) during which repeated, brief episodes of I/R are applied. The aim of this study was to evaluate physiological and cellular effects of intestinal I/R-injury and to compare the influence of in-vivo IPC (iIPC) with ex-vivo IPC (eIPC), in which blood derived factors and nerval regulations are excluded. METHODS: Using an established perfused rat intestine model, effects of iIPC and eIPC on physiological as well as cellular mechanisms of I/R-injury (60 min hypoxia, 30 min reperfusion) were investigated. iIPC was applied by three reversible occlusions of the mesenteric artery in-vivo for 5 min followed by 5 min of reperfusion before isolating the small intestine, eIPC was induced by stopping the vascular perfusion ex-vivo 3 times for 5 min followed by 5 min of reperfusion after isolation of the intestine. Study groups (each N = 8-9 animals) were: iIPC, eIPC, I/R (iIPC group), I/R (eIPC group), iIPC+I/R, eIPC+I/R, no intervention/control (iIPC group), no intervention/control (eIPC group). Tissue morphology/damage, metabolic functions, fluid shifts and barrier permeability were evaluated. Cellular mechanisms were investigated using signaling arrays. RESULTS: I/R-injury decreased intestinal galactose uptake (iIPC group: p<0.001), increased vascular perfusion pressure (iIPC group: p<0.001; eIPC group: p<0.01) and attenuated venous flow (iIPC group: p<0.05) while lactate-to-pyruvate ratio (iIPC group, eIPC group: p<0.001), luminal flow (iIPC group: p<0.001; eIPC group: p<0.05), goblet cell ratio (iIPC group, eIPC group: p<0.001) and apoptosis (iIPC group, eIPC group: p<0.05) were all increased. Application of iIPC prior to I/R increased vascular galactose uptake (P<0.05) while eIPC had no significant impact on parameters of I/R-injury. On cellular level, I/R-injury resulted in a reduction of the phosphorylation of several MAPK signaling molecules. Application of iIPC prior to I/R increased phosphorylation of JNK2 and p38δ while eIPC enhanced CREB and GSK-3α/ß phosphorylation. CONCLUSION: Intestinal I/R-injury is associated with major physiological and cellular changes. However, the overall influence of the two different IPC strategies on the acute phase of intestinal I/R-injury is rather limited.

Hypoxia directed migration of human naïve monocytes is associated with an attenuation of cytokine release: indications for a key role of CCL26.

BACKGROUND: Numerous tissue-derived factors have been postulated to be involved in tissue migration of circulating monocytes. The aim of this study was to evaluate whether a defined hypoxic gradient can induce directed migration of naïve human monocytes and to identify responsible autocrine/paracrine factors. METHODS: Monocytes were isolated from peripheral blood mononuclear cells, transferred into chemotaxis chambers and subjected to a defined oxygen gradient with or without the addition of CCL26. Cell migration was recorded and secretome analyses were performed. RESULTS: Cell migration recordings revealed directed migration of monocytes towards the source of hypoxia. Analysis of the monocyte secretome demonstrated a reduced secretion of 70% (19/27) of the analyzed cytokines under hypoxic conditions. The most down-regulated factors were CCL26 (- 99%), CCL1 (- 95%), CX3CL1 (- 95%), CCL17 (- 85%) and XCL1 (- 83%). Administration of recombinant CCL26 abolished the hypoxia-induced directed migration of human monocytes, while the addition of CCL26 under normoxic conditions resulted in a repulsion of monocytes from the source of CCL26. CONCLUSIONS: Hypoxia induces directed migration of human monocytes in-vitro. Autocrine/paracrine released CCL26 is involved in the hypoxia-mediated monocyte migration and may represent a target molecule for the modulation of monocyte migration in-vivo.

Occurrence of Fibrotic Tumor Vessels in Grade I Meningiomas Is Strongly Associated with Vessel Density, Expression of VEGF, PlGF, IGFBP-3 and Tumor Recurrence.

Angiogenesis is a key feature during oncogenesis and remains a potential target of antiangiogenic therapy. While commonly described in high-grade lesions, vascularization and its correlation with prognosis in grade I meningiomas is largely unexplored. In the histological classification, not only the number but also the composition of blood vessels seems to be important. Therefore, tumor vessel density and fibrosis were correlated with clinical and imaging variables and prognosis in 295 patients with intracranial grade I meningioma. Expression of pro-angiogenic proteins within the meningiomas was investigated by proteome analyses and further validated by immunohistochemical staining. Fibrotic tumor vessels (FTV) were detected in 48% of all tumors and strongly correlated with vessel density, but not with the histopathological tumor subtype. Occurrence of FTV was correlated with a 2-fold increased risk of recurrence in both univariate and multivariate analyses. Explorative proteome analyses revealed upregulation of VEGF (vascular endothelial growth factor), PlGF (placental growth factor), and IGFBP-3 (insulin-like growth factor-binding protein-3) in tumors displaying FTV. Immunohistochemical analyses confirmed strong correlations between tumor vessel fibrosis and expression of VEGF, PlGF, and IGFBP-3. Presence of FTV was strongly associated with disruption of the arachnoid layer on preoperative MRI in univariate and multivariate analyses. In summary, the occurrence of fibrotic tumor vessels in grade I meningiomas is strongly associated with vessel density, disruption of the arachnoid layer, expression of VEGF, PlGF, IGFBP-3 and tumor recurrence.

Human monocytes subjected to ischaemia/reperfusion inhibit angiogenesis and wound healing in vitro.

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.

Characterization of the Angiogenic Potential of Human Regulatory Macrophages (Mreg) after Ischemia/Reperfusion Injury In Vitro.

Ischemia/reperfusion- (I/R-) induced organ damage represents one of the main causes of death worldwide, and new strategies to reduce I/R injury are urgently needed. We have shown that programmable cells of monocytic origin (PCMO) respond to I/R with the release of angiogenic mediators and that transplantation of PCMO results in increased neovascularization. Human regulatory macrophages (Mreg), which are also of monocytic origin, have been successfully employed in clinical transplantation studies due to their immunomodulatory properties. Here, we investigated whether Mreg also possess angiogenic potential in vitro and could represent a treatment option for I/R-associated illnesses. Mreg were differentiated using peripheral blood monocytes from different donors (N = 14) by incubation with M-CSF and human AB serum and stimulation with INF-gamma. Mreg cultures were subjected to 3 h of hypoxia and 24 h of reoxygenation (resembling I/R) or the respective nonischemic control. Cellular resilience, expression of pluripotency markers, secretion of angiogenic proteins, and influence on endothelial tube formation as a surrogate marker for angiogenesis were investigated. Mreg showed resilience against I/R that did not lead to increased cell damage. Mreg express DHRS9 as well as IDO and display a moderate to low expression pattern of several pluripotency genes (e.g., NANOG, OCT-4, and SOX2). I/R resulted in an upregulation of IDO (p < 0.001) while C-MYC and KLF4 were downregulated (p < 0.001 and p < 0.05). Proteome profiling revealed the secretion of numerous angiogenic proteins by Mreg of which several were strongly upregulated by I/R (e.g., MIP-1alpha, 19.9-fold; GM-CSF, 19.2-fold; PTX3, 5.8-fold; IL-1ß, 5.2-fold; and MCP-1, 4.7-fold). The angiogenic potential of supernatants from Mreg subjected to I/R remains inconclusive. While Mreg supernatants from 3 donors induced tube formation, 2 supernatants were not effective. We suggest that Mreg may prove beneficial as a cell therapy-based treatment option for I/R-associated illnesses. However, donor characteristics seem to crucially influence the effectiveness of Mreg treatment.

Remote ischemic preconditioning attenuates intestinal mucosal damage: insight from a rat model of ischemia-reperfusion injury.

BACKGROUND: Remote ischemic preconditioning (RIPC) is a phenomenon, whereby repeated, non-lethal episodes of ischemia to an organ or limb exert protection against ischemia-reperfusion (I/R) injury in distant organs. Despite intensive research, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms, especially in the intestine. Aim of this study was to evaluate possible protective effects RIPC on intestinal I/R injury. METHODS: Thirty rats were randomly assigned to four groups: I/R; I/R + RIPC; Sham; Sham + RIPC. Animals were anesthetized and the superior mesenteric artery was clamped for 30 min, followed by 60 min of reperfusion. RIPC-treated rats received 3 × 5 min of bilateral hindlimb I/R prior to surgery, sham groups obtained laparotomy without clamping. After I/R injury serum/tissue was analyzed for: Mucosal damage, Caspase-3/7 activity, expression of cell stress proteins, hydrogen peroxide (H2O2) and malondialdehyde (MDA) production, Hypoxia-inducible factor-1α (HIF-1α) protein expression and matrix metalloproteinase (MMP) activity. RESULTS: Intestinal I/R resulted in increased mucosal injury (P < 0.001) and elevated Caspase-3/7 activity (P < 0.001). RIPC significantly reduced the histological signs of intestinal I/R injury (P < 0.01), but did not affect Caspase-3/7 activity. Proteome profiling suggested a RIPC-mediated regulation of several cell stress proteins after I/R injury: Cytochrome C (+ 157%); Cited-2 (- 39%), ADAMTS1 (+ 74%). Serum concentrations of H2O2 and MDA remained unchanged after RIPC, while the reduced intestinal injury was associated with increased HIF-1α levels. Measurements of MMP activities in serum and intestinal tissue revealed an attenuated gelatinase activity at 130 kDa within the serum samples (P < 0.001) after RIPC, while the activity of MMPs within the intestinal tissue was not affected by I/R injury or RIPC. CONCLUSIONS: RIPC ameliorates intestinal I/R injury in rats. The underlying mechanisms may involve HIF-1α protein expression and a decreased serum activity of a 130 kDa factor with gelatinase activity.

Effects of hydroxyethyl starch (HES 130/0.42) on endothelial and epithelial permeability in vitro.

Hydroxyethyl starch (HES) is employed to sustain normovolemia in patients. Using a perfused organ model, we recently showed that HES impairs the intestinal barrier which is constituted of endothelial and epithelial cell layers. However, the target cells and molecular actions of HES in the intestine are mainly unknown. Employing a model of human endothelial (HUVEC) and intestinal epithelial cells (Caco-2), we investigated the impact of HES, albumin and HES/albumin on cellular integrity/permeability and evaluated underlying molecular mechanisms. Monolayers of HUVEC and Caco-2 were cultured with HES (3%), albumin (3%) or HES/albumin (1.5%/1.5%). Integrity and permeability of the cell layers were evaluated by FITC-dextran transfer, measurements of cell detachment, vitality, cell volume, LDH release and caspase-3/7 activity. Cellular mechanisms were analyzed by Westernblotting for P-akt, P-erk, claudin-3 and I-FABP. HES application resulted in higher numbers of non-adherent/floating HUVEC cells (P<0.05) but did not change vitality or cell volume. Both, HES and HES/albumin increased the permeability of HUVEC monolayers (P<0.001), while LDH release, caspase-3/7 activity, akt/erk phosphorylation and claudin-3 expression were not affected. HES and HES/albumin did not change any of the parameters in cultures of Caco-2 cells. HES is able to disturb the integrity of the endothelial but not the epithelial barrier in vitro. HES effects are unrelated to cell damage and apoptosis but may involve reduced cell-cell or cell-matrix adhesion.

Neuroprotective strategies following perinatal hypoxia-ischemia: Taking aim at NOS.

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.

Allogeneic transplantation of programmable cells of monocytic origin (PCMO) improves angiogenesis and tissue recovery in critical limb ischemia (CLI): a translational approach.

BACKROUND: Employing growth factor-induced partial reprogramming in vitro, peripheral human blood monocytes can acquire a state of plasticity along with expression of various markers of pluripotency. These so-called programmable cells of monocytic origin (PCMO) hold great promise in regenerative therapies. The aim of this translational study was to explore and exploit the functional properties of PCMO for allogeneic cell transplantation therapy in critical limb ischemia (CLI). METHODS: Using our previously described differentiation protocol, murine and human monocytes were differentiated into PCMO. We examined paracrine secretion of pro-angiogenic and tissue recovery-associated proteins under hypoxia and induction of angiogenesis by PCMO in vitro. Allogeneic cell transplantation of PCMO was performed in a hind limb ischemia mouse model in comparison to cell transplantation of native monocytes and a placebo group. Moreover, we analyzed retrospectively four healing attempts with PCMO in patients with peripheral artery disease (PAD; Rutherford classification, stage 5 and 6). Statistical analysis was performed by using one-way ANOVA, Tukey's test or the Student's t test, p < 0.05. RESULTS: Cell culture experiments revealed good resilience of PCMO under hypoxia, enhanced paracrine release of pro-angiogenic and tissue recovery-associated proteins and induction of angiogenesis in vitro by PCMO. Animal experiments demonstrated significantly enhanced SO2 saturation, blood flow, neoangiogenesis and tissue recovery after treatment with PCMO compared to treatment with native monocytes and placebo. Finally, first therapeutic application of PCMO in humans demonstrated increased vascular collaterals and improved wound healing in patients with chronic CLI without exaggerated immune response, malignant processes or extended infection after 12 months. In all patients minor and/or major amputations of the lower extremity could be avoided. CONCLUSIONS: In summary, PCMO improve angiogenesis and tissue recovery in chronic ischemic muscle and first clinical results promise to provide an effective and safe treatment of CLI.

Effects of propofol on wound closure and barrier function of cultured endothelial cells: An in vitro experimental study.

BACKGROUND: Propofol is widely used in routine clinical practice for the induction and maintenance of anaesthesia. Although propofol is regarded as a well tolerated anaesthetic, its effect on intact or damaged endothelial cells has not yet been elucidated. OBJECTIVE: The aim of this study was to investigate the effects of different concentrations of propofol on cell damage, metabolic activity, barrier function and wound healing capacity of human endothelial cells. DESIGN: An in vitro investigation. SETTING: Research Laboratory of the Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Kiel, Germany. MATERIALS: In vitro cultures of primary human umbilical vein endothelial cells (HUVECs). INTERVENTIONS: Intact HUVEC or wounded HUVEC monolayers were incubated with or without different concentrations of propofol (10, 30 and 100 µmol l). MAIN OUTCOME MEASURES: Cell damage, metabolic activity, monolayer permeability, wound healing capacity, protein phosphorylation. RESULTS: Propofol did not alter the morphology, induce cell damage or influence metabolic activity of intact HUVEC cells. Permeability of a HUVEC monolayer was increased by propofol 100 µmol l (P < 0.05). Wound closure was inhibited by the addition of propofol 30 and 100 µmol l (P < 0.05 and P < 0.01). This effect was associated with increased phosphorylation of extracellular signal regulated kinases (Erk) 1/2 (30 and 100 µmol l; both P < 0.05) and decreased phosphorylation of Rho kinase (Rock) (100 µmol l; P < 0.05). CONCLUSION: Propofol does not damage intact endothelial cells, but increases permeability of an endothelial cell monolayer at high concentrations and inhibits wound closure in vitro. Further experimental and clinical in vivo research should be performed to clarify the influence of propofol on endothelial wound healing.

Signalling mechanisms in PAF-induced intestinal failure.

Capillary leakage syndrome, vasomotor disturbances and gut atony are common clinical problems in intensive care medicine. Various inflammatory mediators and signalling pathways are involved in these pathophysiological alterations among them platelet-activating factor (PAF). The related signalling mechanisms of the PAF-induced dysfunctions are only poorly understood. Here we used the model of the isolated perfused rat small intestine to analyse the role of calcium (using calcium deprivation, IP-receptor blockade (2-APB)), cAMP (PDE-inhibition plus AC activator), myosin light chain kinase (inhibitor ML-7) and Rho-kinase (inhibitor Y27632) in the following PAF-induced malfunctions: vasoconstriction, capillary and mucosal leakage, oedema formation, malabsorption and atony. Among these, the PAF-induced vasoconstriction and hyperpermeability appear to be governed by similar mechanisms that involve IP3 receptors, extracellular calcium and the Rho-kinase. Our findings further suggest that cAMP-elevating treatments - while effective against hypertension and oedema - bear the risk of dysmotility and reduced nutrient uptake. Agents such as 2-APB or Y27632, on the other hand, showed no negative side effects and improved most of the PAF-induced malfunctions suggesting that their therapeutic usefulness should be explored.

Doxycycline protects human intestinal cells from hypoxia/reoxygenation injury: Implications from an in-vitro hypoxia model.

Intestinal ischemia/reperfusion (I/R) injury is a grave clinical emergency and associated with high morbidity and mortality rates. Based on the complex underlying mechanisms, a multimodal pharmacological approach seems necessary to prevent intestinal I/R injury. The antibiotic drug doxycycline, which exhibits a wide range of pleiotropic therapeutic properties, might be a promising candidate for also reducing I/R injury in the intestine. To investigate possible protective effects of doxycycline on intestinal I/R injury, human intestinal CaCo-2 cells were exposed to doxycycline at clinically relevant concentrations. In order to mimic I/R injury, CaCo-2 were thereafter subjected to hypoxia/reoxygenation by using our recently described two-enzyme in-vitro hypoxia model. Investigations of cell morphology, cell damage, apoptosis and hydrogen peroxide formation were performed 24h after the hypoxic insult. Hypoxia/reoxygenation injury resulted in morphological signs of cell damage, elevated LDH concentrations in the respective culture media (P<0.001) and increased protein expression of proapoptotic caspase-3 (P<0.05) in the intestinal cultures. These events were associated with increased levels hydrogen peroxide (P<0.001). Preincubation of CaCo-2 cells with different concentrations of doxycycline (5µM, 10µM, 50µM) reduced the hypoxia induced signs of cell damage and LDH release (P<0.001 for all concentrations). The reduction of cellular damage was associated with a reduced expression of caspase-3 (5µM, P<0.01; 10µM, P<0.01; 50µM, P<0.05), while hydrogen peroxide levels remained unchanged. In summary, doxycycline protects human intestinal cells from hypoxia/reoxygenation injury in-vitro. Further animal and clinical studies are required to prove the protective potential of doxycycline on intestinal I/R injury under in-vivo conditions.

2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro 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 (HIE), but additional pharmacological strategies are being explored to further reduce the damage after perinatal asphyxia. The aim of this study was to evaluate whether 2-iminobiotin (2-IB) superimposed on hypothermia has the potential to attenuate hypoxia-induced injury of neuronal cells. In vitro hypoxia was induced for 7 h in neuronal IMR-32 cell cultures. Afterwards, all cultures were subjected to 25 h of hypothermia (33.5°C), and incubated with vehicle or 2-IB (10, 30, 50, 100, and 300 ng/ml). Cell morphology was evaluated by brightfield microscopy. Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS) was measured using fluorometric assays. Western blotting for PARP, Caspase-3, and the phosphorylated forms of akt and erk1/2 was conducted. To evaluate early apoptotic events and signaling, cell protein was isolated 4 h post-hypoxia and human apoptosis proteome profiler arrays were performed. Twenty-five hour after the hypoxic insult, clear morphological signs of cell damage were visible and significant LDH release as well as ROS production were observed even under hypothermic conditions. Post-hypoxic application of 2-IB (10 and 30 ng/ml) reduced the hypoxia-induced LDH release but not ROS production. Phosphorylation of erk1/2 was significantly increased after hypoxia, while phosphorylation of akt, protein expression of Caspase-3 and cleavage of PARP were only slightly increased. Addition of 2-IB did not affect any of the investigated proteins. Apoptosis proteome profiler arrays performed with cellular protein obtained 4 h after hypoxia revealed that post-hypoxic application of 2-IB resulted in a ≥ 25% down regulation of 10/35 apoptosis-related proteins: Bad, Bax, Bcl-2, cleaved Caspase-3, TRAILR1, TRAILR2, PON2, p21, p27, and phospho Rad17. In summary, addition of 2-IB during hypothermia is able to attenuate hypoxia-induced neuronal cell damage in vitro. Combination treatment of hypothermia with 2-IB could be a promising strategy to reduce hypoxia-induced neuronal cell damage and should be considered in further animal and clinical studies.

Insights into the neuroprotective mechanisms of 2-iminobiotin employing an in-vitro model of hypoxic-ischemic cell injury.

Several animal models have been used to simulate cerebral hypoxia-ischemia and suggested neuroprotective effects of the biotin analogue 2-iminobiotin (2-IB). The aims of this study were to employ a human in-vitro hypoxia model to confirm protective effects of 2-IB on neuronal cells, determine the optimal neuroprotective concentrations of 2-IB and scrutinize underlying cellular effects of 2-IB. Neuronal IMR-32 cells were exposed to hypoxia employing an enzymatic hypoxia system and were thereafter incubated with various concentrations of 2-IB (10 to 300ng/ml). Cell damage, metabolic activity and generation of reactive oxygen species were quantified using colorimetric/fluorometric lactate dehydrogenase (LDH), tetrazolium-based (MTS) and reactive oxygen species assays. Proteome profiling arrays were performed to evaluate the regulation of cell stress protein expression by hypoxia and 2-IB. Seven hours of hypoxia led to morphological changes in IMR-32 cultures, increased neuronal cell damage (P<0.001), reduction of metabolic activity (P<0.01) and enhanced reactive oxygen species production (P<0.05). Post-hypoxic application of 2-IB (30ng/ml) attenuated hypoxia-induced LDH release (P<0.05) and increased metabolic activity of IMR-32 cells (P<0.05), while reactive oxygen species production was only by trend decreased. Array-based protein expression profiling revealed that 2-IB attenuated the expression of several hypoxia-induced cell stress-associated proteins by more than 25% (pp38α, HIF2α, ADAMTS1, pHSP27, PON2, PON3 and p27). Hypoxia-induced neuronal cell damage can be simulated using the described in-vitro model. 2-IB inhibits hypoxia-mediated neurotoxicity most efficiently at 30ng/ml and the underlying mechanisms involve a downregulation of stress-associated protein expression. Our results suggest 2-IB as a potential drug for the treatment of perinatal hypoxia-ischemia.

Adverse effects of hydroxyethyl starch (HES 130/0.4) on intestinal barrier integrity and metabolic function are abrogated by supplementation with Albumin.

BACKGROUND: Volume resuscitation with hydroxyethyl starch (HES) is controversially discussed and we recently showed that HES perfusion impairs endothelial and epithelial intestinal barrier integrity. Here we investigated whether Albumin containing HES solutions are superior to HES alone in maintaining intestinal barrier function. METHODS: An isolated perfused model of the mouse small intestine was used to investigate the effects of: (i) 3 % Albumin (Alb), (ii) 3 % HES or (iii) 1.5 % HES/1.5 % Albumin (HES/Alb). Intestinal morphology, cell damage, metabolic functions, fluid shifts and endothelial/epithelial barrier permeability were evaluated. Potentially involved signaling mechanisms (Erk1/2, Akt and Stat5 phosphorylation) were screened. RESULTS: HES induced histomorphological damage (p < 0.01 vs. Alb), by trend elevated the amount of luminal intestinal fatty acid binding protein and reduced galactose uptake (p < 0.001 vs. Alb). Luminal and lymphatic flow rates were increased (p < 0.001 vs. Alb), while vascular flow was decreased (p < 0.001 vs. Alb) during HES perfusion. HES also increased the vascular to luminal FITC-dextran transfer (p < 0.001 vs. Alb), pointing towards a fluid shift from the vascular to the luminal and lymphatic compartments during HES perfusion. Addition of Alb (HES/Alb) reversed all adverse effects of HES (p < 0.05 vs. HES), restored barrier integrity (p < 0.05 vs. HES) and improved metabolic function of the intestine (p < 0.001 vs. HES; p < 0.05 vs. Alb). Mechanistically, HES/Alb perfusion resulted in an increased phosphorylation of Erk1/2 and Akt kinases (p < 0.001 vs. HES), while Stat5 remained unchanged. CONCLUSIONS: Albumin supplementation abrogates the adverse effects of HES in the intestine and underlying mechanism may function via phosphorylation of Erk1/2 and Akt. Albumin containing HES solutions are superior to HES alone and may improve the suitability of HES in the clinic.