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

Abstract 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.


| INTRODUC TI ON
Ischaemic organ injury is one of the major causes of death and disability. 1 Main events leading to ischaemic organ injury in the clinical routine are thrombotic or arteriosclerotic vascular occlusions and/or stenosis, shock, inflammation and major trauma. 2 The imbalance of oxygen supply and demand causes a temporal loss of function and change of cellular morphology within the affected tissue and can result in irreversible cell death. 3,4 To prevent further organ damage, an early restoration of blood flow is essential. 5 Paradoxically, reperfusion often amplifies the initial tissue damage, resulting in so called ischaemia/reperfusion (I/R) injury. I/R injury is characterized by the release of metabolic intermediates and reactive oxygen species, which provoke an inflammatory immune response. 2,6 The infiltration of leucocytes and the release of cytokines within the damaged tissue further promote a specific local microenvironment characterized by cell degradation, tissue remodelling and neoangiogenesis. 2,3,5 Due to their involvement in human immune defense, inflammation and tissue regeneration, cytokine production, antigen processing and cell transformation, circulating monocytes play a crucial role in I/R injury. [7][8][9][10] Although, high cell plasticity as well as their capability to differentiate into various subtypes under certain conditions are well-documented characteristics of monocytes, little is known about the precise role of monocytes in I/R injury, and thus, monocyte function in neoangiogenesis, tissue regeneration and inflammatory response. 7,10-12 It has been assumed that changing levels in oxygen supply may influence differentiation, maturation and function of circulating and migrating monocytes. 7,10 Moreover, it was shown that neoangiogenesis correlates positively with the number of monocytes/macrophages in various ischaemic injury models, including myocardial infarction and stroke. Whether adverse or protective influences of the monocyte/macrophage axis predominate in injured tissue has been controversially discussed in various studies. 7,13,14 Former experimental studies have assumed that ischaemic injury is significantly reduced in the absence of monocytes/macrophages, while other reports described positive effects of monocyte transplantation for tissue repair in cardiovascular and neural ischaemic disease and suggested a high cell plasticity, pluripotency and resilience of monocytes with a high regenerative potential. 7,9 Especially the pluripotency of monocytes and their putative capability for further cell differentiation, cell-cell transformation and neoangiogenesis has been discussed controversially. [7][8][9]11 It has also been hypothesized that the monocyte phenotype and thus release of cytokines, proangiogenic factors and enzymes change dynamically from a proteolytic and pro-inflammatory M1 phenotype to an anti-inflammatory and regenerative M2 phenotype during I/R injury. 15 The balance between proteolytic and regenerative monocyte function throughout the temporospatial course of ischaemia and reperfusion could potentially play a crucial role in the determination of final tissue injury and subsequent tissue regeneration. Therefore, a thorough understanding of the cellular and molecular mechanisms of I/R injury and monocyte function is essential for the development of novel treatment strategies for I/R injury and related diseases. 15 Although, most studies have investigated the influence of short, isolated hypoxic episodes on monocyte phenotype and function, the current in vitro study was designed to analyse the whole period of initial ischaemia and following reperfusion, representing I/R injury in vivo. Accordingly, we employed an in vitro monocyte cell culture model representing the two phases (ischaemia and reperfusion) of I/R injury to analyse the cellular and molecular effects of I/R on monocytes. Further, the effects of culture media from human monocytes that were subjected to I/R in vitro (conditioned culture media) were evaluated on endothelial tube formation as indicator for angiogenesis as well as endothelial scratch assays for endothelial cell migration and recovery.

| Ethics
The study was approved by the local ethics committee of the University Medical Center Schleswig-Holstein, Kiel, Germany (protocol identification: D519/18 and D518/13). All procedures were in accordance with the Helsinki Declaration of 2000.

| Experimental setting
Briefly, peripheral blood monocytes were obtained from leukapheresis products from the Department of Transfusion Medicine (University Hospital Schleswig-Holstein, Kiel, Germany) and isolated by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation and selective adherence to cell culture surfaces according to established protocols. 16 Monocytes (160 000 cells/cm 2 ) were cultured in RPMI-1640 medium containing 10% human AB-serum, 2 mmol/L L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. For all experiments, cells were allowed to adhere to the respective cell culture plates overnight. In vitro I/R was induced by using our previously described two-enzyme system with minor modifications. 17,18 Briefly, for induction of ischaemia, cell culture medium was exchanged by medium containing 240 U/mL catalyse (Sigma-Aldrich) and 4 U/ mL glucose oxidase (Sigma-Aldrich), resulting in a rapid decrease in partial pressure of oxygen (pO 2 ) below 10 mm Hg. Moreover, using this system pH and glucose concentration in the culture medium decrease, resembling well the in vivo situation of I/R. 18,19 Hypoxic conditions were verified by using a tissue oxygen pressure monitor (LICOX CMP Oxygen Catheter; Integra). Pretests considering the increase in ischaemia-inducible factors in monocytes and progression of cell damage during I/R were performed to evaluate the optimal duration of ischaemia (3 hours) and reperfusion (24 hours beginning of ischaemia, T(0) directly after ischaemia and T(24) the end of reperfusion ( Figure 1). Ischaemia was terminated by replacing the hypoxic medium with standard culture medium, resulting in an immediate reoxygenation as well as increase in pH and glucose concentration in the cultures. Control experiments were performed under non-ischaemic conditions by omitting the hypoxia inducing enzymes (glucose oxidase and catalase) from the respective culture media ( Figure 1).

| LDH cytotoxicity assay
The colorimetric Cytotoxicity Detection Kit PLUS (Roche) was used for the quantification of cell damage by measuring lactate dehy-

| Protein isolation and Western blotting
Protein isolation and Western blotting were performed as described earlier. 20 Briefly, 28 μg of protein concentrate was mixed Signaling Technology), STAT5 (1:1000; R&D Systems) and phospho-STAT5 (1:1000; R&D Systems). Signals from peroxidase-conjugated secondary antibodies (anti-rabbit; 1:20 000; Cell Signaling Technology) were detected using the ECL kit (ECL-Plus Western blotting Detection Reagents; Amersham Pharmacia Biotech). Membranes were exposed to X-ray-films and intensities of the respective protein bands were analysed using the imagej software 1.41 (National Institutes of Health NIH).

| Flow cytometry
To analyse putative functional and phenotype differentiation under

| Immunofluorescence
Monocytes were seeded in 12-well plates containing glass coverslips

| Proteome profiling arrays
Proteome profiling was performed using human angiogenesis arrays (ARY007; R&D Systems) and human XL cytokine arrays (ARY022B; R&D Systems) according to manufacturer's protocol provided with the assay kit. After culturing monocytes as described above, 200 μg of pooled samples of protein concentrate (intracellular proteins) and 500 µL (cytokine array) or 600 µL (angiogenesis array) of pooled cell culture medium (secreted proteins) was applied to the respective array membrane (angiogenesis array: N = 8; XL cytokine array: N = 13).
Expression levels of 55 angiogenesis associated proteins and 160 cytokines were evaluated by densitometric analyses of the arrays using the imagej 1.41 software (NIH). For each spot on the membrane, the optical density was measured and the cut-off signal level was set to 10% of the respective reference spots. Only regulations of more than 20% were considered as relevant and were further analysed.

| Isolation of human umbilical vein endothelial cells (HUVEC)
Human umbilical vein endothelial cells were isolated from umbilical cords as described previously 21 and cultured in endothelial cell growth medium ECGM (PromoCell) supplemented with 4 μL/mL of endothelial cell growth supplement, 0.1 ng/mL epidermal growth factor, 1 ng/ mL basic fibroblast growth factor, 90 μg/mL heparin, 1 μg/mL hydrocortisone (all from PromoCell) and 10% foetal bovine serum (Thermo Fisher). The cells were maintained in a humidified atmosphere (5% carbon dioxide/95% air) at 37°C. For further experiments, cells were detached using a mild cell detachment solution (Accutase; Innovative Cell Technologies) and seeded in respective cell culture plates.

| Endothelial tube formation assays
Human umbilical vein endothelial cells were harvested as described to test the hypothesis that MMP-9 inhibition leads to significant more HUVEC tube formation.

| Endothelial scratch assays
To assess the effect of cell culture media from monocytes that were

| Gelatin zymography
Gelatin zymography was performed as previously described. 24 Briefly, 10 µL of cell culture supernatants was loaded and separated on 7% SDS polyacrylamide gels (containing 1 mg/mL gelatin) under non-reducing conditions. After electrophoresis, gels were first incubated in 2.5% Triton X-100 for 30 minutes to remove SDS, followed by incubation in Tris-HCl (50 mmol/L, pH 7.5) containing CaCl 2 (5 mmol/L) and ZnCl 2 (1 mmol/L) overnight at 37°C. After Coomassie blue staining, white bands of lysis indicated digestion of gelatin by matrix metalloproteinases (MMPs). Densitometric analysis was performed using the imagej 1.41 software (NIH).

| Statistical analyses
All values are expressed as mean ± standard deviation (SD).
Categorical variables are presented as frequency distributions (n) and percentages (%). Data were analysed with graphpad prism ver-

| I/R in vitro increases resilience of monocytes and induces phosphorylation of prosurvival molecules without major changes in monocyte subtypes
To 0.47 ± 0.23 au; P < .001, Figure 2C) and Akt (I/R: 0.62 ± 0.40 au; normoxia: 0.25 ± 0.14 au; P < .01, Figure 2C). There was also a by trend represented in the investigated monocyte population (Appendix S1).
After being subjected to I/R, there was only a minor shift towards the CD14+/CD16− monocyte subtype (Appendix S1).

| I/R in vitro results in major changes of cytokine release and angiogenesis-related protein expression
Cytokine proteome profiler arrays were performed with total cell protein of monocytes (intracellular cytokines) as well as cell culture supernatants (secreted cytokines), as seen in Figure 3A

| Cell culture media derived from monocyte cultures subjected to I/R in vitro suppress angiogenesis and endothelial migration in vitro
Tube formation assays for angiogenesis were performed with human umbilical vein cells (HUVEC) that were cultured on Matrigel-coated dishes. Endothelial tube formation assays showed that cell culture media from monocytes that were subjected to I/R in vitro (I/R con-

F I G U R E 3
Profiling of secreted and intracellular cytokines. A, Analysed cytokines and location on the respective array membrane. B, Red font and rectangles represent intracellular and secreted cytokines that were more than two-fold upregulated by I/R, while green font and rectangles indicate a more than two-fold downregulation. C, Top ten most regulated proteins. All proteins are presented as duplicate spots on the respective array membrane. For a detailed description of the array proteins please refer to Appendix S2 demonstrated less HUVEC migration into the scratched area compared to monocytes that were subjected to normoxia (CoNM) (CoIRM: 1.14 ± 0.14; CoNM/CM: 1.95 ± 0.18, P < .05; Figure 5B).

| Secretome from monocytes subjected to I/R in vitro contain increased activity of MMP-9
Matrix metalloproteinases (MMPs) play a crucial role in remodelling, neoangiogenesis and wound healing. 25 In order to further To determine the viability of monocytes after I/R, apoptosis and cell damage were analysed. Interestingly, 3 hours of ischaemia followed by 24 hours of reperfusion did not influence apoptosis but resulted in significantly lower LDH levels in the culture supernatants compared to normoxia, suggesting an increased resilience of monocytes after I/R. Strong resistance of monocytes to I/R is essential to maintain specific cellular functions within the hypoxic microenvironment which is characterized by inflamed and damaged tissues enriched with cytotoxic inflammatory mediators and low levels of oxygen as well as vital substrates. 31 In general, monocytes possess a limited life span and undergo spontaneous apoptosis during circulation. 32 It is a well-known fact that in the presence of specific pro-inflammatory cytokines (TNF-α, GM-CSF) monocytes can interrupt their apoptotic programme and promote cellular survival by activation of the prosurvival kinase Akt. 32,33 Roiniotis et al demonstrated that monocytes exposed to 24 hours of hypoxia (without reperfusion) undergo a metabolic shift towards anaerobic glycolysis and show reduced apoptosis by activation of Akt. 30 Accordingly, the positive effect of I/R on monocyte survival in our study was also associated with an increased phosphorylation of the prosurvival kinase Akt. In addition, we found an increased phosphorylation of Erk1/2, which has been suggested to also function as prosurvival kinase in various tissues and conditions. 34,35 The results of the necrosis and apoptosis assays did not fully conditions. [43][44][45] Especially, TNF-α and Il-6 mediated processes have been closely associated with ambivalent effects (detrimental vs beneficial) on tissue injury and tissue renewal in I/R injury. Accordingly, several authors described a protective effect for low levels of TNF-α for the myocardium but also an increase in myocardial injury, contractile dysfunction, hypertrophy and fibrosis for high levels of TNFα after I/R injury. 43,44 Likewise, IL-6 activity and its pro-inflammatory pathways, depending on the cellular and temporal context, exert a wide range of diverse and competing effects including anti-apoptotic, proliferative, growth-inhibitory and differentiation-inducing effects during I/R injury. 45 Accordingly, the "Janus face" of inflammation after I/R injury is best exemplified by the presumption that TNF-α and Il-6 may contribute to tissue damage or protection depending on the temporospatial context of its expression. 45 Our current results demonstrated that monocytes secret high levels of TNF-α and IL-6 during the first 24 hours of I/R pointing towards a putative predominantly detrimental role of monocytes in the initial phase of I/R injury. Therapeutic approaches targeting the inflammatory response of tissue injury are therefore promising treatment strategies attenuating the detrimental effects of I/R injury but further research on molecular mechanisms of the identified cytokines and the temporospatial context of their expression is necessary. [46][47][48][49][50] Interestingly, proteome profiling arrays revealed that the release We also show that culture media from monocytes that were subjected to I/R in vitro contain increased MMP-9 activity. MMP-9, a gelatinase that contributes to degradation of extracellular matrix, is highly expressed in disturbed wound healing and remodelling processes. However, inhibiting MMP-9 activity or addition of recombinant MMP-9 to HUVEC cultures did not affect angiogenic parameters.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are available on request due to privacy/ethical restrictions.