In pathological situations such as ischemia-reperfusion and acute respiratory distress syndrome, reactive oxygen species (ROS) are produced by different systems which are involved in endothelial cells injury, ultimately leading to severe organ dysfunctions. The aim of this work was to study the effect of ROS produced by hypoxanthine-xanthine oxidase (Hx-XO) on the adhesion of human umbilical vein endothelial cells (HUVEC) and on the signaling pathways involved. Results show that Hx-XO-derived ROS induced an increase in HUVEC adhesion in the early stages of the process (less than 30 min), followed by a decrease in adhesion in the later stages of the process. Interestingly, Hx-XO-derived ROS induced the same biphasic effect on the phosphorylation of the focal adhesion kinase (FAK), a nonreceptor tyrosine kinase critical for cell adhesion, but not on ERK1/2 phosphorylation. The biphasic effect was not seen with ERK1/2 where a decrease in phosphorylation only was observed. Wortmannin, a PI3-kinase inhibitor, inhibited ROS-induced cell adhesion and FAK phosphorylation. Orthovanadate, a protein tyrosine phosphatase inhibitor, and Resveratrol (Resv), an antioxidant agent, protected FAK and ERK1/2 from dephosphorylation and HUVEC from ROS-induced loss of adhesion. This study shows that ROS could have both stimulatory and inhibitory effects on HUVEC adhesion and FAK phosphorylation and suggests that PI3-kinase and tyrosine phosphatase control these effects.
In pathological situations such as ischemia-reperfusion and acute respiratory distress syndrome, large amounts of reactive oxygen species (ROS) are produced by different enzymatic systems such as xanthine oxidase, mitochondria, and the phagocyte NADPH oxidase NOX2. These ROS are believed to be involved in endothelial cell injury leading to severe tissue and organ dysfunctions [
Oxidative stress, resulting from an imbalance between oxidant production and antioxidant systems, has been reported to induce alterations in signaling pathways leading to modulation of cellular functions, apoptosis, and necrosis [
Phosphorylation and dephosphorylation events play a critical role in the signal transduction pathways that regulate various processes in living cells. ROS have been reported in various cells to alter the phosphorylation of several key proteins involved in signaling pathways [
Hank’s balanced salt solution, with or without Ca2+ and Mg2+ (HBSS or HBSS wo), was obtained from GIBCO (Invitrogen, Paisley, UK). Bacto-gelatin was from DIFCO (Detroit, Michigan, USA). Reagents for cell culture were supplied by GIBCO (Invitrogen, Paisley, UK); 100 mm dishes and 6- and 24-well plates were from Costar (Polylabo, Strasbourg, France). Mouse monoclonal FITC-conjugated antihuman factor VIII, mouse monoclonal antibody anti-human focal adhesion kinase (anti-p125FAK), and rabbit polyclonal antibodies anti-human ERK1 and anti-ERK2 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibody anti-human active phosphorylated ERK1/2 was from New England Biolabs (Beverly, MA). Mouse monoclonal anti-phosphotyrosine (anti-Tyr(P)) antibody was from Upstate Biotechnology (Lake Placid, NY). The rainbow markers (high range), sheep anti-mouse IgG conjugated horseradish peroxidase antibody, and ECL Western blot detection system were from Amersham Biosciences (GE Healthcare Europe Gmbh, Orsay, France). Alkaline phosphatase conjugated goat anti-rabbit IgG antibody was from Jackson Laboratories. Nitrocellulose membranes were from Schleicher and Schuell (Dassel, Germany). PD098059, wortmannin, and LY294002 were from Calbiochem (La Jolla, CA, USA). Protein G Sepharose 4 fast flow media were provided by Amersham Biosciences (GE Healthcare Europe Gmbh, Orsay, France). Calcein-acetoxymethyl (calcein-AM) was purchased from Molecular Probes (Invitrogen, Paisley, UK). Resveratrol was kindly donated by Dr. Bagchi (Creighton University School of Pharmacy and Allied Health Professions, Omaha, USA). All other products were from Sigma (St. Louis, Mo, USA).
Endothelial cells from human umbilical cords veins (HUVECs) were harvested by 0.05% collagenase treatment for 15 minutes at 37°C, as previously described [
HUVECs were washed with HBSS and incubated with hypoxanthine (Hx, 2 × 10−4 M) and xanthine oxidase (XO, 4.5 mU/mL) for various times (5 to 90 minutes) in HBSS buffer. Hypoxanthine oxidation by xanthine oxidase, generated around 3 nmol/mL/minutes of superoxide anion (
Adhesion to the matrix was measured with a Calcein-AM assay as previously described [
Control or Hx-XO treated HUVECs in 100 mm petri dishes were lysed by incubation for 30 minutes in 500
Immunoprecipitated proteins or whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 9% polyacrylamide gels using standard techniques [
All experiments were performed in triplicate and repeated at least three times. Results are expressed as mean ± standard deviation. Data were compared using analysis of variance (ANOVA) followed by a Fischer test.
To determine the consequences of oxidative stress on HUVECs adhesion to the matrix, HUVECs were incubated for 10, 20, 30, 60, or 90 minutes with Hx-XO, a ROS generating system, and adhesion was measured using a Calcein-AM probe. Results show that HUVECs adhesion was significantly increased within the first 20 min of treatment with Hx-XO-derived ROS (Figure
Hx-XO-derived ROS induced time-dependent biphasic HUVEC adhesion. HUVECs monolayers were treated with Hx-XO (4.5 mU/mL) for different time periods and then thoroughly washed to remove nonadherent cells. Cells adherent to the matrix were loaded with 2.5
As FAK plays a pivotal role in adhesion mechanisms [
Hx-XO-derived ROS induced time-dependent biphasic FAK tyrosine phosphorylation. HUVECs were treated with Hx-XO (4.5 mU/mL) for the indicated times after which cells were harvested and the soluble fraction analysed for FAK activation. (a) FAK was immunoprecipitated using a monoclonal antibody, and its phosphorylation state and expression were analysed by Western blot (WB) with anti-phosphotyrosine and anti-FAK antibodies. (b) Phosphorylated FAK from different experiments was quantified by densitometry and corrected for the amount of FAK which was quantified by densitometry. Results are expressed as mean ± SEM,
Hx-XO-derived ROS induced time-dependent dephosphorylation of ERK1/2. HUVECs were treated with Hx-XO (4.5 mU/mL) for the indicated times after which cells were harvested and the soluble fraction analysed for ERK1/2 phosphorylation. Western blot analysis with anti-phospho-ERK1/2 was performed as described in Materials and Methods. Total ERK1/2 proteins levels at all times points were equivalent as measured by immunoblot of the same membranes using antibodies against total ERK1/2. (b) Phosphorylated ERK1/2 from different experiments was quantified by densitometry and corrected for the amount of ERK1/2 which was quantified by densitometry. Results are expressed as mean ± SEM,
Inhibition of both PI3-kinase activity and the consequent Akt serine phosphorylation accelerated oxidative stress-induced apoptosis [
Effect of wortmannin, a PI-3 kinase inhibitor on the early phase of FAK phosphorylation and HUVECs adhesion. HUVECs were preincubated for 20 minutes with PI-3 kinase inhibitor wortmannin (100 nM) and then treated with Hx-XO (4.5 mU/mL) for 15 minutes. (a) FAK was immunoprecipitated using a monoclonal antibody and its phosphorylation state and expression were analysed by Western blot with anti-phosphotyrosine and anti-FAK antibodies. (b) Phosphorylated FAK from different experiments was quantified by densitometry and corrected for the amount of FAK which was quantified by densitometry. (c) HUVECs adhesion was measured after 15 minutes of treatment with Hx-XO by loading cells with 2.5
The dephosphorylation of FAK and ERK1/2 in the late phase of ROS treatment suggests the involvement of a phosphatase which could be activated by a long-term ROS treatment. We then assessed the effect of orthovanadate, a tyrosine phosphatase inhibitor, on ROS-induced dephosphorylation of FAK and ERK1/2. Results show that incubation of HUVECs with 100 mM orthovanadate prior to Hx-XO restored and even increased the level of FAK and ERK1/2 phosphorylation in HUVECs (Figures
Effect of orthovanadate, a tyrosine phosphatase inhibitor on ROS-induced dephosphorylation of FAK and ERK1/2. HUVECs were preincubated for 30 min with 100 mM of orthovanadate, washed twice, and then treated with Hx-XO (4.5 mU/mL) for the indicated times. (a) The cells were harvested and the soluble fraction was analysed for FAK activation. FAK was immunoprecipitated using a monoclonal antibody, and its phosphorylation state and expression were analysed by Western blot with anti-phosphotyrosine and anti-FAK antibodies. (b) Cells were harvested and the soluble fraction was analysed by Western blot with anti-phospho-ERK1/2. Total ERK1/2 proteins levels at all times points were equivalent as measured by immunoblot of the same membranes using antibodies against total ERK1/2 coupled to alkaline phosphatase substrate. (c) HUVECs adhesion was determined by loading cells with Calcein-AM as described in Materials and Methods after 60 minutes of Hx-XO treatment. Results are expressed as % of control without Hx-XO (mean ± SEM,
To verify that HUVECs alterations were due to oxidative injury caused by oxidative stress induced by the Hx-XO system, HUVECs were pretreated with the antioxidant molecule, trans-3,4′,5-trihydroxystilbene (Resveratrol) [
Resveratrol protected HUVEC from ROS-induced loss of adhesion and from FAK/ERK dephosphorylation. Cells were preincubated with 5 and 10
In this study, we have demonstrated that oxidative stress, produced by the hypoxanthine-xanthine oxidase (Hx-XO) system, modulated both HUVECs adhesion and FAK phosphorylation. During the first twenty minutes of ROS exposure, HUVECs adhesion was significantly increased in parallel with FAK phosphorylation. This early ROS-induced adhesion and FAK phosphorylation were dependent on PI3-kinase. Later, we observed a progressive decrease in cell adhesion, concomitant with a decreased in FAK and ERK1/2 phosphorylation. These latter dephosphorylation events could be related to the ROS-induced activation of one or several tyrosine phosphatases. These events are prevented by the antioxidant agent Resveratrol. These results demonstrated that ROS can have dual contrasting effects on HUVECs adhesion to the matrix depending on the length of exposure. This phenomenon is critical for HUVEC functioning and survival.
ROS induced a biphasic effect on cell adhesion and FAK phosphorylation, following a transient increase during the first 20 minutes, a dephosphorylation of FAK and a decrease of cell adhesion occurred in a time-dependent manner. The early ROS-induced increase of FAK phosphorylation was regulated by PI3-kinase as shown by its inhibition by wortmannin. These results suggest that ROS can modulate PI3-kinase directly or indirectly as previously described [
We also showed the involvement of tyrosine phosphatases in the oxidative stress regulation of these kinases. The very quick-acting dephosphorylation of ERK1/2 suggests the activation of constitutive phosphatases by ROS [
The protective effect of polyphenolic antioxidant agent such as Resveratrol [
In our model of time-dependent oxidative stress we propose that initially the ROS-induced increase of FAK tyrosine phosphorylation is dependent on PI3-kinase. This leads to continued HUVECs adhesion in spite of a rapid dephosphorylation of ERK1/2. In the late phase of this process, ROS-induced activation of different phosphatases may be involved in ERK1/2 as well as FAK dephosphorylation, leading to a decrease in HUVECs adhesion. Kinetic studies should be performed to further investigate the effect of ROS on cell function.
In conclusion, our data has demonstrated a time-dependent biphasic effect of ROS on FAK phosphorylation and endothelial cells adhesion. Under these conditions, FAK phosphorylation plays a critical role in regulating cell adhesion to the matrix and was dependent on equilibrium between PI3-kinase and protein tyrosine phosphatases. Such mechanisms could occur during different pathological inflammatory conditions as a result of the release of ROS in proximity to endothelial cells.
Anti-phosphotyrosine antibody
Extracellular signal regulated kinase
Focal adhesion kinase
Hypoxanthine
Immunoprecipitation
Resveratrol
Reactive oxygen species
Western blot
Xanthine oxidase.
The authors declare that they have no competing interests.