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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2014 Oct 15;7(11):7342–7350.

Ulinastatin inhibits oxidant-induced endothelial hyperpermeability and apoptotic signaling

Guicheng Li 1,2,*, Tao Li 2,*, Yunfeng Li 2,*, Shumin Cai 1, Zhiming Zhang 3, Zhenhua Zeng 1,4, Xingmin Wang 5, Youguang Gao 1,6, Yunfeng Li 2,*, Zhongqing Chen 1
PMCID: PMC4270631  PMID: 25550770

Abstract

Oxidants are important signaling molecules known to increase endothelial permeability. Studies implicate reactive oxygen species (ROS) and the intrinsic apoptotic signaling cascades as mediators of vascular hyperpermeability. Here we report the protective effects of ulinastatin, a serine protease inhibitor with antiapoptotic properties, against oxidant-induced endothelial monolayer hyperpermeability. HUVECs were respectively pretreated with 10,000 and 50,000 u/l ulinastatin, followed by stimulation of 0.6 mM H2O2. Monolayer permeability was determined by transendothelial electrical resistance (TER); Mitochondrial release of cytochrome c was determined by enzyme-linked immunosorbent assay; Caspase-3 activity was measured by fluorometric assay; Adherens junction protein β-catenin was detected by immunofluorescense staining; Ratio of cell apoptosis was evaluated by Annexin-V/PI double stain assay; Mitochondrial membrane potential (Δψm) was determined with JC-1; Intracellular ATP content was assayed by a commercial kit; Bax and Bcl-2 expression were estimated by western blotting; Intracellular reactive oxygen species (ROS) level was measured by DCFH-DA. H2O2 exposure resulted in endothelial hyperpermeability and ROS formation (P < 0.05). The activation of mitochondrial intrinsic apoptotic signaling pathway was evidenced from BAX up-regulation, Bcl-2 down-regulation, mitochondrial depolarization, an increase in cytochrome c release, and activation of caspase-3 (P < 0.05). UTI (50,000 u/l) attenuated endothelial hyperpermeability, ROS formation, mitochondrial dysfunction, cytochrome c release, activation of caspase-3, and disruption of cell adherens junctions (P < 0.05). Together, these results demonstrate that UTI provides protection against vascular hyperpermeability by modulating the intrinsic apoptotic signaling.

Keywords: Endothelial barrier, adherens junctions, apoptosis, oxidative stress, mitochondria

Introduction

An increase in vascular permeability is a key hallmark of inflammation and has been implicated in the pathophysiology of many disease states including acute lung injury, ischemia-reperfusion injury, and sepsis [4,17,18,39]. Oxidants including superoxide and hydrogen peroxide (H2O2) generated by activated neutrophils and endothelial cells in response to inflammatory stimuli increase endothelial permeability [18,22,34].

Studies have demonstrated the involvement of mitochondrial “intrinsic” apoptotic signaling in endothelial cell dysfunction and vascular permeability [8,30,35]. Mitochondrial regulation of apoptosis is mediated through the release of cytochrome c, apoptosis-inducing factor (AIF), and second mitochondrial-derived activator of caspases (smac), ultimately caspases activation [5,26]. Caspase-3 has been shown to cleave β-catenin, thereby disrupting the vascular endothelial (VE)-cadherin-β-catenin complex, which serves as a key role of cell-cell adhesion in endothelial cells. Damage of (VE)-cadherin-β-catenin complex may lead to microvascular hyperpermeability [7,25,27].

Ulinastatin (UTI), a serine protease inhibitor, is mainly used in the treatment of sepsis, shock, pancreatitis, and ischemia-reperfusion injury [16,20,38]. Moreover, recent studies have revealed the protective effects of ulinastatin attenuate oxidation and apoptosis [12,13,31]. In present study, we hypothesized that UTI would improve oxidant-induced endothelial hyperpermeability by regulating the intrinsic apoptotic signaling cascade.

Materials and methods

Monolayer permeability

Primary human umbilical endothelial cell (HUVECs; ScienCell, USA) were maintained in DMEM/F12 containing 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2 -95% air. In all experiments, HUVECs were grown to 90% confluence and starved of serum for 12 hours before being stimulated with H2O2. The monolayers were respectively pretreated with 10,000 u/L and 50,000 u/L of UTI for 60 min, followed by stimulation of 0.6 mM H2O2. DMEM/F12 was used as vehicle control. Finally, endothelial permeability was measured 60 min at 15min-intervals.

Transendothelial electrical resistance (TER)

Transendothelial electrical resistance (TER) of HUVECs monolayer was determined using STX2 electrode and EVOM2 meter according to the instruction manual of manufacture (World Precision Instruments, Sarasota, FL, USA) [32]. HUVECs were seeded with number of 1 × 105/cm2 on fibronectin-coated, 6.5 mm Transwell filters (0.4 mm pore size) and were used until full confluence. Resistance values of multiple Transwell inserts of an experimental group were measured sequentially and the mean was expressed in the common unit (Ω cm2) after subtraction of the value of a blank cell-free filter.

Measurement of cytosolic cytochrome c

Cytosolic cytochrome c content was estimated with a cytochrome c ELISA kit. The cell homogenates were centrifuged (10,000 × g for 60 min at 4°C), and the supernatant (cytosolic fraction) was collected and subjected to protein estimation (bicinchoninic acid [BCA] method). Then, the samples were treated with a conjugate reagent, transferred to a cytochrome c antibody-coated microwell plate, and incubated at room temperature for 60 min. The wells were washed and treated with a substrate and incubated for 30 min, followed by addition of a stop solution. The optical density was read at 450 nm using an automatic microplate reader (SpectraMax M5; Molecular Devices, Sun-nyvale, CA, USA). A serial dilution of cytochrome c calibrator was subjected to the assay along with the samples, the values were plotted, and the concentration of cytochrome c was calibrated from the standard curve.

Measurement of caspase-3 activity

Caspase-3 activity was determined using a caspase-3 activity assay kit (Sigma, USA). The cells were lysed in caspase-3 sample lysis buffer provided in the kit. The homogenates were centrifuged at 10,000 g, and the supernatant was collected for protein estimation (BCA method) and for caspase-3 assay. The cell lysates were exposed to the DEVD substrate conjugate provided in the kit. The sample was measured in an automatic microplate reader (SpectraMax M5) at excitation 400 nm and emission 505 nm.

Measurement of cell apoptosis

Cell apoptosis were detected by an Annexin V-FITC apoptosis detection kit (BD Biosciences, USA). After induction with H2O2, cells were washed twice with PBS and suspended in 1× binding buffer at a concentration of approx 1 × 105 cells/ml. 5 μl of FITC-Annexin V and 10 μl of propidium iodide (PI, 50 μg/ml, Sigma) was added to cell suspension. After incubation at room temperature for 20 minutes at dark, the fluorescence of the cells was determined immediately with a flow cytometer (Becton Dickinson FACScan, San Jose, CA).

Measurement of mitochondrial membrane potential

The mitochondrial membrane potential (Δψm) was determined by flow cytometry, using the potential-sensitive fluorescent dye JC-1. The color of this dual-emission probe changed from red-orange to green as the mitochondrial membrane turned depolarized. The JC-1 (5 μmol/L) was loaded onto RLMVECs for 15 min at 37°C. The stained cells were washed with PBS, and analyzed by flow cytometry (Becton Dickinson FACScan). A minimum of 10,000 cells per sample was analyzed. JC-1 monomers emit at 527 nm and “J-aggregates” emit at 590 nm. The percentage of cells with abnormally low Δψm (green fluorescence) was determined.

Measurement of cellular ATP

Intracellular ATP was determined by a luciferase-based assay (CellTiter-Glo, Madison, WI), according to the manufacturer’s recommendation. After adding 100 μL of the CellTiter-Glo reagent to 100 μL of cells suspension containing 10,000 cells in each well of a standard opaque-walled 96-well plate, the plates were allowed to incubate at room temperature for 10 min and the luminescence was recorded in an automatic microplate reader (SpectraMax M5).

Measurement of ROS levels

Intracellular ROS levels were assessed usingDCFH-DA probe (Sigma, USA). Cells were treated with DCFH-DA (10 μM), following sham/burn serum, for 20 mins at 37°C. After incubation, the cells were washed and analyzed using an automatic microplate reader (Spectra Max, M5). The relative intensity of DCF fluorescence was determined at a wavelength of 535 nm as compared to sham group cells.

Measurement of Bcl-2 and Bax expression

The cells were homogenated and analyzed for Bcl-2 and Bax by western blotting. Protein concentrations were determined using the BCA method. An equal amount of protein was loaded onto 10% sodium dodecyl sulphate polyacrylamide gel for electrophoresis. After electrophoresis, proteins were electroblotted onto polyvinylidene fluoride membranes and blotted with primary antibodies against smac (Abcam, UK). Membranes were then incubated with the horseradish peroxidase-tagged secondary antibody (Tianjin Sungene Biotech Co., Ltd. Tianjin, China), and protein expression was detected using an enhanced chemiluminescence reagent.

Immunofluorescence staining for β-catenin

HUVECs were rinsed quickly with ice-cold PBS, and fixed with 4% paraformaldehyde. Cells were permeabilized for 30 min with 0.1% Triton X-100 and then incubated with β-catenin antibody (Abcam, UK) for 24 h at 4°C, followed by incubation with FITC-labeled secondary antibody (Tianjin Sungene Biotech Co.) for 2 h at room temperature. Cell nuclei were labeled with Hoechst33258 (Sigma, USA). Cells were examined using a confocal microscope (LSM780; Zeiss Microsystems, Jena, Germany). Images were collected using the software (Zeiss Microsystems, Germany).

Statistical analysis

All variables are presented as Means ± SD Differences between groups were determined using one-way ANOVA with the LSD multiple-comparison test and Student’s t-test when appropriate. Values were considered significant when P < 0.05.

Results

UTI attenuates oxidant-induced endothelial hyperpermeability

As shown in Figure 1, oxidant-induced monolayer hyperpermeability was evidenced by an obvious decrease in the TER of the cell monolayer treated with H2O2 compared to that of the cell monolayer in the control group; the hyperpermeability was significantly attenuated by pretreatment with 50,000 u/l of UTI but not 10,000 u/l of UTI. At 60 min, TER (normalized) of the HUVECs monolayer decreased from 1.03 ± 0.04 in the control group to 0.68 ± 0.07 in the vehicle group (Figure 1; P < 0.05); however, the TER were improved to 0.73 ± 0.05 (Figure 1; P > 0.05) and 0.90 ± 0.06 (Figure 1; P < 0.05) respectively by 10,000 u/l and 50,000 u/l of UTI.

Figure 1.

Figure 1

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UTI attenuates oxidant-induced endothelial hyperpermeability. HUVECs were respectively pretreated with 10.000 and 50,000 u/l of UTI for 60 min, followed by H2O2 stimulation. The TER was recorded for 60 min at 15-min intervals. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

UTI prevents oxidant-induced cytochrome c release

Cytosolic cytochrome c levels were markedly increased in the vehicle group (49.3 ± 7.5 ng/mg protein) compared with the control group (18.1 ± 3.9 ng/mg protein) (Figure 2; P < 0.05). In contrast, cytochrome c levels in the UTI (50,000 u/l) treatment group were significantly lower (31.5 ± 6.1 ng/mg protein) than those in the vehicle group (Figure 2; P < 0.05). The findings indicate that UTI attenuated oxidant-induced release of cytochrome c from mitochondria to the cytosol.

Figure 2.

Figure 2

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UTI inhibits oxidant-induced cytochrome c release. Cytosolic cytochrome c levels were estimated using a cytochrome c ELISA kit. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

UTI decreases oxidant-induced caspase-3 activation

Caspase-3 activity was obviously increased in the vehicle group compared to the control group (309% ± 42%, Figure 3; P < 0.05), which was prevented by UTI (50,000 u/l) treatment (192% ± 35%, Figure 3; P < 0.05).

Figure 3.

Figure 3

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UTI decreases oxidant-induced caspase-3 activation. The caspase-3 activity was measured using the caspase-3 fluorometric assay kit. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

UTI inhibits oxidant-induced disruption of adherens junctions

Control cells showed strong and continuous β-catenin immunofluorescence at the cell-cell junctions, indicating an intact cell barrier. In contrast, treatment with H2O2 disrupted the cell junctions among cells as evidenced by the irregular and scattered β-catenin fluorescence observed. However, the alterations induced by H2O2 were improved by UTI (50,000 u/l) pretreatment (Figure 4).

Figure 4.

Figure 4

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UTI inhibits oxidant-induced disruption of endothelial cell adherens junction. Adherens junction protein β-catenin was detected by immunofluorescense staining. Red arrow: disruption of the junctions evidenced by irregular and scattered β-catenin fluorescence.

UTI reduces oxidant-induced endothelial cell apoptosis

Rates of cell apoptosis were markedly increased in the vehicle group (19.4% ± 4.7%) compared with the control group (5.1% ± 1.1%) (Figure 5; P < 0.05). In contrast, the alterations were improved by the UTI (50,000 u/l) treatment group (10.4% ± 2.1%) (Figure 5; P < 0.05).

Figure 5.

Figure 5

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UTI inhibits oxidant-induced cell apoptosis. Ratio of cell apoptosis was evaluated by Annexin-V/PI double stain assay and analyzed by flow cytometry. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

UTI inhibits oxidant-induced loss of mitochondrial transmembrane potential

The percentage of low Δψm cells increased from 12.8 ± 3.8% in the control group to 57.3 ± 8.5% following H2O2 stimulation (Figure 6; P < 0.05). The percentage of cells with low mitochondrial potential decreased to 24.2 ± 6.6% by UTI (50,000 u/l) pretreatment (Figure 6; P < 0.05).

Figure 6.

Figure 6

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UTI inhibits oxidant-induced loss of mitochondrial membrane potential (Δψm). The Δψm was measured using the fluorescent probe JC-1 and analyzed by flow cytometry. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

UTI improves oxidant-induced mitochondrial dysfunction

In the vehicle group, the intracellular ATP level was found to be 56.0 ± 6.4% of that of the control group (Figure 7; P < 0.05 vs. control group), indicating HUVECs mitochondrial dysfunction following H2O2 stimulation. Following pretreatment with the UTI (50,000 u/l), the ATP level increased to 81.4 ± 10.3% of that of the control group (Figure 7; P < 0.05 vs. vehicle group).

Figure 7.

Figure 7

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UTI improves oxidant-induced mitochondrial dysfunction. HUVECs’ ATP level was determined by a luciferase-based assay. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

UTI improves oxidant-induced Bax up-regulation and Bcl-2 down-regulation

In vehicle group cells, Bax and Bcl-2 expression were respectively 376% ± 51.8% and 45% ± 8.1% of normal values (Figure 8; P < 0.05). In UTI (50,000 u/l) pretreated cells, Bax and Bcl-2 expression were respectively185% ± 31.6% and 87% ± 13.3% of normal values (Figure 8; P < 0.05).

Figure 8.

Figure 8

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UTI improves oxidant-induced Bax up-regulation and Bcl-2 down-regulation. The Bax and Bcl-2 expression of HUVECs were detected by Western blot. Left, Representative Western blots for Bax and Bcl-2. Right, Protein quantification by densitometry. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

UTI reduces oxidant-induced ROS production

The ROS levels reflected by intensity of DCF fluorescence were decreased from 361% ± 49% of normal values in the vehicle group to 211% ± 32.0 % in UTI (50,000 u/l) treatment group (Figure 9; P < 0.05).

Figure 9.

Figure 9

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UTI reduces oxidant-induced oxidative stress. Intracellular ROS levels were assessed using DCFH-DA probe. Data are presented as mean ± SD (n = 6 in each group). *P < 0.05, versus the control group; #P < 0.05, versus the vehicle group.

Discussions

In present study, we have demonstrated that UTI attenuates oxidant-induced endothelial monolayer hyperpermeability. Further studies showed that UTI inhibited oxidant-induced ROS formation, preserved mitochondrial function, decreased the release of cytochrome c from mitochondria into the cytoplasm, inhibited the activation of caspase-3, and prevented disruption of adherens junctions.

The protective effects against inflammation, oxidative stress, and apoptosis have been well described [12,19,29,38]. Moreover, UTI has been shown prevented vascular endothelial injury in patients undergoing open heart surgery with cardiopulmonary bypass [21]. However, the effects of UTI on endothelial barrier functions and the underlying cellular and physiological mechanisms are not known. In this study, HUVECs were respectively pretreated with 10,000 and 50,000 u/l of UTI, followed by H2O2 stimulation. Results shown that UTI (50,000 u/l) significantly attenuates H2O2-induced endothelial hyperpermeability.

Studies demonstrated the activation of intrinsic apoptotic signaling, release of cytochrome c from mitochondria, and activation of caspase 3 in association with vascular hyperpermeability [8,28]. Thus, the main purpose of the present study was to test the effectiveness of UTI on oxidative stress and intrinsic apoptotic signaling. Our results suggest that UTI protected mitochondria, which is evident from the prevention of leakage of JC-1 dye from mitochondria into the cytoplasm, increase of the intracellular ATP level. The observation that UTI prevented oxidant-induced cytochrome c release further shows the protective effects of UTI on mitochondrial function. In normal cells, cytochrome c is located in the mitochondrial intermembrane/intercristae spaces. The mitochondrial outer membrane permeabilization that occurs in response to several proapoptotic stimuli can mobilize cytochrome c from cardiolipin leading to the release of cytochrome c to the cytosol [3,28,37]. Our results have demonstrated UTI treatment prevented membrane potential collapsed and cytochrome c release. After the release to the cytosol, cytochrome c mediates the allosteric activation of apoptotic protease activating factor 1, which is required for the proteolytic maturation of caspase 9 and caspase 3 [15,28,40]. So, the caspase 3 activation observed in our study may be explained as a result of an increased mitochondrial release of cytochrome c.

Our results show that UTI inhibited oxidant-induced ROS formation. Oxidative stress is one of the most important mediators of apoptotic signaling and subsequent cell death. Continuous mitochondrial oxidative stress caused by ROS is known to regulate mitochondrial release of cytochrome c [1,24]. The mitochondrial transition pore (mPT) is known to be activated by Ca2+ and ROS. Exogenous ROS also induced mPT opening and cytochrome c release in isolated mitochondria [10,33]. It is quite possible that in oxidant-induced endothelial permeability, mitochondrial oxidative stress played an important role in the release of cytochrome c to the cytoplasm. UTI, by its antioxidant activity, might have prevented this effect.

The intrinsic or mitochondrial-dependent mechanism of caspase activation depends on the Bcl-2 family of proteins, consists of anti-apoptotic (Bcl-2, Bcl-xL) and proapoptotic (BAK, BAX) factors, regulate mitochondrial outer membrane permeabilization [2,9,23]. The pro-apoptotic members of this family trigger the release of mitochondrial apoptogenic factors into the cytoplasm by regulating mPT. While the antiapoptotic members play a contrast role to prevent apoptosis [6,9]. In present study, BAX and Bcl-2 expression has been detdcted by western blotting. Results suggest that H2O2-induced BAX up-regulation and Bcl-2 down-regulation, which were improved by UTI treatment.

The activation of caspase 3 leads to the cleavage of a variety of cell adhesion proteins [25,36]. The major components of endothelial cell adherens junctions are the cadherin family of proteins, α-, β-, and γ-catenins. A stable cell-cell adherens junction requires the close interaction of the cytoplasmic domain of the cadherins with a group of intracellular proteins, the catenins [11]. β-Catenin, a member of the Armadillo repeat protein family, functions as a regulator of cadherin-mediated cell-cell adhesion in endothelial cells, and its absence may lead to fluid leakage [8,14]. Proteolytic cleavage of β-catenin occurs after the activation of procaspase 3, 6, or 8 [8]. Results of immunofluorescence staining showed that oxidant stimulation caused irregular and scattered β-catenin fluorescence, which was improved by UTI treatment. Thus, UTI mediated inhibition of caspase 3 activation, and subsequent prevention of microvascular endothelial cell-cell detachment is one of the possible mechanisms by which it protected barrier integrity and prevented oxidant-induced endothelial monolayer hyperpermeability.

In conclusion, our findings show that oxidant stimulation activates intrinsic apoptotic signaling and ultimately activates caspase-3, which leads to the disruption of cell adherens junctions and hyperpermeability. The protective effect of UTI on vascular barrier functions may be caused by its inhibitory effects on ROS formation and intrinsic apoptotic signaling pathway.

Disclosure of conflict of interest

None.

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