NOS3 (sc-654) and DUSP4 (sc-1200) antibodies were obtained from Santa Cruz (Santa Cruz, CA, USA); p-ERK1/2 (9101S), ERK1/2 (9102S), p-p38 (4511S), p38 (9212S), GAPDH (2118S), p-JNK (4671S), JNK (3708S), cleaved caspase-3 (9664), and MK2 sampler kit antibodies (9329) from Cell Signaling (Cambridge, MA, USA); GSH (101-A-250) from ViroGen (Watertown, MA, USA). Secondary anti-rabbit (NA934V) and anti-mouse (NXA931) IgG-HRP antibodies were purchased from GE Life Sciences (Piscataway, NJ, USA). RNA was isolated using TRI Reagent^® purchased from Ambion (Carlsbad, CA, USA). cDNA was synthesized with a High-Capacity cDNA Reverse Transcription kit from Applied Biosystems (Foster City, CA, USA) and real-time PCR conducted using LightCycler 480 SYBR Green I from Roche (Mannheim, Germany).

The maintenance of bovine aortic endothelial cells (BAECs) culture was performed, as previously described by our laboratory (7, 18, 21, 23). For DUSP4 overexpression, cells were seeded to a confluency of 50–60% on six-well dishes and transfected with DUSP4 the next day. Seventy-two hours post-transfection, cells were subjected to H/R treatment. Cells used for immunostaining were grown on sterile glass coverslips coated with attachment factor (Gibco S-006-100, Life technology). First, glass coverslips were coated with attachment factor, and then the excess attachment factor was removed and followed by 30 min incubation in 37°C in the incubator.

Dual-specificity phosphatase 4 (mouse) overexpression plasmid with CMV promoter was purchased from OriGene (Rockville, MD, USA). For DUSP4 overexpression, cells were seeded to confluency of 50–60% on six-well dishes a day before the transfection. Attractene transfection reagent from Qiagen (Hilden, Germany) was used for DUSP4 transfection. Twelve hours post-transfection, medium was removed and replenished with fresh medium to prevent cytotoxicity from the transfection reagents (7). At 72 h post-transfection, cells were then subjected to H/R treatment to investigate the molecular mechanism of DUSP4 on the modulation of H/R-induced oxidant stress.

Seventy-two hours after DUSP4 transfection, the cell media was removed, cells were gently washed with phenol red-free Dulbecco’s Modified Eagle Medium (DMEM), and fresh phenol red-free DMEM was added to the cells. BAECs were then either incubated in normoxic conditions or placed in a hypoxia chamber (Billups-Rothenberg, CA, USA). The hypoxic chamber was flushed with argon for 30 min (24, 25), and the cells were incubated at 37°C under the hypoxic environment for 1 h. After hypoxia, cells were incubated at normoxic (21% O₂) conditions for 30 min. After H/R, cells were collected and processed for protein analysis via SDS-PAGE and immunoblotting and mRNA quantification via qPCR. Cells exposed to only normoxic conditions served as controls and were compared to those subjected to H/R (18, 21).

Bovine aortic endothelial cells were cultured on sterile glass coverslips coated with attachment factor to an 80–90% confluence or 72 h post-transfection. Cells were subjected to H/R treatment (1 h hypoxia and 30 min reoxygenation), cell media was removed, and coverslips were washed 2× with PBS, fixed for 20 min at RT in 4% paraformaldehyde and subsequently washed 3× with PBS containing 0.1% BSA. Cells were then blocked for 45 min at RT in PBS containing 5% BSA and 0.3% TritonX-100 and then incubated overnight at 4°C in anti-cleaved caspase-3 or anti-DUSP4 diluted 1/400 in PBS with 1% BSA and 0.3% TritonX-100. After overnight incubation, coverslips were rinsed 3× with PBS containing 0.1% BSA, and incubated with Alexa Fluor 488 secondary antibody (green) for cleaved caspase-3 and Alexa Fluor 594 secondary antibody (red) for DUSP4 (Cell Signaling, Cambrdige, MA, USA) for 1 h at RT. Finally, coverslips were washed 3× with PBS containing 0.1% BSA, mounted onto a glass slide with ProLong Gold antifade reagent with DAPI (Life Technologies, Grand Island, NY, USA), and fluorescent images (40×) obtained using an Olympus FluoView FV1000 confocal microscope, and data were captured digitally and analyzed (18, 21).

The procedure for the immunoblotting was followed, as previously described (18, 21, 23, 26). Samples were first separated on an 8% Tris-glycine polyacrylamide gel and then electrophoretically transferred to a nitrocellulose membrane. The extent of p38 phosphorylation was determined using the ratio of p-p38 to total p38. Similarly, the extent of ERK1/2 or JNK phosphorylation was determined using the ratio of p-ERK1/2 to total ERK1/2 or p-JNK to total JNK. MK2 phosphorylation at T334 and T222 was used to determine the activity of p38. The extent of eNOS expression was also determined, and GAPDH served as the loading control marker.

The level of superoxide generation from control BAECs and BAECs with DUSP4 overexpression or treated with H/R was measured using DHE-HPLC analysis with a slight modification (27). After with or without H/R treatment, cells were washed once with PBS and incubated in Krebs-HEPES buffer. DHE was added to a final concentration of 25 μM and incubated at 37°C for 30 min. The medium was then removed and replenished with fresh Krebs-HEPES buffer for an additional 1 h incubation. After incubation, cells were collected and lysed in 500 μL cold methanol and centrifuged. 2-hydroxyethidium, DHE, and ethidium were separated using a gradient HPLC system (Shimadzu LC-2010A) with a Hypersil Gold column (250 mm × 4.6 mm, ThermoFisher Scientific, Waltham, MA, USA) and detected with a fluorescence detector using an emission wavelength at 580 nm and an excitation at 480 nm. A linear gradient at a flow rate of 0.5 mL/min was developed from mobile phase A (0.1% trifluoroacetic acid) to mobile phase B (acetonitrile) over 23 min from 37 to 47% acetonitrile (18, 21).

Bovine aortic endothelial cells were cultured on sterile glass coverslips coated with attachment factor to an 80–90% confluence or 72 h post-transfection. Cells were subjected to H/R treatment (1 h hypoxia and 30 min reoxygenation). After with or without H/R treatment, cells were washed once with PBS and incubated in PBS with Ca²⁺ and Mg²⁺. 4,5-Diaminofluorescein diacetate (DAF-2) was added to a final concentration of 5 μM and incubated at 37°C for 60 min (28). After incubation, coverslips were washed once with PBS, mounted onto a glass slide with ProLong Gold antifade reagent with DAPI (Life Technologies, Grand Island, NY, USA), and fluorescent images (20×) obtained using a Zeiss Axiovert 135 microscope.

After transfection with DUSP4 or H/R treatment, cells were first washed with PBS and directly lysed in TRI Reagent^®. In brief, total RNA was isolated from BAECs via the TRI Reaent^®-chloroform extraction per the manufacturer’s instructions. Extracted RNA was quantified via spectrophotometric analysis using absorption spectra at wavelengths of 230, 260, and 280 nm. A total of 1 μg of RNA was reverse transcribed according to the kit’s instructions. Gene expression was detected in triplicate via quantitative real-time PCR on a Roche LightCycler480 thermal cycler as follows: 95°C for 10 min (95°C for 10 s, 60°C for 20 s, 72°C for 20 s with signal detection), for 45 cycles. Ct values were calculated by the LightCycler480 Software using the second derivative max method. Data were calculated using the 2^−ΔΔCt method (29) and are expressed as target gene transcript fold expression relative to control, following normalization to β-actin (18, 21). Primer sequences are as follows: β-actin forward 5′-TGCCCATCTATGAGGGGTACG-3′, reverse 5′-GGACGATTTCCGCTCGGC-3′; DUSP4 forward 5′-ATTCCGCCGTCATCGTCTAC-3′, reverse 5′-ATAGCCACCTTTCAGCAGGC-3′; NOS3 forward 5′-TACCAGCCGGGGGACCACATAGGC-3′, reverse 5′-CTCCAGCTGCTCCACAGCCACAGAC-3′; NOX4 forward 5′-CAGGGGTCTGCATGGTACTG-3′, reverse 5′-CAGCAGCCCTCCTGATACAT-3′. All primers detected bovine targets, while DUSP4 primers also detected mouse targets.

Results were expressed as mean ± SEM, n ≥ 3. Statistical significance of difference between results was calculated using two sample t-tests with an alpha of 0.05. A P-value (two-tailed) <0.05 was considered statistically significant.

Cells were seeded in six-well dishes or on sterile glass coverslips coated with attachment factor to a confluency of 50–60% and transfected with DUSP4 expression plasmids. After 72 h of DUSP4 transfection, cells were then placed inside a modular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA, USA) and subjected to 1 h of hypoxia. Upon completion of the hypoxic period, cells were reoxygenated at normoxia for a total of 30 min and were subsequently processed for either imaging, immunohistochemistry, or protein analysis. From immunostaining (Figure 1A), DUSP4 is overexpressed in endothelial cells 72 h post-transfection. The population of BAECs overexpressed DUSP4 is 72.67 ± 5.53%, as determined by immunostaining with DUSP4 antibody (Figure 1A). The level of DUSP4 overexpression in BAECs is increased by 1.28 ± 0.02-fold compared to control cells (P < 0.05, n = 3). Under H/R treatment, cleaved caspase-3 was activated in endothelial cells contributing to apoptosis (Figure 1B). The percentage of apoptotic cells under H/R is increased by 41.08 ± 9.87% compared to the control (P < 0.05, n ≥ 3). DUSP4 overexpression in endothelial cells suppressed the activation of cleaved caspase-3 to the level of the control and thus prevented apoptosis.

p38 is a stress-activated MAPK and plays a critical role in deciding cell fate under oxidative stress. After 1 h hypoxia and 30 min reoxygenation treatment, the phosphorylation of p38 (Figures 2A,B) was over-activated in response to the increased oxidant stress. Overexpression of DUSP4 in endothelial cells prevented p38 overactivation and protected against H/R-induced oxidant stress. The activity of p38 was further determined by measuring the extent phosphorylation of the downstream target of p38, MK2, using immunoblotting. The phosphorylation of MK2 at T334 was dramatically increased consequent to p38 activation (Figures 2A,B). Overexpression of DUSP4 negatively regulated p38 activity under H/R-induced oxidant stress, which further prevented the activation of MK2. There was no effect on the phosphorylation MK2 at T222.

The phosphorylation of ERK was also increased in response to H/R treatment; however, overexpression of DUSP4 did not significantly change the extent of phosphorylation of ERK after H/R (Figure 2C). JNK is another stress-activated MAPK that responds to oxidative stress. There was no significant difference on the phosphorylation of JNK when cells underwent H/R treatment or DUSP4 overexpression (Figure 2D).

The level of superoxide generated from cells after being subjected to H/R was determined using DHE-HPLC method. The results indicated that DUSP4 overexpression in cells decreases H/R-induced superoxide generation (1.56 ± 0.14 versus 1.19 ± 0.05, *P < 0.05) (Figure 3A) and thus reduces oxidant stress. The level of total protein S-glutathionylation is an indicator of cellular oxidant stress. The total protein S-glutathionylation was probed using anti-GSH antibody. The results demonstrated that overexpression of DUSP4 decreases cellular ROS generation after H/R treatment and thus decreases total protein S-glutathionylation (Figure 3B).

eNOS is an important enzyme in endothelium that is responsible for the production of NO. It is a critical small molecule involved in regulating vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. Under H/R, eNOS is degraded in response to oxidant stress (Figure 4A), which is consistent with our previous study (26). There is no significant difference in eNOS expression when DUSP4 was overexpressed in the control endothelial cells. More interesting, under H/R treatment, overexpression of DUSP4 reduces H/R-induced oxidant stress and thus improves the stability and expression of eNOS (Figure 4A).

Subsequently, it is necessary to demonstrate whether the upregulation of eNOS by DUSP4 correlates with increased NO production. In this experiment, DAF-2 was used to determine the extent of NO generated from cells. The fluorescent intensity of adduct of NO and DAF-2 was measured using a Zeiss Axiovert 135 microscope and quantified using ImageJ. Clearly, DUSP4 overexpression promotes NO production (4,595.24 ± 780.73 versus 1,788.76 ± 566.71, *P < 0.05 compared to control group) from cells. H/R treatment does not alter NO generation in endothelial cells. However, overexpression of DUSP4 in cells and H/R treatment enhance NO production (10,501.38 ± 3,503.89 versus 1,747.39 ± 780.73, **P < 0.05 compared to H/R group) and thus provide the beneficial effect against H/R-induced oxidant stress (Figure 4B).

To further determine the molecular mechanism and beneficial effects of DUSP4 overexpression against H/R-induced oxidant stress, qPCR was used to analyze gene expression. At the end of 72 h post-transfection, DUSP4 overexpression (5.16 ± 0.37 versus control; *P < 0.001) downregulates NOX4 gene expression (0.22 ± 0.01 versus control; *P < 0.001). However, overexpression of DUSP4 increases eNOS gene expression (5.10 ± 0.19 versus control; *P < 0.001) 72 h post-transfection (Figure 5A). The increase in eNOS mRNA expression is consistent with the increase in eNOS protein expression and NO generation (Figure 4), thus providing a beneficial effect against oxidative stress. After 1 h hypoxia and 30 min reoxygenation treatment, there is no significant change in NOX4 expression in normal cells. However, NOX4 gene expression is repressed in cells with DUSP4 overexpression after H/R (0.34 ± 0.04 versus control; **versus control and ^#versus H/R P < 0.001). Conversely, overexpression of DUSP4 upregulates eNOS gene expression (5.86 ± 0.37 versus control; **versus control and ^#versus H/R P < 0.001), thus increasing NO production as a survival mechanism against oxidative stress (Figure 5B).