VEGF and other chemicals of reagent grade were obtained from Sigma (St. Louis, MO, USA). The primary antibodies for PI3K p85, Akt, phospho-Akt (Ser 473), ERK1/2, phospho-ERK1/2 (Thr 202/Tyr 204), eNOS, and phospho-eNOS (Ser 1177) were purchased from Cell Signaling (Danvers, MA, USA). Primary antibodies for PLZF and GAPDH were respectively purchased from Calbiochem (Darmstadt, Germany) and AbFrontier (Seoul, Korea).

The HUVEC line was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan), and cultured in 90% medium 199 with 25 U/ml heparin and 30 µg/ml endothelial cell growth supplement (ECGS) adjusted to contain 1.5 g/L sodium bicarbonate with 10% fetal bovine serum. They were grown until the monolayer became confluent. The medium for culturing cells was then changed to serum-free medium, and cells were incubated overnight before the experiment. Cell culture reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

A ceramic FIR generator, a WS TY301 FIR emitter (WS Far Infrared Medical Technology, Taipei, Taiwan), was used to provide FIR exposure. This FIR emitter generates electromagnetic waves with wavelengths in the range of 3∼25 µm. HUVECs were cultured in a 6-cm Petri dish with 3 ml medium for FIR exposure. During FIR exposure, an experimental group and a negative control covered with aluminum foil were set up in a culture chamber of a LiveCell™ system (Pathology Devices, Westminster, MD, USA) at 37°C with a 5% CO₂ atmosphere. The FIR intensity was adjusted by changing the distance between the FIR emitter and culture chamber. The effective FIR intensity and wavelength received by HUVECs in culture dishes were detected and calculated by China National Infrared & Industrial Electrothermal Products Quality Supervision & Test Center (Wuhan, China). The effective primary FIR emission that the culture cells received ranged 3∼5 µm. Additionally, as a result of FIR exposure, there was secondary emission from the Petri dish lids with a peak wavelength of approximately 10 µm, calculated according to Wein's law.

HUVECs (10⁴ cells/well) were cultured in a 96-well microtiter plate in a final volume of 100 µl/well of culture medium. After FIR exposure, cells were incubated at 37°C overnight. Cell proliferation was analyzed using a Quick Cell Proliferation Assay kit according to the instructions provided by the manufacturer (BioVision, Mountain View, CA, USA), and results are presented as the absorbance of each sample at 440 nm.

In total, 15 µg of HUVEC lysate proteins was applied to each lane and analyzed by Western blotting. Peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G (IgG) (at a 1∶5000 dilution) was used as the second antibody to detect primary antibodies by enhanced chemiluminescence (Thermo Scientific, Rockford, IL, USA). Data of protein bands on Western blots were also quantitated with QuantiScan software (Biosoft, Cambridge, UK).

Nitrate/nitrite in the culture medium was measured as described in a previous report [17]. In brief, a sample of medium was deproteinized with two volumes of 99% ethanol at 4°C, centrifuged (3,000 g for 10 min), and then injected into a collection chamber containing 5% VCl3. This strong reducing environment converted both nitrate and nitrite to nitric oxide (NO). A constant stream of helium gas carried the NO into an NO analyzer (Seivers 270B NOA; Seivers Instruments, Boulder, CO, USA), where the NO reacted with ozone, resulting in the emission of light. The light emitted was proportional to the quantity of NO formed; standard amounts of nitrate were used for calibration.

Intracellular ROS production was detected with a fluorescent microscope using the fluorescent dye, 2′,7′-dichlorofluorescein diacetate (DCF-DA) (Molecular Probes, Eugene, OR, USA). Cells were cultured overnight on 4-well chamber slides (Nalge Nunc International, Naperville, IL, USA) at a concentration of 5×10³ cells per 400 µl culture medium. Before the experimental treatment, slides were preincubated with 10 µM DCF-DA at 37°C for 30 min in the dark, and then subjected to experiments. Fluorescence was observed by fluoromicroscopy with excitation and emission wavelengths of 485 and 530 nm, respectively.

Nitrotyrosine formation was measured using Nitrotyrosine ELISA kits (Millipore, Billerica, MA, USA), following the manufacturer's instructions. A standard curve was used to determine the absolute concentration. Values were standardized to micrograms of protein for each sample.

HUVECs were lysed at 4°C in lysis buffer (50 mM Tris, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM NaCl, protease inhibitors). PTEN was collected by using immunoprecipitation kits (Roche Molecular Biochemicals, Mannheim, Germany) with specific antibodies and protein-G-agarose, following the manufacturer's instructions. Precipitates were washed with lysate buffer, and the activity of PTEN was analyzed by using PTEN Malachite Green Assay Kit (Upstate, Lake Placid, NY, USA). Absorbance was detected at 600 nm. Released phosphate was determined relative to a standard curve.

Total RNA was extracted from HUVECs using the TRIzol method according to the protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA, USA), and used to synthesize single-stranded complementary (c)DNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). PI3K p85 subunit messenger (m)RNAs were quantified using TaqMan Gene Expression Master Mix (Applied Biosystems) with specific primers in an ABI 7300 Real-Time PCR System (Applied Biosystems). TaqMan Gene Expression Assay kits containing specific primers for PI3K p85-α (cat. no. Hs00933163_m1), PI3K p85-β (cat. no. Hs00178181_m1), and GAPDH (cat. no. Hs99999905_m1) were obtained from Applied Biosystems. Specific primers for GAPDH were used to normalize the amount of sample added. Relative amounts of PI3K p85 mRNA were quantitated using the comparative Ct method. All quantifications were performed on triplicate samples of three separate experiments.

HUVECs were suspended in cold buffer A (containing 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 1 mM PMSF) for 15 min, lysed by adding 10% NP-40, and then centrifuged at 5000×g to obtain nuclear pellets. The nuclear pellets were resuspended in cold buffer B (containing 20 mM HEPES, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 0.4 mM NaCl), vigorously agitated, and then centrifuged.

PLZF siRNA (cat. no. sc-37149) and control siRNA (cat. no. sc-44234) were purchased from Santa Cruz Biotechnology. Cells were grown to 70% confluence, and PLZF siRNA and control siRNA were transfected using the TurboFect reagent according to the manufacturer's instructions (Fermentas, Glen Burnie, MD, USA). The final concentration of PLZF siRNA for transfection was 100 nM.

A Student's t-test was used in all statistical tests. Distributions of continuous variables in groups were expressed as the mean±S.D. A value of p of <0.05 was considered to indicate statistical significance.

To evaluate the biological effects of FIR, VEGF-pretreated HUVECs were exposed to 0.13 mW/cm² of FIR for 30 min and then cultured overnight. A cell proliferation analysis revealed that both VEGF and FIR exposure significantly increased cell proliferation in HUVECs (Fig. 1A). However, FIR exposure significantly inhibited VEGF-induced proliferation in HUVECs. In an intensity course analysis, effective FIR intensities of 0.13, 0.8, and 1.80 mW/cm² significantly reduced VEGF-induced proliferation (Fig. 1B). This inhibitory effect of FIR peaked at the effective intensity of 0.13 mW/cm² and decreased at higher FIR intensities. When the effective intensity rose to 7.20 mW/cm², FIR failed to reduce VEGF-induced proliferation in HUVECs. Therefore, we applied 0.13 mW/cm² as the working intensity in subsequent experiments. In a time course analysis, the inhibitory effect of FIR peaked at 30 min and decreased with longer exposure times (Fig. 1C). A western blot analysis was also used to evaluate the influence of FIR exposure on VEGF-activated extracellular signal-regulated kinase (ERK)1/2. As shown in Fig. 1D, FIR exposure significantly inhibited the phosphorylation of ERK1/2. Similar to the inhibitory effect of FIR on cell proliferation, the inhibitory effect of FIR on the phosphorylation of ERK1/2 peaked at 30 min of FIR exposure, and gradually decreased at longer exposure times.

A western blot analysis revealed that both VEGF and FIR exposure induced the phosphorylation of eNOS in HUVECs (Fig. 2A). FIR exposure further increased the phosphorylation of eNOS in VEGF-treated HUVECs. This induction peaked at 30 min and gradually decreased at longer exposure times. When we monitored the concentration of NO in the culture medium, FIR exposure further induced NO generation by HUVECs although VEGF also induced NO generation (Fig. 2B). These increases in NO generation were inhibited by NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS. In cell proliferation tests, FIR exposure slightly increased the cell proliferation of HUVECs without VEGF treatment, but mitigated VEGF-induced proliferation (Fig. 2C). In L-NAME-treated HUVECs, VEGF-induced proliferation significantly decreased, and there was no further decrease due to FIR exposure. L-NAME also reduced VEGF-induced phospho-ERK1/2, which was not influenced by FIR exposure (Fig. 2D).

A thermal effect of FIR on the cell culture system was also examined in this study. With FIR exposure at different effective intensities, we detected the temperature of 3 ml of culture medium in a 6-cm plate at different time points. The effective FIR intensity of 0.13 mW/cm² only mildly heated the culture system, and raised the temperature of the medium from 37.0±0.1 to 37.1±0.1°C in 30 min (Fig. 3A). The higher effective FIR intensity of 7.2 mW/cm² had a strong heating effect, which raised the temperature of the medium from 37.0±0.1 to 38.4±0.1°C in 30 min. The temperature of the medium rose along with the FIR intensity and exposure time. To evaluate the thermal effect on cell proliferation, we cultured VEGF-pretreated HUVECs at different temperatures for 30 min without FIR exposure, and detected VEGF-induced cell proliferation after overnight culture. As shown in Fig. 3B, pretreatment at 38∼39°C slightly increased VEGF-induced proliferation in HUVECs. A western blot analysis revealed that pretreatment at 38∼39°C did not influence VEGF-induced expression of phospho-ERK1/2 or phospho-eNOS although 39°C pretreatment induced eNOS expression (Fig. 3C). These expression patterns of phospho-ERK1/2 and phospho-eNOS differed from FIR-induced events.

Intracellular ROS detection showed that VEGF-induced ROS generation in HUVECs was significantly inhibited by an NADPH oxidase inhibitor, apocynin, and a hydroxyl radical scavenger, dimethylthiourea (DMTU), but was not influenced by FIR exposure (Fig. 4A). Both apocynin and DMTU inhibited VEGF-induced proliferation in HUVECs (Fig. 4B). In ROS scavenger-treated HUVECs with VEGF treatment, FIR exposure significantly increased cell proliferation (Fig. 4B). This result reveals that VEGF-induced ROS generation plays a role in the inhibitory effect of FIR on VEGF-induced proliferation in HUVECs. Additionally, NO reacts with superoxide anion radical to form peroxynitrite. Intracellular peroxynitrite can modify proteins by interacting with and nitrating tyrosine residues to form 3-nitrotyrosine. We also monitored nitrotyrosine formation in HUVECs treated with VEGF and FIR. As shown in Fig. 4C, nitrotyrosine formation significantly increased in HUVECs treated with VEGF and FIR together. Neither VEGF nor FIR alone influenced nitrotyrosine formation in HUVECs.

To understand the molecular mechanisms underlying FIR-induced eNOS activation, we focused on PI3K-dependent activation of Akt, a known eNOS kinase. As shown in Fig. 5A, both FIR and VEGF increased Akt phosphorylation in HUVECs, and FIR further increased Akt phosphorylation in VEGF-treated cells. The PI3K/Akt pathway inhibitor, wortmannin, abolished Akt phosphorylation in VEGF-treated HUVECs with or without FIR exposure. Similar to the expression of Akt phosphorylation, wortmannin also significantly inhibited eNOS phosphorylation in VEGF-treated HUVECs with or without FIR exposure (Fig. 5B).

The influence of FIR on PI3K signaling pathways was investigated by monitoring phosphatase and tensin homolog (PTEN) protein activity and PI3K expression. PTEN is a phosphatase which inhibits Akt signaling pathways. However, there was no influence of FIR on PTEN activity in HUVECs (Fig. 6A). A western blot analysis revealed that FIR but not VEGF significantly increased expression levels of PI3K p85 (Fig. 6B). The results of the qPCR showed that mRNA levels of PI3K p85-α and p85-β subunits significantly increased during FIR exposure, but gradually decreased after FIR exposure (Fig. 6C). This result reveals that FIR exposure upregulates the transcription of the PI3K gene in HUVECs.

PI3K-p85 is known to be positively regulated by PLZF [18]. We further monitored the influence of FIR exposure on PLZF activation. As shown in Fig. 7A, FIR exposure dramatically induced the translocation of PLZF to nuclei in HUVECs. This translocation almost disappeared after FIR exposure for 30 min. VEGF and treatments at 38 and 39°C did not induce PLZF translocation. We used PLZF siRNA transfection to block PLZF expression in HUVECs, and then found that FIR exposure failed to elevate mRNA levels of the PI3K p85-α and p85-β subunits (Fig. 7B). PLZF siRNA transfection blocked FIR-induced proliferation and the inhibitory effect of FIR on VEGF-induced proliferation in HUVECs (Fig. 7C). PLZF siRNA transfection also reduced FIR-induced eNOS phosphorylation, but did not influence VEGF-induced eNOS phosphorylation (Fig. 7D). These results reveal that PLZF activation plays a critical role in the biological effects of FIR.