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Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation.

AIM
To investigate the protective effects of simvastatin (Sim) combined with nifedipine (Nif) on endothelial cells and elucidate the action mechanism.


METHODS
Human umbilical vein endothelial cells (HUVEC) were used. mRNA and protein levels were measured by using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. Intracellular calcium and reactive oxygen species (ROS) were detected using confocal microscopy. The Griess assay was used to evaluate nitric oxide (NO) release.


RESULTS
Treatment of HUVEC with H(2)O(2) 100 micromol/L for 30 min inhibited the mRNA and protein expression of endothelial nitric oxide synthase (eNOS). With increased concentrations of Nif, eNOS mRNA and protein levels increased (P<0.05). Combined treatment with Sim 1.0 micromol/L and Nif 1.0 micromol/L significantly increased the mRNA and protein expression of eNOS and NO release compared with Sim or Nif alone (P<0.05). The combination significantly lowered the intracellular ROS level (P<0.05), which was correlated with the increase in eNOS and NO, but there was no visible change in intracellular calcium (P>0.05). Compared with individual drug treatment, Akt phosphorylation and the ratio of p-eNOS/eNOS were up-regulated in the combination group, and this effect was inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002.


CONCLUSION
The Sim-Nif combination effectively protects HUVEC against H(2)O(2) injury by inhibiting intracellular ROS generation, increasing the ratio of p-eNOS/eNOS and up-regulating Akt phosphorylation.


Introduction
Hypertension and arteriosclerosis are common maladies among aged people in China and remain the most important risk factors for cardiovascular disease. Aging hypertensive patients often co-present with arteriosclerosis [1] . Functional impairment of vascular endothelial cells is involved in the formation of hypertension and arteriosclerosis [2] . Therefore, maintenance of vascular endothelial function is important to prevent hypertension and arteriosclerosis [3,4] .
Endothelial nitric oxide synthase (eNOS) and the nitric oxide (NO) excreted by eNOS are important regulatory factors of endothelial cells because they protect against the development of hypertension and arteriosclerosis by expanding the blood vessels, inhibiting platelet coagulation and preventing conglutination between blood cells and between blood cells and endothelial cells [5,6] . A variety of anti-hypertension drugs have also shown anti-arteriosclerosis activities, independent of blood pressure control [7] . Both clinical [8] and experimental [9] studies have indicated that drugs commonly used to control blood pressure, such as nifedipine (Nif), protect the blood vessel wall, inhibit the progress of arteriosclerosis via exerting anti-oxidation and anti-inflammation functions. Statins, another class of drugs used in blood lipid regulation, enhance the expression of eNOS [10] and reduce reactive oxygen species (ROS) damage [11] . Elderly people with hypertension invariably present with arteriosclerosis. There have been no studies on whether a drug combination therapy can better protect endothelial cells against damage than individual drugs or on the specific mechanisms of such a combination therapy. Here, we used human umbilical vein endothelial cells (HUVEC) to explore the effect and the mechanism of action of combined simvastatin (Sim) and Nif treatment in endothelial cells.
Simvastatin combined with nifedipine enhances endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation Xiao-niao CHEN 1  The expanded products were photographed after electrophoresis in 1.5% agarose. The grayscale of all bands was analyzed under a gel imaging system (ALPHA IMAGER2200).
Western blot analysis HUVEC were grown to reach about 80% confluence, followed by serum-starving synchronization for 12 h and then applying the treatments. Each well was rinsed two times in HBSS, and total protein was extracted with 200 μL RIPA lysis solution (0.5 mmol/L Tris pH 7.4, 1.5 mmol/L NaCl, 10% Np-40, 10 mmol/L EDTA, 100 μg/mL PMSF), 2 μg/mL aprotinin, and 2 μg/mL leupeptin. A BCA protein reagent kit (Pierce, US) was used to measure protein concentration. Electrophoresis was carried out by loading 100 μg total protein in 10% polyacrylamide gels. Proteins were transferred to cellulose acetate membranes, blocked with 10% non-fat milk, and detected with 1:200 eNOS, 1:200 Akt, 1:200 p-Akt and 1:200 β-actin antibodies overnight at 4 °C. Anti-IgG secondary antibody (1:2000) conjugated to horseradish peroxidase was then added at room temperature for 1 h. After washing in TBST, the membrane was labeled with ECL reagents, and grayscale analysis was conducted using an image analysis system (Alphaimager 2200; Alpha Innotech Corp, San Leandro, CA, USA). The level of protein expression was analyzed by normalizing to β-actin.

Fluorescence measurement of intracellular calcium changes
The cultured HUVEC were cultured in 35-mm culture chambers designed for confocal microscopy (MatTek Corp, US). The cultured endothelial cells were synchronized with nonserum media for 12 h. After cells reached the dish walls, they www.chinaphar.com Chen XN et al Acta Pharmacologica Sinica npg were rinsed two times with 37°C HBSS and incubated for 15 min at 37 °C in 7.5 µmol/L Fluo-3 and 0.02% Pluronic F-127 diluted in HBSS containing Ca 2+ and Mg 2+ . Cells were rinsed with HBSS three times, placed in a dark room for 10 min, and observed under the 60×oil-immersion objective of a Radiance 2000 laser scanning confocal microscope (LSCM) (Zeiss). The 49-mW argon ion laser was used, with an excitation wavelength of 488 nm. The fluorescent image of each layer of Fluo-3-stained cells was observed and collected. The scanning employed a time-course procedure with an interval of 5 min for collection of dynamic scanning data for seven cycles (ie, continuous detection for 30 min). The dynamic fluorescent image observed by high-speed computer was drawn in curved lines of fluorescence intensity changes of Ca 2+ . Lasersharp 2000 software (version 6.0) attached to the LSCM was used to analyze the data. At least 40 cells were selected for each experiment. The Ca 2+ concentration in each cell was represented by mean fluorescence intensity (in arbitrary units).

Fluorescence measurement of intracellular reactive oxygen species (ROS)
The membrane-permeable CM-H 2 DCFDA entered endothelial cells and produced a fluorescent signal after intracellular oxidation by ROS, such as hydrogen peroxide and hydroxyl radical. Intracellular oxidant stress was monitored by measuring changes in fluorescence resulting from intracellular probe oxidation [14,15] . The cultured HUVEC were plated in the 35-mm confocal microscopy culture dishes and synchronized with non-serum media for 12 h. After cells reached the dish walls, they were rinsed two times with 37 °C HBSS. CM-H 2 D-CFDA diluted in HBSS to a final concentration of 5.0 µmol/ L was added to the cells, which were incubated at 37 °C for 30 min. After three HBSS washes, cells were placed in a dark room for 10 min and observed by LSCM. Using the 49-mW argon ion laser, the excitation wavelength was 488 nm, and the emission wavelength was 505-530 nm. The intracellular ROS changes were observed in a dynamic state under the 60× oil-immersion lens (in a dark room). The scanning employed a time-course procedure with a 5-min interval for collection of dynamic scanning data and nine data collection points (ie, continuous detection for 40 min). The dynamic fluorescence image was analyzed as above. At least 35 cells were selected for each experimental group.
Measurement of NO levels NO secreted from cells is rapidly oxidized to nitrite in culture medium; therefore, determination of nitrite concentrations was used as a measurement of NO production. A colorimetric Griess assay (Jiancheng Ins, Nanjing, China) was used. Briefly, HUVEC were cultured at 1×10 6 /mL in a 24-well plate, 1 mL/well, at 37 °C and 5% CO 2 . The cells had grown to the wall after 24 h. The clear supernatant medium was removed and replaced with 1 mL fresh culture medium. The cells were divided according to the aforementioned experimental groups and collected 4 h after drug treatment. Following the manufacturer's instructions, the clear supernatant medium was col-lected to detect the NO level by the Griess method.

Statistical analysis
All data are presented as mean±SEM. Statistical significance was assessed using the Student's paired t-test and variance. All data analyses were performed using the software SPSS 10.0. P<0.05 was considered statistically significant.  Simvastatin combined with nifedipine enhances NO secretion more than individual treatments in endothelial cells

Results
The above results showed that Nif and Sim enhanced the expression of eNOS protein and mRNA. However, the increased eNOS does not automatically imply a protective effect of these two drugs for the cells. Therefore, further investigation was carried out by using the Griess assay to examine NO secretion in the cells upon different drug treatments.

Stronger inhibitory effect on ROS generation from joint Sim and Nif treatment compared with separate treatments
The above results show that the expression of eNOS mRNA and protein was reduced by 100 μmol/L H 2 O 2 in endothelial cells and that Nif and Sim inhibited this damaging effect. It has been reported [7,8] that calcium channel blockers and statins affect eNOS expression via many pathways, such as ROS signaling, calcium signaling and the bradykinin pathway.
To investigate the mechanism of protection provided by Sim and Nif, intracellular ROS were marked with the specific fluorescent probe CM-H 2 DCFDA in HUVEC, and we carried out dynamic real-time detection by LSCM. The fluorescence intensity of CM-CDFDA increased remarkably from stimulation with 100 μmol/L H 2 O 2 , reaching a peak after 30 min, which meant that H 2 O 2 stimulation was able to increase ROS in cells. Moreover, ROS generation was inhibited by Nif in a dose-dependent manner (P<0.05 vs B group, n=35-45 cells) (

Impact of simvastatin and nifedipine on Ca 2+ concentration changes in endothelial cells
Calcium ion is the most physiologically important second messenger in endothelial cells, and it serves as the primary medium for communicating information in the cells. Nifedipine is a calcium antagonist, but whether its protective effect on endothelial cells results from inhibiting Ca 2+ increases remains unclear. We first stimulated HUVEC with 100 μmol/L H 2 O 2 , which induced increased intracellular Ca 2+ concentration ([Ca 2+ ] i ) in endothelial cells (P>0.05, n=40-45 cells, Figure 7). This effect on [Ca 2+ ] i was significant after 10

Combination treatment of nifedipine and simvastatin enhances Akt phosphorylation and the ratio of p-eNOS/eNOS in endothelial cells
We wanted to investigate the mechanism by which increased eNOS regulates specific cellular functions. A previous report showed that ROS regulates the expression and activity of eNOS via the PI3K-Akt signaling pathway [16] . Akt activation led to activation of eNOS, increased eNOS phosphorylation (p-eNOS), and greater NO release. Here, we observed that, compared with the individual drug groups, combined Sim and Nif treatment up-regulated the level of Akt phosphorylation (without affecting total Akt) (P<0.05 vs groups 2 and 3), enhanced p-eNOS (P<0.05 vs groups 2 and 3) and total eNOS levels (P<0.05 vs groups 2 and 3), and increased the proportion of p-eNOS/eNOS (P<0.05 vs groups 2 and 3). These effects were inhibited by the PI3K inhibitors wortmannin and LY294002 (P<0.05 vs groups 1, 5, and 6). Taken together, these results indicate that combination treatment of Nif and Sim

Discussion
Endothelial cells are involved in many aspects of the generation and development of many cardiovascular maladies, such as hypertension and arteriosclerosis. Nitric oxide plays a role in stretching blood vessels, inhibiting the conglomeration of blood platelets and preventing conglutination between endothelial cells and between blood cells and endothelial cells. Nitric oxide plays a protective role in preventing the formation of hypertension and arteriosclerosis. Moreover, the expression of eNOS is the key factor for NO generation in endothelial cells. The synthesis and release of NO are markedly reduced in vascular endothelial cells of hypertension patients. Therefore, regulating eNOS expression and enhancing NO secretion will be important methods of reducing hypertension and preventing and controling arteriosclerosis [17][18][19] . A typical model of endothelial cell damage is H 2 O 2 -induced damage. The use of this model helps in the study of the pathogenesis of cardiovascular diseases and in screens for drugs that protect blood vessels. The present investigations of eNOS mRNA and protein levels demonstrated that Nif enhances the expression of eNOS in endothelial cells and participates in the protection of endothelial cells. The expression of eNOS was also up-regulated by treatment with Sim alone. Moreover, we showed that the protective effect on endothelial cells was enhanced by combination treatment of Sim and Nif compared with either drug treatment separately. As shown here and previously [20,21] , 1.0 µmol/L Nif and 1.0 µmol/L Sim are not cytotoxic to endothelial cells; on the contrary, they have a protective effect on endothelium.
For the regulatory mechanism of eNOS expression, it has been reported that an increased ROS level in endothelial cells inhibits the expression of eNOS [22,23] , which is one of the reasons for hypertension in elderly people. The results of our study show that stimulation of endothelial cells with H 2 O 2 can enhance the ROS level in endothelial cells. Nifedipine inhibited this ROS generation in a dose-dependent manner. The fluorescence intensity was further reduced in the cells with the combination treatment of Sim and Nif, which was concomitant with increased eNOS expression and NO level. This indicates  Although Nif serves as an inhibitor of cell membrane L-type calcium channel, it is still disputed whether it protects endothelial cells via inhibiting extracellular calcium ion influx. It has been reported that increased intracellular Ca 2+ may inhibit the expression of eNOS. Therefore, we asked whether the combination treatment of Nif and Sim could also enhance eNOS expression by inhibiting intracellular Ca 2+ changes. We found no visible Ca 2+ changes in HUVEC after stimulation with H 2 O 2 with or without Nif and/or Sim. This indicates that enhanced eNOS expression by Nif and Sim was not a result of the inhibition of calcium influx, in agreement with the results of Aono et al [24] . A recent study indicated that L-type Ca 2+ channels did not exist in the endothelial cell membrane [25] . It was reported in other studies [26] that Nif combines with cell membrane lipids directly to inhibit ROS production, due to the specificity of its molecular structure. Moreover, endothelial cells stimulated with H 2 O 2 may induce a series of biological effects via the release of endoplasmic reticulum Ca 2+ to increase Ca 2+ concentration in the cytoplasm [27] . Here, Nif was unable to block the increase of influx of extracellular Ca 2+ into the cells. Therefore, this result is not in contradiction with Nif serving as an antagonist of the calcium signaling pathway. Nif protects endothelial cells by directly inhibiting ROS generation.
PI3K/Akt signaling regulates the activity of eNOS and enhances NO release from endothelial cells [28] . The excessive production of ROS damages the cells, but lower concentrations of ROS are able to activate the PI3K/Akt/eNOS pathway and enhance the activity of eNOS (raise the level of eNOS phosphorylation), thereby protecting the endothelial cells. The results of this study also support this conclusion, as the combination treatment of Nif and Sim reduced ROS levels, up-regulated Akt phosphorylation relative to total Akt, enhanced the levels of both phosphorylated and total eNOS, and raised the proportion of p-eNOS/eNOS. This effect was more significant from the combination treatment than from each drug separately. Our results are consistent with a mechanism in which Nif combines with cell membrane lipids directly to resist ROS over-production. Yamagishi et al [29,30] . demonstrated that Nif directly abrogates vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity in endothelial cells. Furthermore, Sim inhibits ROS (O 2 -, OH -) production, possibly by decreasing NADPH oxidase and increasing peroxiredoxin 3/thioredoxin 2 antioxidant activities [31] .
In summary, we discovered that combination treatment of Sim and Nif enhanced the expression of eNOS mRNA and protein, up-regulated the ratio of p-eNOS/eNOS, increased NO secretion and enhanced the protective effect of the two drugs individually on endothelial cells. The combination treatment works by reducing the generation of ROS in the cells and up-regulating Akt activation, rather than via inhibition of calcium signaling. These results provide the basis for their use in clinical practice, and they indicate that Sim combined with Nif will be useful in reducing hypertension and preventing/ treating arteriosclerosis.