A total of 30 wild-type, 3- to 5-month-old male Sprague-Dawley (SD) rats, weighing 180–220 g, provided by the Experimental Animal Center of Beijing Academy of Military Medical Sciences (Beijing, China), were used in this study. The rats were housed individually at a temperature of 18–24°C and given ad libitum access to food and water. The care and use of animals in the experiments met the standards set out in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (Bethesda, MD, USA) and closely complied with the Animal Care and Use Committee of the Tianjin Institute of Health and Environmental Medicine (Tianjin, China). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Tianjin Institute of Health and Environmental Medicine (permit no. 2013-D-3604). All efforts were made to lower the number of animals used and to reduce animal stress. Choline chloride (ChCl), ACh and ET-1 were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

The rats were randomly divided into a normoxic control group (no treatment, n=16) and a CIH group (treatment with CIH, n=16). Half the rats in each group were used to study arterial endothelial activity and the other half to study the selective dilatation effects of choline.

Animals from the CIH group were separately housed in cages, which were placed into a Plexiglas chamber (40×30×30 cm). At the beginning of hypoxia, nitrogen was pumped into the chamber to reduce the oxygen concentration. This was continuously measured using an oxygen concentration monitor (CYES-II, Shanghai Scientific Instruments, Shanghai, China) and was stabilized at a level of 10±0.5% (which mimics the hypoxic environment of 6,000 m above sea level) for 8 h (8:00 a.m. to 4:00 p.m.) via an exhaust valve controlled by an automatic computer system. The chamber contained a small hole that allowed the pressure inside to remain consistent with the external environment. Carbon dioxide and water vapor in the chamber were absorbed with soda lime and anhydrous calcium chloride, respectively. Intermittent hypoxia (8 h/day, 6 days/week) lasted for 12 weeks. The treatment of animals from the normoxic control group was the same as that of the CIH group except that they were not given CIH treatment. At the end of the CIH exposure duration, animals were administered 1–2% isoflurane through a face mask for anesthesia prior to sacrifice by decapitation and surgical procedures were performed.

After the rats were sacrificed by decollation, intestinal and brain tissues were rapidly removed and placed into culture dishes containing Krebs-Ringer nutrient solution (118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl_2, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 1.2 mM MgSO₄, 0.026 mM EDTA and 11.1 mM glucose) at 4°C. Under a light microscope, the rat basilar arteries (300-µm diameter) and the third-order branches of mesenteric arteries (250-µm diameter) were carefully dissected using microtweezers and cut into ~2-mm microarterial rings. The arterial rings were then mounted in a wire myograph (Model 610M; Danish Myo Technology AS, Aarhus, Denmark) as described in a previous study (21) and fixed into the 37°C thermostatic bath of a microvascular tension meter (Danish Myo Technology AS). When the tension index became stable, 6 nM ET-1 was added into basilar arteries and 40 µM potassium chloride into mesenteric arteries to induce a precontracted model. The arterial endothelial activity was evaluated by ACh with single or cumulative concentration, which is a classical tool used in medicine to assess whether the vascular endothelium is intact or impaired. In another experiment, ChCl was cumulatively mixed into the 37°C thermostatic bath in accordance with concentration-response relationships to observe the effects of the drug on the dilation of cerebral basilar or mesenteric arteries.

A total of 48 rats were used in the analysis of acute hypoxic exposure. As described above, the rats were housed individually under a 12-h light/dark cycle at a temperature of 18–24°C and relative humidity of 56±10% and provided ad libitum access to food and water under normoxic conditions. After the rats were anesthetized with 1–2% isoflurane, they were sacrificed by decapitation, the mesenteric arteries were dissected and cut into ~2-mm microarterial rings. The arterial rings were randomly divided into three groups: Normoxic control (no treatment, n=16), H1 (pretreatment with hypoxia for 1 h, n=16) and H3 (pretreatment with hypoxia for 3 h, n=16). All the arterial rings were fixed into the 37°C thermostatic bath of the microvascular tension meter and arterial tension measurements were conducted. This was carried out as described above, except that the arterial rings of the H1 and H3 groups were placed into a hypoxic incubator containing 1% O₂, 92% N₂ and 5% CO₂, for 1 and 3 h, respectively, before they were fixed into the 37°C thermostatic bath of the microvascular tension meter.

A total of 5 wild-type, 3- to 5-month-old male Wistar rats, weighing 180–220 g, provided by the Experimental Animal Center of Beijing Academy of Military Medical Sciences (Beijing, China), were used for the RAECs acquisition. The rats were housed individually under a 12-h light/dark cycle at a temperature of 18–24°C and relative humidity of 56±10% and provided ad libitum access to food and water. The care and use of animals in the experiments met the standards set out in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (Bethesda, MD, USA) and closely complied with the Animal Care and Use Committee of the Tianjin Institute of Health and Environmental Medicine (Tianjin, China). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Tianjin Institute of Health and Environmental Medicine (permit no. 2013-D-3806). Animals were administered 1–2% isoflurane through a face mask for anesthesia prior to sacrifice by cervical dislocation. The chest was opened under sterile conditions and the aortas were quickly removed and washed twice using D-Hanks solution (Beijing Leagene Biotechnology Co., Ltd., Beijing, China; pH=7.2) to wash away the blood. The connective tissues surrounding blood vessels were excluded, and the blood vessels were longitudinally cut open, and sliced into 1 mm² sections, which were affixed to the bottom of a Petri dish. M199 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 20% fetal bovine serum (Hyclone; GE Healthcare, Logan, UT, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.) was slowly added to the Petri dish, once the chips were firmly affixed to the bottom. The Petri dish was transferred to an incubator at 37°C and 5% CO₂ for 72 h. When a small amount of RAECs were visible, half the medium was replaced. The medium was completed replaced after 5 days, when a large number of cells had proliferated. Finally, Rat aortic endothelial cells (RAECs) were identified using an antibody against CD31 labeled with FITC (ab33858, 1:200; Abcam, Cambridge, UK). The cells from 4 to 10 generations were selected to be used in the experiments, and when cells at the logarithmic growth phase were digested into a single cell suspension and plated into 96-well plates with 102–104 cells/well, in an M199 medium supplemented with 20% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin in a 37°C, 5% CO₂ incubator until they reached ~90% confluence. The cells were then randomly divided into two groups and all of them were cultured in the serum-free M199 medium. One group remained in the normoxic incubator containing 5% CO₂ at 37°C, and the other was placed into a 37°C incubator containing 1% O₂, 92% N₂ and 5% CO₂.

The proliferation and the survival rates of RAECs cultured in normoxic or hypoxic conditions were measured by MTS assay (ab197010; Abcam) in accordance with the manufacturer's instructions. In brief, cells at the logarithmic growth phase were digested into a single cell suspension and plated into 96-well plates with 102–104 cells/well, 100 µl per well. Cells were cultured in 37°C normoxic or hypoxic incubator for 48 h. 20 µl MTS solution was added per well, and incubated at 37°C for 2 h. Finally, the 96-well plates were measured under 490 nm wavelength, and the cell growth curve was drawn.

LDH is an important glycolytic enzyme widely present in the cytoplasm, so determining its activity levels in the cell culture supernatant indicates the degree of cell damage (22). The activity of extracellular LDH was measured using an LDH Detection kit (Sigma-Aldrich; Merck KGaA). In brief, RAECs at the logarithmic growth phase were digested and plated into 96-well plates with 102–104 cells/well, in an M199 medium supplemented with 20% fetal bovine serum in the 37°C normoxic or hypoxic incubator. Cells were cultured until they adhered to the bottom, then the medium was replaced with a serum-free medium. Once the cells were treated under the normoxic or hypoxic conditions as described previously, they were collected with 1% bovine serum albumin analysis solution. Following this, 200 µl analysis solution containing 104 cells was added to 96-well plates and incubated for 2 h at 37°C in a normoxic or hypoxic incubator. Cells were then centrifuged for 10 min at 250 × g, and 100 µl supernatant per well was immediately transferred to new 96-well plates. To each well, 100 µl reaction mixture was added and incubated 30 min at room temperature. Finally, the absorbance of all samples was measured under 490 nm wavelength.

After RAECs were treated with normoxic or hypoxic conditions as described above, the serum-free culture supernatant was collected (as described in LDH assay to determine the degree of cell damage), and the levels of VEGF in the supernatant were measured using commercially available ELISA kits (Cell Signaling Technology, Inc., Danvers, MA, USA), which contain the Wash Solution, Detection Antibody, Streptavidin solution and Stop Solution, according to a standard enzyme immunoassay procedure. In brief, when the serum-free cell culture supernatant was collected, 100 µl of each standard and sample was immediately added into appropriate wells. The plates was covered and incubated for 2.5 h at room temperature with gentle agitation. The solution was discarded and washed 4 times with 1X Wash Solution. Following this, 100 µl of 1X prepared Detection Antibody was added to each well and the plates were incubated for 1 h at room temperature with gentle agitation. The wash procedure was repeated and 100 µl of prepared Streptavidin solution was added to each well followed by 45 min incubation at room temperature and the wash procedure. TMB One-Step Substrate Reagent (100 µl) was added to each well. Finally, following 30 min incubation at room temperature in the dark with gentle agitation, 50 µl Stop Solution was added, and the absorbance of all samples was measured under 450 nm wavelength immediately.

All data are presented as the mean ± standard error. Results were analyzed using the Student's t-test and analysis of variance with SPSS version14.0 software (SPSS, Inc., Chicago, IL, USA), and P<0.05 was considered to indicate a statistically significant difference.

The endothelial activity of cerebral basilar and mesenteric arterioles of rats exposed to normoxic or CIH conditions were studied using 10 µM ACh. The results showed that the dilation of the normoxic control group was 21.41±7.18% when it was treated with 10 µM ACh, and that of the CIH group was 12.27±4.09% (Fig. 1A). The relaxation rate of the cerebral basilar arterioles was significantly lower in the CIH group compared with the normoxic control group (P<0.01), which suggested that the rat brain VECs had been injured by CIH treatment, and their endothelium-dependent relaxation response to ACh weakened.

Likewise, vascular endothelial damage caused by CIH was also found in the mesenteric arterioles (Fig. 1B). The dilation of the normoxic control group was 46.03±15.34% after treatment with 10 µM ACh, and that of the CIH group was 31.13±10.37%. The relaxation rate of the mesenteric arterioles was significantly lower in the CIH group compared with the normoxic control group (P<0.01). This suggested that CIH exposure caused rat systemic peripheral resistance vessel endothelium injuries and weakened the endothelium-dependent relaxation response to ACh.

The maximum relaxation rates of cerebral basilar arterioles in the normoxic control and CIH groups after treatment with choline were 8.06±2.68 and 19.55±6.51%, respectively (P<0.01; Fig. 2A). The relaxation rate was significantly higher in the hypoxia group compared with the control group (P<0.05 for choline concentrations 10–10, 10–9 and 10–8 mol/l, P<0.01 for choline concentrations 10–7, 10–6, 10–5 and 10–4 mol/l). This indicated that the vasodilator effect of choline on the cerebral basilar arterioles was stronger in the CIH group than in the control group, suggesting that choline could have protective effects against cerebral ischemia induced by hypoxia and improve cerebral circulation.

The vasodilator effects of choline on the mesenteric arterioles showed the same trend. The maximum relaxation rates of the normoxic control and the CIH groups after treatment with choline were 13.32±4.44 and 29.18±9.72%, respectively (P<0.05; Fig. 2B). The relaxation rate was significantly higher in the hypoxia group compared with the control group at choline concentrations of 10⁻⁶, 10⁻⁵ and 10⁻⁴ mol/l (P<0.05). This indicated that the vasodilator effect of choline on the mesenteric arterioles was greater in the CIH group than in the normoxic control group, suggesting that choline could have vasodilator effects against peripheral resistance vascular injuries induced by hypoxia.

The vasodilator effect of choline on normal arterioles was weaker than that on arterioles injured by hypoxia. The selectivity may contribute to the function of choline, currently used for treating stroke, hypertension and other acute and chronic diseases caused by hypoxia, with the advantage of less adverse reactions to the normal human. Therefore, it suggests that choline may be a potential treatment for these diseases.

In the aforementioned experiments, it was found that the endothelial activity of rat cerebral basilar and mesenteric arterioles was affected by CIH, and choline showed protective effects against vascular endothelium damage caused by CIH. The current study subsequently investigated whether these effects were due to CIH treatment specifically, or hypoxia treatment in general.

Healthy rat isolated mesenteric arteries were exposed to acute hypoxic conditions for 1 h (H1 group) or 3 h (H3 group) to observe the effects of acute hypoxic exposure on their vascular endothelial activity. To investigate the effect of acute hypoxic exposure on the endothelial activity of rat isolated mesenteric arterioles, then the arterioles was treated with 10 µM ACh. After treatment with ACh, the vascular endothelium-dependent relaxation of the H1 group was found to have no significant change compared with the control group (Fig. 3A). The relaxation response slightly decreased in the H3 group compared with the control group, but no significant difference was noted. The ACh half maximal effective concentration (EC₅₀) values of the control, H1 and H3 groups were 3.049×10⁻⁸, 3.721×10⁻⁸ and 5.490×10⁻⁸ M, respectively.

Furthermore, the vasodilator effect of choline on the control, H1 and H3 groups was investigated. The response was found to be greater for the H1 group than for the control group, and the strongest effect was found in the H3 group. However, the differences between groups were not significant (Fig. 3B). The choline EC₅₀ values of the control group, H1 group and H3 group were 7.995×10⁻⁷, 3.899×10⁻⁷ and 1.176×10⁻⁷ M, respectively. The results suggested that the vasodilator effects of choline on the mesenteric arterioles under acute hypoxic conditions gradually increased with time, but no significant differences were identified.

The effects of choline on promoting endothelial cell proliferation under hypoxic conditions and protecting endothelial cells against the damage caused by hypoxia were observed.

RAECs were treated with choline for 24 h under normoxic conditions. A choline dosage of 1,000 µM was found to significantly increase the proliferation of endothelial cells compared with the control (P<0.01; Fig. 4A), but the LDH content in the supernatant did not increase significantly at any choline concentration tested (Fig. 4B). The same results and trends were found in the cells treated with choline under normoxic conditions for 48 h, although a significant increase in endothelial cell proliferation was observed from concentrations of 0.1 to 1,000 µM (P<0.01; Fig. 4C and D).

The effects of choline on promoting cell proliferation and protecting against damage under hypoxic conditions were determined (Fig. 5). Under hypoxic conditions, choline with a concentration from 0.1 to 10 µM was able to significantly increase proliferation of RAECs, when choline treatment was for 24 (P<0.05; Fig. 5A and C). Choline treatment for both 24 and 48 h significantly reduced LDH activity in the cell supernatant, compared with the control (P<0.05; Fig. 5B and D).

The mechanisms by which choline promotes endothelial cell proliferation were explored. First, the effect of choline on VEGF secretion of RAECs under hypoxic conditions was investigated. It was found that the quantity of VEGF in the endothelial cell culture supernatant significantly increased under hypoxic conditions for 24 h (P<0.05; Fig. 6A). When the RAECs were incubated with 10 µM choline, the quantity of VEGF in the supernatant increased further (P<0.01; Fig. 6A). Though there was no significant difference between the choline-treated RAECs and the hypoxic group (P>0.05; Fig. 6A), but there was a slight increase. This indicated that hypoxia promoted VEGF secretion from endothelial cells, and choline further increased the VEGF secretion of endothelial cells under hypoxic conditions, suggesting that the mechanism of choline stimulating endothelial cell proliferation might be related to promoting VEGF secretion.

The effect of MLA, an antagonist of α7 nAChR, on choline was also observed. It was found that the effect of choline on promoting rat artery endothelial cell proliferation under hypoxic conditions was significantly inhibited by MLA (P<0.01; Fig. 6B). This suggested that choline may increase cell proliferation by activating α7 nAChR.