Dulbecco’s modified Eagle’s medium (DMEM), penicillin, streptomycin, trypsin, dimethyl sulfoxide (DMSO) and foetal bovine serum were purchased from Gibco, Singapore. 3-(4, 5-dimethylthialzal-z-yl)-2,5-diphenylterazolium bromide (MTT), Hoechst 33258 dye and DHA (22:6n-3) were procured from Sigma-Aldrich (Singapore). Caspase-3 Fluorescence Assay kit was from Cayman chemicals (Michigan, USA). The other reagents were all from Sigma Chemical Co (Singapore). DHA was dissolved in 99.5% ethanol and stocked as aliquots at -80˚C. As DHA is highly susceptible to oxidation, every aliquot was used not more than two times.

Rat aortic VSMC purchased from American Type Culture Collection (ATCC, Manassas, USA) was used for this study. Cells were cultured in DMEM consisting of 5.5 mmol/L glucose, 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37˚C (humidified atmosphere with 5% CO2). Cells at passages 7-9 were used for the experiments.

VSMCs were trypsinized into cell suspension and seeded in each well of 96-well plate with density of 10⁴ cells per well. When the cells became confluence (70-80%), serum-free DMEM was used to replace the medium and cells were allowed to synchronize for 24 hrs. Synchronized cells were then split into eight different categories: normal control group (Con), SHG group, IHG group, DHA control group (DHA-Con), DHA-SHG group, DHA-IHG group and osmotic control groups (OS1 and OS2). In the Con group, cells were exposed to normal concentration of glucose (5.5 mmol/L). The cells in SHG group were treated with stable high concentration of glucose (25 mmol/L) whereas those in IHG group, they were treated with intermittent concentrations (fluctuating between 5.5 mmol/L and 25 mmol/L within 12 hrs) and were incubated for 72 hrs. In DHA-SHG group, the cells were pretreated with 20 µM DHA and exposed to stable glucose whereas in DHA-IHG group, the cells were pretreated with 20 µM DHA and exposed to intermittent concentrations of glucose for indicated period. The cells in DHA-Con group were incubated with identical amount of DHA without any further treatment. Among the two osmotic controls, one was treated with mannitol (44.4 mM) plus normal glucose (5.5 mmol/L) as a common high osmotic control; the other represents an intermittent high osmotic control (alternating between 5.5 mmol/L glucose or mixture of mannitol and 5.5 mmol/L glucose every 12 hrs). Equal quantities of ethanol were used to incubate the control groups and the concentration in the medium was maintained at less than 0.1%. Cells in all different categories were treated under identical conditions and all experiments were conducted three times at least.

Cell proliferation was evaluated by estimating the metabolism of 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). In brief, VSMCs seeded in 96-well microtiter plates were pretreated with 20 µM DHA and exposed to either constant or intermittent high glucose for 72 hrs. After the treatment, 20 μL of MTT solution (5 mg/mL) was mixed in all the wells and cells were incubated at 37°C for another 4 hrs. The solution was disposed of and 150 μL of solubilisation solution (DMSO) was added into each well followed by 10 mins shaking. Absorbance of the solubilised blue formazin dye was read at 570 nm using a microplate reader. Cell viability was represented as a percentage of cytoprotection versus control group, fixed at 100%.

For morphological evaluation of nuclei, VSMCs were stained using nuclear dye Hoechst 33258. Hoechst 33258 fluorochromes have been used to define the nuclear morphological features that act as a quantitative index of apoptosis in vast majority of cell culture systems [24, 25]. After treatment, cells were stained with Hoechst 33258 (10 μg/mL) at room temperature in the dark for 30 mins and washed three times with PBS. Finally, cells were illuminated with ultraviolet light and nuclear morphological transformations, such as condensed and disintegrated nuclei with intense bright fluorescence, were visualized under fluorescence microscopy.

Apoptosis was estimated quantitatively through the analysis of propidium iodide (PI) staining in flow cytometry. In brief, VSMC grown in 25 cm² culture flasks, after being treated with glucose, were harvested by trypsin and washed with PBS. Cells were then fixed with 70% ice cold ethanol at 4°C overnight. Next day, cells were washed twice with cold PBS followed by incubation with 100 µg/ml RNase at 37°C for 20 mins. After that, cells were centrifuged (10 min, 1800 rpm at room temperature) and the pellets were subjected to incubation with PBS containing 50 µg/ml PI at 37°C in the dark for 30 mins. Samples were analysed using flow cytometer (Becton Dickinson FACScan) and flowing software.

Caspase 3 activity was assessed through monitoring the fluorescence of t N-Ac-DEVD-N’-MC-R110 substrate with respect to the manufacturer’s instructions (Cayman Chemical, USA). VSMCs seeded in 96-well plate were treated as described above, and the cell lysates were prepared by adding 100 µl cell lysis buffer (Cayman Chemical) followed by shaking for 30 min at room temperature. After centrifugation at 800 x g for 10 min, 100 µl of supernatant was shifted to another plate followed by incubation at 37°C with 100 µl of N-Ac-DEVD-N’-MC-R110, a caspase 3 substrate. Fluorescence of the cleaved substrate was measured at an excitation wavelength at 485 nm and an emission wavelength at 535 nm using a microplate reader.

Paired student’s t-test was used to analyse the data obtained. Discrepancies of p values less than 0.05 were taken as statistically significant and the results were expressed as mean ± standard deviation (SD) attained from at least three experiments.

It is known that IHG is more detrimental than SHG in hastening abnormal proliferation of VSMCs in diabetes [6, 26]. To test whether DHA modifies IHG-induced proliferation of VSMCs, quiescent cultures were pretreated with DHA and exposed to constant or intermittent high glucose concentrations for 72 hrs. After treatment, cell viability was measured using the MTT reduction assay.

As depicted in Fig. (1), cell viability of the Con group was fixed at 100%. The cell viability of VSMCs exposed to SHG was significantly increased to 160.35 ±1.88% (p<0.001) of the Con value. Furthermore, IHG was found to induce significant increase in cell viability more than SHG (192.25±1.71%, p<0.001). Pre-treatment of cells with DHA reduced the cell viability to 107.31±4.21% (p<0.001) in DHA-SHG group and to 108.93±6.74% (p<0.001) in DHA-IHG treated group. However, the Con groups pre-treated with DHA did not show any significant difference in cell viability. Also, there was no substantial discrepancy in the cell viability noted among the mannitol-treated and control group. These results indicated that the VSMCs exposed to IHG showed increased cell proliferation than SHG and pre-treatment with DHA significantly attenuated the induced proliferation in both SHG and IHG group.

Fig. (1). Effect of DHA on the proliferation of VSMCs induced by IHG. Quiescent cultures of VSMC were pretreated with DHA and exposed to constant or intermittent high glucose level for 72 hrs. After treatment, cell survival was examined using the MTT reduction assay. Cell viability was calculated as the percentage of the control group. The data represent mean ± SEM of 3 individual experiments. *P < 0.001 versus Con, # P < 0.001 versus SHG, ** P < 0.001 versus IHG.

Cell proliferation and apoptosis represent two different cellular events, however, they are often found intimately connected under physiological conditions. As reported by previous studies, any factor which restrains the viability of cell could suscitate the apoptosis of that cell or vice versa [27]. The effects of DHA on apoptotic index of VSMCs treated with HG was assessed by monitoring apoptotic morphological differentiations using bis-benzimide (Hoechst 33258) staining under a fluorescent microscope.

As illustrated in Fig. (2), the nuclei of Con cells contained evenly dispersed chromatin with sparse fluorescence signal showing almost no signs of morphological nuclear damage. Cells exposed to SHG and IHG also exhibited no chromatin disintegration or margination. In marked contrast, DHA treatment in SHG and IHG group demonstrated enhanced DNA fragmentation and condensation. However, treatment with mannitol did not instigate the apoptosis of VSMCs in both control groups.

Fig. (2). Effects of DHA on the nuclear morphology of HG-treated VSM cells stained with Hoechst 33258. The Con group showed cells with uniformly dispersed chromatin and weak fluorescence signals. DHA-treated SHG and IHG group demonstrated hypercondensed chromatin with increased number of apoptotic cells.

The above results revealed that DHA treatment could result in increase of apoptotic cells in both SHG and IHG groups, further affirming that DHA induced cell death through apoptosis.

To quantitatively gain insight into the effect of DHA on apoptosis of VSMCs exposed to IHG, the apoptosis rate of VSMCs was computed by FACS assay using PI staining (Fig. 3a). As shown in Fig. (3b), Con group revealed lower apoptosis rate of 6.62 ± 1.48%. Similarly SHG and IHG groups also showed reduced percentage of apoptotic cells. On the contrary, DHA treatment in SHG and IHG group triggered significant elevations of apoptosis with the apoptosis rate of 31.54 ± 1.39% and 39.89 ± 5.39% respectively. However, mannitol treatment did not show any changes in both control groups.

Cell apoptotic events, triggered by a diverse array of stimuli, focalize on the stimulation of caspase family, premier among which is caspase-3. Caspase-3 enacts an inevitable role in turning on the apoptotic processes in a wide range of cell types including smooth muscle cells [28]. In order to investigate whether DHA mediates its apoptotic effects through caspase-3, caspase-3 activity in VSMC was assessed after being exposed to SHG and IHG. The results revealed that high glucose treatment in both cases did not affect the basal caspase-3 activity when compared to the control Fig. (4). Besides, mannitol treatment in both osmotic control groups did not influence the basal caspase-3 activity. However, addition of DHA resulted in a 2.95-fold raise in caspase-3 activity in the SHG group (P<0.001) and a 3.65-fold increase in the IHG group (p<0.001), while no significant effects in the Con group could be shown.