Vitamin D Reduces Oxidative Stress-Induced Procaspase-3/ROCK1 Activation and MP Release by Placental Trophoblasts

Abstract

Context: Increased microparticle (MP) shedding by placental trophoblasts contributes to maternal vascular inflammatory response and endothelial dysfunction in preeclampsia. Vitamin D has beneficial effects in pregnancy; however, its effect on trophoblast MP release has not been investigated.

Objective: To investigate if vitamin D could protect trophoblasts from oxidative stress-induced MP release.

Design: Placental trophoblasts were isolated from uncomplicated and preeclamptic placentas. Effects of vitamin D on MP release induced by oxidative stress inducer CoCl2 were studied.

Main outcome measures: Annexin V+ MPs were assessed by flow cytometry. Expression of caveolin-1, endothelial nitric oxide synthase (eNOS), procaspase-3, cleaved caspase-3, and Rho-associated coiled-coil protein kinase 1 (ROCK1) in trophoblasts and trophoblast-derived MPs were determined by Western blot.

Results: Trophoblasts from preeclamptic pregnancies released significantly more MPs than cells from uncomplicated pregnancies (P < 0.01). CoCl2-induced increase in MP release was associated with upregulation of caveolin-1 and downregulation of eNOS expression in trophoblasts (P < 0.05), which could be attenuated by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 could also inhibit CoCl2-induced procaspase-3 cleavage and ROCK1 activation in trophoblasts. Consistently, CoCl2-induced upregulation of procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs were also reduced in cells treated with 1,25(OH)2D3.

Conclusions: Placental trophoblasts from preeclamptic pregnancies released more MP than cells from uncomplicated pregnancies. Oxidative stress-induced increase in MP shedding is associated with upregulation of caveolin-1 and downregulation of eNOS expression in placental trophoblasts. Inhibition of caspase-3 cleavage and ROCK1 activation, together with upregulation of eNOS expression, could be the potential cellular/molecular mechanism(s) of vitamin D protective effects on placental trophoblasts.

Figure 1.

MP release and superoxide generation by placental trophoblast cells. (a) MP shedding by trophoblasts from uncomplicated and preeclamptic placentas. Annexin V⁺ MPs were assessed by flow cytometry. Data are expressed as counts per microgram of protein per well. Annexin V⁺ MP shedding was significantly more by trophoblasts from preeclamptic placentas (PE-TC) than those from uncomplicated placentas (Nor-TC). **P < 0.01. (b) CoCl₂-induced increase in trophoblast MP shedding could be attenuated when cells were pretreated with 1,25(OH)₂D₃ (VD). Trophoblasts from uncomplicated placentas were used. The upper panel shows a representative flow cytometry measurement of annexin V⁺ MPs released by control cells, cells treated with VD, cells treated with CoCl₂, and cells treated with VD + CoCl₂. APC-H, allophycocyanin-height. The middle panel box and whisker plots show data from six independent experiments. **P < 0.01 (CoCl₂ treated versus control); #P < 0.05 (VD + CoCl₂ versus CoCl₂). The lower panel shows a dose response effect of CoCl₂ on MPs release by trophoblasts. *P < 0.05 and **P < 0.01 (CoCl₂ treated versus control). Data are from trophoblasts isolated from three placentas, each cultured in duplicate. (c) Consistent with MP shedding, CoCl₂-induced increase in trophoblast superoxide generation could also be attenuated when cells were pretreated with 1,25(OH)₂D₃. **P < 0.01 (CoCl₂ treated versus control); #P < 0.05 (VD + CoCl₂ versus CoCl₂). (d) Trophoblasts and MPs assessed by electron microscopy. Panel a shows an image of trophoblast with membrane blebbing and MPs (magnification, 30,000×; arrows, MPs). Panel b shows and enlargement of the insert of panel a, showing membrane blebbing. Panel c shows an image of MPs, showing that isolated trophoblast MPs are intact and with sizes between 0.1 and 1.0 µm in diameters (magnification, 150,000×; bar, 200 µm).

Figure 2.

Effects of 1,25(OH)₂D₃ (VD) on altered caveolin-1 and eNOS expression induced by CoCl₂ in trophoblasts and trophoblast-derived MPs. Trophoblasts from uncomplicated pregnancies were used. (a) Protein expression for caveolin-1 and eNOS in trophoblasts treated with different concentrations of CoCl₂. The box and whisker plots show relative protein expression after being normalized against β actin from six independent experiments. CoCl₂ induced a dose-dependent increase in caveolin-1 expression, but a dose-dependent decrease in eNOS expression in trophoblasts. *P < 0.05; **P < 0.01 (treated versus control cells). (b) Protein expression for caveolin-1 and eNOS in trophoblast-derived MPs from cells treated with different concentrations of CoCl₂. The box and whisker plots show relative protein expression after being normalized by total cellular protein per well. MP expression of caveolin-1 and eNOS were dose-dependent increased in cells treated with CoCl₂. *P < 0.05; **P < 0.01. (c) Caveolin-1 and eNOS expression in trophoblast cells treated with CoCl₂ in the presence or absence of 1,25(OH)₂D₃. VD prevented CoCl₂-induced increased caveolin-1 expression and decreased eNOS expression in trophoblasts. The box and whisker plots show relative protein expression after being normalized against β actin. **P < 0.01 (CoCl₂ treated versus control); #P < 0.05 (VD + CoCl₂ versus CoCl₂ treated). (d) Expression of caveolin-1 and eNOS in trophoblast-derived MPs in cells treated with CoCl₂ in the presence or absence of 1,25(OH)₂D₃. The increased MP caveolin-1 and eNOS expression induced by CoCl₂ was reduced when cells were treated with VD. The box and whisker plots show relative protein expression after being adjusted by total cellular protein per well from 6 independent experiments. **P < 0.01 (CoCl₂ treated versus control); #P < 0.05 and ##P < 0.01 (VD + CoCl₂ versus CoCl₂ treated). (e) Representative imaging of immunofluorescent staining of caveolin-1 and eNOS in trophoblasts treated with CoCl₂ in the presence or absence of 1,25(OH)₂D₃. Consistent with Western blot results, VD could inhibit CoCl₂-induced increased caveolin-1 expression and decreased eNOS expression in placental trophoblasts.

Figure 3.

Effects of 1,25(OH)₂D₃ (VD) on altered caspase-3 and ROCK1 expression induced by CoCl₂ in trophoblasts and trophoblast-derived MPs. Trophoblasts from uncomplicated pregnancies were used. (a) Protein expression for procaspase-3, cleaved caspase-3, and ROCK1 in trophoblasts treated with different concentrations of CoCl₂. The box and whisker plots show relative expression after being normalized with β actin in each sample. CoCl₂ induced a dose-dependent decrease in procaspase-3 expression, but a dose-dependent increase in cleaved caspase-3 expression. Two bands were detected for ROCK1, inactive form at 160 kDa and a cleaved form at 130 kDa. Although expression of inactive form of ROCK1 was reduced, and activated form was increased in cells treated with CoCl₂, total ROCK1 expression was also dose-dependent decreased in cells treated with CoCl₂. **P < 0.01. (b) Protein expression for procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs from trophoblasts treated with different concentrations of CoCl₂. The box and whisker plots show relative expression after being normalized with total cellular protein per well. A 130-kDa band for ROCK1 was detected in trophoblast-derived MPs. CoCl₂-induced dose-dependent increases in procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs. **P < 0.01. (c) Expression of procaspase-3, cleaved caspase-3, and ROCK1 in trophoblast cells treated with CoCl₂ in the presence or absence of 1,25(OH)₂D₃. The box and whisker plots show relative expression after being normalized with β-actin expression. **P < 0.01 (CoCl₂ treated versus control); #P < 0.05 and ##P < 0.01 (VD + CoCl₂ versus CoCl₂ treated). (d) Expression of procaspase-3, cleaved caspase-3, and ROCK1 in trophoblast-derived MPs from cells treated with CoCl₂ in the presence or absence of 1,25(OH)₂D₃. VD inhibited CoCl₂-induced increases in procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs. The box and whisker plots show relative protein expression after being adjusted by total cellular protein per well. **P < 0.01 (CoCl₂ treated versus control); ##P < 0.01 (VD + CoCl₂ versus CoCl₂ treated). Data are from six independent experiments.

Figure 4.

Effects of ROCK1 inhibitor on MP release and ROCK1 expression by placental trophoblasts. Trophoblasts from uncomplicated pregnancies were used. (a) Effects of ROCK1 inhibitor Y27632 on trophoblast MP shedding. Trophoblasts were treated with CoCl₂ in the presence or absence of Y27632. Annexin V⁺ MPs were assessed by flow cytometry, and MP counts were normalized by total cellular protein. Cells treated with Y27632 released significantly less annexin V⁺ MPs in both control cells and in cells treated with CoCl₂. *P < 0.05 and **P < 0.01 (treated versus control); #P < 0.05 (Y27632 + CoCl₂ versus CoCl₂ alone). (b) ROCK1 expression in trophoblasts treated with CoCl₂ in the presence or absence of Y27632. The box and whisker plots show relative expression after being normalized with β-actin expression in each sample. Y27632 could preserve ROCK1 expression in trophoblasts. *P < 0.05 (treated versus control); #P < 0.05 (Y27632 + CoCl₂ versus CoCl₂ treated alone). Data are from six independent experiments. Increased ROCK1 expression was correlated with reduced MP release in Y27632-treated cells with or without CoCl₂ treatment.