Emerging evidence showed that microparticles (MPs) derived from placental trophoblasts (TC) contribute to maternal vascular dysfunction and promote endothelial inflammatory response in preeclampsia (PE). Vitamin D insufficiency has been linked to placental dysfunction in pregnancy disorders such as PE and IUGR.
Objective
To determine: (1) if TC from PE placentas release more MPs than TC from normal placentas; (2) if vitamin D could suppress oxidative stress-induced MP release by placental TC; and (3) potential mechanism of vitamin D protective effects on placental TCs.
Methods
TC were isolated from normal and PE placentas by trypsin digestion and purified with Percoll gradient centrifugation. Freshly isolated TC (5 × 06 cells/well) were seeded into 6 well plates and cultured with DMEM containing 5% FBS and antibiotics for 72 h. For the oxidative stress experiment, cobalt chloride (CoCl2, a hypoxic mimicking agent) was used to induce TC oxidative stress and 1,25(OH)2D3 was used as bioactive vitamin D. MPs were isolated from culture supernatants by a two-step centrifugation procedure (420 g for 20 min and then 20,000 g for 60 min) and quantified by annexin V positive staining measured by flow cytometry with TruCount beads to determine absolute counts of MP for each sample. Data are expressed as annexin V positive MP/μg cell protein/well. Total cellular protein and MP protein were also collected and protein expression for caveolin-1, eNOS, pro-caspase-3, cleaved caspase-3, and ROCK1 were determined by Western blot. Data were analyzed by paired or unpaired t-test or ANOVA with Newman-Keuls test as a post hoc test. A p level <0.05 was considered statistically significant.
Results
TC isolated from PE placentas (n = 6) released significantly more MP than cells isolated from normal placentas (n = 6), 295.20 ± 47.67 vs. 117.20 ± 18.02/μg protein/well, p < 0.01. MP release was significantly increased in cells treated with CoCl2 compared to control TC, p < 0.01. The increased MP released induced by CoCl2 was significantly reduced in TC treated with 1,25(OH)2D3 + CoCl2, p < 0.05. TC expression of caveolin-1 was increased and eNOS expression was decreased, but MP expression of caveolin-1 and eNOS was increased, in cells treated with CoCl2. This phenomenon could be reversed when cells were treated with 1,25(OH)2D3. Moreover, reduced pro-caspase-3 and ROCK1 expression in TC induced by CoCl2 could also be attenuated in cells treated with 1,25(OH)2D3.
Conclusions
MP release was significantly increased in TC from PE placenta. 1,25(OH)2D3 suppresses oxidative stress-induced MP release by placental TC. Caveolin-1 is an essential structural component of caveolae and eNOS is associated with plasma membrane caveolae. Therefore, reduced caveolin-1 and increased eNOS expression together with reduced MP release in TC treated with 1,25(OH)2D3 demonstrate that vitamin D could protect placental TC from oxidative stress-induced injury during pregnancy. Since caspase-3 cleavage and ROCK1 activation are key steps of MP formation and release, suppression of caspase-3 cleavage and ROCK1 activation by vitamin D could be a plausible mechanism of vitamin D inhibition of increased MP release induced by oxidative stress.
