Reduction in CgA-Derived CST Protein Level in HTR-8/SVneo and BeWo Trophoblastic Cell Lines Caused by the Preeclamptic Environment

Abstract

One of the most dangerous complications of pregnancy is preeclampsia (PE), a disease associated with a high risk of maternal and fetal mortality and morbidity. Although its etiology remains unknown, the placenta is believed to be at the center of ongoing changes. One of the hormones produced by the placenta is chromogranin A (CgA). Thus far, its role in pregnancy and pregnancy-related disorders is enigmatic, yet it is known that both CgA and its derived peptide catestatin (CST) are involved in the majority of the processes that are disturbed in PE, such as blood pressure regulation or apoptosis. Therefore, in this study, the influence of the preeclamptic environment on the production of CgA using two cell lines, HTR-8/SVneo and BeWo, was investigated. Furthermore, the capacity of trophoblastic cells to secrete CST to the environment was tested, as well as the correlation between CST and apoptosis. This study provided the first evidence that CgA and CST proteins are produced by trophoblastic cell lines and that the PE environment has an impact on CST protein production. Furthermore, a strong negative correlation between CST protein level and apoptosis induction was found. Hence, both CgA and its derived peptide CST may play roles in the complex process of PE pathogenesis.

Keywords: catestatin; chromogranin A; hypertension; preeclampsia; pregnancy.

Figure 1

A comparison of relative CHGA gene expression between HTR-8/SVneo (a–d) and BeWo cells (e–h) under different stimulation regimens. The results are expressed as mean ± SEM (n = 3). The results from the cells cultured in 8%O₂ (control) and 2%O₂ were compared using Student’s t-test. One-way ANOVA with post hoc Bonferroni’s correction was used to compare the variants and the controls. p < 0.05 was considered as significant, *** p < 0.001 as compared to control. C—Control (8%O₂), HC—Hypoxic conditions (2%O₂), ILPE1—Inflammatory-like preeclamptic environment 1 (2%O₂ + 1 ng/mL IL6), ILPE2—Inflammatory-like preeclamptic environment 2 (2%O₂ + 10 ng/mL IL6), OLPE1—Oxidative stress-like preeclamptic environment 1 (2%O₂ + 10 µM H₂O₂), OLPE2—Oxidative stress-like preeclamptic environment 2 (2%O₂ + 50 µM H₂O₂), PLE—Preeclamptic-like environment 1 (2%O₂ + 1 ng/mL IL6 + 10 µM H₂O₂), PLE2—Preeclamptic-like environment 2 (2%O₂ + 10 ng/mL IL6 + 50 µM H₂O₂). It was found that the production of CgA protein by the trophoblastic cells depended on the environment. All stimulated variants of both HTR-8/SVneo and BeWo cells demonstrated lowered CgA levels compared to controls. However, only the HTR-8/SVneo cell line yielded significantly different outcomes (p < 0.01). Stimulation with variants reflecting a more or less-intense preeclamptic-like environment resulted in lower relative CgA protein levels compared to the control gene, i.e., GAPDH, and compared to the control culture (8%O₂; normoxia) (Figure 2B). Additionally, CgA protein level was found to be depleted by hypoxia (2% of oxygen), independently of the applied stimulants (Figure 2A). In addition, no significant difference in relative CgA protein level was observed between the other stimulants and controls, in either HTR-8/SVneo or BeWo cells.

Figure 2

Relative CgA protein level compared to GAPDH level (housekeeping gene) in HTR-8/SVneo cells under different stimulation regimens, as indicated by Western blot analysis. (A) All cells were cultured in medium with 2.5%FBS in normoxic (control) or hypoxic conditions. The 8%O₂ (control) and 2%O₂ cultures were compared using the Student’s t-test. (B) CgA expression in cells incubated in medium including IL6 and H₂O₂ under hypoxia (preeclamptic) conditions were compared with untreated controls using one-way ANOVA with Bonferroni’s correction. The results are expressed as mean ± SEM (n = 3). The level of significance was assumed as p < 0.05; ** p < 0.01, * p < 0.05 compared to control. (C) The representative results of Western blot analysis showing bands for CgA and the control GAPDH protein; C—Control (8%O₂), HC—Hypoxic conditions (2%O₂), ILPE1—Inflammatory-like preeclamptic environment 1 (2%O₂ + 1 ng/mL IL6), ILPE2—Inflammatory-like preeclamptic environment 2 (2%O₂ + 10 ng/mL IL6), OLPE1—Oxidative stress-like preeclamptic environment 1 (2%O₂ + 10 µM H₂O₂), OLPE2—Oxidative stress-like preeclamptic environment 2 (2%O₂ + 50 µM H₂O₂), PLE—Preeclamptic-like environment 1 (2%O₂ + 1 ng/mL IL6 + 10 µM H₂O₂), PLE2—Preeclamptic-like environment 2 (2%O₂ + 10 ng/mL IL6 + 50 µM H₂O₂).

Figure 3

A comparison of CST secreted into the cell medium by hypoxic (2%O₂) and normoxic (8%O₂) trophoblastic cells, i.e., HTR-8/SVneo (A) or BeWo (B). CST level was determined in the cell culture medium to test whether CST is secreted by the cells into environment. Thus, the level of CST was determined as ng of studied protein included in 1 mL of cell cultured medium. The results are expressed as ± SEM; n = 3 experiments; the 8%O₂ and 2%O₂ cells were compared using Student’s t-test. The variants and controls were compared using one-way ANOVA with Bonferroni’s multiple comparison test. * p < 0.05 was considered as significant, ** p < 0.01 as compared to control.

Figure 4

The representative results of the apoptosis profile obtained by flow cytometry with the Apoptosis Assay for Muse Cell Analyzer of differently stimulated HTR-8/SVneo (A) and BeWo (B) cells.

Figure 5

Spearman’s rank-order correlation between CST level and total apoptotic index (index for early and late apoptosis): (A) HTR-8/SVneo; (B) BeWo cell line.