Conditioned Media of Choroid Plexus Epithelium Cells Attenuates High Pi-Induced Calcification of MOVAS Cells by Inhibiting ROS-Mediated Signal Pathways

Abstract

Vascular calcification was an independent risk of cardiovascular and cerebrovascular diseases (CCDs). Studies reported that conditioned media of choroid plexus epithelium cells (CPECs-CM) showed potential neuroprotective effects. However, the protective effect of CPECs-CM against vascular calcification (VC) has not been reported yet. Herein, high phosphate (HPi)-induced calcification model in mouse aortic vascular smooth muscle cells (MOVAS) was established, and the protective effects and underlying mechanism of CPECs-CM against HPi-induced calcification were explored. The results indicated that CPEC cells were successfully isolated and cultured, and CPECs-CM co-treatment significantly inhibited HPi-induced calcification of MOVAS cells through blocking alkaline phosphatase activity and expression. CPECs-CM co-treatment also suppressed reactive oxide species-mediated DNA damage in HPi-treated MOVAS cells. Moreover, dysfunction of MAPKs and PI3K/AKT pathways both contributed to HPi-induced calcification of MOVAS cells, and CPECs-CM co-treatment attenuated HPi-induced calcification by normalizing MAPKs and PI3K/AKT expression. Taken together, our findings provide evidence that CPECs-CM had the potential to inhibit vascular calcification with potent application in chemoprevention and chemotherapy of human CCD.

Keywords: DNA damage; MAPKs and PI3K/AKT; ROS; choroid plexus epithelium cells; vascular calcification.

FIGURE 1

Isolation, culture, and identification of CPECs. (A1,A2) Identification of choroid plexus in rat brain. (B1,B2) CPECs were isolated and cultured with cultured with DMEM/F12 medium. The morphology of CPECs (B1: low concentration, B2: high concentration) was observed by phase microscope. Morphology of isolated CPECs. (C1,C2) Identification of isolated CPECs. CPECs were labeled by immunofluorescent staining using a transthyretin (TTR) antibody, a CPEC marker.

FIGURE 2

CPECs-CM attenuates high Pi-induced calcification of MOVAS cells. (A) CPECs-CM inhibited high Pi-induced calcification of MOVAS cells. MOVAS cells seeded in a 6-well plate were cultured with CPECs-CM and treated with 3 mM high Pi for 14 days. Calcium nodules were stained by alizarin red. (B) Absorbance of alizarin red. Alizarin red dye was eluted with 10% formic acid and quantified by a microplate reader at 420 nm. (C) Measurement of calcium content. Protein was quantified by BCA kit, and O-cresolphthalein complexone method was used to examine calcium content as previously described. Ca content was expressed as micrograms per milligram of protein. All data were obtained from three independent experiments. Bars with different letters indicate significance at P < 0.05 level.

FIGURE 3

CPECs-CM suppressed high Pi-induced ALP activity and expression. (A) ALP staining of MOVAS cells. MOVAS cells seeded in a 6-well plate were cultured with CPECs-CM and treated with 3 mM high Pi for 14 days. ALP staining was conducted by an ALP staining kit. (B) Measurement of ALP activity. Total protein was extracted and quantified by BCA kit. ALP activity was examined by an ALP activity kit according to the manufacturer’s instructions. (C,D) ALP expression was detected by western blotting method. All data were obtained from three independent experiments. Bars with different letters indicate significance at P < 0.05 level.

FIGURE 4

CPECs-CM prevents high Pi-induced ROS generation and DNA damage. (A) Real-time imaging of ROS generation and superoxide anion. MOVAS cells were seeded in a 6-well plate and pre-loaded with DCFH-DA or DHE probes for 15 min in the dark. Then cells were washed with PBS and treated with 3 mM high Pi or/and CPECs-CM for 90 min. The ROS generation (green fluorescence) and superoxide anion (red fluorescence) were imaged by a fluorescent microscope. (B) Quantitative analysis of superoxide anion. Intracellular superoxide anion generation was quantified by a microreader. (C) Real-time DNA damage. MOVAS cells were seeded in a 9-cm plate and treated with 3 mM high Pi for 1, 2, 4, 8, 10, 12, and 14 days. (D) CPECs-CM inhibited high Pi-induced DNA damage. MOVAS cells seeded in a 9-cm plate were cultured with CPECs-CM and treated with 3 mM high Pi for 14 days. Protein expression was examined by western blotting. All experiments were done at least three times.

FIGURE 5

CPECs-CM improves high Pi-induced dysfunction of MAPKs and PI3K/AKT. Time-dependent effect of high Pi on MAPKs (A) and PI3K/AKT (B) expression. MOVAS cells seeded in a 9-cm plate were treated with 3 mM high Pi for 1, 2, 4, 8, 10, 12, and 14 days. (C) CPECs-CM improved the expression of MAPKs and PI3K/AKT pathways in high Pi-treated cells. (D) U0126 (an ERK inhibitor) inhibited high Pi-induced calcification of MOVAS cells. MOVAS cells seeded in a 9-well plate were cultured with CPECs-CM and treated with 3 mM high Pi for 14 days. (E) Measurement of calcium content. Protein expression was examined by western blotting. All data were obtained from three independent experiments. Bars with different letters indicate significance at P < 0.05 level.

FIGURE 6

Proposed signal pathways. CPECs were isolated, cultured, and identified. CPECs-CM co-treatment effectively inhibited HPi-induced ROS generation, DNA damage, and dysfunction of MAPKs and PI3K/AKT pathways, and eventually attenuated HPi-induced calcification of MOVAS cells.