Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise

Abstract

Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16^INK4A, p18^INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16^INK4A, p18^INK4C, CDK4 and CDK6 expressions in physiological term villous explants. PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16^INK4A, p18^INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay. We reported significantly increased p16^INK4A and p18^INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16^INK4 and p18^INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast. We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise.

Keywords: cell cycle; mesenchymal stromal cells; placenta; preeclampsia; pregnancy.

Figure 1.

p16^INK4A and p18^INK4C gene and protein expression in normal vs preeclamptic PDMSCs. (a) mRNA (left panel) and protein (right panel) expression of p16^INK4A in normal (N, n = 20) and PE-PDMSCs (PE, n = 24) (b) mRNA (left panel) and protein (right panel) expression of p18^INK4C in normal (N, n = 20) and PE-PDMSCs (PE, n = 24). Statistical significance (*) has been considered as p < 0.05.

Figure 2.

CDK4 and CDK6 gene and protein expression levels in normal vs preeclamptic PDMSCs. (A) mRNA (left panel) and protein (right panel) expression of CDK4 in normal (N, n = 20) and PE-PDMSCs (PE, n = 24) (B) (b) mRNA (left panel) and protein (right panel) expression of CDK6 in normal (N, n = 20) and PE-PDMSCs (PE, n = 24). Statistical significance (*) has been considered as p < 0.05.

Figure 3.

PDMSCs conditioned media effect on physiological term placental villous explants viability. Positive control (+) was provided by the kit. Explants treated by Triton X-100 and by unconditioned culture media for 8 h were used as high (H) and low controls respectively. Cytotoxicity in 72h supernatant of physiological villous explants treated with unconditioned media (CTRL) or media conditioned by normal (N CM) and preeclamptic (PE CM) PDMSCs was assessed by LDH assay. Statistical significance (*) has been considered as p < 0.05.

Figure 4.

JunB and Cyclin D1 gene and protein expression levels in physiological placental villous explants treated with culture media conditioned by normal or PE-PDMSC. (A)JunB mRNA (left panels) and protein (right panels) expression levels in physiological villous explants treated with unconditioned media (CTRL, n = 16 explants) or media conditioned by normal (N CM, n = 16 explants) and preeclamptic (PE CM, n = 16 explants) PDMSCs as assessed by Real Time PCR and Western Blot analysis. B) Cyclin D1 mRNA (left panels) and protein (right panels) expression levels in physiological villous explants treated with unconditioned media (CTRL, n = 16 explants) or media conditioned by normal (N CM, n = 16 explants) and preeclamptic (PE CM, n = 16 explants) PDMSCs as assessed by Real Time PCR and Western Blot analysis. Statistical significance (*) has been considered as p < 0.05.

Figure 5.

P16^INK4A and p18^INK4C gene and protein expression levels in physiological placental villous explants treated with culture media conditioned by normal or PE-PDMSC. (A) P16^INK4A mRNA (left panels) and protein (right panels) expression levels in physiological villous explants treated with unconditioned media (CTRL, n = 16 explants) or media conditioned by normal (N CM, n = 16 explants) and preeclamptic (PE CM, n = 16 explants) PDMSCs as assessed by Real Time PCR and Western Blot analysis. B) and p18^INK4C mRNA (left panels) and protein (right panels) expression levels in physiological villous explants treated with unconditioned media (CTRL, n = 16 explants) or media conditioned by normal (N CM, n = 16 explants) and preeclamptic (PE CM, n = 16 explants) PDMSCs as assessed by Real Time PCR and Western Blot analysis. Statistical significance (*) has been considered as p < 0.05.

Figure 6.

CDK4and CDK6 gene and protein expression levels in physiological placental villous explants treated with culture media conditioned by normal or PE-PDMSC. (A) CDK4 mRNA (left panels) and protein (right panels) expression levels in physiological villous explants treated with unconditioned media (CTRL, n = 16 explants) or media conditioned by normal (N CM, n = 16 explants) and preeclamptic (PE CM, n = 16 explants) PDMSCs as assessed by Real Time PCR and Western Blot analysis. B) CDK6 mRNA (left panels) and protein (right panels) expression levels in physiological villous explants treated with unconditioned media (CTRL, n = 16 explants) or media conditioned by normal (N CM, n = 16 explants) and preeclamptic (PE CM, n = 16 explants) PDMSCs as assessed by Real Time PCR and Western Blot analysis. Statistical significance (*) has been considered as p < 0.05.

Figure 7.

Cyclin D1 spatial localization in physiological placental villous explants treated with culture media conditioned by normal or PE-PDMSC. (A) Absence of positive immunoreactivity for Cyclin D1 in section stained with control IgG. B) Cyclin D1 spatial localization in physiological villous explants treated with unconditioned media (CTRL, n = 4 explants) assessed by immunofluorescent staining. C) Cyclin D1 spatial localization in physiological villous explants treated with media conditioned by normal (N CM, n = 4 explants) assessed by immunofluorescent staining. C) Cyclin D1 spatial localization in physiological villous explants treated media conditioned by with preeclamptic (PE CM, n = 4 explants) assessed by immunofluorescent staining. Cell nuclei are showed in blue by DAPI signal. TR, trophoblast cells; M, mesenchyme; V, vessel. Original magnifications, x40.