In our experiment, we used a primary trophoblastic cells obtained from normal pregnancy that has been exposed to either serum of normal pregnancy, EOPE, or LOPE.(7,27)
Many of in vitro experiments were done as prelude for translational researches. Cell lines have limitations for preelampsia model due to the difficulties in interpretation to in vivo conditions. Many experiments, such as by Petroff et al(29) found experimental techniques to isolate human trophoblastic cells then cultured as primary trophoblastic cells as an approach for in vivo situation. Successful isolation of cytotrophoblastic cells has been proven to resemble human condition for several placental functions, such endocrinology and immunology functions of the placenta, placental differention, and apoptotic conditions.(26,28-30)
Previously, others have used cultured primary trophoblastic cells given serum of preeclamptic patients as models for preeclampsia condition. Pramatirta (26) found expression of TNF-α and caspase-3,and apoptotic index in preeclampsia serum-induced trophoblast cells were higher than normal and controls. In other studies, they found structural derrangement of vessels resembling disruption in interaction of trophoblastic cells with endothelial cells. (25,31) To conclude, these evidences supported the method of serum preeclampsia treatment in vitro is suitable for preeclampsia model.
The mechanisms responsible for the genesis of the preeclamptic syndrome are poorly understood(1,7,32) although roles for abnormal placentation(3-6), uteroplacental ischemia(5,7-9), endothelial cell dysfunction(10-11), and exaggerated maternal inflammatory response to deported trophoblast(3-4,33) have been proposed.
Peroxisome Proliferator-activated Receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily. Included as transcription factors, they are members of the ligand-activated nuclear hormone receptor superfamily, and play major roles in diverse aspects of energy metabolism, inflammation, and development. Following ligand binding, PPARs form heterodimers with retinoid X receptors (RXRs), and bind to PPAR-response elements (PPREs) of target genes to activate transcription.(34-36)
Three subtypes of PPAR has been identified; PPAR α, β, and γ.(20-21) All three subtypes are expressed in placenta with different levels. PPAR-γ has been reported as crucial part of normal placental development as deletion of PPAR-γ in mice is 100% lethal, with no surviving embryos born. Homozygous PPAR-γ-deletion mice embryos die due to severe placental dysfunction.(37-38)
Disrupted expression of PPAR-γ were implicated in several vital placental functions including regulation of inflammatory reactions that resulted in severe gestational disorders. Effect of PPAR-γ in the regulation of pro-inflammatory mediators such as IL-6, IL-8, and TNF-α is important for normal pregnancy to ensue.(20,38) Other evidence proofed the involvement of PPAR-γ in regulation of throphoblasts functions. Antagonists PPAR-γ used in vitro of first trimester extravillous trophoblasts resulted in increase throphoblast invasion, while agonist inhibited it. These data support the role of PPAR-γ in the regulation of trophoblasts invasion to decidua.(21,38)
Previously studies found PPAR-γ was expressed in human placenta.(40-41) PPAR-γ mRNA was expressed in term pregnancy.(39) Previously, others have found the primary culture of isolated trophoblasts from first trimester pregnancy expressed PPAR-γ. This same research strengthen the possible involvement of PPAR-γ in normal pregnancy because they found PPAR-γ expression was preserved until third trimester.(42)
In our study we proved the possible involvement of PPAR-γ in preeclampsia. We used Western Blot technique to analyze expression of PPAR-γ in primary trophoblastic cells treated by serum of normal pregnancy, EOPE, and LOPE. Treatment of the primary trophoblastic cells with LOPE was shown to induce high expression of PPAR-γ. Whereas, treatment of trophoblastic cells by EOPE resulted in similar result as treatment by normal pregnancy, with no induction of PPAR-γ expression. Handschuh et al.(43) stated PPAR-γ activity depend on the trophoblast subpopulation, gestational age, and types of stimulating ligands. Therefore, different content of serum in EOPE and LOPE has different effect to PPAR-γ expression..
In LOPE, maternal factors such as obesity, hyperlipidemia, diabetes, chronic hypertension contribute much for emergence of the disease.(44-47) In our experiment the high expression of PPAR-γ induced by treatment with serum of LOPE may indicate PPAR-γ expression inversely correlates with its activity. As Levistka et al.(48) showed agonist PPAR-γ rosiglitazone induced reduction in the receptor expression in the human choriocarcinoma derived cell line BeWo, an established model of synctitiotrophoblast formation in vitro, and primary trophoblast cells. In contrast, inhibition of PPAR-γ activity using T0070907 causing extreme enhancement of receptor expression. Thus further study is necessary to proof the PPAR-γ expression increasement in our study is affected by the protein activity.
Above evidence suggested the activity of PPAR-γ is modulated by negative feedback.(48). Similar with Levitska et al.(48) other study by Knabl et al.(49) also stated PPAR-γ as transcription factor plays important roles in fat and glucose metabolism, an in cell growth and differentiation, so that thight autoregulation is necessary to even out its activity. A high activity will induce lower expression, while low activity result in high receptor expression.
The mechanism of EOPE in pregnancy was according to the “two-stage theory of preeclampsia”. In this theory, in the first stage of disease development, defective trophoblastic invasion result in shallow placentation, and the impair remodeling of muscular layer in spiral arteries causing the spiral arteries unable to fully dilated to supply normal pregnancy development. Further mechanism was at the second stage resulting from failure to establish an adequate uteroplacental blood flow leads to relative hypoxia in trophoblastic tissue, thereby eliciting an oxidative stress response, thus releasing placental debris that in maternal circulation and emerged as symptomatic disease. As a consequence for abnormal trophoblastic invasion at early placental development will result in earlier disease manifestation (< 34 weeks), or EOPE.(44-47) Based on this theory, in EOPE abnormal trophoblast activity may be regulated by PPAR-γ, however high activity of this receptor result in diminish receptor expression.(48-49)