Assessment of the relation between angiogenesis factors and plasm proteome in preeclampsia

Preeclampsia (pregnancy toxemia) is a pregnancy complication that threatens the life of mother and baby and results in compulsory preterm delivery. Although it is thought to occur due to the disruptions in the vasculogenesis and angiogenesis processes that occur during the formation of the placenta in the first trimester of pregnancy, its pathophysiology has not yet been fully elucidated. The aim of this study is to investigate the level of angiogenesis and anti-angiogenesis factors and plasma proteome in pregnant women with preeclampsia and in healthy pregnant women, to detect proteins with altered protein expression and to obtain new information on the molecular mechanism of the disease by examining their relationship with angiogenesis. In the first stage of the study, whole blood samples were taken from pregnant women diagnosed with preeclampsia (experimental group) and healthy ones (control group) into tubes containing anticoagulants, and plasma samples were separated by centrifugation. Plasma PlGF (placental growth factor, angiogenic factor) and sFlt-1 (soluble fms-like tyrosine kinase-1, anti-angiogenic factor) levels of preeclampsia and control groups were determined by ELISA method and compared. In the second stage, plasma samples of preeclampsia and control groups were pooled and analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). Spots corresponding to differentially expressed proteins between the two groups were cut from the gel and analyzed by MALDI-TOF/TOF MS. Two proteins with increase and decrease in protein amount were confirmed by Western blotting. The amount of sFlt-1 per unit protein (0.83 ± 0.063 ng/mg protein) in the preeclampsia group was significantly higher than the control (0.067 ± 0.010 ng/mg protein). PlGF level was determined as 0.0026 ± 0.0005 ng/mg protein in the preeclampsia group and 0.0023 ± 0.0004 ng/mg protein in the control group, so there was no significant difference between the two groups in terms of this factor. The sFlt-1/PlGF ratio in the PE group (0.20 ± 0.02) was significantly higher than the control group (0.13 ± 0.006). As a result of the proteomic analyzes, it was found that the regulation was different in 17 spots. In the preeclampsia group, haptoglobin (6.8-fold), complement C3 (5.34-fold), apolipoprotein A-1 (1.82-fold), keratin type 1 cytoskeleton 10 (9.71-fold) were up-regulated while albumin (1.3 fold), fibrinogen beta chain (2.59 fold), α-1-antitrypsin (4.83 fold), and fibrinogen alpha chain (68.88 fold) were down-regulated. In the final stage of the study, the change in expression of the two proteins (increase in haptoglobin and decrease in fibrinogen β) was validated by Western blotting. Haptoglobin, one of the acute phase proteins, is associated with inflammation, oxidative stress and infection. Since this protein functions by binding to free hemoglobin causing endothelial dysfunction, vascular damage and inflammation, it may be one of the possible factors trying to reverse the anti-angiogenesis associated with increased sFlt-1 level in preeclampsia. It is thought that down-regulation of fibrinogen β in patients with preeclampsia is related to the stage of pregnancy and the severity of the disease. Since there are different findings related to this coagulation factor in the literature, it has been concluded that in order to make a comment on its role in angiogenesis, it should be considered together with other factors and examined at the gene level. In conclusion, this study provided important clues about the relationship between angiogenic factors and plasma proteome in preeclampsia. However, it is clear that sampling procedures and proteomic methods are need to be standardized, and research at the gene level are required in order to elucidate the molecular mechanism of preeclampsia. December 2021, 101 pages. Keywords: Preeclampsia, proteomic, angiogenesis factors, sFlt-1, PlGF, haptoglobin