The Potential for Placental Activation of PPARγ to Improve the Angiogenic Profile in Preeclampsia

Abstract

Preeclampsia (PE) is one of the most common causes of maternal-fetal morbidity and mortality world-wide. While the underlying causes of PE remain elusive, aberrant trophoblast differentiation and function are thought to cause an imbalance of secreted angiogenic proteins resulting in systemic endothelial dysfunction and organ damage in the mother. The placental dysfunction is also characterized by a reduction of the transcription factor, peroxisome proliferator activated receptor γ (PPARγ) which normally promotes trophoblast differentiation and healthy placental function. This study aimed to understand how placental activation of PPARγ effects the secretion of angiogenic proteins and subsequently endothelial function. To study this, healthy and PE placental tissues were cultured with or without the PPARγ agonist, Rosiglitazone, and a Luminex assay was performed to measure secreted proteins from the placenta. To assess the angiogenic effects of placental activation of PPARγ, human umbilical vein endothelial cells (HUVECs) were cultured with the placental conditioned media and the net angiogenic potential of these cells was measured by a tube formation assay. This is the first study to show PPARγ's beneficial effect on the angiogenic profile in the human preeclamptic placenta through the reduction of anti-angiogenic angiopoietin-2 and soluble endoglin and the upregulation of pro-angiogenic placental growth factor, fibroblast growth factor-2, heparin-binding epidermal growth factor, and follistatin. The changes in the angiogenic profile were supported by the increased angiogenic potential observed in the HUVECs when cultured with conditioned media from rosiglitazone-treated preeclamptic placentas. The restoration of these disrupted pathways by activation of PPARγ in the preeclamptic placenta offers potential to improve placental and endothelial function in PE.

Keywords: PPARγ; angiogenesis; placenta; preeclampsia.

Figure 1

Angiopoeitin-2 secretion is reduced in Rosiglitazone-treated preeclamptic placentas. Angiopoeitin-2 (Ang-2) levels were measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). There was no significant difference in Ang-2 secretion between PE and control placentas (A, n > 10). Rosi-treated PE placentas show a significant reduction of Ang-2 secretion in comparison to the vehicle control (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 2

Soluble Endoglin secretion is increased in the preeclamptic placenta but reduced after Rosiglitazone treatment. Soluble Endoglin (sEng) levels were measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). There was a significant upregulation of sEng secretion in PE compared to control placentas (A, n > 10). Rosi-treated PE placentas show a reduction of sEng secretion however this was not statistically significant when compared to the vehicle control (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 3

There are no significant differences in Endothelin-1 secretion from healthy or preeclamptic placentas with or without drug treatment. Secretion of Endothelin-1 was measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). There was no significant change in ET-1 secretion between PE and control placentas (A, n > 10) and between vehicle and Rosi-treated PE placentas in the PE compared to control placentas however this was not statistically significant (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 4

There is a decreasing trend in placental growth factor secretion in the preeclamptic placenta that is partially rescued by Rosiglitazone treatment. Secretion of Placental growth factor (PlGF) were measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). There was a trending decrease of PlGF secretion in the PE compared to control placentas however this was not statistically significant (A, n > 10). Rosi-treated PE placentas show an increase of PlGF secretion however this was not statistically significant when compared to the vehicle control (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 5

Fibroblast Growth Factor 2 shows reduced secretion from the preeclamptic placenta that is reversed by Rosiglitazone treatment. Secretion of Fibroblast Growth Factor 2 (FGF-2) was measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). FGF-2 secretion appears to be reduced in preeclamptic placenta however this decrease is not statistically different from control placentas (A, n > 10). Rosi-treated PE placentas show a significant increase in FGF-2 secretion compared to the vehicle control (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 6

There are no significant changes in Epidermal Growth Factor secretion between healthy and preeclamptic placentas treated with or without Rosiglitazone. Secretion of Epidermal Growth Factor (EGF) was measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). There was no significant change in EGF secretion between PE and control placentas (A, n > 10) and between vehicle and Rosi-treated PE placentas in the PE compared to control placentas however this was not statistically significant (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 7

Heparin-Binding Epidermal Growth Factor shows reduced secretion from the preeclamptic placenta but is reversed by Rosiglitazone treatment. Secretion of Heparin-Binding Epidermal Growth Factor (HB-EGF) was measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). There was a significant reduction of HB-EGF secretion from the PE placenta compared to control (A, n > 6). Rosi-treated PE placentas show a significant increase in HB-EGF secretion compared to the vehicle control (B, n = 10). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, ** p < 0.01, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 8

Follistatin shows reduced secretion from the preeclamptic placenta but is reversed by Rosiglitazone. Secretion of Follistatin (FST) was measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). FST secretion was significantly reduced in the PE placenta compared to control placentas (A, n > 10) however, Rosi treatment led to a significantly increased secretion of FST from the PE placenta compared to vehicle-treated PE placentas (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 9

There are no significant changes in Leptin secretion between healthy and preeclamptic placentas with or without drug treatment. Secretion of Leptin was measured via Luminex assay from conditioned media from non-treated control and preeclamptic (PE) placentas (A) and vehicle- or Rosiglitazone (Rosi)-treated PE placentas (B). Although there was an increasing trend of Leptin secretion from PE placentas, this was not statistically different from the control placentas (A, n > 10). There were no significant changes in Leptin secretion between vehicle and Rosi-treated PE placentas (B, n = 14). (Protein secretion was measured by a Luminex assay where experimental values were determined relative to a standard curve. Statistical analysis was performed by student’s t-test to determine significant differences between groups, bar plots and data reported are reported as mean pg/mL values ± SEM).

Figure 10

There is a significant reduction in the number of nodes present in the HUVECs cultured with preeclamptic conditioned media, but this is reversed in Rosiglitazone-treated placentas. HUVECs were cultured with conditioned media on matrigel, and the number of nodes present was calculated by the Image J Angiogenesis Analyzer tool (30). HUVECs cultured with conditioned media from preeclamptic (PE) placentas show significantly reduced number of nodes as compared to healthy control placentas (A, n =6). Conditioned media from Rosiglitazone (Rosi)-treated PE placentas led to a significant increase in the number of nodes present in the HUVECs compared to the conditioned media from the vehicle-treated PE placentas (B, n = 6). Rosi and vehicle were cultured in placental media without any tissues for 24 h then applied to the HUVECs. Additionally, HUVECs cultured with standard full-serum media (HUVEC Only) all served as controls for this experiment. There were no significant changes in the number of nodes present among the HUVEC Only, Rosi, and vehicle conditioned media controls (C, n = 6). (Statistical analysis was performed by student’s t-test to determine significant differences between groups, ** p < 0.01, *** p < 0.001, bar plots and data reported are reported as numerical values ± SEM).

Figure 11

There is a reduction of junctions present in HUVECs cultured with preeclamptic placental conditioned media and this was significantly increased after culture with Rosiglitazone-treated preeclamptic placentas. HUVECs were cultured with conditioned media on matrigel, and the number of junctions present was calculated by the Image J Angiogenesis Analyzer tool (30). Conditioned media from preeclamptic (PE) placentas show significantly reduced number of junctions as compared to healthy control placentas (A, n = 6). Conditioned media from Rosiglitazone (Rosi)-treated PE placentas led to a significant increase in the number of junctions present in the HUVECs compared to the conditioned media from the vehicle-treated PE placentas (B, n = 6). Rosi and vehicle were cultured in placental media without any tissues for 24 h then applied to the HUVECs. Additionally, HUVECs cultured with standard full-serum media (HUVEC Only) all as controls for this experiment. There were no significant changes in the number of junctions present among the HUVEC Only, Rosi, and vehicle conditioned media controls (C, n = 6). (Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, *** p < 0.001, bar plots and data reported are reported as numerical values ± SEM).

Figure 12

There is a reduction of the total branching length present in HUVECs cultured with preeclamptic placental conditioned media, but this was significantly increased after culture with Rosiglitazone-treated preeclamptic placentas. HUVECs were cultured with conditioned media on matrigel, and the total branching length was calculated by the Image J Angiogenesis Analyzer tool (30). Conditioned media from preeclamptic (PE) placentas show significantly reduced total branching length as compared to healthy control placentas (A, n = 6). Conditioned media from Rosiglitazone (Rosi)-treated PE placentas led to a significant increase in the total branching length present in the HUVECs compared to the conditioned media from the vehicle-treated PE placentas (B, n = 6). Rosiglitazone and vehicle were cultured in placental media without any tissues for 24 h along with HUVECs cultured with standard full-serum media (HUVEC Only) all served as controls for this experiment. There were not any significant changes in the total branching length among the HUVEC Only control, Rosi, and vehicle conditioned media controls (C, n = 6). (Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, ** p < 0.01, bar plots and data reported are reported as numerical values ± SEM).

Figure 13

There is a reduction of the total number of meshes present in HUVECs from culture with conditioned media from preeclamptic placentas, but this was significantly increased after culture with Rosiglitazone-treated preeclamptic placentas. HUVECs were cultured with conditioned media on matrigel, and the number of meshes was calculated by the Image J Angiogenesis Analyzer tool (30). Conditioned media from preeclamptic (PE) placentas show significantly reduced the number of meshes as compared to healthy control placentas (A, n = 6). Conditioned media from Rosiglitazone (Rosi)-treated PE placentas led to a significant increase in the number of meshes present in the HUVECs compared to the conditioned media from the vehicle-treated PE placentas (B, n = 6). Rosi and vehicle were cultured in placental media without any tissues for 24 h along with HUVECs cultured with standard full-serum media all served as controls for this experiment. There were not any significant changes in the number of meshes among the HUVEC Only control, Rosi, and vehicle conditioned media controls (C, n = 6). (Statistical analysis was performed by student’s t-test to determine significant differences between groups, * p < 0.05, ** p < 0.01, bar plots and data reported are reported as numerical values ± SEM).

Figure 14

Representative images of HUVEC tube formation assays. Conditioned media from non-treated control (A) and preeclamptic (PE) (B) placentas, vehicle-treated PE placentas (C), Rosiglitazone (Rosi)-treated PE placentas (D), HUVEC Only control (E) and Rosi (F) and vehicle conditioned media controls (G) were cultured with HUVECs on matrigel and images were captured after 18 h of culture. Images were captured using a 4× magnification and then uploaded to the ImageJ Angiogenesis Analyzer to measure various parameters of tube formation to indicate which conditions permit the greatest angiogenic potential of the HUVECs. Scale bar represents 500 μm.