The Regulation of Endothelin-1 in Pregnancies Complicated by Gestational Diabetes: Uncovering the Vascular Effects of Insulin

Figures

• 

  Figure 1. Schematic diagram of the endothelin-1 system, including its vasoconstrictive and dilatory actions on arteries. This figure highlights the antagonism of the endothelin-1 receptors, ETA and ETB, using BQ123 and BQ788 antagonists, respectively (indicated by the red X).

• 

  Figure 2. Maternal circulating ET-1 levels and vascular mRNA expression are similar at term in pregnancies complicated by GDM, compared to healthy controls. Circulating ET-1 concentrations were measured by ELISA in samples of plasma (A) from patients with pregnancies complicated by GDM who were treated with diet (n = 12, green squares) or insulin (n = 12, blue squares) and from uncomplicated pregnancies at matched gestation controls (n = 42, dark grey circles). The mRNA expression of ET-1 was measured in omental arteries (B) collected from pregnancies complicated by GDM who were diagnosed and subsequently treated with either diet (n = 5) or insulin (n = 8), compared to gestation-matched healthy term controls (n = 15). Individual points represent individual patients, and the data is expressed as mean ± SEM for ELISA data, and qPCR expressed as the log fold change of 2−ΔΔCT Geometric (95% CI) relative to control.

• 

  Figure 3. Omental arteries collected from pregnancies complicated by gestational diabetes mellitus (GDM) and treated with insulin constrict less in response to endothelin-1 (ET−1). Constriction curves (A), analysis of the area under the curve (B), and maximum constriction (C) generated by increasing logarithmic doses of a known vasoconstrictor, ET-1, in omental arteries collected from pregnancies complicated by GDM, insulin- (blue circles) and diet-treated (green circles), and from uncomplicated pregnancies at matched gestation (normal controls, black circles). The percentage of constriction was normalized to the maximum constriction induced by 50 mM potassium salt solution (KPSS). Individual points represent individual patients (n = 5–7). The data is expressed as mean ± SEM. Significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

• 

  Figure 4. Antagonism of the ET−1 receptors alters ET−1-induced vasoconstriction of omental arteries from pregnancy. Omental arteries were collected from healthy-term pregnancies (A,B), or those complicated by GDM with insulin management as treatment (C,D). Prior to constriction with recombinant ET−1, arteries were incubated with ETA antagonist, BQ123 (1 µM, blue circles), and/or ETB antagonist, BQ788 (5 µM, green circles indicate BQ788 alone and purple circles indicate a combination), or vehicle control (black circles). (A,C) Constriction curves were generated by increasing logarithmic doses of ET−1, and the percent of constriction was normalized to a maximum constriction induced by 50 mM of potassium salt solution (KPSS). (B,D) The area under the curve analysis is expressed as a percentage of the vehicle treatment constriction. Individual points represent individual patients. The data is expressed as mean ± SEM. Significance is indicated as follows: * p < 0.05, ** p < 0.01 and **** p < 0.0001.

• 

  Figure 5. The mRNA expression of endothelin receptors and markers of endothelial dysfunction were not altered in omental arteries collected from GDM pregnancies. ET-1 receptors ETA (A), ETB (B), and markers of endothelial dysfunction, intercellular adhesion molecule-1 (ICAM1; (C)) and vascular cell adhesion molecule-1 (VCAM1; (D)), were examined by qPCR in omental vessels collected from pregnancies complicated by GDM managed with diet intervention (n = 5, green squares) or insulin treatment (n = 8, blue squares), compared to healthy gestation-matched controls (n = 15, grey circles). Data is expressed as the log fold change of 2−ΔΔCT Geomean (95% CI) relative to normal control.

• 

  Figure 6. Insulin treatment decreases the maximum constriction induced by ET−1 in healthy maternal omental arteries. Constriction dose-response curves (A), analysis of the area under the curve (B), and maximum constriction (C) generated by increasing logarithmic doses of ET-1 in omental arteries collected from healthy pregnancies (term) incubated with 1 mU/mL insulin (n = 7, purple circles) or 10 mU/mL insulin (n = 5, blue circles), compared to vehicle control (n = 12, white circles) for 30 min. The percent constriction was normalised to the maximum constriction induced by 50 mM potassium salt solution (KPSS). Individual points represent artery reactivity from individual patients. The data is expressed as mean ± SEM. Significance is as follows; * p < 0.05 (control vs. insulin 10 mU/mL).