Abstract 177: Antenatal Betamethasone Exposure Reduces Ang-(1-7) but Enhances Angiotensin-Converting Enzyme and a Thiol Peptidase to Metabolize Ang-(1-7) in the Cerebrospinal Fluid

Abstract

Antenatal betamethasone (BM) therapy accelerates lung development in preterm infants but may induce early programming events with long-term cardiovascular consequences. To elucidate these programming events, we developed a model of programming whereby pregnant ewes are administered BM (2 doses of 0.17 mg/kg) or vehicle at the 80th day of gestation and offspring are delivered at term. The BM-exposed (BMX) offspring develop elevated blood pressure, decreased baroreflex sensitivity, and alterations in the circulating, renal, and brain renin-angiotensin systems (RAS) by 6-months of age. While components of the RAS are present in cerebrospinal fluid (CSF), it is not known if BM exposure influences the CSF RAS to the same extent as other tissue compartments. Therefore, we characterized the CSF RAS in 6-month old male sheep, and established the impact of antenatal exposure on the RAS. Consistent with our previous findings of predominant ACE2 activity in the choroid plexus, CSF Ang-(1-7) levels are significantly higher than Ang I (320.8 ± 96.0 vs. 19.3 ± 4.6 pM; p<0.01, N = 12) or Ang II (1.4 ± 0.5 pM; p<0.01, N = 12). Moreover, Ang-(1-7) was 70% lower in the CSF of the BMX group (97.3 ± 31.4 pM; p<0.05) without changes in Ang I or Ang II. We next assessed the metabolism of Ang-(1-7) in the CSF and demonstrate a trend for increased enzyme activity in BMX males. Further analysis revealed two peptidases that contribute to Ang-(1-7) degradation in CSF of both groups. ACE accounted for approximately 15% (85 out of 100) of the overall Ang-(1-7) metabolism that hydrolyzed the peptide to Ang-(1-5) in CSF and ACE was 1.4-fold higher in the BMX group (6.5 ± 0.5 vs. 8.8 ± 0.5 fmol/min/ml; p<0.02, N = 4). We also identified a novel thiol peptidase activity (Ki = 4 μM PCMB) as the predominant activity that metabolized Ang-(1-7) to Ang-(1-4) in CSF; kinetic analysis of this peptidase in pooled CSF revealed Km and Vmax constants of 5.4 μM and 54 nmol/min/mg for control, and 4 μM and 60 nmol/min/mg for BMX. In summary, CSF levels of Ang-(1-7) were markedly lower following antenatal exposure. The reduced expression of central Ang-(1-7) potentially through an enhanced metabolism pathway may contribute to the long-term increase in blood pressure and an attenuated baroreflex in this model of fetal programming.