Fetal Betamethasone Exposure Markedly Attenuates the Protein Expression of Angiotensin Converting Enzyme 2 but not Dipeptidyl Peptidase 3 within the Brain Dorsomedial Medulla of Adult Female Sheep

Abstract

Premature infants receiving antenatal glucocorticoids (GC) to accelerate lung development and improve survival may exhibit negative long‐term consequences regarding autonomic dysregulation. We have established a model of antenatal steroid exposure in sheep where the pregnant ewes receive an identical dose of the GC agoninst betamethasone as that given clinically and at a comparable gestational age. Previous studies show that adult sheep with betamethasone exposure (BMX) in utero exhibit elevated mean arterial pressure (MAP), decreased baroreflex sensitivity (BRS) for control of heart rate and insulin resistance associated with a higher Ang II to Ang‐(1‐7) peptide ratio and lower Mas receptor expression in brain dorsomedial medulla (DMM).We hypothesized that alterations in renin‐angiotensin system (RAS) enzymes locally within the DMM may contribute to lower Ang‐(1‐7) expression. Pregnant sheep were administered BM (2 doses of 0.17 mg/kg, 24 hours apart) or vehicle during the 80th day of gestation and delivered at term. DMM was excised from ~12‐mo‐old BMX and control female offspring (n=3–5 per group). Tissue was homogenized in PBS containing a protease cocktail inhibitor and centrifuged at 10,000 × g for Western blot analysis. The resultant supernatant was fractionated in 10% mini‐PROTEAN TGX gels and electrophoretically transferred onto polyvinylidene difluoride membranes. Immunodetection was performed with antibodies against ANG I‐intact rat angiotensinogen (Aogen), Dipeptidyl‐Peptidase 3 (DPP3), a peptidase that sequentially metabolizes Ang‐(1‐7) to Ang‐(3‐7) and to Ang‐(5‐7) and angiotensin converting enzyme 2 (ACE2). The protein bands were normalized to total protein in each lane using ChemiDoc Touch Imaging System and ImageLab 5.2.1 and compared using Student's t‐test on GraphPad Prism 6.The DMM from BMX female sheep exhibited 2.75‐fold higher expression of Aogen as compared to control (BMX: 47 ± 2.1 × 10−3vs. 17.0 ± 4.0 × 10−3; P = 0.05), consistent with previous reports at this age in males. In contrast, protein expression of ACE2, the major enzyme that converts Ang II to Ang‐(1‐7), was reduced by 90% (BMX: 5.0 ± 0.9 × 10−3 vs 50.0 ± 2.4 × 10−3; P = 0.05). However, despite our previous findings that DPP3 activity was increased in the cerebrospinal fluid (CSF) of the BMX animals, the protein expression of DPP3 in the DMM was not different between female BMX and controls (16.0 ± 2.0 × × 10−3vs 19.2 ± 7.0 × 10−3; P=0.68).In summary, our analysis of RAS protein expression reveal lower ACE2, unchanged DPP3 and elevated Aogen in BMX adult female sheep. These findings support the overall concept that the elevated Ang II to Ang‐(1‐7) ratio in DMM of BMX animals may reflect the consequence of both reduced generation of Ang‐(1‐7) and attenuated metabolism of Ang II as the mechanism for the altered ratio of these peptides. The BMX‐induced alterations in the RAS observed in previous studies include higher activity of the Ang‐(1‐7) degrading enzymes ACE and DPP3, with or without alterations in Aogen or the Ang‐(1‐7) forming activity (ACE2 and Neprilysin) in the proximal tubules of the kidneys and CSF. Therefore, in this model of fetal programmed hypertension we conclude that antenatal steroid treatment results in consistent findings of lower Ang‐(1‐7) and its receptor mas in several key sites for blood pressure regulation via alterations within the RAS pathway.