324 - Inhaled Ciclesonide, a brain-sparing glucocorticoid, attenuates hyperoxia-induced lung injury in neonatal rats

Publication Number: 1308.324

Heather Menden, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Ying Wang, The Lundquist Institute, Torrance, CA, United States; Jie Liu, Lundquist Institute, Torrance, CA, United States; Celia Yu, The Lundquist Institute for Biomedical Innovation, Torrance, CA, United States; Gourav Chandan, Lundquist Institute at Harbor UCLA, Torrance, CA, United States; Dhanesh Sivadasan Bindu, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Virender K.. Rehan, Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, United States; Venkatesh Sampath, Children's Mercy Kansas City, kansas city, KS, United States

Background: There is a lack of safe, efficacious glucocorticoid (GC) therapy to prevent bronchopulmonary dysplasia (BPD) in preterm infants. We have shown that ciclesonide (CIC), a synthetic GC, does not impair somatic or brain growth in neonatal rodents. Herein, we evaluated the efficacy of inhaled CIC (i-CIC) to rescue hyperoxia-induced acute lung injury (ALI). We hypothesized that CIC would attenuate hyperoxia (HOX)-induced inflammation and cellular injury in neonatal rats as well as human primary lung epithelial (HPEpiC) and microvascular endothelial (HPMEC) cells.

Objective: To test the efficacy of: a) i-CIC in a neonatal rat model of HOX-induced ALI and b) CIC in rescuing HOX-induced HPEpIC and HPMEC injury.

Design/Methods: P4 rat pups exposed to HOX (60% O2) or room air (RA) for 72 h were administered inhaled i-CIC (2.5mg/kg) or vehicle once daily using the SCIREQ InExpose system (Figure 1). Lungs harvested were used to investigate immune cell influx, vascular permeability (albumin levels), pro-inflammatory signaling, and markers of alveolar remodeling. Rat lung epithelial (EPCAM) and endothelial (PECAM) cell pull-downs were done to assess cell-type specific inflammation and injury. HPEpiC and HPMEC derived from 18-19 week neonatal human lungs (ScienCell, CA) were exposed to HOX or RA for 24 h ± CIC (0.1µM), followed by assessment of cell viability and inflammation.

Results: Lung immune cell influx and albumin levels induced by HOX were suppressed by i-CIC (Figure 1). HOX-induced expression of cytokine and pro-apoptotic genes (Bak1, Bax) was inhibited by i-CIC, with i-CIC inducing the anti-apoptotic gene, Bcl2, in parallel with GR-target genes, Gilz and Fam107a in RA and HOX (Figure 2). I-CIC inhibited induction of regulators of inflammation and alveolar remodeling, NFKB (pp65), TGFβ, and cleaved caspase-3 seen in HOX. In rat lung EC, HOX-induced Angpt2, while suppressing markers of angiogenesis, lumenized vessels and cell proliferation (Icam2, and Ki67), which was rescued by i-CIC. In rat lung EpiC, HOX-induced Lcn2 (inflammatory marker) and suppressed Ki67, which was rescued by i-CIC. HOX-mediated depletion of AT1 cells (AGER+) was rescued by i-CIC (Figure 2). Studies in HPEpiC and HPMEC revealed that i-CIC inhibited cytokine and pro-apoptotic gene induction, while rescuing cell differentiation (VEGF, SFTPC) and viability in HOX (Figure 3).

Conclusion(s): Inhaled CIC rescues HOX-induced cellular injury and ALI. In conjunction with our prior work revealing the somatic- and brain toxicity-sparing effects of CIC, our results highlight the promise of i-CIC as a safe, efficacious therapy for preventing BPD.

Figure 1. Inhaled CIC inhibits HOX-induced lung immune cell influx and vascular permeability (albumin levels) in newborn rats:
A) Illustration of nose-only inhaled treatments with i-CIC to neonatal rats through the SCIREQ InExpose system. B-D) Rat pups (P4) exposed to HOX (60%O2) or RA from 72 h received daily nebulization of CIC (2.5mg/kg) or vehicle through the SCIREQ InExpose system. B-C) Flow cytometric analysis was done on bronchoalveolar fluid to estimate proportion of neutrophils and macrophages. N=3-4/group. * P<0.05, ANOVA with Tukey's test. D) Albumin levels were quantified in bronchoalveolar lavage fluids using a Bradford assay. N=3-4/group. * P<0.05, ANOVA with Tukey's test.

Figure 2. HOX-induced lung inflammatory, matrix remodeling and cellular injury is rescued by i-CIC:
A-E) Rat pups (P4) exposed to HOX (60%O2) or RA from 72 h received daily nebulization of CIC (2.5mg/kg) or vehicle through the SCIREQ InExpose system. A) Whole lung RNA expression of cytokines, pro-apoptotic genes (Bak1, Bax1) induced by HOX was inhibited by i-CIC, while i-CIC induced the anti-apoptotic gene, Bcl2, and known GR target genes, Gilz and Fam107a. B) Whole lung protein expression of activated NFKB (pp65, RelA), TGFβ, and cleaved caspase 3 (immunoblotting), induced by HOX was suppressed by i-CIC. C) HOX induced expression of Lcn2, and repression of Aqp5 and Ki67 in rat epithelial cells (EPCAM pull down assay) was inhibited by i-CIC. D) HOX induced expression of Angpt2, and repression of Icam2 and Ki67 in rat endothelial cells (PECAM pull down assay) was inhibited by i-CIC. E) Immunofluorescence of lung sections after HOX or RA exposure ± I-CIC treatments as above showing loss of the type I AEC population (AGER) in HOX that is rescued by i-CIC. N=3-6/group. *P <0.05.ANOVA with Tukey's test.

Figure 3. i-CIC rescues HOX-induced inflammation, pro-apoptotic signaling and cell viability in human primary alveolar epithelial cells (HPAEpiC) and pulmonary microvascular endothelial cells (HPMEC):
HPMEC (A-B) and HPAEpiC (C-D) derived from 18 weeks human fetal lung were exposed to HOX (60% O2 ) or RA for 24 h with or without CIC (0.1µM) treatment. A proportion of cells were used to assess cell viability (A, C) using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, while the majority of cells were used to evaluate RNA expression of cell differentiation markers, inflammatory cytokines and pro-apoptotic (BAK1) and anti-apoptotic gene (BCL2) by qRT PCR. N=4 experiments. *P <0.05. ANOVA with Tukey's test.