326 - Decreased Pulmonary RAMP2 Expression Characterizes Bronchopulmonary Dysplasia (BPD) in Infants and Its Genetic Augmentation Protects Against Experimental BPD in Mice

Publication Number: 1310.326

Shyam Thapa, Baylor College of Medicine, Houston, TX, United States; Denise G. Lanza, Bayl, Houston, TX, United States; Jason D. Heaney, Baylor College of Medicine, Houston, TX, United States; Binoy Shivanna, Baylor College of Medicine, Houston, TX, United States

Background: Bronchopulmonary dysplasia (BPD) is a morbid lung disorder of preterm infants with no effective therapies. Adrenomedullin (Adm) is shown to mitigate experimental BPD. However, whether Adm signaling is disrupted in the lungs of BPD infants is unclear. It is also unclear if the Adm receptor, receptor activity-modifying protein 2 (Ramp2), can be targeted to mitigate experimental BPD.

Objective: To quantify Ramp2 expression in the lungs of BPD infants and to test the hypothesis that Ramp2 knock-in (KI) will mitigate experimental BPD in mice.

Design/Methods: Human studies: Postmortem lung RNA samples and unstained lung section slides were obtained from BPD infants (n=8) and controls (n=10) from the BRINDL biorepository (University of Rochester). Controls included infants with pathologist-certified normal lung histology. RNA samples were analyzed by RT-PCR using gene-specific primers, and lung sections were immunostained with antibodies against Ramp2 and CD31 to assess Ramp2 protein expression.
Mouse studies: We generated Ramp2 KI (Rosa26 CAG-LSL-Ramp2) mice using CRISPR-Cas9 technology, enabling conditional Ramp2 expression in Cre-expressing cell types. Genomic integration was confirmed by sequencing and genotyping. Rosa26 CAG-LSL-Ramp2 mice were bred with tamoxifen (TAM)-inducible global Cre mice (R26-CreERT2), and their offspring received TAM (50 µg i.p.) or vehicle on postnatal days (P) 1 and 2, while they were exposed to normoxia (21% O₂) or hyperoxia (70% O₂) from P1 to P14 (n=3-4/group). Alveolarization was quantified at P28 by measuring radial alveolar counts (RAC) and mean linear intercepts (MLI). Data was tested for normality and expressed as mean ± SD. Data were compared using unpaired t-tests (BPD vs. non-BPD infants) and ANOVA (mice; RAC and MLI).

Results: The postmenstrual age of controls and BPD infants was comparable (controls: 34.2 ± 6.5; BPD: 36.3 ± 7.5; p value = 0.52). The sex ratio of the groups was also similar. Ramp2 mRNA levels were reduced in BPD lungs compared to controls (Fig. 1A). Immunofluorescence analysis confirmed diminished Ramp2 protein expression, predominantly within the lung vasculature, in BPD infants (Fig. 1C). In the mouse model, hyperoxia exposure decreased RAC and increased MLI, consistent with alveolar simplification (fewer and larger alveoli). Notably, Ramp2-KI mice exhibited attenuated alveolar simplification following hyperoxia exposure (Fig. 2).

Conclusion(s): Our findings indicate that infants with BPD exhibit reduced lung Ramp2 expression, consistent with reduced Adm signaling, and that genetic Ramp2 overexpression via knock-in mitigates experimental BPD in mice.

Figure 1: Pulmonary Ramp2 Expression is Decreased in BPD Infants
Adrenomedullin signaling is reduced in the lungs of BPD infants.

Figure 2. Hyperoxia-Induced Alveolar Simplification is Reduced in Ramp2-KI Mice
Ramp2-KI Abrogates Experimental BPD in Mice.