243 - Neonatal hyperoxia causes a durable pericyte loss and compromises proangiogenic signaling in small vessels

Bronchopulmonary dysplasia (BPD), a chronic lung disease of infancy, is characterized by an arrest of secondary septation, compromised angiogenesis, and, often, pulmonary hypertension (PH). Though pericytes, mesenchymal cells (MC) that provide structural support to capillaries, are vital to angiogenesis and pulmonary vascular maturation, how pericyte number and function might be altered by neonatal hyperoxia remains unknown.

Objective:

To test the hypothesis that in a murine model of hyperoxia-induced neonatal lung injury,

pericytes are uniquely vulnerable to hyperoxia-induced neonatal lung injury.

Design/Methods:

Cells were isolated from mice at early alveolar (P7) and late alveolar (P21) stages of development. Mice were raised in either (i) room air (control; FiO₂=0.21); (ii) hyperoxia from birth (H; FiO₂=0.8 x 7d); or (iii) hyperoxia for 14 d with room air recovery for 7d (hyperoxia recovery), a preclinical model of BPD. Single cell libraries were prepared via SmartSeq2 and were sequenced on Illumina NovaSeq at a depth of ~10⁷ reads per cell. Cell type populations and genes of interest were validated with in situ hybridization.

Results:

Pericytes were identified by coexpression of Cox4i2, Gucy1a1, and Pdgfrb.  Compared to P7 and P21 controls, in hyperoxic mice pericyte abundance was markedly decreased, both absolutely and relative to other MC populations. Pericyte loss was confirmed by fluorescence in-situ hybridization microscopy.  Cdkn1a, Nfkbia, and Trp53inp1, genes associated cell quiescence and Col4a1 and Col4a2, extracellular matrix genes, were all upregulated in hyperoxia. Vtn, a positive regulator of angiogenesis, was downregulated in hyperoxia. Expression of both Timp3 and Flt1,  inhibitors of angiogenesis, was increased in hyperoxia. 

Conclusion(s):

Neonatal hyperoxia markedly and durably decreased lung pericyte abundance, to a degree far greater than other MC populations, marking pericytes as uniquely vulnerable to hyperoxia. Moreover, hyperoxia increased expression of molecules associated with cell-cycle arrest, extracellular matrix organization, and decreased expression of proangiogenic signaling molecules that can inhibit endothelial cell growth and development. These results identify a previously undescribed role for pericytes in the decreased angiogenesis that characterizes BPD. Development of pericyte-specific protective strategies may represent a novel approach to preserve lung angiogenesis and alveolarization.