Prenatal VEGF Nano-Delivery Reverses Congenital Diaphragmatic Hernia-Associated Pulmonary Abnormalities

Congenital diaphragmatic hernia (CDH) affects 1-2 in 5,000 neonates who are born with hypoplastic lungs leading to significant morbidity and mortality. In severe cases tracheal occlusion (TO) can be offered to promote lung growth through airway stretch, but such growth remains limited. Vascular endothelial growth factor (VEGF), a key mediator of normal lung development, is downregulated in animal models of CDH, but its role in CDH-associated lung pathology in humans is not known. Using 18-22 post-conception week fetal lungs from CDH patients, we demonstrated for the first time that lung hypoplasia in this setting is associated with impaired VEGF expression and reduced pulmonary epithelial cell proliferation. We then developed a human ex vivo model of fetal lung compression relevant to CDH, in which a mechanical constraint was added to cultured lung fragments. Our model recapitulated impaired branching morphogenesis observed in CDH fetal lungs, and scRNAseq revealed significant reduction of the proliferative potential of terminal bud epithelial progenitors with concomitant reduction in expression of VEGF. Importantly, the exogenous supplementation of recombinant VEGF in this setting induced a significant increase of terminal bud progenitor cell proliferation, comparable to non-compressed control tissue. Finally, we developed a novel therapeutic agent delivery platform by conjugating recombinant VEGF on the surface of nanoparticles (nanodiamonds; ND-VEGF). In utero, intratracheal administration of ND-VEGF into the in vivo TO Nitrofen rat model of CDH resulted in lung growth as well as pulmonary arterial remodelling leading to histological appearances comparable to healthy controls. The beneficial effects of ND-VEGF were abrogated by co-administration of a selective VEGF-receptor-2 (KDR/Flk1) inhibitor and were not observed when unconjugated VEGF was delivered. This innovative approach could have a significant impact on the treatment of CDH. We combined culture of whole embryonic mouse lungs with a recently developed technology that can generate stiff barriers through selective crosslinking of Cumarin HCC-conjugated 8-arm-polyethylene glycol gel (PEG-HCC) under a two-photon microscope. Mouse lungs were freshly isolated from fetuses at 12.5 days of gestation (E12.5) and embedded in Matrigel drops placed on top of a porous cell culture insert in Air-liquid Interphase (ALI). The next day (Day 0), a 20% PEG-HCC solution was dispersed into the Matrigel drop and selectively crosslinked to surround the lungs and inhibit their growth over a total of 2 days. Control lungs had the same amount of PEG-HCC solution added to the Matrigel, but this was not crosslinked with the two-photon laser. 4 replicates were analyzed by bulk RNA-seq at Day 0, Day 2 under control conditions, Day 2 under compression conditions.