Abstract
Purpose :
Preterm infants are at risk of aberrant angiogenesis and can develop Retinopathy of Prematurity (ROP), a leading cause of acquired visual impairment. Risk factors for ROP are hyperoxia and inflammation. Surfactant protein A (SP-A) is deficient in preterm infants and we previously showed that deficiency of SP-A in mice is associated with decreased retinal vessel growth in early development and in oxygen-induced retinopathy, with abnormal elongation of retinal endothelial cells (EC’s). Actin and cytoskeleton function are critical for EC migration and function. We hypothesize that lack of SP-A impairs retinal EC function via decreased actin polymerization, and cytoskeleton dysregulation.
Methods :
Gene expression in SP-A-/- and WT P6 mouse retina tissue was evaluated by RNAseq and protein by mass spectroscopy (MS). Confirmation of targets was done by Cytoskeleton PCR Array. Retina flat mounts were evaluated by IHC with staining for actin and cd31 (EC marker). HRECs were treated with control HEPES (HREC+Con) or human SP-A protein (HREC+SPA). Actin expression and organization was analyzed by IHC. Migration and proliferation assays were assessed in HREC’s. Data was analyzed by students t-test or one-way ANOVA, with p< 0.05.
Results :
We show differential expression of factors mapped to signaling pathways and transcriptional regulation of cytoskeleton-dependent functions, including actin polymerization (actin polymerization complex related proteins Arpc2, Arcp4, and WasL), microtubule formation and cell cycle division (Ktn1 and Map4). HRECs+SP-A had more than twice as much cell migration compared to HRECs+Con (p< 0.0001) and increased proliferation (p< 0.0001). HRECs+SP-A exhibited elongation of actin filaments and protrusion of filopodia compared to HREC+Con, Actin was more peripherally organized in ECs and tip cells of blood vessels in WT compared to SP-A-/- mice.
Conclusions :
Deficiency of SP-A alters expression of genes related to actin filament interactions, organization & biogenesis, microtubules, cell shape and size. Furthermore, HREC’s exposed to purified SP-A exhibit morphological changes including elongation of cells and migration of actin to endothelial cell borders with an increase in migration and proliferation. Future studies will determine underlying mechanisms of these observed findings which will allow for development of therapy targeting aberrant vessel growth in ROP.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.