The role of type I interferon on trisomy 21 endothelial angiogenesis

Abstract

Rationale: Down syndrome (DS), resulting from Trisomy 21 (T21), is the most common chromosomal disorder worldwide. Cardiopulmonary complications are a leading cause of morbidity and mortality in DS. Pulmonary disorders include pulmonary hypertension and pulmonary capillaritis, which can result from a dysregulated lung endothelium. One of the major pathways consistently activated in DS is the type I interferon (IFN-I) pathway. High IFN-I inhibits VEGF induced development of capillary like structures, thus playing an anti-angiogenic role. Therefore, we aim to characterize the effects of IFN-I on T21 and euploid iPSC-derived endothelial cells (iPSC-ECs). Methods: Endothelial cell populations from single cell sequencing data of age matched T21 and Non-T21 prenatal lungs were carefully analyzed. Four iPSC lines were derived from age and sex matched T21 and non-T21 human fetal lung fibroblasts and used for endothelial cell differentiation using standardized kits and protocols from Stem Cell Technologies. FACs analysis confirmed endothelial cell purity and differentiation success: CD31+CD34+CD144+CD45-. For angiogenesis assays, endothelial cells were plated onto Cultrex in Ibidi angiogenesis 96-well plates with varying concentrations (0 to 10 4 pM) of IFNβ for 24 hours. Phase contrast images were taken with a 2.5X lens and analyzed in FIJI with the polygon shape tool to measure continuous mesh areas (CMA), defined here as a continuous endothelial cell layer undisrupted by branching structures. Results: Pathway analysis from our scRNAseq data demonstrated an upregulation of the IFN-I pathway across T21 endothelial clusters. T21 and non-T21 iPSC lines exhibited markers of pluripotency (OCT4, NANOG, SOX2, LIN28) and successfully generated pure populations of endothelial cells (~98% CD31+CD34+CD144+CD45-) by passage 2. Quantification demonstrated that at baseline T21 iPSC-ECs consistently had a significantly larger mean CMA than that of non-T21 iPSC-ECs (p< 0.01, n=4). High concentrations of IFN treatment (10 4 pM) resulted in an increased CMA in non-T21 cultures (p=0.14; n=4), trending towards baseline T21 cultures (p=0.28, n=4). Additionally, we observed a significant increase in the CMA of T21 cultures treated with 10 4 pM IFNβ (p=0.04, n=4). Conclusion: Our data suggest decreased angiogenesis in T21 iPSC-ECs may be caused by hyperactive IFN-I signaling. Further studies are needed to determine the mechanisms by which IFN-I regulates angiogenesis in T21 lungs. NIH/NHLBI/OD HL155104-01A1, NIH/NHLBI HL141856, NIH/NHLBI R21HL165411-01, NIH/NICHD 2R24HD000836-52 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.