VEGF-A isoforms induce the expression of APLN in endothelial cells during human prenatal lung development.

INTRODUCTION: Single-cell RNA-sequencing analyses have revealed the existence of two distinct capillary cell populations in the human lung: general capillary cells (CAP1) and alveolar capillary cells (CAP2). Studies in mouse have shown that the splicing of Vegf-a evolves during embryonic development, creating a temporal pattern of expression for different isoforms, which contributes to the formation of pulmonary capillaries. Moreover, it was demonstrated that murine Vegf-a188 isoform promotes the emergence of CAP2 in vitro. Human homologs of these VEGF-A isoforms exist; however, their role in this process remains elusive. This study investigates the role of VEGF-A and its isoforms in the differentiation of lung capillaries during human prenatal development. METHODS: A cohort of human prenatal tissues, aged from the late pseudoglandular to early canalicular stages of development (10-20 weeks of gestation), was used to study the emergence of CAP2 markers (TBX2, SOSTDC1, EDNRB, HPGD, APLN) in correlation with the expression of the different VEGF-A isoforms (VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A189). RESULTS: RT-qPCR analyses revealed a simultaneous expression of certain VEGF-A isoforms with several CAP2 markers, which peaked at around 18-20 weeks of gestation. Human prenatal lung explants were then treated with recombinant proteins of the different VEGF-A isoforms to study their impact on EC proliferation, as well as on the expression of CAP2 markers. While most of the isoforms did not impact EC proliferation, except for VEGF-A189 which downregulated it, almost all of them upregulated the expression of APLN, a major CAP2 marker. By using fluorescence in situ hybridization, we showed that this increase of expression was specific to the ECs. However, most of the isoforms induced a downregulation of EDNRB and HPGD. They also did not impact the expression of SOSTDC1 and TBX2. DISCUSSION: Our study shows that the different VEGF-A isoforms do not have the same effect on human lung capillary differentiation as those observed with their homologs in mice, highlighting the importance of studying this process in the human model. Moreover, while it demonstrated that VEGF-A isoforms can induce APLN expression in ECs, it also revealed that CAP2 differentiation is most likely a multifactorial process, not only involving VEGF-A.