The effect of VEGF-A isoforms on capillary endothelial cell differentiation in human fetal lung development

Abstract

Rationale: Single-cell RNA-sequencing has revealed two distinct capillary cell populations in the lung - general capillary cells (gCap) and alveolar capillary cells (aCap). Studies in mouse have demonstrated that VEGF-A188 promotes the formation of aCaps in vitro. Our study investigates the role of VEGF-A in endothelial cell (EC) differentiation into functional pulmonary capillaries during human lung development. We hypothesize that VEGFA-189, the human equivalent of murine VEGFA-188, promotes the formation of aCaps in the developing human lung. Methods: We examined the expression of the different VEGF-A isoforms ( VEGF-A121, 145, 165 and 189) on human fetal lungs aged 10-20 weeks gestation. RT-qPCR was performed to correlate the expression of VEGF-A isoforms to the expression of aCap makers ( CA4, EDNRB, SOSTDC1, HPGD, TBX2 and IL1RL1). In parallel to these analyses, we examined the effects of VEGF-A isoforms by culturing human fetal lung explants (13-20 weeks gestation) for either 2 or 7 days in the presence or absence of either recombinant human protein VEGF-A165 (20 ng/mL) or VEGF-A189 (80 ng/mL) (n=8). To verify this hypothesis of VEGF-A189, we selected VEGF-A165 which has one exon less (exon 6) than VEGF-A189. Explants were collected and analyzed for gCap ( EDN1) and aCap ( TBX2, EDNRB, SOSTDC1, HPGD, and CA4) markers expression by RT-qPCR. Immunofluorescence (IF) staining was also performed on explants to analyze the effect of VEGF-A isoforms on the proliferation of endothelial cell. Results: In native human fetal lung, we demonstrated a simultaneous expression of VEGF-A-121, 145 and 189 isoforms with several aCap markers ( SOSTDC1, EDNRB, HPGD), that peaked at around 18 weeks of gestation. Based on this, we decided to culture human fetal lung explants within this same developmental stage in the presence or absence of either VEGF-A165 or -189. Following 2 days treatment with VEGF-A165, RT-qPCR demonstrated that human fetal lung explants exhibited a significant increase in the expression of EDN1 (p<0.005), accompanied by a significant decrease in expression of CA4 (p<0.005). Interestingly, whereas VEGF-A189 also demonstrated decreased expression of several aCap markers ( CA4, EDNRB, SOSTDC1, and HPGD (p<0.01)) it also showed increased expression of TBX2 and EDN1 (p<0.05). Furthermore, IF staining displayed decreased Ki67/CD31 double positive cells on explants treated with VEGF-A189 with a more elongated and flattened morphology, much like what is observed in aCaps. Prolonging the treatment to 7 days yielded comparable results to the 2 days treatment, with VEGF-A165 and VEGF-A189 downregulating several aCap markers. Conclusion: Our study revealed that the VEGF-A189 isoform does not have the same effect on human lung capillary differentiation as observed in mouse. We noted a contrasting impact of VEGF-A isoforms on the expression of gCap and aCap markers and highlighted the significance of specific VEGF isoforms in capillary EC differentiation. Additional investigations are required to elucidate the underlying mechanisms. California Institute for Regenerative Medicine-CIRM. This is the full abstract presented at the American Physiology Summit 2024 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.