Aquaporins enriched in endothelial vacuole membrane regulate the diameters of microvasculature in hyperglycemia

Background: In patients with diabetic microvascular complications, decreased perfusion or vascular occlusion, caused by reduced vascular diameter, is a common characteristic that will lead to insufficient blood supply. However, the regulatory mechanism remains undetermined and the treatment approach is lacking. Methods: Gene expression of Aquaporin 1 (AQP1) was compared between diabetic and non-diabetic human retina samples. Confocal live imaging analysis of zebrafish was utilized to investigate the hyperglycemia-induced alterations in vascular morphology. Transcriptomic and single-cell RNA sequencing, in situ hybridization, and quantitative PCR were used to analyze gene expressions. Gene knock-out zebrafish and stable transgenic lines were established for functional analysis. Human embryonic stem cells (H1 line), H1-derived endothelial cells (ECs), human umbilical vein endothelial cells (HUVECs), and human retinal microvascular endothelial cells (HRMECs) were used for functional analysis. Results: Firstly, we found that the expression of human AQP1 was downregulated in diabetic human retina samples (49 healthy vs. 54 diabetic samples) and high-glucose-treated human retinal microvascular endothelial cells. Then, we observed the reduction of vascular diameter and compromised perfusion in high glucose-treated zebrafish embryos. Two aquaporins (aqp1a.1 and aqp8a.1), highly enriched in ECs, were significantly down-regulated under hyperglycemic conditions. Aqp1a.1 and/or aqp8a.1 loss-of-function leaded to reduction in intersegmental vessel diameters, recapitulating the phenotype of the hyperglycemic zebrafish model. Overexpressing aqp1a.1/aqp8a.1 in zebrafish ECs promoted the enlargement of microvascular diameters. Moreover, the reduced vessel diameters induced by high-glucose treatment was rescued by aquaporin overexpression. In addition, both aqp1a.1 and apq8a.1 were localized in the intracellular vacuoles in cultured ECs as well as the ECs of sprouting ISVs, and loss of Aqps caused the reduction of those vacuoles, which was required for lumenization. Loss of AQP1 had no influence on EC differentiation from human stem cells but strongly inhibited the vascular tube formation of differentiated ECs. Conclusions: EC-enriched aquaporins regulate intracellular vacuole-mediated blood vessel diameter hyperglycemia. All of these suggest that EC-expressed aquaporins might be potential targets for gene therapy to cure diabetes-related vascular perfusion defects.