Serial intravital imaging captures dynamic and functional endothelial remodeling with single-cell resolution

Abstract

Endothelial cells are important in the maintenance of healthy blood vessels and in the development of vascular diseases. However, the origin and dynamics of endothelial precursors and remodeling at the single-cell level have been difficult to study in vivo due to technical limitations. We aimed to develop a direct visual approach to track the fate and function of single endothelial cells over several days-weeks in the same vascular bed in vivo using multiphoton microscopy (MPM) of transgenic Cdh5-Confetti mice and the kidney glomerulus as a model. Individual cells of the vascular endothelial lineage were identified and tracked due to their unique color combination, based on the random expression of cyan/green/yellow/red fluorescent proteins. Experimental hypertension, hyperglycemia, and laser-induced endothelial cell ablation rapidly increased the number of new glomerular endothelial cells that appeared in clusters of the same color, suggesting clonal cell remodeling by local precursors at the vascular pole. Furthermore, intravital MPM allowed the detection of distinct structural and functional alterations of proliferating endothelial cells. No circulating Cdh5-Confetti + cells were found in the renal cortex. The heart, lung, and kidneys showed more significant clonal endothelial cell expansion compared to the brain, pancreas, liver and spleen. Serial MPM of Cdh5-Confetti mice in vivo is a powerful new technical advance to study endothelial remodeling and repair in the kidney and other organs under physiological and disease conditions.

Intravital Multiphoton Microscopy as a Tool for Studying Renal Physiology, Pathophysiology and Therapeutics

“…The relative ease of generating unique transgenic mice has also been an important advantage. The Peti-Peterdi laboratory has used this approach to follow endothelial and glomerular epithelial regeneration using serial intravital multi-photon microscopy ( Hackl et al, 2013 ; Schiessl et al, 2020 ; Desposito et al, 2021 ). These studies have shed light on the dynamic alterations, spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli.…”

New Endothelial Mechanisms in Glomerular (Patho)biology and Proteinuria Development Captured by Intravital Multiphoton Imaging

“…Rich in proteoglycans and secreted glycosaminoglycans (e.g., heparan sulfate and hyaluronic acid), the GEnC glycocalyx has important roles in several processes including restricting albumin passage through the GFB, binding chemokines and growth factors, and immune cell adhesion (2,24,25). Intravital MPM imaging approaches have successfully visualized these GEnC structures and functions in the intact living kidney including the bulk fluid flow in fenestrated capillaries (11,26) and the presence and alterations in GEnC glycocalyx in various disease models (15,(19)(20)(21).…”

“…This same approach was recently applied to GEnCs with single-cell resolution to shed light on the dynamic and functional endothelial remodeling mechanisms and vascular regeneration in healthy glomeruli and in disease models. Compared to the slow turnover observed in other organs and vascular beds, the rapid, and clonal expansion of single GEnC precursors were quantitatively visualized in response to hypertensive, diabetic, and laser-induced GEnC injuries ( 20 ). Interestingly, GEnC progenitor cells were locally residing at the glomerular vascular pole, mostly in the terminal afferent and/or efferent arteriole segments rather than derived from a circulating progenitor pool.…”

“…However, recent major improvements in laser and microscopy technologies have made it possible to routinely image deep cortical glomeruli in the intact mouse kidney (17). Most of the new knowledge and topics reviewed here were derived from using these recent state-of-the-art MPM imaging approaches in intact adult mouse kidneys up to 8months of age (Figures 1, 2) (18)(19)(20)(21)(22). Importantly, MPM has been able to directly visualize (patho)physiological processes of the entire glomerulus, the many elements of the GFB simultaneously as parts of the whole functional unit rather than just focusing on a single cell type (12,14).…”

“…This integrative and holistic pathophysiology approach has successfully identified multiple levels of cell-to-cell interactions between individual cells and cell types of the GFB. Intravital imaging was able to shed light on the consequences of manipulating a single GFB layer on overall glomerular structure and function including albumin leakage ( 10 , 12 , 14 , 15 , 18 , 20 , 21 ). This review highlights important functions and roles of GEnCs in the development of glomerular injury and proteinuria based on recent in vivo MPM studies in both physiological and disease models ( Figure 1 ).…”