Shailey Gale Twamley

Alveolar epithelial cells (AEC) are covered apically by the alveolar lining layer (ALL). The alveolar epithelial glycocalyx (AEGCX), which is part of the ALL, consists of a mixture of glycoproteins, proteoglycans and glycosaminoglycans. In comparison to the endothelial glycocalyx (GCX), much less is known about the composition and structure of the AEGCX although it is presumed to be involved in many cellular processes. The challenges of in vivo visualization of the AEGCX, coupled with the distorting effects of aldehyde fixation, make it difficult to accurately determine its structural organization and molecular composition. In this study, we aimed to improve the preservation of the ultrastructure of the AEGCX by using high-pressure freezing combined with freeze substitution (HPF/FS) for transmission electron microscopy (TEM) analysis. HPF is a type of cryopreservation that uses both liquid nitrogen and high pressure. By using HPF/FS to preserve commercially available human primary AECs, human alveolar-like organoids, monolayers of human organoid-derived alveolar-like epithelial cells, and ex vivo tissue from both mice and human lungs, we observed two distinct regions on the apical surface via TEM: an inner GCX-like layer along the cell membrane followed by a loosely arranged outer layer. Our data shows a bi-layered structure on the surface of AECs. To minimize freeze induced damage during HPF, a protein-rich cryoprotectant was introduced to stabilize the sample. By processing control samples of protein-rich cryoprotectants lacking cells, we found that HPF/FS preserves components of the cryoprotectant, as confirmed by TEM analysis. This implies that HPF/FS preserves not only cellular derived constituents but also components found in the cryoprotectant which may interfere with interpretation of the outer surface layer.

The study investigates the role of cell-surface glycosaminoglycans during the rapid uptake of Synomag®-COOH, a promising MPI-tracer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.