(25d) Endothelial Glycocalyx of Human Lung Microvascular Endothelial Cells As a Regulator of Vascular Permeability in a Fibrotic Lung Environment

Introduction: A fibrotic lung is known to recruit inflammatory cells, cancer cells, and viruses, possibly due to its mechanical environment ^1-6. A fibrotic lung experiences high lung matrix stiffness (20 kPa-100 kPa) compared to healthy lung stiffness (1-5 kPa)⁷. Stiffness may influence vascular permeability. The endothelial cell (EC) glycocalyx (GCX), primarily on the luminal side of ECs, is the first vascular barrier. It is made primarily of glycosaminoglycan chains (GAGs) such as heparan sulfate (HS) and hyaluronic acid (HA), along with negatively charged sialic acid (SA) 8, 9. GCX composition and barrier function are influenced by its mechanical environment and are understudied in human lung microvascular ECs (HPMECs). By modeling healthy and diseased lung stiffness under dynamic flow, we aim to understand how stiffness regulates the GCX of HPMECs. We hypothesize that high matrix stiffness decreases overall GCX expression on HPMECs by producing less GAG-synthesizing enzymes, increasing GCX barrier permeability.

Methods: Gelatin methacrylate (GelMA) hydrogels and a parallel plate flow chamber are used to model soft (healthy) and stiff (fibrotic) conditions. HPMECs on GelMA hydrogels of ~5 kPa vs ~30 kPa stiffness are subjected to 12 dyn/cm² physiological shear stress for 6 hours. GCX expression (apical/basal/junctional thickness, percent area coverage, and mean fluorescence intensity) are determined through fluorescence labeling of the whole GCX structure with wheat germ agglutinin (WGA), HS with 10E4-epitope mouse monoclonal antibody, HA with HA binding protein, and α2,6-linked SA with sambucus nigra (SNA) lectin. Confocal microscopy captures fluorescence Z-stacks which are translated into 3D images and analyzed via a custom-designed python script. The impact of matrix stiffness on GCX synthesizing proteins (EXTL1 and EXTL3 for HS, HAS1 for HA, α-2,6-sialyltransferase for SA) are similarly examined, while GCX barrier permeability is assessed with nanoparticle uptake studies.

Results: WGA-labeled structures indicated significant decreases in whole GCX percent area coverage of ECs for the stiff matrix (73.76 ± 2.30%) compared to the soft matrix (85.88 ± 2.14%). WGA thickness at EC junctions showed significant reduction on stiffer matrices (1.64 ± .03 µm) compared to soft matrices (2.32 ±.10 µm). HA coverage and junctional thickness slightly decreased on stiffer matrices (11.56 ± 5.82%, 0.17 ±.09 µm) compared to softer matrices (23.76 ±8.91%, 0.38 ± .14 µm). α2,6-linked SA residue was found to be more prominent than HA and was sensitive to stiffness. SA displayed less coverage on stiff matrices (80.80 ± .71%) than on soft matrices (91.37 ± 1.91%), and less thickness on stiff matrices (1.47 ± .15 µm) compared to soft matrices (2.38 ± .28 µm). Work is ongoing to further quantify the structure of WGA-labeled GCX, HA, and HS; expression of GCX synthesizing enzymes; and related GCX permeability.

Conclusion: This work progresses our understanding of how the mechanical environment regulates HPMECs’ GCX in health and fibrosis. Fibrotic stiffness inhibits overall GCX coverage of ECs along with thickness at EC-to-EC junctions, which may contribute to increased vascular permeability in fibrosis10. Also, the GCX loss could lead to release of chemokines and cytokines that the GCX typically store, creating a chemokine/inflammation gradient 11. The data shows that SA residue is a major GCX component that is significantly reduced in fibrosis. SA may play a dual role in regulating EC surface adhesiveness and the physical trans-endothelial barrier 12. Examining the GCX composition changes occurring in the diseased state may open a path to understanding its role in vascular permeability and discovering a possible therapeutic option to limit various pathological diseases.

Acknowledgements: We thank the Institute for Chemical Imaging of Living Systems (RRID:SCR_022681) at Northeastern University for support. This work was funded by NSF CAREER Award CMMI 1846962 (to E. E. Ebong) and the NSF LSAMP-BD STARS Award HRD 1812412 (to Northeastern Univ.; C. Okorafor was supported as a predoctoral fellow).

References: (1) Zhang, C. et al., Oncotarget 2016, 7 (29), 45702-45714. (2) Cox, T. R. et al., Breast Cancer Management 2014, 3 (6), 453-455. (3) Barkan, D. et al., Cancer Research 2010, 70 (14), 5706-5716. (4) Cox, T. R. et al., Cancer Res 2013, 73 (6), 1721-32. (5) Stramer, B. M. et al., Journal of Investigative Dermatology 2007, 127 (5), 1009-1017. (6) Warheit-Niemi, H. I. et al., JCI Insight 2022, 7 (4). (7) Hinz, B., Proceedings of the American Thoracic Society 2012, 9 (3), 137-147. (8) Harding, I. C. et al., Biorheology 2019, 56 (2-3), 131-149. (9) Harding, I. C. et al., Journal of Translational Medicine 2018, 16 (1), 364. (10) Probst, C. K. et al., Eur Respir J 2020, 56 (1). (11) Queisser, K. A et al., J Histochem Cytochem 2021, 69 (1), 25-34. (12) Zaib, T. et al., Int J Mol Sci 2023, 24 (3).