K3 - Modeling Host–Pathogen Interactions Using an Orbital Shaker-Based Model of Endovascular Infection

Introduction: : Endothelial cell (EC) activation plays a critical role in the pathogenesis of cardiac device infections, particularly in the setting of Staphylococcus aureus bloodstream infection. However, most in vitro models fail to incorporate physiologic wall shear stress (WSS), a critical determinant of EC signaling and inflammatory response. This limits their utility in modeling interfaces where shear stress may influence host-pathogen interactions. To address this gap, we developed an orbital shaker-based infection model that mimics shear stress conditions relevant to the vascular interface of implanted devices. We examined EC responses to microbial agonists and prototypical pathogens to evaluate the utility of this model as a novel platform for studying host-pathogen interactions in endovascular infections.

Materials and

Methods: : We engineered a custom orbital shaker platform using 6-well plates combined with a surface patterning technique that localized EC adhesion to the outer perimeter of each well, thereby targeting cells to laminar shear stress zones (1–15 dynes/cm²). Human umbilical vein endothelial cells (HUVECs) were cultured on collagen-coated, micropatterned substrates for 48 hours under static or shear conditions. To model infection, cells were stimulated with microbial cell wall agonists (lipopolysaccharide (LPS), lipoteichoic acid (LTA), or zymosan), or challenged with prototypical bloodstream pathogens (Staphylococcus aureus, Escherichia coli, and Candida albicans). EC response was assessed by RT-qPCR and immunofluorescence, focusing on flow-responsive and inflammatory markers relevant to vascular dysfunction in bloodstream infections.

Results, Conclusions, and Discussions:: Exposure to shear stress enhanced the expression of flow-responsive genes (KLF2, NRF2, TIE2, NOS3) and induced EC alignment in the direction of flow within 24–48 hours. Consistent with in vivo conditions, stimulation with microbial agonists triggered a transient upregulation of leukocyte adhesion molecules (E-selectin, VCAM-1, ICAM-1), peaking at 5 hours and declining by 18 hours. Expression increased in a dose-dependent manner, with E-selectin showing the most pronounced upregulation across all agonists tested. This is consistent with its de novo, stimulus-inducible expression and critical role in mediating early neutrophil-endothelial interactions. In contrast, components of the Ang/TIE signaling axis showed only modest, non-significant changes, potentially due to the model's restriction to laminar flow. Preliminary studies with live GFP-S. aureus demonstrated notable EC adhesion and internalization, yet only modest inflammatory activation. This finding is clinically relevant, as intracellular persistence is a recognized etiology of microbiologic failure in endovascular infections despite appropriate antibiotic therapy. Taken together, these results demonstrate that the orbital shaker platform is a promising model system to investigate endovascular infection pathogenesis. Future studies will assess the effect the elevated shear and disturbed flow on EC responses thereby advancing the design of preclinical systems for cardiovascular infection research.

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