Abstract Body: Background: The vascular endothelium mediates many vital physiological functions. Endothelial cells experience various hemodynamic forces such as shear stress, pressure, and tensile strain which profoundly influence their structure, function, and signaling. Yet, key gaps remain in understanding how these flow–derived forces influence vascular biology and contribute to thrombosis and atherosclerosis. Prior studies often use syringe or peristaltic pump systems that fail to reproduce clinically observed multiphasic hemodynamic complexity. Consequently, systematic investigation of endothelial mechanosensing under human blood flow waveforms has been limited. We developed a novel in vitro perfusion system, Hemadyne, that produces vessel and patient-specific waveforms to enables such studies.

Objective: This study deployed Hemadyne to 1) determine whether diastolic rest duration governs endothelial homeostasis, and 2) recapitulate clinically observed diastolic retrograde flow–associated vascular dysfunction

Methods: Human flow waveforms captured by clinical Doppler ultrasound were programmed to Hemadyne. Human aortic (HAECs) and umbilical vein endothelial cells (HUVECs) were cultured on collagen-I coated microfluidic vessel-chips and exposed to the corresponding waveforms for 24 hours. The cells were then fixed, immunostained for cadherin junctions, nitric oxide synthase, and intercellular adhesion molecule 1 (ICAM–1), followed by imaging and quantitative analysis.

Results: Hemadyne faithfully reproduced multiphasic clinical flow profiles (Pearson correlation > 0.97). After exposure, both HAECs and HUVECs maintained intact adherens junctions only when the flow included a 0.5 s diastolic rest period. Either doubling this rest time or eliminating it compromised endothelial barrier integrity in both cell types. Analysis of diastolic retrograde flow interactions with HAECs showed that transient reversal lasting over 300ms induced endothelial dysfunction in a duration dependent manner, evidenced by disrupted cadherin junctions, reduced eNOS production, elevated ICAM–1 expression, and loss of actin cytoskeletal organization.

Conclusions: Hemadyne enabled physiologically relevant study of hemodynamic–endothelial interactions and replicated clinical retrograde flow associated vascular dysfunction in vitro. This system may facilitate discovery of novel mechanosensory pathways and enable discovery of targeted therapeutic intervention strategies for vascular disorders.