Associate Professor Texas A&M University College Station, Texas, United States
Introduction:: The vascular endothelium plays a crucial role in mediating a range of vital physiological functions. Endothelial cells are continuously subjected to diverse hemodynamic forces, including shear stress, pressure, and tensile strain, which significantly influence their function, structure, and signaling pathways. These factors ultimately affect vascular health and disease progression. Blood flow, inherently pulsatile due to the rhythmic heartbeat, exhibits unique transient waveforms depending on the physiological status and anatomical location of vessels. However, current in-vitro perfusion pumps inadequately replicate hemodynamic flow patterns at physiological time scales due to low flow responsivity. Consequently, clinical hemodynamics has remained irreproducible in experimental models. To address this limitation, we have developed a novel tissue perfusion system capable of modeling the transient vessel-specific flow patterns with high temporal resolution. As a proof-of-concept, we employed this system to examine the impact of diastolic flow reversal, a phenomenon associated with sedentary lifestyles and aging, on endothelial function and vascular health.
Materials and Methods:: Our perfusion system features a custom-built pump that pneumatically drives flow in microfluidic channels according to pre-programmed waveforms. An inline pressure sensor connected to the pump facilitates closed-loop control for maintaining precise flow conditions. We derived waveforms from published clinical Doppler ultrasound data and converted blood velocity-time plots into wall shear-time plots based on Poiseuille's flow assumption. We seeded human umbilical vein endothelial cells (HUVECs) into collagen-I and fibronectin-coated vessel-chips and cultured them under hydrostatic pressure-driven flow until confluence. Subsequently, we connected the chips to the pump and applied either atheroprotective (no-flow reversal) or atheroprone (diastolic flow reversal) waveforms. The cells were stained with CellROX fluorescent probe and imaged using live-cell timelapse microscopy to study the oxidative stress. After the experiment, we fixed and stained the cells for eNOS, VE-Cadherin, and actin to assess their morphology and function. Additionally, we lysed the cells to perform quantitative PCR (qPCR) to examine the transcriptomic expression of genes associated with endothelial dysfunction including VCAM-1, ICAM-1, MCP-1, VEGF, and PECAM-1.
Results, Conclusions, and Discussions:: We began by characterizing the pump's performance and observed a linear (R2 >0.99) displacement-pressure response and high responsivity (20 ms), essential for modeling physiological waveforms, but not possible to achieve in syringe, peristaltic or commercial pulsatile pumps. We programmed the pump with in-vivo human flow waveforms obtained from Doppler ultrasound of internal carotid artery, external carotid artery, superior mesenteric artery (both pre- and post-prandial), and common femoral vein. The pump successfully modeled all waveforms with high fidelity (Pearson correlation >0.95), demonstrating its ability to capture vessel-specific hemodynamic heterogeneity. Similar results were observed when using patient-specific waveforms for brachial artery flow patterns in individuals with and without diastolic flow reversal. Upon applying these waveforms to the endothelialized vessel-chip, we discovered that one-hour exposure to the diastolic reversal waveform significantly increased oxidative stress in HUVECs. This was accompanied by a reduction in eNOS production and loss of barrier integrity, indicative of endothelial dysfunction. Conversely, exposure to the waveform without diastolic reversal exhibited an atheroprotective effect, reducing endothelial oxidative stress and maintaining the barrier integrity. These findings were cross-validated using qPCR, revealing similar trends.
In summary, we have introduced a novel perfusion system capable of accurately modeling transient hemodynamic changes in-vitro, providing a powerful tool for investigating the effects of varying flow patterns on vascular health. This technology has allowed the examination of the long-term effects of diverse flow patterns on the development and progression of vascular diseases, such as atherosclerosis, to gain deeper insights into underlying mechanisms and identify potential therapeutic targets.
Acknowledgements (Optional): : This material is based upon work partially supported by the NASA, BARDA, NIH, and USFDA, under Contract No. 80ARC023CA002
