Shear Stress Induces Morphology Changes in Human Lung Microvascular Endothelial Cells via Calcium Influx

• Published in: Physiology (Bethesda, Md.) Vol. 39; no. S1
• Main Authors: Croglio, Michael P., Kolb, Todd M, Yan, Simin, Philip, Nicolas M, Niedermeyer, Shannon, Suresh, Karthik, Damarla, Mahendra
• Format: Journal Article
• Language: English
• Published: 01.05.2024
• ISSN: 1548-9213; 1548-9221
• DOI: 10.1152/physiol.2024.39.S1.816

Abstract only Introduction: The pulmonary vascular endothelium is in contact with laminar blood flow, resulting in a physiologic degree of shear stress exerted on endothelial cells (ECs). In vitro cell culture of ECs is typically performed under static conditions, leading to underappreciation of the impact of shear stress. Calcium signaling is among the most ubiquitous cell signaling mechanisms, and changes in intracellular calcium concentration ([Ca 2+ ] i ) have been linked to shear stress. The impact of shear stress on human lung microvascular endothelial cell (hLMVEC) morphology and cell signaling is not well described. We hypothesized that hLMVECs exposed to shear stress would undergo Ca 2+ -dependent morphologic changes. Methods: The effect of shear stress on [Ca 2+ ] i was first tested using ratiometric measurement. Early passage (p≤8) primary hLMVECs from three unique donor lots were plated in gelatin-coated Ibidi 0.2 uM depth plates for 16 h, then incubated with the calcium indicator Fura-2 AM for 1 h. Measurement of [Ca 2+ ] i was obtained over 15 minutes under static and physiologic shear stress (0 and 12 dyn/cm 2 , respectively), using validated flow rates from the Ibidi system. Measured [Ca 2+ ] i was compared between cells treated vehicle or an inhibitor of the mechanosensitive Ca 2+ -permeable channel TRPV4 (HC-067047). HC treatment was for 1 h prior to and throughout the duration of shear exposure. Morphology changes were then assessed in hLMVECs from the same three cell lots grown in standard 6-well plates; one plate kept in static culture, while another was exposed to 24 h of 12 dyn/cm 2 shear stress using an orbital shaker. Treatments in each plate were control, vehicle, and HC. Representative photos of each well were taken at 0 h and at 24 h. Average ratios of shortest to longest dimensions from 30 cells/field were measured using ImageJ and compared between treatments and shear conditions. A 2-way ANOVA with post-test was used to test for statistically significant changes in morphology. Results: Shear stress increased [Ca 2+ ] i within 15 minutes, and both baseline [Ca 2+ ] i and observed change in response to shear stress were decreased by TRPV4 inhibition with HC. Morphologic changes were observed after exposure to shear stress on microscopic examination, with cells grown in static culture having typical cobblestone appearance and shear-adapted cells having an elongated, spindle like appearance aligned in the direction of flow. The calculated ratio (shortest to longest dimension) of all wells at 0 h was ~0.70 with no significant differences between groups. 24 h of shear stress caused a nearly 80% decrease in ratio compared to static culture in untreated cells (p<0.05), with a decrease of only 30% in HC-treated cells after 24 h of shear (p<0.05). Conclusions: Exposure to shear stress at physiological levels results in a rapid increase [Ca 2+ ] i through mechanosensitive, Ca 2+ -permeable TRPV4 channels as well as notable morphologic changes to hLMVECs with prolonged exposure to shear stress that appear to be mediated in large part by TRPV4. Further investigation into the impact of shear stress on hLMVECs is warranted to elucidate biochemical pathways and other downstream functional consequences of increased [Ca 2+ ] i . NIH-T32HL007534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.