Raghuveer Lalitha Sridhar

Sprint (United States)

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Michael R. Moreno

Texas A&M University

Caleb A. Davis

Texas A&M University

David C. Zawieja

Texas A&M Health Science Center

Sanjukta Chakraborty

Texas A&M Health Science Center

Lindsey B. Bezek

Los Alamos National Laboratory

Andrew B. Robbins

The University of Texas at Tyler

John R. Tumbleston

Carbon180

Cameron Miller

National Institute of Standards and Technology

Callie I. Higgins

National Institute of Standards and Technology

David Walker

Columbia University

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Texas A&M University

National Institute of Standards and Technology

Mitchell Institute

National Institute of Standards

Walker (United States)

Georgia Institute of Technology

Material Measurement Laboratory

University of Pittsburgh

Government of the United States of America

Physical Measurement Laboratory

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A Bioreactor for in Vitro Studies of Lymphatic Endothelial Cells with Simultaneous Fluid Shear Stress and Membrane Strain

Caleb A. Davis Byron Zambrano-Roman Raghuveer Lalitha Sridhar A. Jastram Sanjukta Chakraborty

Reproducing the in vivo physiologic conditions and biomechanical environment to stimulate natural growth and behavior of lymphatic endothelial cells (LECs) is critical in studying the lymphatic system and its response to stimuli. In vitro studies that deconstruct the biomechanical environment, e.g. independently incorporate flow-induced shear stress or membrane strain have demonstrated the significance of mechanotransduction in LECs (and vascular endothelial cells). Such studies have facilitated the investigation of intracellular signaling pathways stimulated by a particular mechanical cue but do not accurately reproduce natural physiologic behavior of in vivo LECs given the absence of other natural mechanical cues. In this study, we present a novel experimental device designed to reconstruct the in vivo biomechanical environment, i.e. a device that enables the simultaneous application of flow-induced shear stress and cyclic stretching of LECs in vitro. The device is uniquely capable of simulating physiologically-relevant conditions for lymphatic endothelial cells, such as low-flow, high-strain scenarios. Using this device, we observed that, like vascular ECs, LECs aligned in the direction of fluid shear stress when steady flow was applied. In our case the behavior was observed under conditions closer to the physiological mean flow in the lymphatic vessels than vascular levels of shear stress. When concurrent cyclic stretching was applied, the alignment in the direction of flow and perpendicular to the uniaxial stretch was detected in a substantially shortened timeframe. Additionally, the distribution of alignment angles was more closely clustered around 90 degrees under the flow/stretch scenario after 6 hours than the 24 hour flow only scenario, perhaps indicating a greater sensitivity to cyclic stretching than to fluid shear stress in the morphological alignment response of LECs. We also observed alignment of cell nuclei and F-actin filaments in HDLECs after only 6 hours of combined flow and stretch. These observations underscore the importance of including both sources of mechanical stress when studying the growth and behavior of LECs.

Strain (injury)

Lymphatic Endothelium

10.2139/ssrn.4562304

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A versatile biaxial testing platform for soft tissues

Journal of the mechanical behavior of biomedical materials/Journal of mechanical behavior of biomedical materials (2020)

Mingliang Jiang Raghuveer Lalitha Sridhar Andrew B. Robbins Alan D. Freed Michael R. Moreno

10.1016/j.jmbbm.2020.104144

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Citations (23)

Results of an interlaboratory study on the working curve in vat photopolymerization

Additive manufacturing (2024)

Thomas J. Kolibaba Jason P. Killgore Benjamin W. Caplins Callie I. Higgins Uwe Arp

Stereolithography

Curve fitting

Light Field

10.1016/j.addma.2024.104082

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Citations (7)

Experimental Investigation of the Anisotropic Mechanical Response of the Porcine Thoracic Aorta

Annals of Biomedical Engineering (2022)

Manoj Myneni Raghuveer Lalitha Sridhar Κ. R. Rajagopal Chandler C. Benjamin

Thoracic aorta

10.1007/s10439-022-02931-2

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Quantifying Lymphatic Endothelial Cell Morphological Changes in Response to Fluid Shear Stress, Cyclic Strain, or Combined Stress and Strain In Vitro

The FASEB Journal (2021)

Caleb A. Davis Raghuveer Lalitha Sridhar Sanjukta Chakraborty David C. Zawieja Michael R. Moreno

The lymphatic system performs vital transport of fluid, lipids, immune cells, and macromolecules. Lymphatic endothelial cells (LECs), which are highly mechanosensitive, are involved in modulating lymphatic contraction, permeability, etc. LECs are sensitive to the fluid shear stress (FSS) caused by lymph flow and the cyclic strain caused by wall contraction and relaxation. The effects of these mechanical forces on LECs are less understood compared to blood vascular endothelial cells, (BVEC), yet the relative changes in these forces on LECs are considerably greater in situ. To the best of our knowledge, no previous in vitro studies have applied simultaneous FSS and cyclic stretching to LECs. To address this information gap, we tested the hypothesis that both FSS and cyclic stretching will alter LEC structure and function. Toward this goal, we developed a novel bioreactor to apply simultaneous, independently-controlled FSS and cyclic uniaxial stretching to LECs on silicone membranes, and a second bioreactor for applying only uniaxial cyclic stretch. After subjecting LECs to FSS alone, stretch alone, or combined FSS and stretch in these systems, we imaged the cells using conventional or confocal fluorescence techniques and measured alterations in orientation and actin fiber alignment of the cultured LECs. Our results show statistically significant changes in LEC orientation compared to controls, aligning in the direction of low steady FSS (0.2 dynes/cm^2) after 12-24 hours. LECs subjected to the low steady flow combined with a 7.5% cyclic stretch, exhibited a more dramatic alignment that was parallel to flow and perpendicular to stretch after only 6 hours. These data indicate that, like BVEC, LEC morphological alignment is sensitive to both FSS and stretching, with a synergistic response when both are applied simultaneously. Similar levels of alignment were seen in LECs subjected to only cyclic stretch after 6 or 12 hours, but rather than aligning perpendicular to stretch, the distribution of cell angles was bimodal, with most cells aligning about +/- 15 degrees from perpendicular. This slightly off-axis alignment could be a function of applying stretch without fluid flow, or may be a response to the higher magnitude of stretch in those studies (40% compared to 7.5%). There was some visual indication of alignment of actin fibers in LEC subjected to combined FSS and stretching, but it was more difficult to determine clear trends due to spatial variation when imaging different regions of the membrane, and further study is therefore required. Taken together, our results show that application of FSS and strain differentially alter LEC alignment and morphology and provides an important platform to further investigate the mechanisms underlying the LEC response to complex physiologically relevant mechanical environments.

Lymphatic Endothelium

10.1096/fasebj.2021.35.s1.03528

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