Poster V10 - Hypoxia-driven glycolysis contributes to vascular permeability

Introduction: Secondary lymphedema affects 1 in 1000 people in the United States. It is caused when a portion of the lymphatic system is damaged through surgery, injury, or radiation. Lymphedema is a progressive disease which leads to edema, fibrosis, adipose deposition, and loss of mobility in the affected limb(s). Disease progression is currently attributed to a feedback loop of inflammation, hypoxia, and tissue remodeling; however, this model ignores the impact of metabolic changes on vascular function. Hypoxia, like that found in lymphedematous tissues, shifts cell metabolism in favor of glycolysis, while glycolytic bursts have been found to drive cell rearrangement and migration in aortic and venous endothelial cells through the Rho/ROCK pathway. Supporting evidence of this is found in mouse models of lymphedema, where lymphatic-specific ROCK inhibition reduced swelling and restored barrier integrity.
Here, we culture lymphatic endothelial cells (LECs) at 21%, 5%, and 1% oxygen with or without reactive oxygen species (ROS) inhibitor N-Acetyl Cysteine (NAC), additional glutamine, or hypoxia mimicking agent CoCl2 in order to further clarify the link between hypoxia, metabolism, migration, and vascular leakiness in LECs. We hypothesize that treating with CoCl2 or hypoxia will increase glucose uptake, cell migration, and permeability, while this result will be alleviated by NAC and glutamine. Elucidating these interactions will be an essential step forward in developing effective therapeutic interventions for lymphedema.

Materials and

Methods: Human Juvenile Lymphatic Endothelial Cells (LECs; Promocell) were expanded in Endothelial Cell Growth Media MV2 with supplement at 37°C, 5% CO2. Treatments included culture for 24 hours at 21% and 5% oxygen, with or without a total concentration of 5mM NAC, 12mM glutamine, and 100μM CoCl2 in supplemented MV2 (Figure 1). To visualize changes in ROS, cells were stained with CellTracker Red CMTPX (Invitrogen), CellROX (Thermo Fisher) and DAPI; the average fluorescence of the nuclei and cytoplasm was then quantified using FIJI imaging software. Glucose uptake was measured using Promega’s Glucose Uptake-Glo™ Assay. To examine changes in gene expression, qPCR was performed for glucose transporter genes SLC2A1 and SLC2A3, glycolysis marker HK2, and transmembrane receptor EPHB4, which is involved in a signaling pathway that controls vascular maturation and cell junction. These results will be confirmed with western blot using tubulin as a control. Lastly, LEC migration and permeability will be measured using an Ibidi wound-healing assay and interruptions to the cell monolayer will be visualized using a Sigma-Aldrich In Vitro Vascular Permeability Imaging Assay. All assays will be repeated in triplicate, and statistical analysis will be performed using Prism 10, with significance levels set to P less than or equal to 0.05.

Results, Conclusions, and Discussions: Currently, we have performed a preliminary glucose uptake assay on cells cultured in 21% and 5% oxygen, as well as ROS analysis and qPCR for 21% and 5% groups with or without treatment with 5mM NAC.
In the glucose uptake assay, the cells cultured for 24 hours in 5% oxygen had a significantly larger luminescence than cells cultured in 21% oxygen, indicating that they internalized more 2-deoxyglucose (Figure 2). ROS analysis found that ROS increased significantly in both the nucleus and the cytoplasm of the 5% group compared to the 21% group. Additionally, NAC was found to significantly reduce ROS production in the nuclei and cytoplasm of cells cultured at 5% oxygen. However, at 21% oxygen NAC caused no change in nuclear ROS and an increase in ROS in the cytoplasm (Figure 3). These results were reflected when examining gene expression via qPCR, as the expression of glucose transporter proteins SLC2A1 and SLC2A3 significantly increased (i.e. had a decreased Cq) in the 5% group compared to the 21% group. NAC treatments decreased their expression in both groups. HK2, an enzyme responsible for the commitment step in glycolysis, did not significantly change between 21% and 5%, the Cq was slightly lower in the 5% group, indicating a higher expression. NAC also decreased HK2 expression significantly in the 21% group. EPHB4 expression decreased in the 5% group, indicating a decrease in vascular integrity (Figure 4).
Our initial results draw a correlation between hypoxia, increased ROS, and increased glycolysis which results in lowered vascular integrity. Further experiments will confirm these findings via western blot, as well as explore changes in cell behavior caused by glycolysis in the form of permeability and wound healing assays.

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