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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I2 - Pericyte Uncoupling and Bridging in Prolonged Hyperglycemia

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Yashasvisai Veeramasu

Undergraduate Student
University of Virginia Biomedical Engineering Department
Glen Allen, Virginia, United States

Co-Author(s)

Kareem El-Ghazawi

PhD Candidate
University of Virginia Biomedical Engineering Department, United States
Anthony C. Bruce, MS

Lab Manager
University of Virginia Biomedical Engineering Department
Charlottesville, Virginia, United States

Primary Investigator(s)

Shayn M. Peirce, PhD (she/her/hers)

Investigator
University of Virginia
Charlottesville, Virginia, United States

Introduction: Microvascular complications are a significant aspect of diabetes, with diabetic retinopathy (DR) being the most common of these complications. This sight-threatening condition is characterized by capillary leakage and degeneration. Capillaries, essential for nutrient, oxygen, and waste exchange between blood and tissues, are supported by pericytes – specialized cells that wrap around the abluminal surface of capillary endothelial cells (ECs) to regulate blood flow and provide structural stability. Our laboratory has previously demonstrated evidence of pericyte-EC uncoupling in the Streptozotocin (STZ)-induced murine model of DR, suggesting this may be a cellular mechanism contributing to capillary leakage. In our previous study, we observed pericyte-EC uncoupling 14-days after a single, high-dose intraperitoneal injection of STZ, which causes hyperglycemia. Here, we test the hypothesis that pericyte-EC uncoupling will persist out to a more progressed stage of DR, at 21-days post STZ-injection.

Materials and

Methods: Genetically engineered Myh11-CreERT2 ROSA STOP-floxed TdTomato tamoxifen-inducible pericyte lineage tracing mice, whose pericytes and their progeny are permanently labeled with the fluorescent TdTomato reporter following a 2-week tamoxifen diet, were injected with Streptozotocin (STZ) to induce hyperglycemia or with vehicle buffer solution to generate controls (N=1 mouse per group). Mice retinas were harvested 14-days and 21-days post injection, after glucose tolerance testing confirmed sustained hyperglycemia. Whole-mounted retinas were immunostained with fluorophore-conjugated anti-CD-31 to label for ECs. Confocal microscope images (n=4 fields of view) (Figure 1A) were obtained and evaluated with ImageJ to quantify the frequency of pericyte-EC uncoupling. Bridging phenomena of these pericytes was observed. Bridging pericytes were defined as pericytes whose cell bodies or processes connected two capillaries, or by cell bodies on one capillary with processes extending to another (Figure 1C). These were quantified relative to the total number of pericytes in each field of view to determine the ratio of bridged pericytes for each mouse. In addition to bridging pericytes, uncoupled pericytes were defined as those with cell bodies unattached to the capillary (Figure 1E). The same method of quantification and ratio calculation was applied to both uncoupled and bridging pericytes.

Results, Conclusions, and Discussions: An analysis of pericyte dynamics in vehicle and STZ-treated mice over time was studied. A quantitative analysis of the total number of pericytes shows a higher total number of pericytes at the Day 14 timepoint compared to the Day 21 timepoint in both the STZ and vehicle group, suggesting age as a potential cause of pericyte dropout with time (Figure 1B). The ratio of bridging pericytes was higher in the STZ-treated group compared to the vehicle group in both the 14-day and 21-day timepoints (Figure 1D). At day 14, the STZ-treated group also showed a higher fraction of uncoupled pericytes compared to the vehicle group. These findings both indicate early disruption of pericyte-EC interactions due to hyperglycemia. However, by day 21, the fraction of uncoupled pericytes was lower in the STZ-treated group than in the vehicle group, a possible result of lower total pericyte numbers that warrants further investigation (Figure 1F). The overall results highlight the impact of hyperglycemia on pericyte interactions with ECs, but require further investigation to understand the extent of these altered interactions. In conclusion, this study shows hyperglycemia-driven pericyte dynamics such as bridging and uncoupling. We also report a lower total pericyte count at 21 days as compared to 14 days, in both STZ and vehicle injected mice. However, due to the limitation of using 1 mouse per group, we plan to repeat these experiments in larger groups (n = 5 mice per group as determined by a power analysis). By repeating the experiment in more mice, we can make stronger inferences on pericyte dropout and the alterations in pericyte behavior through disease progression. This work will serve to identify the cellular time-course of DR, allowing for improved diagnostic signs and therapeutic targets.

Acknowledgements (Optional):