Role of Innate Immune Cells in Vascular Graft Regeneration: Effects of Host Age

Introduction: Cardiovascular diseases (CVDs) are a leading cause of deaths across the globe, often requiring surgical replacement of blood vessels that are damaged or occluded. Historically, autologous vessels like saphenous veins have served as the gold standard for vascular grafts. However, their limited availability, high morbidity at donor site and high rate of long-term failure have prompted the search for alternative strategies. These include acellular tissue engineered vessels (ATEVs), which are often designed to recruit host cells to their lumen in order to promote tissue regeneration post-implantation. Our group reported that innate immune cells like monocytes (MC) and macrophages (Mϕ) play crucial roles during vascular regeneration and endothelialization of ATEVs [1]. Furthermore, we designed a novel fusion peptide, H2R5, to functionalize the ATEV lumen and recruit MC and Mϕ from the blood via integrin α4β1 (Figure 1A) [2]. At 4 weeks post-implantation in the arterial system of young mice, H2R5-ATEVs remained patent and their lumens were populated by cells that co-expressed MC and anti-inflammatory Mϕ (M2) markers. These grafts also demonstrated tissue organization resembling that of native vessels. Aging, however, often dysregulates the immune responses of MC and Mϕ, as well as increases the risk of acquiring CVDs and so, a significant population requiring vascular grafts are the elderly. Therefore, in this study, we sought to evaluate how H2R5-ATEVs perform in geriatric mice to assess how host age may impact the behavior of MC and Mϕ during tissue regeneration.

Materials and

Methods: H2R5-ATEVs were manufactured by wrapping decellularized porcine small intestinal submucosa (SIS) sheets three times around a perforated silicone tubing (outer diameter = 600 μm) and functionalizing the luminal surface with H2R5 by following previously-established protocols [2]. To provide direct evidence for MC recruitment to the luminal surfaces of H2R5-ATEVs via lineage tracing, the novel CX3CR1-confetti mouse model was previously generated to track the fluorescently-labeled MCs via the CX3CR1 promoter as described in Figure 1B [2]. H2R5-ATEVs (diameter = 800 μm) were implanted interpositionally into the infrarenal aortas of young (~ 3 months-old) and geriatric (22-24 months-old) CX3CR1-confetti mice. Patency and blood flow of the ATEVs were monitored bi-weekly via doppler ultrasound and were explanted at 4 weeks post-implantation. Explanted ATEVs were embedded in OCT either fresh or after fixation in 4% paraformaldehyde following standard protocols. Tissue sections of the explanted ATEVs were then obtained to perform histological analyses, including immunofluorescence for endothelial, MC/ Mϕ, and senescence markers, in order to evaluate and compare the luminal cellularization of H2R5-ATEVs between young and geriatric mice.

Results, Conclusions, and Discussions: At 4 weeks post-implantation, we observed that H2R5-ATEVs remained patent in geriatric mice and were successfully populated by the circulating MCs, as evidenced by the expression of fluorescently labeled-CX3CR1-expressing cells in the ATEV lumen (Figure 1C). Interestingly, the majority of the CX3CR1-fluorescent cells populating the ATEVs were RFP+, regardless of age (Figure 1D). Tissue measurement parameters (as defined in Figure 2A) show that although the lumen area of the ATEVs implanted in geriatric mice were similar to those in young mice (Figure 2B), the ATEVs in geriatric mice displayed slightly greater cell layer area (Figure 2C) and average tissue thickness (Figure 2D) than those in young mice. This observation may be attributed to the increased amounts of MC in circulation that is associated with aging in mice [3], possibly leading to higher amounts of MC being captured by ATEVs implanted in geriatric mice. Additionally, immunostaining of ATEV cross-sections revealed that similar to young mice, the ATEV lumen was populated by cells that co-expressed CD14 and CD206 (Figure 2E-F), showing that the captured MCs differentiated into Mϕ that polarized towards an anti-inflammatory phenotype. However, a greater amount of these cells co-expressed the fatty acid-uptake marker, CD36, in geriatric mice (Figure 2G), indicating potential increase in fatty acid uptake by the MC/Mϕ with age. The exact mechanism for how tissue regeneration in ATEVs may be impacted by age remains unclear, so additional staining with senescence markers like p16, p21, and p53 will be performed to provide further insight into the differences (or similarities) between the cells populating the ATEVs in geriatric vs. young mice. Moreover, we will utilize spatial transcriptomics to evaluate the positional context of gene expression in the explanted ATEVs from both young and geriatric mice, which may provide deeper understanding of the role of MC/Mϕ in ATEV tissue regeneration with age. Overall, our current results highlight an exciting potential of H2R5-ATEVs in future clinical applications, including in geriatric patients requiring bypass surgeries.

Acknowledgements (Optional): This work was supported by a grant from the National Institutes of Health, NHLBI R01 HL151196, to Dr. Stelios T. Andreadis.