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 previously reported that innate immune cells like monocytes (MC) and macrophages (Mϕ) play crucial roles during vascular regeneration and endothelialization of ATEVs. However, aging often dysregulates the immune responses of MC and Mϕ, while also increasing the risk of acquiring CVDs. Since the population mostly in need for vascular grafts are the elderly. Therefore, in this study, we sought to evaluate how H2R5 ATEVs perform in aged hosts using the CX3CR1-confetti mouse that enables tracking of MC/Mϕ as they populate the graft leading to tissue regeneration.
Methods:
We designed a novel fusion peptide, H2R5 to functionalize the ATEV lumen and recruit MC and Mϕ from the blood via integrin α4β1. 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. To provide direct evidence for MC recruitment to the luminal surfaces of these ATEVs via lineage tracing, the novel CX3CR1-confetti mouse model was previously generated to track the fluorescently-labeled MCs via the CX3CR1 promoter. The ATEVs were implanted interpositionally into the infrarenal aortas of young (~ 3 months-old) and old (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 cell (EC), smooth muscle cell (SMC), MC/Mϕ, and senescence markers, to evaluate and compare the luminal cellularization of H2R5-ATEVs between young and old mice.
Results and Conclusion
At 4 weeks post-implantation, the H2R5-ATEVs remained patent in both young and old mice and were successfully populated by the circulating MCs, as evidenced by the presence of fluorescently-labeled CX3CR1-expressing cells on the ATEV lumen. Moreover, no graft stenosis was observed as both the mean ATEV lumen diameter and wall thickness were similar between the old and young mice. To assess the extent of senescence of cells populating the ATEVs in old mice, tissue sections from ATEVs and aortas (controls) from young and old mice were immunostained for senescence markers including p21, HMGB1, Ki67, and SA-β-gal. We observed increased number of senescent cells populating the ATEVs in old mice, as evidenced by expression patterns of HMGB1, Ki67, and SA-β-gal. Surprisingly, p21 was highly expressed in ATEVs from both young and old mice, most likely indicating Mϕ activation/polarization into a pro-inflammatory (M1) phenotype in the arterial microenvironment that is dominated by high shear stress. Indeed, when Mϕ were subjected to shear stress using a microfluidic device in vitro, we observed high expression of pro-inflammatory cytokines e.g., TNFa, IL-1b, and iNOS. Immunostaining of ATEV cross-sections revealed that while cells in the graft lumen of young and old mice expressed the MC-marker CD14, the expression levels and percentage of cells expressing the M1 marker, iNOS, were significantly higher in old mice. Interestingly, ATEVs in both young and old mice contained cells co-expressing, the M2 marker, CD206. This suggests that the lumen of H2R5-ATEVs is populated by circulating MCs, which subsequently differentiate into activated M1 Mϕ before turning into a pro-regenerative M2 phenotype that promotes endothelialization and vascular wall formation. Indeed, after one month, the grafts contained an endothelial monolayer expressing eNOS, and multiple SMC layers expressing aSMA and MYH11. Collectively, our results highlight differences in the inflammatory state of ATEVs in young vs. aged hosts, mostly due to immune cell senescence. They also demonstrate the potential of H2R5-ATEVs for successful transplantation into geriatric recipients, the population mostly in need of arterial replacement therapy.