Interleukin-33 regulates lymphatic function in the ageing heart

As life expectancy has significantly increased, age-related diseases, particularly cardiovascular diseases, have become a primary cause of death. Ageing is the primary risk factor for cardiovascular dysfunction, hence it is of fundamental interest to understand the mechanisms underlying age-related cardiac dysfunction. While the microcirculation has been well studied, the contribution of the lymphatic vasculature, which is responsible for draining excessive tissue fluid and immune cells, to age-related pathologies is currently unknown. Therefore, we addressed the influence of aging on lymphatic functions.

We show that the number of lymphatic vessels is reduced starting at an age of 16 month, with significant declines at 18 months in the sub-endocardial and epicardial area in both sexes (0.28±0.08 fold and 0.68±0.03 fold; p<0.05). 

To understand the mechanisms underlying age-related lymphatic changes, we performed single-cell RNA sequencing on CD31+ endothelial cells obtained from young (3-month-old) and 20-month old mice. Unsupervised clustering revealed one lymphatic endothelial cell cluster enriched for the prototypical marker genes Prox1 and Lyve1. Age induced the differential expression of 197 genes. Among the top targets, we identified interleukin-33 (Il33) which was up-regulated in aged cardiac lymphatic endothelial cells. Histological assessments confirmed the up-regulation of IL-33 in aged cardiac LYVE1+ lymphatics starting at 16 months of age (4.6±0.6 fold; p=0.0009). IL-33 can be present in two distinct isoforms: a nuclear located pro-peptide and as a secreted cytokine. Interestingly, we exclusively detected only the nuclear located form that was present specifically in epicardial lymphatics of aged mouse hearts. This suggests that IL-33 might act as transcriptional regulator of lymphatic ageing. To understand the role of IL-33 in the ageing cardiac lymphatics, we cultured human dermal lymphatic endothelial cells and silenced Il33 expression by siRNAs and assessed the morphology of the cells. A characteristic feature of lymphatic endothelial cells is the junction formation. Lymphatic endothelial cells form loose “button-junctions” in lymphatic capillaries to allow the uptake of immune cells and excessive tissue fluid, while draining collector tubes form tight “zipper-junctions” to prevent leakage. Interestingly, the loss of Il33 resulted in impaired junction formation, as assessed by VE-cadherin staining. In line with these observations, we found that aged cardiac lymphatics reveal an increase in zipper-junctions, while IL-33 was up-regulated (1.7 fold; p<0.05).

These data indicate that cardiac ageing leads to an increased expression of IL-33 in cardiac lymphatics that may affect lymphatic junction formation. Whether this results in impaired lymphatic drainage, is still under investigation.