Age-related decline in cardiac lymphatic vessel density is associated with cardiac edema and immune cell accumulation

Aging is one of the major established risk factor for cardiovascular diseases, but vascular aging itself may contribute to the progressive deterioration of organ function. While the impact of age on the blood vasculature 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.

To address the effect of aging on lymphatics, we examined lymphatic capillary density using LYVE1 and PDPN staining in old (>20 months) and young (3 months) mouse hearts. Aging induced a significant reduction in lymphatic vessel density in the left ventricles, but not in the right ventricle, in both sexes (0.28±0.08 fold and 0.68±0.03 fold; p<0.05). Notably, dilation of lymphatics and increased formation of zipper-junctions were observed in in old mouse hearts, indicating a lymphatic drainage dysfunction.

Indeed, cardiac aging was accompanied by increased numbers of CD68+ macrophages, T-cells and neutrophils, and accumulation of the plasma protein fibrinogen and amyloid into the interstitial space, while the draining cardiac lymph nodes contained less macrophages. Moreover, we observed a significant increase in tissue oedema in the aged hearts (p<0.05). The decline in lymphatic vessels was associated with a reduced expression of the major lymphatic growth factor Vegfc (0.72±0.04 fold; p<0.005) in 20-month-old compared to 3-month-old mouse hearts.

To establish a causal link between lymphatic dysfunction and age-related cardiac changes, we examined hearts from 3-month-old mice after AAV9-mediated overexpression of a soluble VEGFR3 receptor that binds VEGFC and VEGFD (VEGFC/D trap), which has been shown to lead to lymphatic vessel attrition and malfunction.Despite their young age, the mice expressing the VEGF-C/D trap showed increased fibrinogen (4.5±0.3 fold; p<0.05) and macrophage (1.7±0.1 fold; p<0.05) accumulation in the left ventricle as well as significant diastolic dysfunction. Likewise, blocking VEGFC signaling in young mice by using deletion of the Flt4 gene (encoding VEGFR3) in lymphatic endothelial cells showed a similar phenotype, suggesting that the decline of lymphatics induces typical age-associated pathologies.

Finally, we addressed whether restoring Vegfc expression in old mice can rescue age-related cardiac impairment. AAV9-mediated Vegfc gene expression in aged mice increased cardiac lymphatic density by 3-fold and reduced CD68+ macrophage density (0.6±0.1 fold; p<0.05).

In conclusion, our study demonstrates an age-related reduction in left ventricular lymphatic density, with cardiac edema and inflammation. Vegfc overexpression prevented the age-dependent decline of lymphatic vasculature and reduced cardiac inflammation.