Age-related decline in cardiac lymphatic vessels causes cardiac edema and macrophage accumulation

Abstract As life expectancy has significantly increased, age-related diseases, particularly cardiovascular diseases, have become a primary global cause of death. Aging has multiple effects on the heart and is one major risk factor for cardiovascular diseases. The microcirculation has been extensively studied in this context. However, 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 sub-endocardial and –epicardial area of aged left ventricles, in both genders (0.28±0.08 fold and 0.68±0.03 fold; p<0.05), while the right ventricle showed no change. Notably, dilation of (pre-) collector lymphatics was observed in in old hearts, indicating, together with the lymphatic vessel reduction, a lymphatic drainage dysfunction. Indeed, cardiac aging was accompanied by increased numbers of CD68+ macrophages, accumulation of the plasma protein fibrinogen and amyloid into the interstitial space. 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 Vegfc (0.72±0.04 fold; p<0.005), a major lymphatic growth factor, in 20-month-old mouse hearts compared to their 3-month-old counterparts. To establish a causal link between lymphatic dysfunction and age-related cardiac changes, we examined hearts from 3-month-old Flt4;Prox1-CreERT2 mice, that have defective lymphatic vessels. Also these mice, despite their young age, displayed fibrinogen (1.32±0.08 fold; p<0.05) and CD68+ macrophage (1.58±0.08 fold; p<0.05) accumulation compared to wildtype littermates. Apart from these, other age-related changes, such as tendency to cardiac hypertrophy, were also observed; this is currently under further investigation. Likewise, blocking VEGFC signaling in young mice by overexpressing the soluble form of Flt4 impaired cardiac lymphatic capillary density (0.62±0.06 fold; p<0.01), leading to increased macrophage and fibrinogen accumulation (1.69±0.09 fold, 4.60±0.30 fold; p<0.001), suggesting that a decline of lymphatics induces some of the age-associated pathologies. Finally, we addressed whether restoring Vegfc expression in old mice can rescue age-related cardiac impairment. Overexpressing Vegfc via AAV9 in aged mice restored cardiac lymphatic density by 3-fold and reduced CD68+ macrophage density by 1.6-fold, without effect on fibrinogen accumulation. In conclusion, our study demonstrates an age-related reduction in left ventricular lymphatic density, cardiac edema and inflammation. Vegfc overexpression prevented the age-dependent decline of lymphatic vasculature and reduced cardiac inflammation.