Age-associated decline in cardiac lymphatic capillaries causes cardiac edema and macrophage accumulation

Julian Wagner (Frankfurt am Main)1, I. Sultan (Helsinki)2, S. Antila (Helsinki)2, N. Manickam (Frankfurt am Main)1, J. Panthel (Frankfurt am Main)1, H. Kujundzic (Frankfurt am Main)1, A. Fischer (Frankfurt am Main)1, K. Schmitz (Frankfurt am Main)1, K. Alitalo (Helsinki)2, S. Dimmeler (Frankfurt am Main)1

1Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; 2Wihuri Research Institute Helsinki, Finnland


As life expectancy has significantly increased, age-related diseases, particularly cardiovascular diseases, have become a primary global cause of mortality. Aging has multiple effects on cellular and functional processes in heart and displays 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 immunohistochemistry in aged (>20 months) and young (3 months) mouse hearts. Aging induced a significant reduction in lymphatic capillary density in the sub-endocardial area of the aged left ventricle, in both male (0.28±0.08 fold; p<0.005) and female mice (0.68±0.03 fold; p<0.05), while the right ventricle exhibited no significant change. Notably, dilated (pre-) collector tubes were observed in aged mice, indicating, together with lymphatic capillary reduction, a lymphatic drainage dysfunction. Indeed, cardiac aging is accompanied by increased numbers of CD68+ macrophages, accumulation of the plasma protein fibrinogen and amyloid in the interstitial space. Moreover, we observed a significant increase in oedema formation 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 Prox1-CreERT2;Vegfr3 mice, a model with impaired lymphatic vessels. Also these mice, although being young, 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, also other age-related changes, such as mild cardiac hypertrophy was observed which is currently under further investigation. Likewise, blocking VEGFC signaling in young mice by overexpressing the soluble form of Flt4 impaired cardiac lymphatic capillary density, leading to increased macrophage (1.69±0.09-fold; p<0.005) and fibrinogen (4.6±0.6-fold; p<0.005) accumulation, 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. Overexpression of Vegfc via AAV9 in aged mice restored cardiac lymphatic density by 3-fold and reduced CD68+ macrophage density by 0.6-fold. However, fibrinogen accumulation stayed unaffected. 
In conclusion, our study demonstrates an age-related reduction in left ventricular lymphatic density, leading to the development of cardiac edema and inflammation. Vegfc overexpression prevented the age-dependent decline of lymphatic vasculature and reduced cardiac inflammation.
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