Introduction: Aging is a major risk factor for cardiovascular diseases. One prominent hallmark of cardiac aging is inflammation. The microcirculatory system and age-related immune cell infiltration has been extensively studied in this context, however, the contribution of the lymphatic vasculature to age-related pathologies remains elusive.
Methods: To assess the cardiac lymphatic system, whole mount light sheet microscopy and confocal imaging was used. Functional alterations were investigated using AAV9 coding for Vegfc or soluble Ft4 were injected into 18-month-old and 3-month-old mice, respectively. Flt4;prox1-CreERT2 mice were used as additional model. Finally, molecular mechanisms were characterized using snRNA-seq on aged AAV9-Vegfc treated mcie.
Results: We examined lymphatic capillary density in old (>20 months) and young (3 months) mouse hearts using LYVE1 and PDPN staining. Aging induced a significant decline 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. Dilation of peri-arterial lymphatics was observed in old hearts, indicating lymphatic drainage dysfunction. Cardiac aging was accompanied by increased numbers of CD68+ macrophages, accumulation of fibrinogen and amyloid in the interstitial space, and significant tissue edema (p<0.05). The decline in lymphatic vessels was associated with reduced Vegfc expression (0.72±0.04 fold; p<0.005) in aged hearts.
To establish a causal link, we examined hearts from 3-month-old Flt4;Prox1-CreERT2 mice with defective lymphatic vessels. These mice 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. Blocking VEGFC signaling in young mice by overexpressing soluble 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) and diastolic dysfunction.
We then addressed whether restoring Vegfc expression in old mice rescues age-related cardiac impairment. Inducing Vegfc via AAV9 in aged mice restored cardiac lymphatic density by 3-fold and reduced CD68+ macrophage density by 1.6-fold, without affecting fibrinogen accumulation. However, overexpression of Vegfc lead to cardiac fibrosis and transiently impaired diastolic function.
Conclusion: Our study demonstrates an age-related reduction in left ventricular lymphatic density, cardiac edema, and inflammation. Vegfc overexpression prevented age-dependent decline of lymphatic vasculature and reduced cardiac inflammation.