Age-Associated Loss of Lymphatic Vessels Promotes Cardiac Inflammation

J. U. G. Wagner (Frankfurt am Main)¹, H. Gulshan (Frankfurt am Main)¹, I. Sultan (Helsinki)², S. Antila (Helsinki)³, L. Esteve (Bad Nauheim)⁴, D. Rodriguez Morales (Frankfurt am Main)⁵, E. G. Solomonidis (Frankfurt am Main)¹, S.-F. Glaser (Frankfurt am Main)⁵, W. Abplanalp (Frankfurt am Main)⁶, T. Procida-Kowalski (Giessen)⁷, M. Bartkuhn (Giessen)⁷, E. Ullrich (Frankfurt am Main)⁸, S. A. Khan (Frankfurt)⁹, F. Buettner (Frankfurt)⁹, J. Pöling (Bad Rothenfelde)¹⁰, T. Braun (Bad Nauheim)¹¹, K. Alitalo (Helsinki)³, S. Dimmeler (Frankfurt am Main)^5
1Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration Frankfurt am Main, Deutschland; ²Wihuri Research Institute Biomedicum Helsinki Helsinki, Deutschland; ³University of Helsinki Translational Cancer Medicine Program Helsinki, Finnland; ⁴Bad Nauheim, Deutschland; ⁵Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; ⁶Universitätsklinikum Frankfurt Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland; ⁷Giessen, Deutschland; ⁸Goethe Universität Frankfurt am Main Frankfurt Cancer Institute Frankfurt am Main, Deutschland; ⁹Frankfurt, Deutschland; ¹⁰Schüchtermann-Klinik Bad Rothenfelde Kardiologie/Rhythmologie Bad Rothenfelde, Deutschland; ¹¹Max-Planck-Institut für Herz- und Lungenforschung Bad Nauheim, Deutschland

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 effect on 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 podoplanin 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 morphological alterations increased zipper-junctions were observed in in old mouse hearts, indicating a lymphatic drainage dysfunction. Consistent with a declined and impaired lymphatic vasculature, cardiac aging was accompanied by tissue oedema, increased numbers of CD68+ macrophages, T-cells and neutrophils, and accumulation of plasma fibrinogen and amyloid into the interstitial space. Furthermore, draining cardiac lymph nodes contained less macrophages. 
To establish a causal link between lymphatic dysfunction and age-related cardiac changes, we examined hearts from young mice after AAV9-mediated overexpression of a soluble VEGFR3 receptor that binds VEGFC and VEGFD (VEGFC/D trap) and results in lymphatic vessel attrition and malfunction. Despite their young age, the mice expressing the VEGFC/D trap showed increased fibrinogen (4.5±0.3 fold; p<0.05) and macrophages (1.7±0.1 fold; p<0.05) 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 LECs showed a similar phenotype, suggesting that the decline of lymphatics induces typical age-associated pathologies.
ScRNA-seq of young and aged mouse hearts revealed up-regulation of Interleukin-33 (IL-33) in the lymphatic endothelial cell (LEC) cluster.  Interestingly, only the nuclear form of IL33, which acts as transcription factor, was up-regulated in aged cardiac lymphatics. Overexpressing nuclear IL33 in cultured human LECs (hLECs) impaired proliferation and promoted apoptosis.
To rescue IL33-driven lymphatic impairment, we treated hLECs with putative pro-lymphangiogenic factors including VEGFC, Reelin, Apelin and sphingosine-1-phosphate. Of these, only VEGFC and Reelin induced hLEC profileration, while the IL33-induced apoptosis of hLECs was most efficiently rescued by VEGFC.
As Vegfc gene expression was reduced in old mouse hearts (0.72±0.04 fold; p<0.005), we addressed whether restoring Vegfc expression may rescue age-related cardiac impairment. Indeed, 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). SnRNA-seq further confirmed that the the LEC cluster was expanded after AAV9-Vegfc injection and that lymphatic endothelial Il33 expression was reduced.
In conclusion, our study demonstrates an age-related reduction in LV lymphatic density, with cardiac edema and inflammation which can be restored therapeutically using Vegfc overexpression.