Secretomes from human epicardial adipocytes modulate atrial lymphatics and atrial fibrosis

Abstract Background Cardiac lymphatics maintains myocardial fluid and immune cell homeostasis. Downregulation of cardiac lymphatics leads to cardiac dysfunction through impairment of inflammation and edema. On the other hand, it is well known that adipose tissue accumulation causes lymphatic dysfunction and impairs lymphedema. Purpose We evaluated the expression of atrial lymphatics in human left atrial appendage (LAA) histologically and biologically. Subsequently, we examined secretomes from epicardial adipose tissue (EAT)-derived adipocytes and validated if they change atrial lymphatics expression using ex vivo Organo-culture system. Methods Human LAA samples were collected from forty-two patients during cardiovascular surgery. They were categorized into the sinus rhythm (SR) group (n=16, 73.1±10.4 years), the paroxysmal AF (PAF) group (n=13, 69.8±12.6 years) and the persistent AF (PeAF) group (n=13, 71.2±6.5 years). Human EAT were also excised from patients with and without AF (SR; n=5, AF; n=11) during cardiovascular surgery. Preadipocytes were extracted from EAT by homogenization and collagenase treatment, and cultured to confluence. After differentiation and maturation to adipocytes, cell culture supernatant (CCS) was collected, and was dropped onto the epicardial side of left atrial tissues excised from 8 weeks old male rats for 7 days using an ex vivo organo-culture system. Results In human LAA samples, mRNA expression of lymphangiogenesis-related genes (LYVE1, PROX1, VEGFC, and VEGFR3) was significantly lower in the PeAF group compared to the SR group (p=0.0370, <0.0001, 0.0157, and 0.0009). The number of LYVE1-positive atrial lymphatic endothelial cells (LECs) was also significantly lower in the PeAF group compared to the SR group and the PAF group (p=0.0186 and 0.038). There was a negative correlation between the number of LYVE1-positive atrial LECs and the area percentage of atrial myocardial fibrosis (r=-0.5350, p<0.001). In the experiment using organo-culture system, lymphangiogenesis-related genes expression (Lyve1, Prox1, Vegfc, and Vegfr3) of rat atria treated with CCS of adipocytes from AF patients was significantly lower than those from SR patients (p=0.0391, 0.0100, 0.0034, and 0.0338). Furthermore, the CCS of adipocytes from AF patients contained higher protein levels of leptin, which is known to suppress lymphangiogenesis (p=0.0357). Leptin treatment (100ng/ml) on rat atria also decreased lymphangiogenesis-related genes expression such as Lyve1, Prox1, Vegfc, and Vegfr3 (p=0.0413, < 0.0001, <0.0001, and 0.0190). Conclusions Our study with human LAA demonstrated that atrial lymphatics interact with atrial myocardial fibrosis/progression of AF. Furthermore, our findings indicate that secretomes from EAT-derived adipocytes regulate atrial lymphatics expression, and leptin plays an important role in modulating atrial lymphangiogenesis.