Growth differentiation factor 15 (GDF15) is elevated in human plaques, shuffles into the nucleus of ECs during EndMT, and promotes Endothelial-to-Mesenchymal Transition (EndMT)

Growth differentiation factor 15 (GDF15), part of the TGF-β superfamily, is known to be upregulated during injury and stress conditions such as hypoxia. A GDF15 secretion from adipocytes and its impact on the neuronal system have been previously described. However, high serum GDF15 levels also correlate with inflammation, cardiac fibrosis, and an adverse prognosis in cardiovascular diseases. Yet, the functional role of GDF15 in endothelial cells is largely unknown. Here, we investigate a possible role of GDF15 in the transdifferentiation of endothelial cells (EC) into mesenchymal cells (EndMT).

 

GDF15 expression was notably detected in primary human ECs at both mRNA and protein levels through various analyses – qPCR, single-cell sequencing (scSeq), and enzyme-linked immunosorbent assay (mean ± SEM, 743.8±39.3 pg/ml, n=3). Furthermore, spatial transcriptomics analysis of vascular sections from Ldlr^-/- mice that were fed a high-fat diet revealed a significant increase in the expression of Gdf15 in plaque sections. ScRNA-seq analysis of atherosclerotic plaque sections and patient-matched proximal adjacent sections of carotid tissue from patients undergoing carotid endarterectomy showed a 22-fold (p<0.05) increase in GDF15 in ECs located in plaques compared to ECs in control vessel regions. In-vitro experiments using cultured human ECs revealed an upregulation of GDF15 mRNA following EndMT induction as demonstrated by qPCR (+1.7-fold control, p<0.05) and scSeq analysis.

Treatment with increasing levels of recombinant GDF15 (10, 50 and 150 ng/ml) induced EndMT as measured by expression of the EndMT marker calponin (+3.01, 7.73 and 7.93-fold, all p<0.05).  Moreover, measurement of trans-endothelial electrical resistance revealed that GDF15 treatment leads to deterioration of endothelial function.  The use of anti-GDF15 antibodies (1 µg/ml) reduced soluble GDF15 levels by 97.7% as measured by ELISA (p<0.001). Treatment with the same neutralising antibodies against GDF15 reduced EndMT induction by 54% as measured by the EndMT marker calponin (p<0.05). Interestingly, during the process of EndMT a reduction in the levels of extracellular GDF15 by 68.2% (p<0.05) was determined, whereas the intracellular levels were augmented by 3.1-fold in comparison to the control (p<0.05). After performing cell fractionation, the protein amount of GDF15 was measured in cytosol, membrane, and nuclear fractions, revealing that GDF15 is present in the nucleus.

 

GDF15 is elevated in mouse and human plaques, and during EndMT. Treatment with GDF15 induces EndMT, whereas inhibition by neutralizing antibodies reduces EndMT induction. Furthermore, GDF15 shuffles into the nucleus of ECs during EndMT, suggesting a possible transcriptional effect of GDF15. These data are consistent with a functional role of GDF15 in endothelial dysfunction and atherogenesis.