Background: Sodium glucose co-transporter 2 inhibitors (SGLT2i) are approved anti-diabetic drugs with a wide clinical use. Beneficial effects of SGLT2i Dapagliflozin (Dapa) and Empagliflozin (Empa) on heart failure worsening and rate of death due to cardiovascular (CV) events have been reported in multiple clinical trials. However, considering the absence of such effects in patients with type 2 diabetes and atherosclerotic CV disease upon Ertugliflozin treatment, an SGLT2 independent modus operandi in the CV environment is likely. As such, a direct interaction of SGLT2i with the sodium proton exchanger 1 has been identified to alter ion homeostasis in cardiomyocytes leading to various protective downstream processes. Moreover, there is apparent interference of SGLT2i with inflammatory pathways in the vascular context. However, details of the molecular mechanisms still remain largely undisclosed and need to be defined.

Methods and Results: Initially, we examined putative anti-inflammatory potential of selected SGLT2i using a nuclear factor kB (NFkB)-dependent in vitro reporter assay. We detected SGLT2-independent NFkB-repressive activity of Dapa but not Empa. Since endothelial cells (EC) are key mediators of inflammatory processes in the myocardium, we thus monitored NFkB activity in human umbilical vein ECs (HUVECs) measuring expression of interleukins and adhesion molecules. Using multi-dimensional bioinformatic network analysis, we screened for deregulated nodes in mass-spectrometry-based proteomics datasets from HUVECs treated with Dapa or Empa explaining possible underlying molecular mechanisms. Next to altered levels of IL-6 and ICAM1, we discovered a regulation of clusters involved in reactive oxygen homeostasis, mitochondrial respiratory chain components, and vascular endothelial growth factor receptor signaling. Subsequently, we validated the suggested modes of action using various in vitro and ex vivo functional assays as well as transcriptome evaluations. Reactive oxygen species levels were reduced in HUVECs after treatment with Dapa and Empa. Decreased basal and maximal respiratory capacity of mitochondria confirmed the interference of Dapa with the respiratory chain complex. Moreover, Dapa inhibited migration of HUVECs in wound healing assays but interestingly had no effect on their tube formation capacity reflecting angiogenic signaling. Additionally, we employed a high-throughput ex vivo model to monitor inflammation in living myocardial tissue, in which ECs deteriorate rapidly after preparation. IL-6 expression was not altered in rat living myocardial tissue upon SGLT2i treatment supporting an endothelial-specific background of the inflammatory regulation of Dapa.

Conclusion: We screened for the impact of SGLT2i on HUVECs applying bioinformatic processing of proteomics data coupled with various in vitro and ex vivo assays. Our findings indicate a modulation of anti-oxidative potential, mitochondrial respiration and angiogenic capacity triggered by SGLT2i in ECs, the precise underlying mechanisms of which still need to be elucidated. Additionally, direct targeting of the NFkB signaling axis via SGLT2i was not only proven in vitro but also supported by experiments performed ex vivo. The herein described non-renal mediated effects of selected SGLT2i on the vasculature in the healthy and diseased states grant further insights into potential molecular modes of action of these drugs to support clinical decision making.