Empagliflozin reduces endoplasmic reticulum stress associated TXNIP/NLRP3 activation in tunicamycin-stimulated aortic endothelial cells.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to be of therapeutic significance for cardiovascular diseases beyond the treatment of type 2 diabetes. Recent studies have demonstrated the beneficial effects of SGLT2i on endothelial cell (EC) dysfunction, but the underlying cellular mechanisms remain to be clarified. In this study, we sought to understand the effect of empagliflozin (EMPA; Jardiance®) on cell homeostasis and endoplasmic reticulum (ER) stress signaling. ER stress was induced by tunicamycin (Tm) in human abdominal aortic ECs treated with EMPA over 24 h. Tm-induced ER stress caused increases in the protein expression of thioredoxin interacting protein (TXNIP), NLR-family pyrin domain-containing protein 3 (NLRP3), C/EBP homologous protein (CHOP), and in the ratio of phospho-eIF2α/eIF2α. EMPA (50-100 µM) resulted in a dampened downstream activation of ER stress as seen by the reduced expression of CHOP and TXNIP/NLRP3 in a dose-dependent manner. Nuclear factor erythroid 2-related factor 2 (nrf2) translocation was also attenuated in EMPA-treated ECs. These results suggest that EMPA improves redox signaling under ER stress which in turn attenuates the activation of TXNIP/NLRP3.

Empagliflozin mitigates endothelial inflammation and attenuates endoplasmic reticulum stress signaling caused by sustained glycocalyx disruption.

The disruption of the endothelial cell (EC) glycocalyx (GCX) leads to cellular dysfunction promoting inflammation and cardiovascular disease progression. Recent studies have shown that empagliflozin (EMPA; Jardiance), a sodium-glucose cotransporter 2 inhibitor used in the treatment of type 2 diabetes, can improve EC functions impacted by GCX disruption although the exact cellular mechanisms remain to be elucidated. In this study, the effect of EMPA on EC inflammatory response induced by sustained GCX disruption was investigated. Human aortic ECs were cultured under shear (10 dyne/cm2) for 24 h with or without sustained degradation of heparan sulfate (HS). HS degradation increased inflammatory cell adhesion to ECs. EMPA (50 µM) normalized adhesion levels under sustained HS degradation. Protein expressions of eNOS, phospho-eNOS Ser1177 and ICAM-1 remained unchanged between conditions. Transcriptome analysis revealed the induction of the unfolded protein response (UPR) through the increased expression of ATF3, ATF4, DDIT3 (CHOP), EIF2AK3 (PERK), HSPA5 (Grp78), PPP1R15A (GADD34) and TRIB3 which was in part downregulated by EMPA. mRNA and protein expression of thioredoxin interacting protein (TXNIP) was also downregulated by EMPA. Mitigation of oxidative stress with N-Acetyl-L-cysteine resulted in similar reduction in inflammatory cell adhesion compared to EMPA which could indicate a potential mechanism by which EMPA normalized the inflammatory response. In conclusion, this study demonstrated the potential of EMPA to resolve the inflammatory response of ECs caused by sustained GCX disruption while altering UPR signaling under endoplasmic reticulum stress.

Empagliflozin restores the integrity of the endothelial glycocalyx in vitro.

The antihyperglycemic agent empagliflozin not only improves glycemic control but has also been associated with clinically meaningful reductions in cardiovascular events. Studies have shown that empagliflozin significantly reduces cardiovascular death and heart failure-associated hospitalizations. Given that endothelial dysfunction is closely linked with the pathogenesis of atherosclerotic cardiovascular disease, we hypothesized that the cardiovascular benefits observed with empagliflozin may be a result of its positive impact on the health of the endothelial glycocalyx (GCX), a critical component for the endothelium homeostasis. Human abdominal aortic endothelial cells (HAAECs) were either statically cultured or subjected to a steady wall shear stress of 10 dyne/cm2. Empagliflozin (50 µM, 24 h) restored heparinase III-mediated GCX disruption and the normal mechanotransduction responses in GCX-compromised HAAECs while reducing the attachment of all-trans retinoic acid-transformed NB4 cells to HAAECs. The current body of work suggests that the cardioprotective properties previously reported for empagliflozin may in part be due to the ability of empagliflozin to preserve and restore the structural integrity of the GCX, which in turn helps to maintain vascular health by promoting an anti-inflammatory endothelium, in the presence of a pro-inflammatory environment. Further studies are needed to fully understand the mechanisms underlying the cardiovascular benefits of empagliflozin.

Increased MMP activity in curved geometries disrupts the endothelial cell glycocalyx creating a proinflammatory environment.

Wall shear stress gradients (WSSGs) induce an inflammatory phenotype in endothelial cells (ECs) which is hypothesized to be mediated by mechanotransduction through the EC glycocalyx (GCX). We used a three-dimensional in vitro cell culture model with a 180o curved geometry to investigate if WSSGs created by curvature can cause EC inflammation and disruption of the GCX. The hydrodynamics of the model elicited a morphological response in ECs as well as a pattern of leukocyte adhesion towards the inner wall of curvature that was attenuated with enzymatic removal of GCX components. GCX degradation was also observed in regions of curvature which corresponded to increased activity of MMPs. Together, these results support the hypothesis that the EC GCX is involved in mechanotransduction of WSSGs and that components of the GCX are regulated by MMP activity in regions of curvature.

Effect of manufacturing and experimental conditions on the mechanical and surface properties of silicone elastomer scaffolds used in endothelial mechanobiological studies.

BACKGROUND: Mechanobiological studies allow the characterization of cell response to mechanical stresses. Cells need to be supported by a material with properties similar to the physiological environment. Silicone elastomers have been used to produce various in vitro scaffolds of different geometries for endothelial cell studies given its relevant mechanical, optical and surface properties. However, obtaining defined and repeatable properties is a challenge as depending on the different manufacturing and processing steps, mechanical and surface properties may vary significantly between research groups. METHODS: The impact of different manufacturing and processing methods on the mechanical and surface properties was assessed by measuring the Young's modulus and the contact angle. Silicone samples were produced using different curing temperatures and processed with different sterilization techniques and hydrophilization conditions. RESULTS: Different curing temperatures were used to obtain materials of different stiffness with a chosen silicone elastomer, i.e. Sylgard 184®. Sterilization by boiling had a tendency to stiffen samples cured at lower temperatures whereas UV and ethanol did not alter the material properties. Hydrophilization using sulphuric acid allowed to decrease surface hydrophobicity, however this effect was lost over time as hydrophobic recovery occurred. Extended contact with water maintained decreased hydrophobicity up to 7 days. Mechanobiological studies require complete cell coverage of the scaffolds used prior to mechanical stresses exposure. Different concentrations of fibronectin and collagen were used to coat the scaffolds and cell seeding density was varied to optimize cell coverage. CONCLUSION: This study highlights the potential bias introduced by manufacturing and processing conditions needed in the preparation of scaffolds used in mechanobiological studies involving endothelial cells. As manufacturing, processing and cell culture conditions are known to influence cell adhesion and function, they should be more thoroughly assessed by research groups that perform such mechanobiological studies using silicone.