The Purinergic Signaling System Modulates the Inflammatory Response of Human Valvular Endothelial Cells

Objectives: Recent studies describe a prominent role for valvular endothelial cells (VECs) in maintaining valve homeostasis by regulating permeability, inflammatory cell adhesion and paracrine signaling, thereby also partly regulating the hemostasis of valvular interstitial cells. According to current knowledge, inflammatory activation and signaling may represent one of the key events in the initiation of calcific aortic valve disease (CAVD). In this study, we aimed to investigate how the purinergic signaling affects the inflammatory response of human aortic VECs.

Methods: Human VECs (n = 6–8) were isolated from aortic valves of patients undergoing heart transplantation, purified by magnetic cell separation and characterized by immunofluorescent staining. Cells were treated with inhibitors of Ecto-NTPDases (POM-1), ATPases (orthovanadate) and 5′-nucleotidase (CD73, AMP-CP), as well as agonists of purine ionotropic (P2X) receptor (3′-O-(4-Benzoyl)-benzoyl adenosine 5′-triphosphate), adenosine receptors A2A (CGS 21680) and A2B (BAY 60–6583) and AMP-activated protein kinase (AMPK, metformin), respectively with and without additional stimulation with tumor necrosis factor (TNF-α). Expression of vascular cell adhesion protein 1 (VCAM) and intercellular adhesion molecule 1 (ICAM) was analyzed by Western blot. Interleukin (IL) -6 levels in supernatants were measured by ELISA.

Result: Inhibition of ATP-conversing NTPDases neither results in a pro-inflammatory, nor in an anti-inflammatory repercussion of hVECs, while inhibition of ATPases decreases TNF-α induced VCAM (0.71 fold, p < 0.05) and ICAM (0.75 fold, p < 0.05) expression. Activation of P2X receptor decreases TNF-α- induced expression of VCAM (0.42 fold, p < 0.01), but interestingly not ICAM. However, none of those treatments affects IL-6 secretion. Further, neither inhibition of AMP conversion, nor direct activation of AMPK results in pro- or anti-inflammatory effects. Activation of A2A and A2B receptor decreases TNF-α induced VCAM expression (0.65 and 0.83 fold, p < 0.05). Additionally, activation of A2B receptor inhibits secretion of IL-6 (0.73 fold, p < 0.05).

Conclusion: Our findings suggest that purinergic signaling can affect the TNF-α induced inflammatory signaling of hVECs. Activation of P2X-, A2A-, and A2B-receptors results in anti-inflammatory ramifications and could be a possible approach to suppress inflammatory activation of hVECs in order to inhibit CAVD progression.

No conflict of interest has been declared by the author(s).

Publication History

Article published online:
19 February 2021