Endothelial Contribution to Warfarin-Induced Arterial Media Calcification in Mice

Abstract

Arterial media calcification (AMC) is predominantly regulated by vascular smooth muscle cells (VSMCs), which transdifferentiate into pro-calcifying cells. In contrast, there is little evidence for endothelial cells playing a role in the disease. The current study investigates cellular functioning and molecular pathways underlying AMC, respectively by, an ex vivo isometric organ bath set-up to explore the interaction between VSMCs and ECs and quantitative proteomics followed by functional pathway interpretation. AMC development, which was induced in mice by dietary warfarin administration, was proved by positive Von Kossa staining and a significantly increased calcium content in the aorta compared to that of control mice. The ex vivo organ bath set-up showed calcified aortic segments to be significantly more sensitive to phenylephrine induced contraction, compared to control segments. This, together with the fact that calcified segments as compared to control segments, showed a significantly smaller contraction in the absence of extracellular calcium, argues for a reduced basal NO production in the calcified segments. Moreover, proteomic data revealed a reduced eNOS activation to be part of the vascular calcification process. In summary, this study identifies a poor endothelial function, next to classic pro-calcifying stimuli, as a possible initiator of arterial calcification.

Endothelial dysfunction aggravates arterial media calcification in warfarin administered rats

“…The fact that carotid segments of animals exclusively treated with warfarin did not show an altered ACh relaxation compared with carotid segments of control animals, indicate that the stimulated NO response was intact. Compromised basal NO release together with an intact receptor‐stimulated NO response was also seen earlier in organ chamber experiments using aortic segments of Fbn1C1039G/+ (Marfan model) mice, 39 apolipoprotein E‐deficient mice, 40 stroke‐prone spontaneously hypertensive rats 41 and warfarin fed DBA/2J mice 16 …”

“…Compromised basal NO release together with an intact receptor-stimulated NO response was also seen earlier in organ chamber experiments using aortic segments of Fbn1C1039G/+ (Marfan model) mice, 39 apolipoprotein E-deficient mice, 40 stroke-prone spontaneously hypertensive rats 41 and warfarin fed DBA/2J mice. 16 Western blot results showed that eNOS and p-eNOS expression in the infrarenal aorta of L-NAME, warfarin-and combination-treated animals was markedly increased. Increased eNOS expression might represent a compensatory mechanism for the altered NOS activity.…”

“…23 As seen in our results, a disturbance in this balance, characterized by diminished production/availability of NO and/or an imbalance in the relative contribution of endothelium-derived relaxing and contracting factors is known as endothelial dysfunction. 24 Recently, our group described the possible contribution of a dysfunctional endothelium in AMC development in a warfarin mouse model 16 : both at the level of arterial cell function and arterial protein expression, mice with warfarin-induced AMC showed signs of compromised basal NO levels.…”

“…15 Recently, by studying alterations in cellular functionality as well as protein expression during AMC development, our group found strong arguments for the involvement of a dysfunctional endothelium in the process of AMC. 16 In the present study, we attempt to further investigate/prove this involvement. Endothelial dysfunction was induced in rats by oral administration of L-NG-nitro arginine methyl ester (L-NAME).…”

Deletion of the P2Y 2 receptor aggravates internal elastic lamina calcification in chronic kidney disease mice through upregulation of alkaline phosphatase and lipocalin‐2

“…Regarding this, it is worth mentioning that the P2Y 2 receptor is expressed by endothelial cells and regulates its nitric oxide release 33 . Interestingly, our research group has recently observed that reduced basal nitric oxide production stimulates arterial media calcification in warfarin‐exposed mice 44 . Thus, arguing that the P2Y 2 receptor might mediate its effects on arterial media calcification by affecting the behavior of endothelial cells or other vascular cells rather than VSMCs.…”