ß,γ-Methylene-ATP and its metabolite medronic acid affect both arterial media calcification and bone mineralization in non-CKD and CKD rats.

Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PPi/Pi ratio). Also, it has been shown that ATP analogs (ie, ß,γ-methylene-ATP [ß,γ-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of ß,γ-meATP (2 mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that ß,γ-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4 mg/kg ß,γ-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of ß,γ-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of Enpp1 and Enpp3 was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications.

Deletion of the P2Y2 receptor aggravates internal elastic lamina calcification in chronic kidney disease mice through upregulation of alkaline phosphatase and lipocalin-2.

Calcification of the medial layer, inducing arterial stiffness, contributes significantly to cardiovascular mortality in patients with chronic kidney disease (CKD). Extracellular nucleotides block the mineralization of arteries by binding to purinergic receptors including the P2Y2 receptor. This study investigates whether deletion of the P2Y2 receptor influences the development of arterial media calcification in CKD mice. Animals were divided into: (i) wild type mice with normal renal function (control diet) (n = 8), (ii) P2Y2 R-/- mice with normal renal function (n = 8), (iii) wild type mice with CKD (n = 27), and (iv) P2Y2 R-/- mice with CKD (n = 22). To induce CKD, animals received an alternating (0.2-0.3%) adenine diet for 7 weeks. All CKD groups developed a similar degree of chronic renal failure as reflected by high serum creatinine and phosphorus levels. Also, the presence of CKD induced calcification in the heart and medial layer of the aortic wall. However, deletion of the P2Y2 receptor makes CKD mice more susceptible to the development of calcification in the heart and aorta (aortic calcium scores (median ± IQR), CKD-wild type: 0.34 ± 4.3 mg calcium/g wet tissue and CKD-P2Y2 R-/- : 4.0 ± 13.2 mg calcium/g wet tissue). As indicated by serum and aortic mRNA markers, this P2Y2 R-/- mediated increase in CKD-related arterial media calcification was associated with an elevation of calcification stimulators, including alkaline phosphatase and inflammatory molecules interleukin-6 and lipocalin 2. The P2Y2 receptor should be considered as an interesting therapeutic target for tackling CKD-related arterial media calcification.

New Therapeutics Targeting Arterial Media Calcification: Friend or Foe for Bone Mineralization?

The presence of arterial media calcification, a highly complex and multifactorial disease, puts patients at high risk for developing serious cardiovascular consequences and mortality. Despite the numerous insights into the mechanisms underlying this pathological mineralization process, there is still a lack of effective treatment therapies interfering with the calcification process in the vessel wall. Current anti-calcifying therapeutics may induce detrimental side effects at the level of the bone, as arterial media calcification is regulated in a molecular and cellular similar way as physiological bone mineralization. This especially is a complication in patients with chronic kidney disease and diabetes, who are the prime targets of this pathology, as they already suffer from a disturbed mineral and bone metabolism. This review outlines recent treatment strategies tackling arterial calcification, underlining their potential to influence the bone mineralization process, including targeting vascular cell transdifferentiation, calcification inhibitors and stimulators, vascular smooth muscle cell (VSMC) death and oxidative stress: are they a friend or foe? Furthermore, this review highlights nutritional additives and a targeted, local approach as alternative strategies to combat arterial media calcification. Paving a way for the development of effective and more precise therapeutic approaches without inducing osseous side effects is crucial for this highly prevalent and mortal disease.

Endothelial dysfunction aggravates arterial media calcification in warfarin administered rats.

Arterial media calcification is an active cell process. This encompasses osteochondrogenic transdifferentiation of vascular smooth muscle cells followed by the deposition of calcium-phosphate crystals. Increasing evidence suggests a significant role for endothelial cells (ECs) in the development of arterial media calcification. This manuscript explores a role for endothelial dysfunction in the disease progression of arterial media calcification. Male rats were randomly assigned to four different groups. The first group received standard chow. The second group was given L-NAME (≈50 mg kg-1 · d-1 ), to induce endothelial dysfunction, in addition to standard chow. The third group and fourth group received a warfarin-supplemented diet to induce mild calcification and the latter group was co-administered L-NAME. Prior to sacrifice, non-invasive measurement of aortic distensibility was performed. Animals were sacrificed after 6 weeks. Arterial media calcification was quantified by measuring aortic calcium and visualized on paraffin-embedded slices by the Von Kossa method. Arterial stiffness and aortic reactivity was assessed on isolated carotid segments using specialized organ chamber setups. Warfarin administration induced mineralization. Simultaneous administration of warfarin and L-NAME aggravated the arterial media calcification process. Through organ chamber experiments an increased vessel tonus was found, which could be linked to reduced basal NO availability, in arteries of warfarin-treated animals. Furthermore, increased calcification because of L-NAME administration was related to a further compromised endothelial function (next to deteriorated basal NO release also deteriorated stimulated NO release). Our findings suggest early EC changes to impact the disease progression of arterial media calcification.

Endothelial Contribution to Warfarin-Induced Arterial Media Calcification in Mice.

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.