Impaired arterial vitamin D signaling occurs in the development of vascular calcification

Abstract

Conflicting data exists as to whether vitamin D receptor agonists (VDRa) are protective of arterial calcification. Confounding this, is the inherent physiological differences between human and animal experimental models and our current fragmented understanding of arterial vitamin D metabolism, their alterations in disease states and responses to VDRa's. Herein, the study aims to address these problems by leveraging frontiers in human arterial organ culture models. Human arteries were collected from a total of 24 patients (healthy controls, n = 12; end-stage CKD, n = 12). Cross-sectional and interventional studies were performed using arterial organ cultures treated with normal and calcifying (containing 5mmol/L CaCl2 and 5mmol/L β-glycerophosphate) medium, ex vivo. To assess the role of VDRa therapy, arteries were treated with either calcitriol or paricalcitol. We found that human arteries express a functionally active vitamin D system, including the VDR, 1α-hydroxylase and 24-hydroxylase (24-OHase) components and these were dysregulated in CKD arteries. VDRa therapy increased VDR expression in healthy arteries (p<0.01) but not in CKD arteries. Arterial 1α-OHase (p<0.05) and 24-OHase mRNA and protein expression were modulated differentially in healthy and CKD arteries by VDRa therapy. VDRa exposure suppressed Runx2 and MMP-9 expression in CKD arteries, however only paricalcitol suppressed MMP-2. VDRa exposure did not modulate arterial calcification in all organ culture models. However, VDRa reduced expression of senescence associated β-galactosidase (SAβG) staining in human aortic-smooth muscle cells under calcifying conditions, in vitro. In conclusion, maladaptation of arterial vitamin D signaling components occurs in CKD. VDRa exposure can exert vasculo-protective effects and seems critical for the regulation of arterial health in CKD.

Fig 1. The vitamin D signalling system in human arteries from healthy and CKD patients.

A) The VDR and 1α-OHase is expressed in arteries from healthy donors, with strong expression across the medial smooth muscle layer and endothelium. CKD arteries exhibited suppressed VDR and 1α-OHase expression. 24-OHase expression is high in arteries from CKD patients across the medial smooth muscle layer and endothelium but suppressed in healthy arteries. CKD arteries exhibited significant medial calcification; B) RNA and C) protein expression of VDR, 1α-OHase and 24-OHase expression in arterial lysates; D) 1,25-OH-D production is in arteries following 25-OH-D treatment, but production is blunted in CKD arteries. *p<0.05, **p<0.01.

Fig 2. Modulation of the vitamin D signalling system in human arteries following calcifying stress and VDRa therapy.

Arterial rings were treated for 14 days with calcitriol (100nM) or paricalcitol (300nM) in normal or calcifying medium. A) mRNA and B) protein for vitamin D receptor (VDR); C) mRNA and D) protein for 1α-OHase (CYP27B1); E) mRNA and F) protein for 24-OHase (CYP24A1). * refers to comparison within treatment group between control and CKD artery; *p<0.05, **p<0.01, ***p<0.001.

Fig 3. Modulation of arterial phenotype following calcifying stress and VDR therapy.

Arterial rings were treated for 14 days with calcitriol (100nM) or paricalcitol (300nM) in normal or calcifying medium. A) mRNA and B) protein for Runx2; C) mRNA quantification for MMP-2; D) mRNA quantification for MMP-9; E) mRNA of osteocalcin. * refers to comparison within treatment group between control and CKD artery; *p<0.05, **p<0.01, ***p<0.001.

Fig 4. Treatment of arteries with calcifying stress and VDR activators alters vascular inflammatory molecule synthesis.

Arterial rings were treated for 14 days with calcitriol (100nM) or paricalcitol (300nM) in normal or calcifying medium. A) IL-6 mRNA quantification; B) IL-10 mRNA quantification. * refers to comparison within treatment group between control and CKD artery; *p<0.05, **p<0.01.

Fig 5. Treatment of arteries with VDR activators does not alter arterial calcium content.

Arterial rings were treated for 14 days with calcitriol (100nM) or paricalcitol (300nM) in normal or calcifying medium. Arteries were assessed for calcification using the orthocresolphthalein complexone method. Arteries cultured in calcifying medium exhibited increased calcium content. Treatment of arteries with either calcitriol or paricalcitol did not altered calcium content.