Calcifying Human Aortic Smooth Muscle Cells Express Different Bone Alkaline Phosphatase Isoforms, Including the Novel B1x Isoform

Abstract

Background: Vascular calcification, causing cardiovascular morbidity and mortality, is associated with hyperphosphatemia in chronic kidney disease (CKD). In vitro, phosphate induces transdifferentiation of vascular smooth muscle cells to osteoblast-like cells that express alkaline phosphatase (ALP). In vivo, raised serum ALP activities are associated with increased mortality. A new bone ALP isoform (B1x) has been identified in serum from CKD patients. The present study investigated the different ALP isoforms in calcifying human aortic smooth muscle cells (HAoSMCs). Methods: HAoSMCs were cultured for 30 days in medium containing 5 or 10 mmol/l β-glycerophosphate in the presence or absence of the ALP-specific inhibitor tetramisole. Results: All known bone-specific ALP (BALP) isoforms (B/I, B1x, B1 and B2) were identified in HAoSMCs. β-Glycerophosphate stimulated calcification of HAoSMCs, which was associated with increased BALP isoforms B/I, B1x and B2. Tetramisole inhibited the β-glycerophosphate-induced HAoSMC calcification, which was paralleled by the inhibition of the B1x and B/I, but not the other isoforms. Conclusions: HAoSMCs express the four known BALP isoforms. B/I, B1x and B2 could be essential for soft tissue calcification. B/I and B1x were more affected by tetramisole than the other isoforms, which suggests different biological functions during calcification of HAoSMCs.

Alkaline phosphatase: a novel treatment target for cardiovascular disease in CKD

“…All BALP isoforms, including B1x, are also expressed in vascular smooth muscle cells (VSMCs), the calcification of which is associated with a strong increase in BALP activity level 39 . In particular, increased B1x, B/I and B2 activity could be associated with phosphate-induced vascular calcification 40 . In bone, the B1:B2 ratio differs between cortical sites (~1.7) and trabecular sites (~0.7) 35 .…”

“…VSMCs exclusively express BALP in vitro 40 , most studies in human and animal models do not specify the measured TNALP isoform, which does not exclude that other isoforms could be involved in vascular calcification. The highest concentration of BALP is found in matrix vesicles shed by VSMCs 49 .…”

Increased circulating sclerostin levels in end-stage renal disease predict biopsy-verified vascular medial calcification and coronary artery calcification

“…7 Upregulation of ALP and pyrophosphate hydrolysis is increasingly recognized as a potential mechanism of uremic vascular calcification, 27 and serum ALP predicts mortality in ESRD. 28 Even though the increased ALP activity in calcifying vascular smooth muscle cells is exclusive to bALP, 29 it is still unknown to which extent vascular calcification contributes to circulating total ALP or bALP activities. An interaction between Wnt signaling and promoters of mineralization may occur in the process of vascular calcification.…”

Bone alkaline phosphatase isoforms in chronic kidney disease : mineral and bone disorder

“…Vascular calcification involves transdifferentiation of VSMCs to an osteoblast-like phenotype, which demonstrates increased expression of BALP (163). In study II we found that BALP isoforms B/I, B1x and B2 are involved in the calcification of VSMCs in vitro (258). Furthermore, in study I we found an association of B1x with hyperphosphatemia and increased calcium×phosphate product in CKD stages 3-5, both of which are associated with CV mortality in CKD (241).…”