An Investigation Into the Role of Osteocalcin in Human Arterial Smooth Muscle Cell Calcification

Abstract

Osteocalcin (OCN) is a bone-derived protein that is detected within human calcified vascular tissue. Calcification is particularly prevalent in chronic kidney disease (CKD) patients but the role of OCN in calcification, whether active or passive, has not been elucidated. Part 1: The relationship between OCN, CKD and vascular calcification was assessed in CKD patients (n = 28) and age-matched controls (n = 19). Part 2: in vitro, we analyzed whether addition of uncarboxylated osteocalcin (ucOCN) influenced the rate or extent of vascular smooth muscle cell (VSMC) calcification. Human aortic VSMCs were cultured in control media or mineralisation inducing media (MM) containing increased phosphate with or without ucOCN (10 or 30 ng/mL) for up to 21 days. Markers of osteogenic differentiation and calcification were determined [alkaline phosphatase (ALP) activity, total intracellular OCN, Runx2 expression, α-SMA expression, alizarin red calcium staining, and calcium quantification]. Part 1 results: In our human population, calcification was present (mean age 76 years), but no differences were detected between CKD patients and controls. Plasma total OCN was increased in CKD patients compared to controls (14 vs. 9 ng/mL; p < 0.05) and correlated to estimated glomerular filtration rate (p < 0.05), however no relationship was detected between total OCN and calcification. Part 2 results: in vitro, ALP activity, α-SMA expression and calcium concentrations were significantly increased in MM treated VSMCs at day 21, but no effect of ucOCN was observed. Cells treated with control media+ucOCN for 21 days did not show increases in ALP activity nor calcification. In summary, although plasma total OCN was increased in CKD patients, this study did not find a relationship between OCN and calcification in CKD and non-CKD patients, and found no in vitro evidence of an active role of ucOCN in vascular calcification as assessed over 21 days. ucOCN appears not to be a mediator of vascular calcification, but further investigation is warranted.

Serum biomarkers for arterial calcification in humans: A systematic review

“…In total, eighteen studies were performed investigating an association between osteocalcin and arterial calcification ( Barbarash et al, 2016 ; Golovkin et al, 2016 ; Liu et al, 2019 ; Pirro et al, 2013 ; Torii et al, 2016 ; Awan et al, 2010 ; Bernandes et al, 2019 ; Kim et al, 2012 ; Watson et al, 1997 ; Salari et al, 2017 ; Wilund et al, 2008 ; Miyabara et al, 2011 ; Zwakenberg et al, 2020 ; Choi et al, 2015 ; Kim et al, 2016 ; Millar et al, 2020 ; Cahalane et al, 2021 ; Zhelyazkova-Savova et al, 2021 ). Two studies reported a positive association ( Liu et al, 2019 ; Salari et al, 2017 ), five a negative association ( Golovkin et al, 2016 ; Awan et al, 2010 ; Kim et al, 2012 ; Wilund et al, 2008 ; Cahalane et al, 2021 ) and eleven no association ( Barbarash et al, 2016 ; Pirro et al, 2013 ; Torii et al, 2016 ; Bernandes et al, 2019 ; Watson et al, 1997 ; Miyabara et al, 2011 ; Zwakenberg et al, 2020 ; Choi et al, 2015 ; Kim et al, 2016 ; Millar et al, 2020 ; Zhelyazkova-Savova et al, 2021 ). One longitudinal study reported higher osteocalcin in people with a calcified coronary or carotid plaque after five years of follow-up in 1571 participants, ≥60 years of age, without plaque at baseline.…”

The Influence of Calcification Factors and Endothelial-Dysfunction Factors on the Development of Unstable Atherosclerotic Plaques

“…International literature data about the effects of osteocalcin on vascular calcification and stabilization/destabilization of atherosclerotic plaques are contradictory. For instance, the results of Millar et al imply that osteocalcin is not a mediator of vascular calcification [ 17 ]. Rashdan et al showed the colocalization of osteocalcin with the calcification of smooth muscle cells in calcified plaques and pointed out the crucial role of osteocalcin in arterial calcification [ 18 ].…”

Osteocalcin and vascular function: is there a cross-talk?

“…The majority of previous studies have reported the association of OC with the later stages of atherosclerotic plaque development, calcification and CVD events, often with conflicting findings [ 14 , [17] , [18] , [19] ]. However, given the importance of blood vessel dysfunction in the development of atherosclerosis, an increasing number of studies have begun to investigate the association of OC with endothelial and smooth muscle cell function ( Table 1 ) [ [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] ]. These studies will be discussed at length below.…”

“…In contrast, higher tOC was associated with higher PWV in men and women with end-stage renal disease on peritoneal dialysis [ 27 ]. Whereas, in both men and women with stage 3 and 4 chronic kidney disease (CKD), no association was reported between tOC and PWV [ 20 ]. Several studies have also examined the association of tOC with endothelial function ( Table 1 ).…”