Abstract
Vascular calcification, which is common in the elderly and in patients with atherosclerosis, diabetes and chronic renal disease, increases the risk of cardiovascular morbidity and mortality. It is a complex, active and highly regulated cellular process that resembles physiological bone formation. It has previously been established that pharmacological doses of glucocorticoids facilitate arterial calcification. However, the consequences for vascular calcification of endogenous glucocorticoid elevation have yet to be established. Glucocorticoids (cortisol, corticosterone) are released from the adrenal gland, but can also be generated within cells from 11-keto metabolites of glucocorticoids (cortisone, 11-dehydrocorticosterone [11-DHC]) by the enzyme, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In the current study we hypothesized that endogenous glucocorticoids facilitate vascular smooth muscle cell (VSMC) calcification and investigated the receptor-mediated mechanism underpinning this process.In vitro studies revealed increased phosphate-induced calcification in mouse VSMCs following treatment for 7days with corticosterone (100nM; 7.98 fold; P<0.01), 11-DHC (100nM; 7.14 fold; P<0.05) and dexamethasone (10nM; 7.16 fold; P<0.05), a synthetic glucocorticoid used as a positive control. Inhibition of 11β-HSD isoenzymes by 10μM carbenoxolone reduced the calcification induced by 11-DHC (0.37 fold compared to treatment with 11-DHC alone; P<0.05). The glucocorticoid receptor (GR) antagonist mifepristone (10μM) had no effect on VSMC calcification in response to corticosterone or 11-DHC. In contrast, the mineralocorticoid receptor (MR) antagonist eplerenone (10μM) significantly decreased corticosterone- (0.81 fold compared to treatment with corticosterone alone; P<0.01) and 11-DHC-driven (0.64 fold compared to treatment with 11-DHC alone; P<0.01) VSMC calcification, suggesting this glucocorticoid effect is MR-driven and not GR-driven. Neither corticosterone nor 11-DHC altered the mRNA levels of the osteogenic markers PiT-1, Osx and Bmp2. However, DAPI staining of pyknotic nuclei and flow cytometry analysis of surface Annexin V expression showed that corticosterone induced apoptosis in VSMCs.This study suggests that in mouse VSMCs, corticosterone acts through the MR to induce pro-calcification effects, and identifies 11β-HSD-inhibition as a novel potential treatment for vascular calcification.
Highlights
Vascular calcification is a marker of increased cardiovascular disease risk in ageing, including in diabetes, atherosclerosis and chronic kidney disease (CKD) (Mackenzie and MacRae, 2011; Zhu et al, 2012)
Several studies have reported that vascular smooth muscle cells (VSMCs), the predominant cell type involved in vascular calcification, can undergo phenotypic transition to osteoblastic, chondrocytic and osteocytic cells in a calcified environment (Johnson et al, 2005; Zhu et al, 2011)
Glucocorticoids facilitates VSMC calcification We initially examined the effects of physiological glucocorticoids on the calcification of VSMCs, with the synthetic glucocorticoid dexamethasone used as a positive control
Summary
Vascular calcification is a marker of increased cardiovascular disease risk in ageing, including in diabetes, atherosclerosis and chronic kidney disease (CKD) (Mackenzie and MacRae, 2011; Zhu et al, 2012). Several studies have reported that vascular smooth muscle cells (VSMCs), the predominant cell type involved in vascular calcification, can undergo phenotypic transition to osteoblastic, chondrocytic and osteocytic cells in a calcified environment (Johnson et al, 2005; Zhu et al, 2011). Phosphate accelerates this trans-differentiation process, with the loss of characteristic smooth muscle markers and the increased expression of osteoblastic markers Vascular calcification can proceed through mechanisms involving the reciprocal loss of recognised calcification inhibitors including inorganic pyrophosphate (PPi), fetuin A and Matrix Gla Protein (Johnson et al, 2005; Mackenzie et al, 2012; Murshed et al, 2005; Reynolds et al, 2005)
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