Vitamin D analogs down-regulate plasminogen activator inhibitor-1 in human coronary artery smooth muscle cells.

Abstract

End-stage renal disease (ESRD) patients experience a high mortality rate from cardiovascular diseases [1, 2]. Vitamin D analogs that activate vitamin D receptor (VDR) such as paricalcitol and calcitriol are commonly used to manage secondary hyperparathyroidism associated with ESRD [3]. Recent clinical data show that vitamin D analogs provide survival benefit for ESRD patients in the order of paricalcitol > calcitriol > no vitamin D analog therapy, independent of the PTH and calcium levels [4, 5]. Moreover, the survival benefit seems to be associated with cardiovascular causes [6]. Atherosclerosis is a process that involves a complex interplay among different factors and cell types including smooth muscle cells [7, 8]. The high prevalence of atherosclerosis in ESRD has been well documented [9, 10]. Plasminogen activator inhibitor-1 (PAI-1), a mediator of thrombosis and one of the risk markers for coronary heart disease, is enhanced in atherosclerotic plaques and co-localizes with macrophages [11]. It is not known how vitamin D analogs modulate PAI-1 in smooth muscle cells. In this study, we examined the effects of calcitriol and paricalcitol on PAI-1 in human coronary artery smooth muscle cells. Primary cultured human coronary artery smooth muscle cells were purchased from Cambrex (Walkesville, MD, USA), grown to confluence according to the instruction from Cambrex, and used within five passages. Both VDR and PAI-1 were expressed in these cells; the VDR level was approximately 26% of PAI-1. Figure 1 shows that the PAI-1 level was reduced by paricalcitol or calcitriol in a dose-dependent manner. The EC50 of paricalcitol and calcitriol on inhibiting PAI-1 were 16 and 4 nm, respectively. At 1 μm, both paricalcitol and calcitriol reduced PAI-1 by 36%. Plasminogen activator inhibitor-1 (PAI-1) protein expression in smooth muscle cells. Human coronary artery smooth muscle cells were treated with paricalcitol or calcitriol at 0, 10, 100 and 1000 nm for 48 h. Cells (∼1 × 106 cells per sample) were solubilized in 50 μL of SDS-PAGE sample buffer (Sigma, St Louis, MO, USA); the protein content in each sample was determined by the Pierce (Rockford, IL, USA) BCA protein assay. Samples were resolved by SDS-PAGE using a 4–12% NuPAGE gel (Invitrogen, Carlsbad, CA, USA), and proteins were electrophoretically transferred to PVDF membrane for Western blotting. The membrane was blotted for 1 h at 25°C with 5% non-fat dry milk in PBS-T and then incubated with a mouse anti-PAI-1 monoclonal antibody (1000-fold dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS-T overnight at 4 °C. The membrane was washed with PBS-T and incubated with a horseradish peroxidase-labeled anti-mouse antibody for 1 h at 25 °C. The membrane was then incubated with detection reagent (SuperSignal WestPico, Pierce). Specific bands were visualized by exposing the paper to Kodak BioMax films (Eastman Kodak, Rochester, NY, USA). Band intensity was quantified by Quantity One (Bio-Rad, Hercules, CA, USA). The specific bands were visualized and the density of each band was measured and normalized to the protein content in each sample. Values from three independent experiments were averaged and expressed as mean ± SD. Statistical comparisons were performed by paired t-test. The role of VDR in the cardiovascular system is largely unknown. To our knowledge, the only other study on the potential involvement of VDR in regulating PAI-1 is from a recent paper reporting that, in human umbilical vein endothelial cells, calcitriol stimulated the expression of PAI-1, while paricalcitol has no significant effect [12]. Our result from the current study shows that both paricalcitol and calcitriol suppress PAI-1 in human coronary artery smooth muscle cells. One possible explanation for the seemingly inconsistent observation is that the effects of vitamin D analogs on PAI-1 differ in different cell/tissue types. More studies examining other cells and/or tissues will be needed to confirm this possibility. In summary, in this report we show for the first time that vitamin D analogs down-regulate PAI-1 in human coronary artery smooth muscle cells. Our data suggest that suppression of PAI-1 may be one of the factors contributing to the benefits of vitamin D analog therapy on reducing mortality and morbidity risk in ESRD patients.