Abstract
Vascular calcification is an important pathogenic process in atherosclerosis (AS); however, its immediate cause is unknown. Our previous study demonstrated that carbonic anhydrase 1 (CA1) stimulates ossification and calcification in ankylosing spondylitis and breast cancer. The current study investigated whether CA1 plays an important role in AS calcification and whether the CA inhibitor methazolamide (MTZ) has a therapeutic effect on AS. We successfully established an AS model by administration of a high-fat diet to apolipoprotein E (ApoE−/−) mice. The treated animals had significantly increased serum levels of high-density lipoprotein cholesterol (HDL-c) and nitric oxide (NO) and decreased serum concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-c), interleukin (IL-6), interferon (IFN)-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), chemokine (C-X-C motif) ligand 1/keratinocyte-derived chemokine (CXCL1/KC), and C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP-1). The treated mice also had reduced AS plaque areas and fat accumulation, with no clear calcium deposition in the intima of the blood vessels. CA1 expression was significantly increased in the aortic lesions, particularly in calcified regions, but the expression was dramatically lower in the mice that received MTZ treatment or MTZ preventive treatment. CA1 was also highly expressed in human AS tissues and in rat vascular smooth muscle cells (VSMCs) with β-glycerophosphate (㒐β-GP)-induced calcification. Acetazolamide (AZ), a CA inhibitor with a chemical structure similar to MTZ, markedly suppressed calcification and reduced CA1, IL-6, IFN-γ, GM-CSF, and TNF-α expression in cultured VSMCs. Anti-CA1 small interfering ribonucleic acid (siRNA) significantly suppressed calcification, cell proliferation, and migration, promoted apoptosis, and reduced IL-6, IFN-γ, GM-CSF, and TNF-α secretion in cultured VSMCs. These results demonstrated that CA1 expression and CA1-mediated calcification are significantly associated with AS progression. MTZ significantly alleviated AS and suppressed CA1 expression and proinflammatory cytokine secretion, indicating the potential use of this drug for AS treatment.
Highlights
Vascular calcification is involved in the plaque formation of atherosclerosis (AS) (Ross, 1999; Gamble, 2006; Cianciolo et al, 2010)
Many cells in the vascular wall were immunostained with anti-carbonic anhydrase 1 (CA1) antibody in the diseased tissues
No identifiable calcium deposition was found in the healthy samples (Figure 1E). messenger ribonucleic acid (mRNA) levels of CA1, CA2, CA3, CA4, CA5a, CA6, CA7, CA8, CA9, and CA10 were examined in the aortic aneurysm tissues, aortic dissection tissues, and healthy aortic tissues using real-time PCR
Summary
Vascular calcification is involved in the plaque formation of atherosclerosis (AS) (Ross, 1999; Gamble, 2006; Cianciolo et al, 2010). We found that CA1 could promote joint calcification, ossification, and joint fusion by accelerating calcium carbonate deposition (Chang et al, 2012; Zheng et al, 2012). As shown in our previous studies, the induction of calcification in human osteosarcoma Saos-2 cells and murine mammary adenocarcinoma 4T1 cells upregulated CA1 expression. Treatment of these cells with AZ reduced CA1 expression and suppressed cell calcification (Chang et al, 2012; Zheng et al, 2015). Our clinical study showed that MTZ was an effective treatment for patients with active ankylosing spondylitis by suppressing CA1 expression and joint fusion (Chang et al, 2011)
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