Attenuating Effects of Pyrogallol-Phloroglucinol-6,6-Bieckol on Vascular Smooth Muscle Cell Phenotype Changes to Osteoblastic Cells and Vascular Calcification Induced by High Fat Diet

Abstract

Advanced glycation end products/receptor for AGEs (AGEs/RAGEs) or Toll like receptor 4 (TLR4) induce vascular smooth muscle cell (VSMC) phenotype changes in osteoblast-like cells and vascular calcification. We analyzed the effect of Ecklonia cava extract (ECE) or pyrogallol-phloroglucinol-6,6-bieckol (PPB) on VSMC phenotype changes and vascular calcification prompted by a high-fat diet (HFD). HFD unregulated RAGE, TLR4, transforming growth factor beta (TGFβ), bone morphogenetic protein 2 (BMP2), protein kinase C (PKC), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signals in the aorta of mice. ECE and PPB restored the increase of those signal pathways. AGE- or palmitate-treated VSMC indicated similar changes with the animal. HFD increased osteoblast-like VSMC, which was evaluated by measuring core-binding factor alpha-1 (CBFα-1) and osteocalcin expression and alkaline phosphatase (ALP) activity in the aorta. ECE and PPB reduced vascular calcification, which was analyzed by the calcium deposition ratio, and Alizarin red S stain was increased by HFD. PPB and ECE reduced systolic, diastolic, and mean blood pressure, which increased by HFD. PPB and ECE reduced the phenotype changes of VSMC to osteoblast-like cells and vascular calcification and therefore lowered the blood pressure.

Keywords: Ecklonia cava extract; Toll-like receptor 4; pyrogallol-phloroglucinol-6,6-bieckol; receptor of AGEs; vascular calcification.

Figure 1

Comparative analysis of concentration-dependent ECE and PPB administration on the reduction of AGE and its receptor expression level in HFD-fed mice. (A) AGE levels were calculated in HFD-fed mice with or without ECE/PPB administration. (B) Light microscopic images showing RAGE expression (brown, arrow) in the aorta of HFD-fed mice. Quantitative graphs showing RAGE intensity from representative images. Scale bar = 50 μm. (C) Light microscopic images showing TLR4 expression (brown, arrow) in the aorta of HFD-fed mice. Quantitative graphs showing TLR4 intensity from representative images. Scale bar = 50 μm. **, p < 0.01 vs. NFD/Saline group; ##, p < 0.01 vs. HFD/Saline group. AGE, advanced glycation end products; ECE, Ecklonia cava extract; HFD, high-fat diet; NFD, normal fat diet; PPB, pyrogallol-phloroglucinol-6,6-bieckol; RAGE, receptor for advanced glycation end products; TLR4, toll-like receptor 4.

Figure 2

Comparative analysis of concentration-dependent ECE and PPB treatment on the reduction of RAGE and TLR4 receptor expression level in AGE or PA-treated VSMC. (A,B) Confocal fluorescence microscopic images demonstrating RAGE expression (green) and nuclei (DAPI, blue) in AGE or PA-treated VSMC. Quantitative graphs demonstrating RAGE intensity from representative images. Scale bar = 50 μm. (C,D) Confocal fluorescence microscopic images demonstrating TLR4 expression (green) and nuclei (DAPI, blue) in AGE or PA-treated VSMC. Quantitative graphs demonstrating TLR4 intensity from representative images. Scale bar = 50 μm; **, p < 0.01 vs. PBS group; ##, p < 0.01 vs. AGE or PA group. AGE, advanced glycation end products; DAPI, 4′,6-diamidino-2-phenylindole; ECE, Ecklonia cava extract; PA, palmitate; PBS, phosphate-buffered saline; PPB, pyrogallol-phloroglucinol-6,6-bieckol; RAGE, receptor for advanced glycation end products; TLR4, toll-like receptor 4; VSMC, vascular smooth muscle cell.

Figure 3

Comparative analysis of ECE and PPB treatment on the reduction of vascular smooth muscle cell phenotypic switching to osteoblast-like cell in AGE or palmitate-treated VSMC and HFD-fed mice. mRNA levels of the phenotypic switching to osteoblast-like cell-related molecules (A) PKC, (B) TGFβ, (C) BMP2, and (E) CBFα1 were determined using qRT-PCR. (D) Arrows indicate NF-κB protein expression of HFD-fed mice and quantified graph showing intensity. We incubated 25 μg/mL ECE or 1.8 μg/mL PPB with AGE or PA-treated VSMC (cell) and 100 mg/kg ECE or 2.5 mg/kg PPB administered with HFD-fed mice. scale bar = 25 μm. **, p < 0.01 vs. PBS or NFD/Saline group; #, p < 0.05, ##, p < 0.01 vs. AGE, PA or HFD/Saline group. AGE, advanced glycation end products; BMP2, Bone morphogenetic protein 2; CBFα1, core-binding factor alpha 1; ECE, Ecklonia cava extract; HFD, high-fat diet; NFD, normal fat diet; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PA, palmitate; PBS, phosphate-buffered saline; PKC, protein kinase C; PPB, pyrogallol-phloroglucinol-6,6-bieckol; qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; RAGE, receptor for advanced glycation end products; TGFβ, transforming growth factor beta; VSMC, vascular smooth muscle cell.

Figure 4

Comparative analysis of ECE and PPB treatment on the reduction of osteoblast-like cell marker expression in AGE or palmitate-treated VSMC and HFD-fed mice. (A–C) mRNA levels of the osteoblast-like markers CBFα1 in AGE- or PA-treated VSMC (cell) and HFD-fed mice were determined by qRT-PCR. (D–F) mRNA levels of the osteoblast-like markers osteocalcin in AGE- or PA-treated VSMC (cell) and HFD-fed mice were determined by qRT-PCR. (G) Calcium deposition was calculated using the calcium deposition assay and (H) Alizarin red S staining in HFD-fed mice. Positive signal (red dot, arrow) was marked with an arrow in images. Scale bar = 50 μm. (I) ALP activity was measured with the use of the aorta of HFD-fed mice. **, p < 0.01 vs. PBS or NFD/Saline group; #, p < 0.05, ##, p < 0.01 vs. AGE, PA or HFD/Saline group. AGE, advanced glycation end products; ALP, alkaline phosphatase; CBFα1, core-binding factor alpha 1; ECE, Ecklonia cava extract; HFD, high-fat diet; NFD, normal fat diet; PA, palmitate; PBS, phosphate-buffered saline; PPB, pyrogallol-phloroglucinol-6,6-bieckol; qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; VSMC, vascular smooth muscle cell.

Figure 5

Comparative analysis of ECE and PPB administration on the regulation of blood pressure in HFD-fed mice. (A–C) Systolic, diastolic, and mean artery pressures were measured prior to sacrifice. (D) Light microscopic images showing H&E stained blood vessels and (E) Intima-media thickness acquired using representative H&E images. Arrows indicate media thickness. Scale bar = 50 μm. (F) Summary illustration image showing inhibitory effects of ECE and PPB on vascular smooth muscle cell calcification in HFD condition. Means denoted by a different letter indicate significant differences between groups. AGE, advanced glycation end products; BMP2, bone morphogenetic protein 2; CBFα1, core-binding factor alpha 1; ECE, Ecklonia cava extract; HFD, high-fat diet; H&E, hematoxylin and eosin stain; NFD, normal fat diet; PPB, pyrogallol-phloroglucinol-6,6-bieckol; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; RAGE, receptor for advanced glycation end products; TGFβ, transforming growth factor beta; TLR4, toll like receptor 4.