Effects of ghrelin on mitogen-activated protein kinase (MAPK) activation in calcifying vascular smooth muscle cells (CVSMCs).

<p>(A, B) Cells were exposed to 10−6 mol/L ghrelin for 0–60 min, or 50 µM hydrogen peroxide (H2O2) for 15 min as a positive control for p38 and JNK activation. Cell lysates were subjected to western blotting and incubated with antibodies against p-ERK, ERK, p-p38, p38, p-JNK, and JNK. The representative results are shown. (C) Cells exposed to 10−9 mol/L, 10⁻⁸ mol/L, 10⁻⁷ mol/L, and 10⁻⁶ mol/L of ghrelin for 30 min. Cell lysates were subjected to western blotting and incubated with antibody against p-ERK and ERK. The representative results are shown.</p

Effects of ghrelin on alkaline phosphatase (ALP) activity, Runx2 protein expression, bone morphogenetic protein-2 (BMP-2) mRNA expression and calcium deposition in vascular smooth muscle cells (VSMCs) cultured with beta-GP.

<p>(A) Effect of ghrelin on ALP activity. The cells were cultured with or without 10−6 mol/L ghrelin for the indicated time periods. ALP activity was measured by an ALP kit, normalized to the cellular protein contents, and presented as mean ± standard deviation (SD) (<i>n</i> 3). (B, C) Effect of ghrelin on Runx2 expression. The cells were cultured for 6 days with or without 10−6 mol/L ghrelin above and presented as mean ±SD (<i>n</i> = 3). The expression of Runx2 was measured by western blot. (D) Effect of ghrelin on BMP-2 mRNA expression. The cells were cultured with or without 10−6 mol/L ghrelin for the indicated time periods. BMP-2 mRNA expression was determined by real-time quantitative polymerase chain reaction (qPCR). Results are expressed as fold of control. Bars represent mean ±standard deviation (SD) (<i>n</i> = 3). (E) Effect of ghrelin on calcium deposition. The cells were cultured for 20 days with or without 10−6 mol/L ghrelin and presented as mean ±SD (<i>n</i> = 3). The calcium contents of the cell layers were measured by the atomic absorption spectroscopy method.</p

Effects of ghrelin on alkaline phosphatase (ALP) activity, Runx2 protein expression, bone morphogenetic protein-2 (BMP-2) mRNA expression and calcium deposition in calcifying vascular smooth muscle cells (CVSMCs).

<p>(A) Effect of ghrelin on ALP activity. The cells were cultured with or without 10−6 mol/L ghrelin for the indicated time periods. ALP activity was measured by an ALP kit, normalized to the cellular protein contents, and presented as mean ±standard deviation (SD) (<i>n</i> = 3). (B, C) Effect of ghrelin on Runx2 expression. The cells were cultured for 48 h with or without 10−6 mol/L ghrelin and presented as mean ±SD (<i>n</i> = 3). The expression of Runx2 was measured by western blotting. (D) Effect of ghrelin on BMP-2 mRNA expression. The cells were cultured with or without 10−6 mol/L ghrelin for the indicated time periods. BMP-2 mRNA expression was determined by real-time quantitative polymerase chain reaction (qPCR). Results are expressed as fold of control. Bars present mean ±SD (<i>n</i> = 3). (E) A representative entire plate view of the Alizarin Red S staining in 24-well plates for control cells and cells treated with ghrelin in 12-day cultures. (F) Effect of ghrelin on calcium deposition. The cells were cultured for 12 days with or without 10−6 mol/L ghrelin and presented as mean ±SD (<i>n</i> = 3). The calcium contents of the cell layers were measured by the atomic absorption spectroscopy method.</p

Effect of <i>in vitro</i> incubation of calcifying vascular smooth muscle cells (CVSMCs) with insulin on RANKL expression.

<p>Cells were exposed to vehicle or 1–100 nM insulin for 48 h or to 10 nM insulin for 6–48 h. (A) The dose–response of insulin on RANKL mRNA levels measured by real-time polymerase chain reaction (PCR) in cultured CVSMCs. Data are shown as percentage over basal values. (B) The dose–response of insulin on RANKL protein secreted by CVSMCs in the culture medium. (C) Time course of effects of 10 nM insulin on RANKL mRNA levels in cultured CVSMCs. (D) Time course of effects of 10 nM insulin on RANKL protein secretion in the culture medium by CVSMCs. The dots represent the percent of control RANKL levels at various time points. (<i>n</i> = 3, *<i>p</i><0.05 vs. control, **<i>p</i><0.01 vs. control.).</p

Insulin-promoted osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs) through the ERK1/2/RANKL signaling pathway.

<p>(A) Treatment with siRNA-RANKL blocked the expression of RANKL protein in osteoblasts. Cells were treated with siRNA control, or siRNA-RANKL. Total cellular protein was subjected to immunoblot analysis using anti-RANKL antibody. Lane 1, lysate from CVSMCs; lane 2, lysate from CVSMCs treated with siRNA control; lane 3, lysate from CVSMCs treated with siRNA-RANKL. (B) The cells were homogenized for ALP activity assay. Cells were incubated with PD98059 (10 µM), HIMO (10 µM) or LY294002 (10 µM) for 2 h before being treated with 10 nM insulin for 48 h. Cells were also treated with siRNA control or siRNA-RANKL in the presence of 10 nM insulin. (C) Effect of recombinant RANKL protein (15 pM) on the ALP activities decreased by RANKL-siRNA in 10 nM insulin-stimulated CVSMCs after 48 h. (<i>n</i> = 5; **<i>p</i><0.01 vs. insulin-treated control).</p

Insulin increased RANKL mRNA expression through ERK1/2, but not Akt signal pathway in calcifying vascular smooth muscle cells (CVSMCs).

<p>Cell lysates were subjected to western blot and incubated with anti-p-ERK1/2, ERK1/2, p-Akt, and Akt antibodies. (A) Effects of insulin on ERK1/2 and Akt activation in CVSMCs. Representative results are shown for cells exposed to 10 nM insulin for 5–60 min. (B) Cells incubated with PD98059 (10 µM) for 2 h prior to treatment with 10 nM insulin for 30 min. (C) Cells incubated with LY294002 (10 µM) or HIMO (10 µM) for 2 h prior to treatment with 10 nM insulin for 15 min. (D) Cells were incubated with PD98059 (10 µM), LY294002 (10 µM) or HIMO (10 µM) for 2 h before treatment with 10 nM insulin for 48 h. Cells were treated with 10⁻⁷ M 1α,25-dihydroxyvitamin D3 (1,25 vitD), or 1,25 vitD+10 µM PD98059 as another control. RANKL mRNA expression was determined by real-time quantitative PCR. Results are expressed as percent of control. Bars represent mean ± standard deviation (SD) (<i>n</i> = 3; **<i>p</i><0.01 vs. insulin-treated control).</p

Insulin promoted the mineralization of the matrix in calcifying vascular smooth muscle cells (CVSMCs).

<p>CVSMCs were cultured in DMEM that contained 10% fetal bovine serum (FBS) in 24-well plates, in the presence of vehicle or 10 nM insulin for 20 days. Mineralization of the matrix was determined by Alizarin red S staining. (A) Representative microscopic view at a magnification of ×100. Left panel showed CVSMCs incubated with vehicle, and the right panel showed CVSMCs incubated with 10 nM insulin for 12 days. (B) Quantification of Alizarin red S stain through extraction with cetyl-pyridinium chloride. The amount of released dye was quantified by spectrophotometry at 540 nm. The bars represent mean ± standard deviation (SD) (<i>n</i> = 4; **<i>p</i><0.01 vs. control). (C) Cells were treated with vehicle (serum-free medium) or insulin at 1, 5, 10, or 100 nM for 48 h in serum-free DMEM. ALP activity were determined and normalized to cell total protein. The dose–response of insulin on ALP activity in cultured CVSMCs. (D) Cells were treated with vehicle (serum-free medium) or insulin at 1, 5, 10, or 100 nM for 48 h in serum-free DMEM. Osteocalcin secretion were determined and normalized to cell total protein. The dose–response of insulin on osteocalcin secretion in cultured CVSMCs. The bars represent mean ± standard deviation (SD) (**<i>p</i><0.01 vs. control, *<i>p</i><0.05 vs. control, <i>n</i> = 4).</p

Weekly distribution of AFP cases <15 years of age by classification in 2011 and 2012 in Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China.

<p>Note: VDPV, vaccine-derived poliovirus; AFP, acute flaccid paralysis.</p

Rounds, dates, target population, and numbers of persons immunized for SIAs by province, 2012.

<p>Rounds, dates, target population, and numbers of persons immunized for SIAs by province, 2012.</p

Antibody seropositivity of type I, type Π, and type by age group in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China, 2012.

<p>Antibody seropositivity of type I, type Π, and type by age group in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China, 2012.</p