Journal: Nutrients

Article Title: Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

doi: 10.3390/nu16020291

Figure Lengend Snippet: Figure 3. ALP activity in rat aortic A7r5 cells under zinc treatment. Rat aortic A7r5 cells were cultured for 15 d under zinc treatments supplemented with either βGP or NaP as the phosphate source. (A) ALP staining of the ECM using BCIP/NBT as substrate did not show an increase in ALP activity under Zn-deficient conditions with increasing P levels. (B) Measurement of ALP activity using PNPP as substrate did not show a significant effect of zinc (p < 0.05, one-way ANOVA followed by Tukey HSD). (C) Protein expression as assessed through Western blotting showed very weak ALP expression and downregulation under Zn−is only observed in βGP-treated cultures.

Article Snippet: Mouse origin osteoblastic MC3T3-E1 cell subclone 4 (CRL-2593) and rat aortic vascular smooth muscle cells A7r5 (CRL-1444) were obtained commercially from American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Cell Culture, Staining, Expressing, Western Blot

Journal: Nutrients

Article Title: Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

doi: 10.3390/nu16020291

Figure Lengend Snippet: Figure 4. Ca and P deposition in zinc-deficient VSMCs supplemented with increasing concentrations of β-GP or NaP. Rat aortic A7r5 cells were cultured for 15 d under zinc treatments supplemented with either βGP or NaP as the phosphate source. (A) Ca deposition was assessed through Alizarin red staining, while (B) P deposition was assessed through von Kossa. Images are representative from n = 4 samples.

Article Snippet: Mouse origin osteoblastic MC3T3-E1 cell subclone 4 (CRL-2593) and rat aortic vascular smooth muscle cells A7r5 (CRL-1444) were obtained commercially from American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture, Staining

Journal: Nutrients

Article Title: Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

doi: 10.3390/nu16020291

Figure Lengend Snippet: Figure 5. Pit1 expression in rat aortic A7r5and osteoblastic MC3T3-E1 cells under zinc treatment. Both A7r5 and MC3T3-E1 cells were cultured up to 15 d under zinc treatments. GM (normal growth media) was used as control in A7r5 while OSM (normal osteogenic medium) was used as control for MC3T3-E1 cells. Data represent means ± SD (n = 3). The statistical significance between values for each group was assessed through Dunnett’s t-test. * p < 0.05 between the Zn−or Zn + and control groups.

Article Snippet: Mouse origin osteoblastic MC3T3-E1 cell subclone 4 (CRL-2593) and rat aortic vascular smooth muscle cells A7r5 (CRL-1444) were obtained commercially from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Cell Culture, Control

Journal: Nutrients

Article Title: Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

doi: 10.3390/nu16020291

Figure Lengend Snippet: Figure 6. Mineral measurement in extracellular (media) and intracellular (cell) fractions. Aortic A7r5 cells were cultured up to 15 d under zinc treatments. (A) The concentrations of P in both media and cell fractions were not significantly affected by zinc. Upon addition of PFA, media P increased under Zn−while cellular P decreased in Zn−and Zn+. (B) Ca concentrations were also not affected by zinc and, like P, its concentration decreased upon PFA addition. (C) The levels of zinc in the media confirmed our zinc treatment, although the cellular zinc levels remained unchanged. The addition of PFA did not alter cellular zinc levels. The mean with the different superscripts are significantly different by Zn treatment. p < 0.05, one-way ANOVA followed by post hoc Tukey HSD test.

Article Snippet: Mouse origin osteoblastic MC3T3-E1 cell subclone 4 (CRL-2593) and rat aortic vascular smooth muscle cells A7r5 (CRL-1444) were obtained commercially from American Type Culture Collection (Manassas, VA, USA).

Techniques: Cell Culture, Concentration Assay

Journal: Nutrients

Article Title: Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

doi: 10.3390/nu16020291

Figure Lengend Snippet: Figure 7. Inhibition of phosphate uptake via Pit1. Both A7r5 and MC3T3-E1 cells were cultured up to 15 d under zinc treatments. (A) Inhibition of phosphate uptake did not inhibit Pit1 expression in both A7r5 VSMCs and osteobalstic MC3T3-E1 cells. Data represent means ± SD (n = 3). The statistical significance between values for each group was assessed through Dunnett’s t-test. * p < 0.05 between Zn−or Zn + and control groups. (B) However, Ca and P deposition in both cells lines were significantly inhibited by treatment with PFA. (C). In A7r5 VSMCs, treatment with PFA restored the expression of the VSMC markers SM22α and calponin under zinc deficiency.

Article Snippet: Mouse origin osteoblastic MC3T3-E1 cell subclone 4 (CRL-2593) and rat aortic vascular smooth muscle cells A7r5 (CRL-1444) were obtained commercially from American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Cell Culture, Expressing, Control

Journal: Nutrients

Article Title: Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation.

doi: 10.3390/nu16020291

Figure Lengend Snippet: Figure 8. Schematic summary of the molecular mechanisms enabled through Pit1 expression in VSMCs; this is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.

Article Snippet: Mouse origin osteoblastic MC3T3-E1 cell subclone 4 (CRL-2593) and rat aortic vascular smooth muscle cells A7r5 (CRL-1444) were obtained commercially from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Inhibition, Marker