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Inorganic phosphate accelerates the migration of vascular smooth muscle cells: evidence for the involvement of miR-223.

Rangrez AY, M'Baya-Moutoula E, Metzinger-Le Meuth V, Hénaut L, Djelouat MS, Benchitrit J, Massy ZA, Metzinger L - PLoS ONE (2012)

Bottom Line: Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells.Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs.Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1088, Amiens, France.

ABSTRACT

Background: An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs).

Methodology/principal findings: Exposure of human primary VSMCs in vitro to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Krüppel-like factors-4 and -5 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these in vitro findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification.

Conclusions/significance: Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.

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Physiological and molecular effects of Pi.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS in the presence or absence of 3.5 mM Pi. (A) A representative photograph of a calcification assay using alizarin staining, indicating greater calcification in Pi treated cells and the corresponding quantification. Alongside a control with cells incubated at 1.1 mM Pi and to check for non-specific calcium deposition, we also ran a cell-free control with the same Pi concentrations. (B) The VSMCs' metabolic activity was studied in a WST-1 colorimetric assay. Cells treated with 3.5 mM Pi showed low metabolic activity. (C) VSMC proliferation was measured in a BrdU ELISA. (D) The migration rate of VSMCs was determined using Boyden chambers and was found to be twice as high in Pi-treated VSMCs than in control cells. (E) Expression of miR-143, miR-145 and miR-223 was measured by qPCR. Data are represented as the mean of two or three independent experiments in triplicate.
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pone-0047807-g001: Physiological and molecular effects of Pi.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS in the presence or absence of 3.5 mM Pi. (A) A representative photograph of a calcification assay using alizarin staining, indicating greater calcification in Pi treated cells and the corresponding quantification. Alongside a control with cells incubated at 1.1 mM Pi and to check for non-specific calcium deposition, we also ran a cell-free control with the same Pi concentrations. (B) The VSMCs' metabolic activity was studied in a WST-1 colorimetric assay. Cells treated with 3.5 mM Pi showed low metabolic activity. (C) VSMC proliferation was measured in a BrdU ELISA. (D) The migration rate of VSMCs was determined using Boyden chambers and was found to be twice as high in Pi-treated VSMCs than in control cells. (E) Expression of miR-143, miR-145 and miR-223 was measured by qPCR. Data are represented as the mean of two or three independent experiments in triplicate.

Mentions: Hyperphosphatemia is associated with vascular calcification in various cardiovascular disorders[14]. In order to study the direct effect of Pi on VSMCs from the human aorta, we treated the cells for 10 days with 3.5 mM Pi (to reflect hyperphosphatemia [15]) in Dulbecco's modified Eagle Medium (DMEM) with 1% Fetal Bovine Serum (FBS; the concentration needed to obtain Ca*Pi deposits in vitro). We determined the level of calcification by alizarin staining and found that Pi indeed induced calcification in VSMCs. The calcification was 15- to 20-fold higher in Pi treated-cells than in control cells exposed to a physiological concentration of Pi (1.1 mM) (Figure 1A). The same Pi concentration significantly lowered the VSMCs' metabolic activity (by 25%, as measured in a WST-1 assay; Figure 1B) and proliferation activity (by 50%, as measured by BrdU incorporation; Figure 1C). Decreased proliferation induced by high Pi treatment could tentatively be attributed to increased apoptosis. In order to explore this hypothesis, we carried out Annexin V/PI staining. Our results indicate that Pi treatment for 10 days does not affect apoptosis rate in our experimental conditions (figure S1). Other, as yet undetected mechanisms are thus responsible for this phenomenon.


Inorganic phosphate accelerates the migration of vascular smooth muscle cells: evidence for the involvement of miR-223.

Rangrez AY, M'Baya-Moutoula E, Metzinger-Le Meuth V, Hénaut L, Djelouat MS, Benchitrit J, Massy ZA, Metzinger L - PLoS ONE (2012)

Physiological and molecular effects of Pi.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS in the presence or absence of 3.5 mM Pi. (A) A representative photograph of a calcification assay using alizarin staining, indicating greater calcification in Pi treated cells and the corresponding quantification. Alongside a control with cells incubated at 1.1 mM Pi and to check for non-specific calcium deposition, we also ran a cell-free control with the same Pi concentrations. (B) The VSMCs' metabolic activity was studied in a WST-1 colorimetric assay. Cells treated with 3.5 mM Pi showed low metabolic activity. (C) VSMC proliferation was measured in a BrdU ELISA. (D) The migration rate of VSMCs was determined using Boyden chambers and was found to be twice as high in Pi-treated VSMCs than in control cells. (E) Expression of miR-143, miR-145 and miR-223 was measured by qPCR. Data are represented as the mean of two or three independent experiments in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475714&req=5

pone-0047807-g001: Physiological and molecular effects of Pi.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS in the presence or absence of 3.5 mM Pi. (A) A representative photograph of a calcification assay using alizarin staining, indicating greater calcification in Pi treated cells and the corresponding quantification. Alongside a control with cells incubated at 1.1 mM Pi and to check for non-specific calcium deposition, we also ran a cell-free control with the same Pi concentrations. (B) The VSMCs' metabolic activity was studied in a WST-1 colorimetric assay. Cells treated with 3.5 mM Pi showed low metabolic activity. (C) VSMC proliferation was measured in a BrdU ELISA. (D) The migration rate of VSMCs was determined using Boyden chambers and was found to be twice as high in Pi-treated VSMCs than in control cells. (E) Expression of miR-143, miR-145 and miR-223 was measured by qPCR. Data are represented as the mean of two or three independent experiments in triplicate.
Mentions: Hyperphosphatemia is associated with vascular calcification in various cardiovascular disorders[14]. In order to study the direct effect of Pi on VSMCs from the human aorta, we treated the cells for 10 days with 3.5 mM Pi (to reflect hyperphosphatemia [15]) in Dulbecco's modified Eagle Medium (DMEM) with 1% Fetal Bovine Serum (FBS; the concentration needed to obtain Ca*Pi deposits in vitro). We determined the level of calcification by alizarin staining and found that Pi indeed induced calcification in VSMCs. The calcification was 15- to 20-fold higher in Pi treated-cells than in control cells exposed to a physiological concentration of Pi (1.1 mM) (Figure 1A). The same Pi concentration significantly lowered the VSMCs' metabolic activity (by 25%, as measured in a WST-1 assay; Figure 1B) and proliferation activity (by 50%, as measured by BrdU incorporation; Figure 1C). Decreased proliferation induced by high Pi treatment could tentatively be attributed to increased apoptosis. In order to explore this hypothesis, we carried out Annexin V/PI staining. Our results indicate that Pi treatment for 10 days does not affect apoptosis rate in our experimental conditions (figure S1). Other, as yet undetected mechanisms are thus responsible for this phenomenon.

Bottom Line: Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells.Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs.Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.

View Article: PubMed Central - PubMed

Affiliation: INSERM U1088, Amiens, France.

ABSTRACT

Background: An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs).

Methodology/principal findings: Exposure of human primary VSMCs in vitro to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Krüppel-like factors-4 and -5 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these in vitro findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification.

Conclusions/significance: Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.

Show MeSH
Related in: MedlinePlus