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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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Effect of miR-223 up- and down regulation and Pi on miR-223 specific targets Mef2c and RhoB.To up-regulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. For Pi effect, VSMCs were cultured for 2 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, expression of Mef2c and RhoB messengers was measured by RT-qPCR. Data are represented as the mean of 3 independent experiments +/− SD). (B) Immunofluorescence images showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression in VSMCs (magnification 20X). (C) Fluorescence intensity measurements from the immunofluorescence images displayed in Figure 4B, showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression (n = 3 for each marker, data are represented as the mean of 3 independent experiments +/− SD).
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pone-0047807-g005: Effect of miR-223 up- and down regulation and Pi on miR-223 specific targets Mef2c and RhoB.To up-regulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. For Pi effect, VSMCs were cultured for 2 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, expression of Mef2c and RhoB messengers was measured by RT-qPCR. Data are represented as the mean of 3 independent experiments +/− SD). (B) Immunofluorescence images showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression in VSMCs (magnification 20X). (C) Fluorescence intensity measurements from the immunofluorescence images displayed in Figure 4B, showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression (n = 3 for each marker, data are represented as the mean of 3 independent experiments +/− SD).

Mentions: We next investigated the effect of miR-223 regulation on reported targets Mef2c and RhoB. Mef2c has been shown to play a critical role in VSMC differentiation and to regulate myocardin expression [18].Two independent studies have validated that miR-223 directly targets and inhibits Mef2c mRNA [19]–[20]. Accordingly, in our model, miR-223 upregulation induced a strong and significant decrease of Mef2c mRNA (of approximately 80%, Figure 5A) and a concomitant decrease of the protein product, as evidenced by immunofluorescence protein staining (of approximately 35%, Figure 5B and C). Interestingly, high Pi treatment induced comparable changes, suggesting a link between the two treatments. On the other hand, miR-223 knock-down had no effect on the expression of these markers. We also looked at the effect of miR-223 regulation on another recently described miR-223 target, RhoB [21]. RhoB is a member of the Rho guanosine triphosphatases family of proteins which has been shown to increase VSMC contractility and mediate adaptational changes to hypoxia [22]. Like with Mef2c, miR-223 upregulation significantly decreased the amount of mRNA (of approximately 90%, Figure 5A) with a concomitant decrease of the RhoB protein (of approximately 40%, Figure 5B and C). Again, 3.5 mM Pi treatment also induced very similar dramatic changes in RhoB gene expression (90% decrease of mRNA amount, and 40% decrease of the protein). As in the Mef2c experiments, anti-miR-223 treatment had no detectable effect.


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)

Effect of miR-223 up- and down regulation and Pi on miR-223 specific targets Mef2c and RhoB.To up-regulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. For Pi effect, VSMCs were cultured for 2 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, expression of Mef2c and RhoB messengers was measured by RT-qPCR. Data are represented as the mean of 3 independent experiments +/− SD). (B) Immunofluorescence images showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression in VSMCs (magnification 20X). (C) Fluorescence intensity measurements from the immunofluorescence images displayed in Figure 4B, showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression (n = 3 for each marker, data are represented as the mean of 3 independent experiments +/− SD).
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Related In: Results  -  Collection

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pone-0047807-g005: Effect of miR-223 up- and down regulation and Pi on miR-223 specific targets Mef2c and RhoB.To up-regulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. For Pi effect, VSMCs were cultured for 2 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, expression of Mef2c and RhoB messengers was measured by RT-qPCR. Data are represented as the mean of 3 independent experiments +/− SD). (B) Immunofluorescence images showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression in VSMCs (magnification 20X). (C) Fluorescence intensity measurements from the immunofluorescence images displayed in Figure 4B, showing the effect of miR-223 up- and down regulation and Pi on Mef2c and RhoB protein expression (n = 3 for each marker, data are represented as the mean of 3 independent experiments +/− SD).
Mentions: We next investigated the effect of miR-223 regulation on reported targets Mef2c and RhoB. Mef2c has been shown to play a critical role in VSMC differentiation and to regulate myocardin expression [18].Two independent studies have validated that miR-223 directly targets and inhibits Mef2c mRNA [19]–[20]. Accordingly, in our model, miR-223 upregulation induced a strong and significant decrease of Mef2c mRNA (of approximately 80%, Figure 5A) and a concomitant decrease of the protein product, as evidenced by immunofluorescence protein staining (of approximately 35%, Figure 5B and C). Interestingly, high Pi treatment induced comparable changes, suggesting a link between the two treatments. On the other hand, miR-223 knock-down had no effect on the expression of these markers. We also looked at the effect of miR-223 regulation on another recently described miR-223 target, RhoB [21]. RhoB is a member of the Rho guanosine triphosphatases family of proteins which has been shown to increase VSMC contractility and mediate adaptational changes to hypoxia [22]. Like with Mef2c, miR-223 upregulation significantly decreased the amount of mRNA (of approximately 90%, Figure 5A) with a concomitant decrease of the RhoB protein (of approximately 40%, Figure 5B and C). Again, 3.5 mM Pi treatment also induced very similar dramatic changes in RhoB gene expression (90% decrease of mRNA amount, and 40% decrease of the protein). As in the Mef2c experiments, anti-miR-223 treatment had no detectable effect.

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