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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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Related in: MedlinePlus

Over-expression of miR-223 enhances VSMC migration.To upregulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR-223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. The migration rate was studied by (A) wound healing assay and (B) Boyden chambers (n = 3, two independent experiments).
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pone-0047807-g003: Over-expression of miR-223 enhances VSMC migration.To upregulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR-223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. The migration rate was studied by (A) wound healing assay and (B) Boyden chambers (n = 3, two independent experiments).

Mentions: To further study the effects of miR-143 or miR-223, we used a wound-healing assay that mimics cell motility during vessel healing in vivo and enables migration to be studied. We created a “wound” in the monolayer and captured the images of VSMC migration after 48 h in culture. Vascular smooth muscle cells transfected with pre-miR (over-expression) and anti-miR (knock-down) specific for miR-143 and miR-223 were compared with a scrambled RNA control (Figure 3A). Anti-miR-143 treatment did not affect motility, whereas pre-miR-143 treatment was associated with moderately (20%) but significantly greater migration than in control experiments. Pre-miR-223 induced a highly significant 64% increase in cell migration. Accordingly, anti-miR-223 treatment was associated with significantly (28%) lower migration (Figure 3A).


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)

Over-expression of miR-223 enhances VSMC migration.To upregulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR-223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. The migration rate was studied by (A) wound healing assay and (B) Boyden chambers (n = 3, two independent experiments).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047807-g003: Over-expression of miR-223 enhances VSMC migration.To upregulate and knock-down the expression of miR-223, VSMCs were transfected for 48 h with pre-miR-223 and anti-miR-223, respectively. Scrambled, unrelated miRNA was used as a control. The migration rate was studied by (A) wound healing assay and (B) Boyden chambers (n = 3, two independent experiments).
Mentions: To further study the effects of miR-143 or miR-223, we used a wound-healing assay that mimics cell motility during vessel healing in vivo and enables migration to be studied. We created a “wound” in the monolayer and captured the images of VSMC migration after 48 h in culture. Vascular smooth muscle cells transfected with pre-miR (over-expression) and anti-miR (knock-down) specific for miR-143 and miR-223 were compared with a scrambled RNA control (Figure 3A). Anti-miR-143 treatment did not affect motility, whereas pre-miR-143 treatment was associated with moderately (20%) but significantly greater migration than in control experiments. Pre-miR-223 induced a highly significant 64% increase in cell migration. Accordingly, anti-miR-223 treatment was associated with significantly (28%) lower migration (Figure 3A).

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