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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 downregulation on VSMC proliferation, cytoskeleton size and marker gene expression.To upregulate and knock-down the expression of miR-223, VSMCs were transfected using pre-miR-223 and anti-miR-223, respectively. After 48h of transfection, cells were assayed for metabolic activity (A), cell proliferation (B) and marker gene expression levels (with RT-qPCR) (C). Data are presented as the mean ± SD of triplicates. One representative experiment is shown (n = 2). (D) Immunostaining of VSMC protein markers. (E) Quantification of the immunofluorescence data from Fig. 4D. Data are presented as the mean ± SD relative to the control (scrambled RNA, set at 100%) (KLF4 n = 12, MYO n = 11, SMαA n = 5, SRF n = 8). See Fig. 2 for abbreviations.
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pone-0047807-g004: Effect of miR-223 up- and downregulation on VSMC proliferation, cytoskeleton size and marker gene expression.To upregulate and knock-down the expression of miR-223, VSMCs were transfected using pre-miR-223 and anti-miR-223, respectively. After 48h of transfection, cells were assayed for metabolic activity (A), cell proliferation (B) and marker gene expression levels (with RT-qPCR) (C). Data are presented as the mean ± SD of triplicates. One representative experiment is shown (n = 2). (D) Immunostaining of VSMC protein markers. (E) Quantification of the immunofluorescence data from Fig. 4D. Data are presented as the mean ± SD relative to the control (scrambled RNA, set at 100%) (KLF4 n = 12, MYO n = 11, SMαA n = 5, SRF n = 8). See Fig. 2 for abbreviations.

Mentions: We thus evidenced a significant impact of miR-223 on VSMC migration. We decided to focus on this miRNA by modulating its expression and studying the effects on cell proliferation and marker gene expression. Relative to control experiments, over-expression of miR-223 significantly increased the metabolic activity of VSMCs (by 17%, according to the WST-1 assay). However, miR-223 knock-down had no effect (Figure 4A). We found similar results (a 25% increase) for miR-223 upregulation in the BrdU assay, which measures DNA synthesis and thus VSMC proliferation. Again, knock-down had no effect (Figure 4B). We next looked at the effect of miR-223 regulation on several VSMC marker genes (Figure 4C). Interestingly, miR-223 upregulation induced a significant downregulation of SMαA mRNA and a mild increase in Serum Response Factor (SRF) mRNA but did not significantly affect other marker genes. This was further confirmed by immunofluorescence protein staining, which clearly showed a very low level of SMαA and MYO and a high level of SRF when miR-223 was over-expressed in VSMCs (Figure 4D-E). In contrast, anti-miR treatment was associated only with significantly lower levels of SRF (Figure 4D -E).


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 downregulation on VSMC proliferation, cytoskeleton size and marker gene expression.To upregulate and knock-down the expression of miR-223, VSMCs were transfected using pre-miR-223 and anti-miR-223, respectively. After 48h of transfection, cells were assayed for metabolic activity (A), cell proliferation (B) and marker gene expression levels (with RT-qPCR) (C). Data are presented as the mean ± SD of triplicates. One representative experiment is shown (n = 2). (D) Immunostaining of VSMC protein markers. (E) Quantification of the immunofluorescence data from Fig. 4D. Data are presented as the mean ± SD relative to the control (scrambled RNA, set at 100%) (KLF4 n = 12, MYO n = 11, SMαA n = 5, SRF n = 8). See Fig. 2 for abbreviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475714&req=5

pone-0047807-g004: Effect of miR-223 up- and downregulation on VSMC proliferation, cytoskeleton size and marker gene expression.To upregulate and knock-down the expression of miR-223, VSMCs were transfected using pre-miR-223 and anti-miR-223, respectively. After 48h of transfection, cells were assayed for metabolic activity (A), cell proliferation (B) and marker gene expression levels (with RT-qPCR) (C). Data are presented as the mean ± SD of triplicates. One representative experiment is shown (n = 2). (D) Immunostaining of VSMC protein markers. (E) Quantification of the immunofluorescence data from Fig. 4D. Data are presented as the mean ± SD relative to the control (scrambled RNA, set at 100%) (KLF4 n = 12, MYO n = 11, SMαA n = 5, SRF n = 8). See Fig. 2 for abbreviations.
Mentions: We thus evidenced a significant impact of miR-223 on VSMC migration. We decided to focus on this miRNA by modulating its expression and studying the effects on cell proliferation and marker gene expression. Relative to control experiments, over-expression of miR-223 significantly increased the metabolic activity of VSMCs (by 17%, according to the WST-1 assay). However, miR-223 knock-down had no effect (Figure 4A). We found similar results (a 25% increase) for miR-223 upregulation in the BrdU assay, which measures DNA synthesis and thus VSMC proliferation. Again, knock-down had no effect (Figure 4B). We next looked at the effect of miR-223 regulation on several VSMC marker genes (Figure 4C). Interestingly, miR-223 upregulation induced a significant downregulation of SMαA mRNA and a mild increase in Serum Response Factor (SRF) mRNA but did not significantly affect other marker genes. This was further confirmed by immunofluorescence protein staining, which clearly showed a very low level of SMαA and MYO and a high level of SRF when miR-223 was over-expressed in VSMCs (Figure 4D-E). In contrast, anti-miR treatment was associated only with significantly lower levels of SRF (Figure 4D -E).

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