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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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Schematic representation of the hypothetical molecular and cellular consequences of elevated phosphate exposure in VSMCs.Elevated extracellular Pi resulted in the downregulation of VSMC master regulators, MYO and SMαA. The expression of miR-143 and miR-145 was also downregulated in Pi-treated cells. These events affected downstream processes by reducing the size of the actin cytoskeleton, disturbing cell morphology, upregulating miR-143 and miR-145 targets and, ultimately, leading to increased calcification and a greater VSMC migration rate. Up-regulation of miR-223 further enhances VSMC migration and reduces the amount of actin cytoskeleton. ‘-’ indicates downregulation, whereas ‘+’ indicates upregulation of the respective miRNAs or genes. See Fig. 2 for abbreviations.
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pone-0047807-g007: Schematic representation of the hypothetical molecular and cellular consequences of elevated phosphate exposure in VSMCs.Elevated extracellular Pi resulted in the downregulation of VSMC master regulators, MYO and SMαA. The expression of miR-143 and miR-145 was also downregulated in Pi-treated cells. These events affected downstream processes by reducing the size of the actin cytoskeleton, disturbing cell morphology, upregulating miR-143 and miR-145 targets and, ultimately, leading to increased calcification and a greater VSMC migration rate. Up-regulation of miR-223 further enhances VSMC migration and reduces the amount of actin cytoskeleton. ‘-’ indicates downregulation, whereas ‘+’ indicates upregulation of the respective miRNAs or genes. See Fig. 2 for abbreviations.

Mentions: Another group of researchers has already demonstrated that high Pi concentrations are associated with greater calcification in VSMCs[27]. Our data confirm these literature results and further demonstrate that hyperphosphatemia increases the VSMCs' ability to migrate and reduces cell metabolic activity and proliferation. Based on earlier reports, we postulated that the reduced proliferation in Pi treated VSMCs could be explained by increased cytotoxicity and rate of apoptosis. However, we did not observe significant difference in the apoptosis rate in Pi treated or untreated cell in our experimental conditions. Therefore, the increased calcification and some yet unknown mechanism would be involved in lowering the cell proliferation in Pi treated VSMCs. Interestingly though, our results indicate that hyperphosphotemia at least contributes to inducing VSMCs to switch from a differentiated state to a dedifferentiated state by modulating the underlying molecular markers (Figure 7).


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)

Schematic representation of the hypothetical molecular and cellular consequences of elevated phosphate exposure in VSMCs.Elevated extracellular Pi resulted in the downregulation of VSMC master regulators, MYO and SMαA. The expression of miR-143 and miR-145 was also downregulated in Pi-treated cells. These events affected downstream processes by reducing the size of the actin cytoskeleton, disturbing cell morphology, upregulating miR-143 and miR-145 targets and, ultimately, leading to increased calcification and a greater VSMC migration rate. Up-regulation of miR-223 further enhances VSMC migration and reduces the amount of actin cytoskeleton. ‘-’ indicates downregulation, whereas ‘+’ indicates upregulation of the respective miRNAs or genes. See Fig. 2 for abbreviations.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047807-g007: Schematic representation of the hypothetical molecular and cellular consequences of elevated phosphate exposure in VSMCs.Elevated extracellular Pi resulted in the downregulation of VSMC master regulators, MYO and SMαA. The expression of miR-143 and miR-145 was also downregulated in Pi-treated cells. These events affected downstream processes by reducing the size of the actin cytoskeleton, disturbing cell morphology, upregulating miR-143 and miR-145 targets and, ultimately, leading to increased calcification and a greater VSMC migration rate. Up-regulation of miR-223 further enhances VSMC migration and reduces the amount of actin cytoskeleton. ‘-’ indicates downregulation, whereas ‘+’ indicates upregulation of the respective miRNAs or genes. See Fig. 2 for abbreviations.
Mentions: Another group of researchers has already demonstrated that high Pi concentrations are associated with greater calcification in VSMCs[27]. Our data confirm these literature results and further demonstrate that hyperphosphatemia increases the VSMCs' ability to migrate and reduces cell metabolic activity and proliferation. Based on earlier reports, we postulated that the reduced proliferation in Pi treated VSMCs could be explained by increased cytotoxicity and rate of apoptosis. However, we did not observe significant difference in the apoptosis rate in Pi treated or untreated cell in our experimental conditions. Therefore, the increased calcification and some yet unknown mechanism would be involved in lowering the cell proliferation in Pi treated VSMCs. Interestingly though, our results indicate that hyperphosphotemia at least contributes to inducing VSMCs to switch from a differentiated state to a dedifferentiated state by modulating the underlying molecular markers (Figure 7).

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