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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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Elevated Pi affects VSMC phenotypic markers.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, we performed RT-qPCR using specific primers for VSMC phenotypic marker genes. Contractile phenotype markers (such as myocardin (MYO) and Smooth muscle α–actin (SMαA) were highly downregulated), whereas synthetic phenotype markers like Krüppel-like factor 4 (KLF4), KLF5, versican (VSCN) and platelet-derived growth factor receptor-α (PDGFRα) were significantly upregulated (n = 3). (B) Immunofluorescence of microscopic images showing the actin cytoskeleton after Pi treatment of VSMCs (magnification 40X), as also assessed (C) by fluorescence intensity measurements in corresponding histogram. (D) Representative immunoblot indicating the downregulation of NFIA in Pi-treated VSMCs. β-actin was used as endogenous control. One representative experiment shown out of two or three independent experiments.
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pone-0047807-g002: Elevated Pi affects VSMC phenotypic markers.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, we performed RT-qPCR using specific primers for VSMC phenotypic marker genes. Contractile phenotype markers (such as myocardin (MYO) and Smooth muscle α–actin (SMαA) were highly downregulated), whereas synthetic phenotype markers like Krüppel-like factor 4 (KLF4), KLF5, versican (VSCN) and platelet-derived growth factor receptor-α (PDGFRα) were significantly upregulated (n = 3). (B) Immunofluorescence of microscopic images showing the actin cytoskeleton after Pi treatment of VSMCs (magnification 40X), as also assessed (C) by fluorescence intensity measurements in corresponding histogram. (D) Representative immunoblot indicating the downregulation of NFIA in Pi-treated VSMCs. β-actin was used as endogenous control. One representative experiment shown out of two or three independent experiments.

Mentions: Given our observation of significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in cells treated with 3.5 mM Pi, we used RT-qPCR to determine the expression levels of representative VSMC phenotypic marker genes - most of which are targeted by these miRNAs[8], [10]. We found that Pi treatment results in significant downregulation of contractile marker genes (Figure 2A), such as myocardin (MYO) and Smooth Muscle α–actin (SMαA). Nuclear factor IA (NFIA, an experimentally validated miR-223 target[13]) appeared to be downregulated in cells treated with 3.5 mM Pi, as expected (given the increase in miR-223). The low level of NFIA was confirmed by Western blotting (Figure 2D). In contrast, some of the synthetic phenotypic markers (such as Krüppel-like factor 4 [KLF4], KLF5, platelet derived growth factor receptor-α [PDGFR α] and versican [VSCN]) which are all targeted by miR-143 and/or miR-145[7] were significantly upregulated; this was expected, given the observed downregulation of the corresponding miRNAs (Figure 2A).


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)

Elevated Pi affects VSMC phenotypic markers.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, we performed RT-qPCR using specific primers for VSMC phenotypic marker genes. Contractile phenotype markers (such as myocardin (MYO) and Smooth muscle α–actin (SMαA) were highly downregulated), whereas synthetic phenotype markers like Krüppel-like factor 4 (KLF4), KLF5, versican (VSCN) and platelet-derived growth factor receptor-α (PDGFRα) were significantly upregulated (n = 3). (B) Immunofluorescence of microscopic images showing the actin cytoskeleton after Pi treatment of VSMCs (magnification 40X), as also assessed (C) by fluorescence intensity measurements in corresponding histogram. (D) Representative immunoblot indicating the downregulation of NFIA in Pi-treated VSMCs. β-actin was used as endogenous control. One representative experiment shown out of two or three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0047807-g002: Elevated Pi affects VSMC phenotypic markers.VSMCs were cultured for 10 days in DMEM supplemented with 1% FBS, in the presence or absence of 3.5 mM Pi. (A) After RNA extraction, we performed RT-qPCR using specific primers for VSMC phenotypic marker genes. Contractile phenotype markers (such as myocardin (MYO) and Smooth muscle α–actin (SMαA) were highly downregulated), whereas synthetic phenotype markers like Krüppel-like factor 4 (KLF4), KLF5, versican (VSCN) and platelet-derived growth factor receptor-α (PDGFRα) were significantly upregulated (n = 3). (B) Immunofluorescence of microscopic images showing the actin cytoskeleton after Pi treatment of VSMCs (magnification 40X), as also assessed (C) by fluorescence intensity measurements in corresponding histogram. (D) Representative immunoblot indicating the downregulation of NFIA in Pi-treated VSMCs. β-actin was used as endogenous control. One representative experiment shown out of two or three independent experiments.
Mentions: Given our observation of significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in cells treated with 3.5 mM Pi, we used RT-qPCR to determine the expression levels of representative VSMC phenotypic marker genes - most of which are targeted by these miRNAs[8], [10]. We found that Pi treatment results in significant downregulation of contractile marker genes (Figure 2A), such as myocardin (MYO) and Smooth Muscle α–actin (SMαA). Nuclear factor IA (NFIA, an experimentally validated miR-223 target[13]) appeared to be downregulated in cells treated with 3.5 mM Pi, as expected (given the increase in miR-223). The low level of NFIA was confirmed by Western blotting (Figure 2D). In contrast, some of the synthetic phenotypic markers (such as Krüppel-like factor 4 [KLF4], KLF5, platelet derived growth factor receptor-α [PDGFR α] and versican [VSCN]) which are all targeted by miR-143 and/or miR-145[7] were significantly upregulated; this was expected, given the observed downregulation of the corresponding miRNAs (Figure 2A).

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