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Vascular Proteomics Reveal Novel Proteins Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Regulating Proliferation and Inflammatory Response.

Kang DH, Choi M, Chang S, Lee MY, Lee DJ, Choi K, Park J, Han EC, Hwang D, Kwon K, Jo H, Choi C, Kang SW - PLoS ONE (2015)

Bottom Line: The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury.Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane.Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Research Center for Cell Homeostasis, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Neointimal hyperplasia of vascular smooth muscle cells (VSMC) plays a critical role in atherosclerotic plaque formation and in-stent restenosis, but the underlying mechanisms are still incompletely understood. We performed a proteomics study to identify novel signaling molecules organizing the VSMC hyperplasia. The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury. The combination of cellular function assays and a protein network analysis further demonstrated that 27 out of 44 proteins constitute key signaling networks orchestrating the phenotypic change of VSMC from contractile to epithelial-like synthetic. Among the list of proteins, the in vivo validation specifically revealed that six proteins (Rab15, ITR, OLR1, PDHβ, PTPε) are positive regulators for VSMC hyperplasia. In particular, the OLR1 played dual roles in the VSMC hyperplasia by directly mediating oxidized LDL-induced monocyte adhesion via NF-κB activation and by assisting the PDGF-induced proliferation/migration. Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane. Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

No MeSH data available.


Related in: MedlinePlus

In vitro cell assays for functional validation of differentially-expressed protein candidates in HASMCs.HASMCs were transfected with the specific siRNA mixes listed in the S1 Table for 18 hr and stimulated with PDGF-BB (25 ng/ml) or TNF-α (10 ng/ml). Each experiment was performed in triplicate and the data in the graph are means ± S.D. of fold changes from three independent experiments (*P<0.02, **P<0.002). UT, untreated; C, control siRNA-transfected cells; S, cytosolic S-100; N, nuclei; M, membrane. The number designated each bar corresponds to the spot number.
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pone.0133845.g002: In vitro cell assays for functional validation of differentially-expressed protein candidates in HASMCs.HASMCs were transfected with the specific siRNA mixes listed in the S1 Table for 18 hr and stimulated with PDGF-BB (25 ng/ml) or TNF-α (10 ng/ml). Each experiment was performed in triplicate and the data in the graph are means ± S.D. of fold changes from three independent experiments (*P<0.02, **P<0.002). UT, untreated; C, control siRNA-transfected cells; S, cytosolic S-100; N, nuclei; M, membrane. The number designated each bar corresponds to the spot number.

Mentions: A general concern has emerged whereby the expression pattern of a particular gene of interest can be different among the species. Thus, we decided to validate the cellular function of the rodent-origin 43 candidate proteins, except Ba1 (spot: M2), in human aortic VSMCs (HASMCs). The expression of each target gene was knocked down in the HASMCs by a reverse transfection with a mixture of four specific small interfering RNAs (siRNAs), as described in Materials and Methods (S2 Table). The high in vitro transfection efficiency was tested using a red fluorescent siGLO probe. The proliferation and chemotactic migration of aortic VSMCs were induced by PDGF-BB, which is a major VSMC growth factor; whereas, the monocyte adhesion to VSMCs was induced by tumor necrosis factor-α (TNF-α), a major pro-inflammatory cytokine. Both PDGF-BB and TNF-α are presumed to be produced mainly by macrophages in the atherosclerotic and balloon-injured lesions. From the extensive cell-based assays performed in triplicate, we found that depletion of 27 proteins showed positive and negative effects on one or more cellular activities; whereas, the depletion of residual 16 proteins had no effect on all three cellular activities (Fig 2 and Table 1). It supposed that the latter false positive proteins could be identified due possibly to the use of entire carotid vessel, including neointimal and medial layers, for proteomics analysis. Among those showing the effector activity, some of the candidate proteins were likely to be the master regulator in diverse cellular activities of human VSMCs. For example, the knockdown of PKAα catalytic subunit (PKACA) increased all three cellular activities; whereas, the knockdown of six proteins (Rab-15, Lactate dehydrogenase B, Intimal thickness-related receptor (ITR/GPR180), Oxidized LDL receptor (OLR1/LOX-1), and IL-12 receptor β2 (IL-12Rβ2)) significantly reduced three types of VSMC activities. It is noteworthy that the latter 5 positive effector proteins could be targeted for prevention of typical vascular SMC hyperplasia. Furthermore, we performed in-depth validation of two candidates, ITR and PKACA, as representative positive and negative effectors, respectively. RT-PCR showed the almost perfect knockdown of two gene expressions (S3A Fig). The in vitro cell assays confirmed that the depletion of ITR expression consistently reduced the PDGF-induced proliferation and chemotactic migration of human SMCs and inhibited the monocyte adhesion to human SMCs pre-activated by TNF-α treatment (S3B Fig). In contrast, the depletion of PKACA markedly augmented those SMC activities (S3C Fig). Collectively, the data indicate that the in vitro cell-based validation using human VSMCs effectively filtered out human-irrelevant false-positives among a number of candidate proteins screened from rodent disease model.


Vascular Proteomics Reveal Novel Proteins Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Regulating Proliferation and Inflammatory Response.

Kang DH, Choi M, Chang S, Lee MY, Lee DJ, Choi K, Park J, Han EC, Hwang D, Kwon K, Jo H, Choi C, Kang SW - PLoS ONE (2015)

In vitro cell assays for functional validation of differentially-expressed protein candidates in HASMCs.HASMCs were transfected with the specific siRNA mixes listed in the S1 Table for 18 hr and stimulated with PDGF-BB (25 ng/ml) or TNF-α (10 ng/ml). Each experiment was performed in triplicate and the data in the graph are means ± S.D. of fold changes from three independent experiments (*P<0.02, **P<0.002). UT, untreated; C, control siRNA-transfected cells; S, cytosolic S-100; N, nuclei; M, membrane. The number designated each bar corresponds to the spot number.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548952&req=5

pone.0133845.g002: In vitro cell assays for functional validation of differentially-expressed protein candidates in HASMCs.HASMCs were transfected with the specific siRNA mixes listed in the S1 Table for 18 hr and stimulated with PDGF-BB (25 ng/ml) or TNF-α (10 ng/ml). Each experiment was performed in triplicate and the data in the graph are means ± S.D. of fold changes from three independent experiments (*P<0.02, **P<0.002). UT, untreated; C, control siRNA-transfected cells; S, cytosolic S-100; N, nuclei; M, membrane. The number designated each bar corresponds to the spot number.
Mentions: A general concern has emerged whereby the expression pattern of a particular gene of interest can be different among the species. Thus, we decided to validate the cellular function of the rodent-origin 43 candidate proteins, except Ba1 (spot: M2), in human aortic VSMCs (HASMCs). The expression of each target gene was knocked down in the HASMCs by a reverse transfection with a mixture of four specific small interfering RNAs (siRNAs), as described in Materials and Methods (S2 Table). The high in vitro transfection efficiency was tested using a red fluorescent siGLO probe. The proliferation and chemotactic migration of aortic VSMCs were induced by PDGF-BB, which is a major VSMC growth factor; whereas, the monocyte adhesion to VSMCs was induced by tumor necrosis factor-α (TNF-α), a major pro-inflammatory cytokine. Both PDGF-BB and TNF-α are presumed to be produced mainly by macrophages in the atherosclerotic and balloon-injured lesions. From the extensive cell-based assays performed in triplicate, we found that depletion of 27 proteins showed positive and negative effects on one or more cellular activities; whereas, the depletion of residual 16 proteins had no effect on all three cellular activities (Fig 2 and Table 1). It supposed that the latter false positive proteins could be identified due possibly to the use of entire carotid vessel, including neointimal and medial layers, for proteomics analysis. Among those showing the effector activity, some of the candidate proteins were likely to be the master regulator in diverse cellular activities of human VSMCs. For example, the knockdown of PKAα catalytic subunit (PKACA) increased all three cellular activities; whereas, the knockdown of six proteins (Rab-15, Lactate dehydrogenase B, Intimal thickness-related receptor (ITR/GPR180), Oxidized LDL receptor (OLR1/LOX-1), and IL-12 receptor β2 (IL-12Rβ2)) significantly reduced three types of VSMC activities. It is noteworthy that the latter 5 positive effector proteins could be targeted for prevention of typical vascular SMC hyperplasia. Furthermore, we performed in-depth validation of two candidates, ITR and PKACA, as representative positive and negative effectors, respectively. RT-PCR showed the almost perfect knockdown of two gene expressions (S3A Fig). The in vitro cell assays confirmed that the depletion of ITR expression consistently reduced the PDGF-induced proliferation and chemotactic migration of human SMCs and inhibited the monocyte adhesion to human SMCs pre-activated by TNF-α treatment (S3B Fig). In contrast, the depletion of PKACA markedly augmented those SMC activities (S3C Fig). Collectively, the data indicate that the in vitro cell-based validation using human VSMCs effectively filtered out human-irrelevant false-positives among a number of candidate proteins screened from rodent disease model.

Bottom Line: The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury.Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane.Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Research Center for Cell Homeostasis, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Neointimal hyperplasia of vascular smooth muscle cells (VSMC) plays a critical role in atherosclerotic plaque formation and in-stent restenosis, but the underlying mechanisms are still incompletely understood. We performed a proteomics study to identify novel signaling molecules organizing the VSMC hyperplasia. The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury. The combination of cellular function assays and a protein network analysis further demonstrated that 27 out of 44 proteins constitute key signaling networks orchestrating the phenotypic change of VSMC from contractile to epithelial-like synthetic. Among the list of proteins, the in vivo validation specifically revealed that six proteins (Rab15, ITR, OLR1, PDHβ, PTPε) are positive regulators for VSMC hyperplasia. In particular, the OLR1 played dual roles in the VSMC hyperplasia by directly mediating oxidized LDL-induced monocyte adhesion via NF-κB activation and by assisting the PDGF-induced proliferation/migration. Importantly, OLR1 and PDGFRβ were associated in close proximity in the plasma membrane. Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

No MeSH data available.


Related in: MedlinePlus