Induction of MiR133a expression by IL-19 targets LDLRAP1 and reduces oxLDL uptake in VSMC - PubMed (original) (raw)

Induction of MiR133a expression by IL-19 targets LDLRAP1 and reduces oxLDL uptake in VSMC

Khatuna Gabunia et al. J Mol Cell Cardiol. 2017 Apr.

Abstract

The transformation of vascular smooth muscle cells [VSMC] into foam cells leading to increased plaque size and decreased stability is a key, yet understudied step in atherogenesis. We reported that Interleukin-19 (IL-19), a novel, anti-inflammatory cytokine, attenuates atherosclerosis by anti-inflammatory effects on VSMC. In this work we report that IL-19 induces expression of miR133a, a muscle-specific miRNA, in VSMC. Although previously unreported, we report that miR133a can target and reduce mRNA abundance, mRNA stability, and protein expression of Low Density Lipoprotein Receptor Adaptor Protein 1, (LDLRAP1), an adaptor protein which functions to internalize the LDL receptor. Mutations in this gene lead to LDL receptor malfunction and cause the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans. Herein we show that IL-19 reduces lipid accumulation in VSMC, and LDLRAP1 expression and oxLDL uptake in a miR133a-dependent mechanism. We show that LDLRAP1 is expressed in plaque and neointimal VSMC of mouse and human injured arteries. Transfection of miR133a and LDLRAP1 siRNA into VSMC reduces their proliferation and uptake of oxLDL. miR133a is significantly increased in plasma from hyperlipidemic compared with normolipidemic patients. Expression of miR133a in IL-19 stimulated VSMC represents a previously unrecognized link between vascular lipid metabolism and inflammation, and may represent a therapeutic opportunity to combat vascular inflammatory diseases.

Keywords: Cholesterol uptake; LDLRAP1; Vascular smooth muscle cell; miR133a.

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Conflict of interest statement

Disclosures:

The authors declare no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations.

Figures

Figure 1

IL-19 significantly increases expression of miRNA133a. A. Human VSMC, EC, or mouse BMDM were treated with IL-19, RNA extracted at the indicated times, and miR133a abundance was detected by quantitative RT-PCR. * indicate P<0.05, ** P<0.01.

Figure 2

miR133a reduces LDLRAP1 expression. A. Targetscan analysis identifies seed regions of miR133a which target regions of LDLRAP1 mRNA. The locations of the miR133a complementary sites on human LDLRAP1 3′UTR are shown. B. Transfection of miR133a mimic at different concentrations reduce LDLRAP1 protein expression. Cell lysates were harvested 24 hours post-transfection and western blotted using the indicated antibodies. C. Densiometric quantitation of LDLRAP1 protein in VSMC transfected with different concentrations of miR133a mimic. D. Time course showing LDLRAP1 mRNA is significantly decreased after transfection with 25ng/ml miR133a mimic. RNA was extracted at the indicated times after transfection, and LDLRAP1 mRNA abundance detected by quantitative RT-PCR. E. Time course showing LDLRAP1 protein is significantly decreased after transfection with 25ng/ml miR133a mimic. Cell lysates were harvested at the indicated times post-transfection and western blotted using the indicated antibodies. F. Densiometric quantitation of LDLRAP1 protein in VSMC transfected with 25ng/ml miR133a mimic. Results from three independent experiments. G. miR133a significantly reduces LDLRAP1 mRNA stability. Human VSMC transfected with either miR133a or scrambled control were treated with Actinomycin D, and RNA extracted at the indicated times. H. miR133a targets and silences LDLRAP1 3′UTR. NIH 3T3 cells transfected with a luciferase reporter containing LDLRAP1 3′UTR and also miR133a or scrambled control. Asterisks indicate significant difference from control. * indicate P<0.05, *** P<0.001.

Figure 3

LDLRAP1 expression in activated VSMC and injured arteries. A. Increased LDLRAP1 expression localizes to neointima in ligated mouse carotid artery. Area marked “m” is the media, “n” is the neointima. B. Secondary IgG [negative control] staining of “A”. C. LDLRAP1 expression in oxLDL stimulated and TNFα-stimulated human VSMC. VSMC lysates were harvested at the indicated times post stimulation and western blotted using the indicated antibodies. D. Densiometric quantitation of LDLRAP1 protein expression in VSMC treated with oxLDL or TNFα. Asterisks indicate significant difference from unstimulated VSMC. * indicate P<0.05.

Figure 4

miR133a and LDLRAP1 expression in atherosclerosis tissue. A. miR133a expression is significantly increased in aortic arch from LDLR−/− mice fed a HFD compared with controls. B. LDLRAP1 mRNA expression is significantly increased in aortic arch from LDLR−/− mice fed a HFD compared with controls. C. LDLRAP1 mRNA is significantly reduced in aortic arch from LDLR−/− mice fed a HFD and also injected with IL-19 compared with saline controls. D. LDLRAP1 protein is expressed in plaque VSMC from LDLR−/− mice. E. higher magnification of “D” showing increased LDLRAP1 expression in medial and cap VSMC in proximity to the plaque. F. Secondary IgG [negative control] staining of “E”. G. higher magnification of “D” showing little to no LDLRAP1 expression in VSMC on the opposite side of the aorta from the plaque. H. higher LDLRAP1 expression in myelofibrotic plaque region compared with medial cells in human atherosclerotic plaque. I. Secondary IgG (negative control) staining of “H”. * indicate P<0.05.

Figure 5

IL-19 reduces LDLRAP1 expression. A. LDLRAP1 mRNA expression is significantly reduced by IL-19. Human VSMC were treated with IL-19, RNA extracted at the indicated times, and LDLRAP1 mRNA abundance detected by quantitative RT-PCR. B. IL-19 significantly reduces LDLRAP1 protein expression. VSMC lysates were harvested at the indicated times post IL-19 stimulation and western blotted using the indicated antibodies. C. Densiometric quantitation of LDLRAP1 protein in VSMC treated with IL-19. Times from 4 to 48 hours were significantly lower compared with untreated cells. D. anti-miR133a prevents IL-19 from reducing LDLRAP1 protein abundance. 25ug anti-miR133a or scrambled control were transfected into human VSMC and stimulated with IL-19. E. densiometric quantitation of LDLRAP1 protein expression in VSMC transfected with anti-miR133a. Asterisk indicates significant reduction in the scrambled versus anti-miR transfected VSMC. * indicate P<0.05, **P<0.01, *** P<0.001.

Figure 6

Reduction in uptake of fluorescently labeled oxLDL in VSMC by IL-19 and miR133a. A. Human VSMC were treated with IL-19 for 16 hours, then uptake quantitated by flow cytometry. B. mouse VSMC were treated with IL-19 for 16 hours, then uptake quantitated by flow cytometry. C. VSMC from IL-19−/− mice take up significantly more oxLDL than wild type VSMC. D. VSMC from IL-19−/− mice express significantly more LDLRAP1 mRNA than wild type VSMC. E. VSMC from IL-19−/− mice express significantly more LDLRAP1 protein than wild type VSMC. F. Densiometric quantitation of LDLRAP1 protein expression in IL-19−/− and wild-type VSMC. Asterisks indicate significant reduction in the treated versus control VSMC. * indicate P<0.05, **P<0.01, *** P<0.001.

Figure 7

miR133a and LDLRAP1 regulate oxLDL uptake in human VSMC. Oxidized LDL uptake was quantitated by flow cytometry 48 hours after transfection. A. transfection of miR133a mimic significantly reduces oxLDL uptake in human VSMC. IL-19 does not further reduce this uptake. B. transfection of LDLRAP1 siRNA significantly reduces oxLDL uptake in human VSMC. IL-19 does not further reduce this uptake. C. anti-miR133a prevents IL-19 from reducing oxLDL uptake in human VSMC. Anti-miR133a also prevents IL-19 from reducing LDLRAP1 protein expression. D. Densiometric quantification of effect of anti-miR133a on LDLRAP1 expression shown in “C”. Asterisks indicate significant reduction in the treated versus control VSMC. * * indicate P<0.01.

Figure 8

miR133a and LDLRAP1 regulate human VSMC proliferation. A. miR133a reduces VSMC proliferation. Human VSMC were transduced with 4 MOI of Adscrambled control vector or AdmiR133a, equal numbers seeded, and VSMC counted at 3 and 6 days post-seeding. B. Relative expression of miR133a in adenoviral transduced VSMC. VSMC were infected with AdmiR133a or Adcontrol vector, RNA isolated and miR133a abundance detected by quantitative RT-PCR. C. LDLRAP1 knockdown reduces VSMC proliferation. VSMC were transfected with LDLRAP1 or scrambled siRNA, equal numbers seeded, and VSMC counted at 3 and 6 days postseeding with a BioRad TC20 automated cell counter, n=3. D. western blot verifying that LDLRAP1 siRNA reduces LDLRAP1 protein expression in human VSMC. Asterisks indicate significant reduction in the treated versus control VSMC. E. miR133a levels are significantly increased in plasma from hyperlipidemic patients. Plasma was isolated from normal, or patients with plasma cholesterol >200mg/dl. RNA was isolated, and miR133a levels quantitated by quantitative RT-PCR. N=9 each. ** indicate P<0.01, *** P<0.001.

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Induction of MiR133a expression by IL-19 targets LDLRAP1 and reduces oxLDL uptake in VSMC - PubMed (original) (raw)