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. 2019 Sep 27:19:872-882.
doi: 10.1016/j.isci.2019.08.044. Epub 2019 Aug 27.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation

Affiliations

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation

Xiujie Xie et al. iScience. .

Abstract

Vascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

Keywords: Molecular Biology; Molecular Mechanism of Behavior; Pathophysiology; Vascular Remodeling.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Perivascular Administration of DSF Reduces IH In Vivo in a Rat Angioplasty Model Rat carotid artery balloon angioplasty was performed followed by periadventitial administration of disulfiram (DSF, 9 mg/rat) or vehicle control (equal amount of DMSO). The animals were euthanized 14 days later, and the arteries were collected for histological analyses. Shown are representative H&E-stained carotid artery cross sections and quantification of the intima versus media area ratio (I/M, measure of IH). The data were generated by averaging six to eight sections from each animal. The averages from six animals in each treatment group were then averaged (see the scatterplot). *p < 0.05, n = 6 animals, Student's t test. A pair of arrows define the neointima thickness. A, adventitia; M, media; N, neointima. Scale bar: 100 μm.
Figure 2
Figure 2
The ALDH1A3 Isoform Predominantly Accounts for Cytokine-Stimulated Increase of VSMC Total ALDH Activity (A) Increase of ALDH activity in AoSMCs (human) in response to treatment with cytokines. (B) Increase of ALDH activity in MOVAS (mouse) in response to treatment with cytokines. (C) Effects of cytokines on mRNA levels of 19 ALDH isoforms. (D) Effects of cytokines on protein levels of ALDH1A3. Cultured AoSMCs and MOVAS cells were starved for 24 h and then treated with PDGF-BB (40 ng/mL), IL1β (10 ng/mL), TGFβ1 (20 ng/mL), TNFα (20 ng/mL), or solvent control (4 mM HCl/0.1% BSA) for another 24 h. ALDH activity was determined using fluorescence-activated cell sorting analysis (FACS, see Figure S1) and presented as the percentage of the fluorescently positive cell number versus the total cell number. The mRNA expression of each ALDH isoform was determined using real-time qPCR. For Western blot replicates, samples of equal amount of total protein were loaded, and the exposure time length was kept consistent for the same protein to be detected. The densitometry of ALDH1A3 was normalized to that of beta-actin. Data are expressed as mean ± SE derived from three independent experiments. Student's t test: *p < 0.05, **p < 0.01, ***p < 0.001, each compared with solvent control; r.u., relative unit.
Figure 3
Figure 3
ALDH1A3 Knockdown Reduces ALDH Activity, VSMC Proliferation, and the Expression of ESM1 and TNC1 (A) Western blots indicating efficient ALDH1A3 knockdown. (B) ALDH1A3 knockdown abolishes the PDGF stimulation of total ALDH activity. (C) ALDH1A3 knockdown abolishes the PDGF stimulation of AoSMC proliferation (CellTiter-Glo assay). (D) ALDH1A3 knockdown reduces protein and mRNA levels of ESM1 and TNC1. (E and F) ESM1 or TNC1 knockdown inhibits AoSMC proliferation. Human AoSMCs were cultured and treated with solvent control or PDGF-BB as described in Figure 1. For ALDH1A3 knockdown, shRNA was initially used (in A and C) and then siRNA was used throughout (B and D). ALDH activity was determined using fluorescence-activated cell sorting analysis (FACS, Figure S2) and presented as the percentage of fluorescently positive cells versus total cells. mRNA expression of individual ALDH isoform was determined using real-time qPCR. Values were expressed as means ± SE derived from three independent experiments. Student's t test: **p < 0.01, ***p < 0.001, each compared with scrambled control.
Figure 4
Figure 4
Effects of Gene Silencing of ALDH1A3, ESM1, or TNC1 on the AKT/mTOR and MEK/ERK Signaling Pathways in AoSMCs (A and B) Western blots of phospho-AKT, phospho-mTOR, and phospho-Ribosomal protein S6 (RPS6) in AoSMCs transfected with a scrambled siRNA (Scr.) or an siRNA to knock down ALDH1A3, ESM1, or TNC1. (C and D) Western blots of p-MEK, p-p38, and CyclinD1 in AoSMCs transfected with a scrambled siRNA or an siRNA to knock down ALDH1A3, ESM1, or TNC1. In both (A) and (B), bar graphs show the quantification of protein levels. For western blot replicates, samples of equal amount of total protein were loaded, and the exposure time was kept consistent for the same protein to be detected. Values are expressed as means ± SE derived from three independent experiments. Student's t test: *p < 0.05, ***p < 0.001, each compared with scrambled control. P, phosphoprotein; T, total protein.
Figure 5
Figure 5
Pan-ALDH Inhibitor DSF Reverses PDGF-stimulated VSMC Phenotypic Changes AoSMCs, MOVAS cells, or rat SMCs were cultured, starved, and then treated with solvent control or PDGF-BB (as described in Figure 1), with or without pretreatment with 10 μM DSF for 3 h. (A) DSF inhibits PDGF-stimulated cell proliferation (determined by CellTiter-Glo viability assay). Mean ± SD, n = 3. (B) DSF inhibits PDGF-stimulated cell migration (scratch assay). Shown are calcein-stained cells 20 h after PDGF-BB stimulation. (C) DSF does not induce MOVAS cell apoptosis (flow sorting). (D) DSF inhibits PDGF-stimulated activation of AKT and MEK signaling pathways. Shown are representative blots from at least two similar experiments. P, phosphoprotein; T, total protein.
Figure 6
Figure 6
ALDH1A3 Upregulation in Injured Rat Carotid Arteries Rat carotid artery balloon angioplasty and periadventitial administration of DSF were performed as described in Figure 1. Artery cross sections were immunostained for ALDH1A3 (A) and TNC1 and ESM1 (B). Negative control staining was performed with the same procedures except that primary antibody was omitted. Positive staining was measured as colorimetric intensity per image field and quantified by averaging six to eight sections from each animal, and the averages from all of the six animals in each treatment group were then averaged (see the scatter plots). Student's t test: *p < 0.05, ***p < 0.001. A, adventitia; M, media; N, neointima. Scale bar: 100 μm.

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