Expression and suppressive effects of interleukin-19 on vascular smooth muscle cell pathophysiology and development of intimal hyperplasia

Abstract

Anti-inflammatory cytokines may play a protective role in the progression of vascular disease. The purpose of this study was to characterize interleukin (IL)-19 expression and function in the development of intimal hyperplasia, and discern a potential mechanism of its direct effects on vascular smooth muscle cells (VSMCs). IL-19 is an immunomodulatory cytokine, the expression of which is reported to be restricted to inflammatory cells. In the present study, we found that IL-19 is not expressed in quiescent VSMCs or normal arteries but is induced in human arteries by injury and in cultured human VSMCs by inflammatory cytokines. Recombinant IL-19 significantly reduced VSMC proliferation (37.1 +/- 4.8 x 10(3) versus 72.2 +/- 6.1 x 10(3) cells/cm(2)) in a dose-dependent manner. IL-19 adenoviral gene transfer significantly reduced proliferation and neointimal formation in balloon angioplasty-injured rat carotid arteries (0.172 +/- 29.9, versus 0.333 +/- 71.9, and 0.309 +/- 56.6 microm(2)). IL-19 induced activation of STAT3 as well as the expression of the suppressor of cytokine signaling 5 (SOCS5) in VSMCs. IL-19 treatment significantly reduced the activation of p44/42 and p38 MAPKs in stimulated VSMCs. Additionally, SOCS5 was found to interact with both p44/42 and p38 MAPKs in IL-19-treated human VSMCs. This is the first description of the expression of both IL-19 and SOCS5 in VSMCs and of the functional interaction between SOCS5 and MAPKs. We propose that through induction of SOCS5 and inhibition of signal transduction, IL-19 expression in VSMCs may represent a novel, protective, autocrine response of VSMCs to inflammatory stimuli.

Figure 1

Expression of IL-19 in VSMCs. A: Representative immunoblot of human coronary artery VSMCs that were serum-starved for 48 hours and then stimulated with the factors shown for 48 hours. Extracts were immunoblotted with anti-IL-19 or GAPDH antibody. TCM, T-cell conditioned media; IFN-γ, interferon-γ; PDGF, platelet-derived growth factor; TNF-α, tumor necrosis factor-α. B: Immunohistochemical analysis of IL-19 expression in injured arteries. Sections from vessels obtained from a normal heart without coronary artery disease, and a rejected heart from a patient with transplant vasculopathy stained with IL-19 antibody. Red-brown staining indicates antibody recognition. Sections were counterstained with hematoxylin. C: IL-19 co-localizes with VSMCs and leukocytes. Immunofluorescence immunohistochemistry of an artery from a human patient with transplant vasculopathy co-stained with anti-IL-19 (red), SMC-α actin, or CD45 leukocyte common antigen (both green). Scale bar = 50 μm. Original magnifications: ×400 (B); ×600 (C).

Figure 2

Kinetics of IL-19 expression in injured arteries. Rat carotid arteries were subject to balloon angioplasty, and harvested at different times after the procedure. IL-19 expression was detected by immunohistochemistry. Red-brown staining indicates positive staining, and sections were counterstained with hematoxylin. Medial VSMC, m; neointimal, ni. Scale bar = 50 μm.

Figure 3

IL-19 is anti-proliferative for VSMCs. A: Equal numbers of human VSMCs were seeded into 24-well trays and stimulated with the indicated amounts of IL-19 for the indicated times. B: VSMCs were infected with 50 MOI of AdLacZ or IL-19. The next day, VSMCs were seeded into 24-well trays, and counted at the designated times. *P < 0.05, **P < 0.01, ***P < 0.0001 versus control cells for all experiments (n = 3). C: Relative expression of IL-19 in adenoviral-infected VSMCs. Extracts from adenoviral-infected VSMCs were separated by SDS-PAGE, and IL-19 or GAPDH protein was detected by Western blot.

Figure 4

Modulation of IL-19 expression influences neointimal hyperplasia. Adenoviral delivery of IL-19 to balloon angioplasty-injured rat carotid arteries 1 × 10⁸ AdLacZ, AdIL-19, or phosphate-buffered saline (vehicle), 15 minutes dwell time. A–C: Representative sections harvested 14 days after injury stained with H&E. D–F: Representative sections harvested 7 days after injury immunostained with anti-IL-19 antibody. Red-brown indicates positive immunoreactivity. G: IL-19 Western blot of arteries excised from rats treated as described. Morphological and statistical analysis of effect of modulation of IL-19 expression on neointima formation. H: Neointimal area; I: intimal/medial ratio; J: lumen area. Data shown are means ± SEM. Significance versus AdLacZ determined by analysis of variance (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars: 150 μm (A–C); 50 μm (D–F). Original magnifications: ×40 (A–C); ×600 (D–F).

Figure 5

Modulation of IL-19 expression influences neointimal VSMC proliferation. A: Ki-67-positive (red nuclei), SMC-α actin-positive (green cytoplasm) VSMCs in the media and neointima were counted from merged images from at least four representative stained tissue sections at least 100 to 150 μm apart per carotid artery, from three different rats. B: Data shown are means ± SEM. Significance versus AdLacZ and PBS determined by analysis of variance (n = 3). ***P < 0.001. Scale bar = 50 μm. Original magnifications: ×400.

Figure 6

IL-19 induces STAT3 phosphorylation. A: Representative immunoblot of human VSMCs that were serum-starved and then stimulated with 120 nmol/L recombinant IL-19 for the indicated times. Extracts were blotted with anti-phospho STAT3 (Tyr705), or total STAT3. B: Densiometry was performed and values normalized to total protein from at least three experiments. Asterisk indicates significant difference from unstimulated (P < 0.05). C: For STAT3 translocation, VSMCs were serum-starved for 24 hours and then stimulated with IL-19 for the times indicated followed by analysis of compartmental lysates by immunoblot. D: IL-19 induces SOCS5 protein expression. Representative immunoblot of human VSMCs that were serum-starved and then stimulated with IL-19 for the indicated times. Extracts were blotted with SOCS5 antibody. Anti-GAPDH was used as a loading control.

Figure 7

IL-19 decreases MAPK activation. A: Representative immunoblot of human coronary artery VSMCs that were serum-starved and then pretreated with IL-19 for 8 hours. VSMCs were then stimulated with 10% FCS for 15 minutes, and extracts blotted with anti-phospho p44/42 or p38, and total p44/42 or p38. Anti-GAPDH was used as a loading control. B: Densiometry was performed and values normalized to total protein. Values and means from at least three experiments. Asterisk indicates significant difference from control (P < 0.05). Figures are representative of at least four performed. C: SOCS5 interacts with MAPKs. Immunoblot of human coronary artery VSMCs that were serum-starved, pretreated with IL-19 for 8 hours to induce endogenous SOCS5 expression, and then PDGF 15 minutes to induce phosphorylation of MAPKs. Both forward and reverse immunoprecipitation was performed on the same gel. Lysates were incubated with either SOCS5, p44/42, or p38 antibody, and immunoprecipitated. These antibodies used to immunoprecipitate are indicated above the blot. Immunoprecipitates were separated by SDS-PAGE in an individual lane on the same gel. The membrane was cut so that proteins of different molecular weight could be immunoblotted from the same gel, and immunoblotted with either SOCS5, p44/42, or p38 antibody, which are indicated under the blot. The input consisted of 10% of the IP lysate volume run in separate lanes on the same gel, and served as a positive control for the antibody. Representative of at least three experiments.