Stainless steel ions stimulate increased thrombospondin-1-dependent TGF-beta activation by vascular smooth muscle cells: implications for in-stent restenosis

Abstract

Background/aims: Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release of metal ions. Stainless steel (SS), a metal alloy widely used in stents, releases ions to the vessel wall and induces reactive oxygen species, inflammation and fibroproliferative responses. The molecular mechanisms are unknown. TGF-beta is known to be involved in the fibroproliferative responses of vascular smooth muscle cells (VSMCs) in restenosis, and TGF-beta antagonists attenuate ISR. We hypothesized that SS ions induce the latent TGF-beta activator, thrombospondin-1 (TSP1), through altered oxidative signaling to stimulate increased TGF-beta activation and VSMC phenotype change.

Methods: VSMCs were treated with SS metal ion cocktails, and morphology, TSP1, extracellular matrix production, desmin and TGF-beta activity were assessed by immunoblotting.

Results: SS ions stimulate the synthetic phenotype, increased TGF-beta activity, TSP1, increased extracellular matrix and downregulation of desmin in VSMCs. Furthermore, SS ions increase hydrogen peroxide and decrease cGMP-dependent protein kinase (PKG) signaling, a known repressor of TSP1 transcription. Catalase blocks SS ion attenuation of PKG signaling and increased TSP1 expression.

Conclusions: These data suggest that ions from stent alloy corrosion contribute to ISR through stimulation of TSP1-dependent TGF-beta activation.

Fig. 1

TSP1 and active TGF-β (pSmad 2) are expressed in arteries with ISR. a Left circumflex artery showing restenotic remodeling from a patient who received a SS drug-eluting stent (Taxus) at least 1 year prior to harvesting of vessels with ISR at the time of cardiac transplant. There is staining for both TSP1 and nuclear pSmad 2 in the endothelium and in the restenotic neointimal VSMCs (NI). There are also macrophages (*) and VSMCs in a region of atheroma (A) adjacent to the restenotic remodeling that also stain for pSmad 2 and TSP1, respectively. L = Lumen; M = media. b Section from a proximal left anterior descending artery implanted with 4 Cypher stents 12 months prior to autopsy. The VSMCs in the mature, fibrous neointima (NI) are positive for both TSP1 and nuclear pSmad 2. The rectangle denotes the area shown at 20×. Control sections incubated with nonimmune IgG show no staining. L = Lumen.

Fig. 1

TSP1 and active TGF-β (pSmad 2) are expressed in arteries with ISR. a Left circumflex artery showing restenotic remodeling from a patient who received a SS drug-eluting stent (Taxus) at least 1 year prior to harvesting of vessels with ISR at the time of cardiac transplant. There is staining for both TSP1 and nuclear pSmad 2 in the endothelium and in the restenotic neointimal VSMCs (NI). There are also macrophages (*) and VSMCs in a region of atheroma (A) adjacent to the restenotic remodeling that also stain for pSmad 2 and TSP1, respectively. L = Lumen; M = media. b Section from a proximal left anterior descending artery implanted with 4 Cypher stents 12 months prior to autopsy. The VSMCs in the mature, fibrous neointima (NI) are positive for both TSP1 and nuclear pSmad 2. The rectangle denotes the area shown at 20×. Control sections incubated with nonimmune IgG show no staining. L = Lumen.

Fig. 2

SS ions stimulate increased TGF-β activity, TSP1, extracellular matrix production and reduce desmin expression. VSMCs (p3–4) were cultured in the presence of 0.1–1.0 ppm SS ion cocktail in dilute acid, TGF-β (200 pmol/l) or TSP1 (32 nmol/l) for 24 h. Pooled cell lysates from triplicate samples were harvested and proteins were immunoblotted for pSmad 2 and total Smad 2/3 (n = 3) (a) or type I collagen (n = 4), TSP1 (n = 3) (b) and ED-A fibronectin (n = 3) or α-SMA (n = 3) (c). Blots were stripped and reprobed for β-tubulin as a loading control. d Pooled cell lysates of triplicate samples were harvested at 48 h of treatment and immunoblotted for desmin (n = 4). Blots were stripped and reprobed for β-tubulin.

Fig. 3

SS ion treatment induces the synthetic phenotype in VSMCs. VSMCs were cultured for 24 h in presence of dilute acid (a, c) or 0.1 ppm SS ions (b, d). Cells were fixed and permeabilized and then stained for calponin (a, b) and vimentin (c, d) by indirect immunofluorescence with an Alexa Fluor 488 secondary antibody. 40×.

Fig. 4

Blockade of TSP1-dependent TGF-β activation reduces TGF-β signaling and matrix expression due to SS ions and increases desmin expression. VSMCs (p3) were treated with 0.1 ppm SS ions for 24 h in the presence or absence of LSKL or SLLK (1 μmol/l), GGWSHW (20 μmol/l) or Mab 133 monoclonal antibody to TSP1 or nonimmune mouse IgG (25 μg/ml). Active TGF-β (200 pmol/l) was used as a positive control. Pooled cell lysates from triplicate samples were harvested, separated by SDS-PAGE and proteins analyzed by immunoblotting for phospho-Smad 2 (n = 3) (a) or ED-A fibronectin (n = 3) (b). Membranes were stripped and reprobed for β-tubulin. c In separate experiments, cells were treated with 0.1 ppm SS ions for 48 h in the presence of either 1 μmol/l LSKL or SLLK peptide and cell lysates were harvested and immunoblotted for desmin (n = 3).

Fig. 5

SS ions increase H₂O₂ in VSMCs. H₂O₂ levels were measured using the Amplex Red Hydrogen Peroxide/Peroxidase assay according to the manufacturer's instructions. a VSMCs (p5) were treated with SS ions (0.1 ppm), TSP1 (32 nmol/l) or active TGF-β (200 pmol/l) for 1–4 h. Hydrogen peroxide was measured as fluorometric emission at 580 nm following excitation at 530 nm using a Cytofluor 4000 microplate reader. Results are expressed as mean fluorometric units ± SD of triplicate samples read at 3 h. b In the same experiment, VSMCs (p5) were treated with SS ions in the presence or absence of 100 units of catalase or with catalase alone. Results are expressed as mean fluorometric units ± SD of triplicate samples read at 4 h. c VSMCs (p5) were treated with cocktails of SS ions, each lacking one of the metal ion components. Results are expressed as mean fluorometric units ± SD of triplicate samples read at 4 h. All experiments were performed in triplicate in at least 2 separate experiments. * p < 0.027 vs. SS ion treatment; ** p < 0.001 vs. SS ion treatment. n.s. = Not significant.

Fig. 6

Catalase reduces SS ion-induced TSP1 and ED-A FN expression and TGF-β activity in VSMCs. VSMCs (p4) were treated with SS ions (0.1 ppm) in the presence or absence of catalase (100 units/ml). Media with dilute acid was used as a control for the SS ion preparation. Cells analyzed for pSmad 2 were treated with both 100 units/ml and 500 units/ml catalase. Active TGF-β (200 pmol/l) or the PKG activator, 8pCPT-cGMP (1 mmol/l), were used as positive controls for stimulation of pSmad 2 and PKG activity, respectively. a Cells harvested after 24 h of treatment were assayed for TSP1 and ED-A FN (n = 5). b Cells harvested at 6 h were analyzed for pSmad 2 (n = 3) by immunoblotting of pooled cell lysates from triplicate samples. Blots were reprobed for β-tubulin.

Fig. 7

SS ions reduce PKG-dependent VASP phosphorylation in VSMCs. VSMCs (p2) were treated with DMEM, dilute acid in DMEM or 0.1 ppm SS ions in dilute acid for 1 or 6 h. Pooled cell lysates of triplicate samples were harvested and immunoblotted with rabbit anti-PKG and rabbit anti-phospho-VASP (ser239) antibodies (n = 3). Cells treated with an activator of PKG, 8pCPT-cGMP (1 mmol/l), were used as a positive control for VASP phosphorylation. Blots were stripped and reprobed for β-tubulin as a loading control.

Fig. 8

Catalase blocks SS ion reduction in PKG-dependent VASP phosphorylation in VSMCs. VSMCS (p4) were treated with SS ions (0.1 ppm) for 6 h in the presence or absence of either 100 units/ml or 500 units/ml catalase. Dilute acid is a control for SS ion preparation. 8pCPT-cGMP (1 mmol/l) is a positive control for PKG-dependent VASP phosphorylation. Cell lysates of pooled triplicate samples were analyzed for phospho-VASP (ser239) by immunoblotting (n = 3). Blots were stripped and reprobed for β-tubulin.