Arterial gene transfer of the TGF-beta signalling protein Smad3 induces adaptive remodelling following angioplasty: a role for CTGF

Abstract

Aims: Although transforming growth factor-beta (TGF-beta) is believed to stimulate intimal hyperplasia after arterial injury, its role in remodelling remains unclear. We investigate whether Smad3, a TGF-beta signalling protein, might facilitate its effect on remodelling.

Methods and results: Using the rat carotid angioplasty model, we assess Smad3 expression following arterial injury. We then test the effect of arterial Smad3 overexpression on the response to injury, and use a conditioned media experimental design to confirm an Smad3-dependent soluble factor that mediates this response. We use small interfering RNA (siRNA) to identify this factor as connective tissue growth factor (CTGF). Finally, we attempt to replicate the effect of medial Smad3 overexpression through adventitial application of recombinant CTGF. Injury induced medial expression of Smad3; overexpression of Smad3 caused neointimal thickening and luminal expansion, suggesting adaptive remodelling. Smad3 overexpression, though exclusively medial, caused adventitial changes: myofibroblast transformation, proliferation, and collagen production, all of which are associated with adaptive remodelling. Supporting the hypothesis that Smad3 initiated remodelling and these adventitial changes via a secreted product of medial smooth muscle cells (SMCs), we found that media conditioned by Smad3-expressing recombinant adenoviral vector (AdSmad3)-infected SMCs stimulated adventitial fibroblast transformation, proliferation, and collagen production in vitro. This effect was attenuated by pre-treatment of SMCs with siRNA specific for CTGF, abundantly produced by AdSmad3-infected SMCs, and significantly up-regulated in Smad3-overexpressing arteries. Moreover, periadventitial administration of CTGF replicated the effect of medial Smad3 overexpression on adaptive remodelling and neointimal hyperplasia.

Conclusion: Medial gene transfer of Smad3 promotes adaptive remodelling by indirectly influencing the behaviour of adventitial fibroblasts. This arterial cell-cell communication is likely to be mediated by Smad3-dependent production of CTGF.

Figure 1

Effects of AdSmad3 on the arterial injury response. (A–C) Representative arterial sections stained with Verhoeff's Elastin Stain 14 days post-injury and treatment with no virus (A), AdNull (B), or AdSmad3 (C). Arrows: medial boundaries. ×40. (D–F) Morphometric analyses of injured arteries. The luminal area (D), EEL area (E), and intima/media ratio (F) were determined by microscopic examination of sections from AdSmad3-treated arteries and compared with those of AdNull controls (n = 6, *P < 0.05).

Figure 2

Effect of AdSmad3 on the adventitia. Sections of uninfected, AdNull-, and AdSmad3-infected arteries harvested 14 days post-injury were assessed for proliferation (A), expressed as the fraction of adventitial nuclei that stained positively for PCNA. Picrosirius staining (B) was used to determine the relative amounts of adventitial collagen. Sections were also immunostained for SMA (C), a marker of myofibroblast transformation, expressed as fraction of adventitial area staining positively (n = 6; *P < 0.05). Arrows: EEL. (A) ×200, (B) ×40, and (C) ×200.

Figure 3

Effects on fibroblast function by media conditioned by SMC in vitro. (A) Western blot for SMA in cell lysate of fibroblasts stimulated by conditioned media taken from SMCs treated as indicated. (B) Proliferation of primary fibroblasts as measured by ³H-thymidine incorporation assay. (C) Collagen production in primary adventitial fibroblasts in response to conditioned media as measured by ³H-proline incorporation assay. (D) COL1A2 promoter activity in an immortalized fibroblast cell line in response to conditioned media, as measured with a luciferase reporter construct (n = 4, *P < 0.05).

Figure 4

Effect of CTGF siRNA on SMC–fibroblast communication. SMCs were infected with AdNull or AdSmad3, allowed to recover for 24 h, and then incubated with siRNA (33 pM) of either a scrambled control or CTGF-specific sequence for 6 h before TGF-β1 stimulation (5 ng/mL, 72 h) (A) Western blot for CTGF in media conditioned by SMCs incubated with a scrambled control siRNA or CTGF-specific siRNA. All SMCs were infected with AdSmad3 and stimulated with TGF-β1. (B–D) Primary fibroblasts were treated with media conditioned by AdSmad3-infected, TGF-β1-stimulated SMCs incubated with no siRNA, scrambled control, and CTGF-specific sequences. Their SMA expression (B), proliferation (C), and collagen production (D) were then evaluated. (E) Activity of COL1A2 promoter in a fibroblast cell line in response to the same media as in (B–D) as measured with a luciferase reporter construct (n = 4, *P < 0.05).

Figure 5

Effects of AdSmad3 on expression of endogenous CTGF. (A–D) Representative sections, harvested 14 days following balloon angioplasty with or without viral infection, immunostained for CTGF. (A) No virus, (B) AdNull, (C) AdSmad3, and (D) AdSmad3 stained with secondary antibody alone (negative control). (E) Quantification of CTGF-positive immunostaining expressed as the fold increase from uninjured control (n = 4, *P < 0.05). ×200.

Figure 6

Effect of periadventitial application of CTGF on the adventitia. Immediately after injury, 400 ng/mL recombinant human CTGF suspended in F127 pluronic gel was applied periadventitially. Fourteen days post-injury, the injured segments of the CTGF-treated arteries were analysed for (A) change in EEL area compared with controls (×40), (B) adventitial collagen content, as measured by Picrosirius Red staining (×40), (C) adventitial SMA expression as measured by immunohistochemistry (×200), and (D) cellular proliferation as measured by PCNA immunostaining. Representative sections of six experiments are shown (×200). Arrows: EEL (n = 6, *P < 0.05).