TGF-β is recognized for its ability to enhance intimal hyperplasia following arterial injury, but conflicting evidence exists regarding its effect on adaptive remodelling.11,13,18,19
Smad3 is a signalling protein known to mediate many of TGF-β's effects, and in these studies, we evaluated its role in arterial response to injury—specifically, in adaptive remodelling.
Using the rat carotid balloon angioplasty model, we first found that arterial injury leads to significant induction of endogenous Smad3. We also found that Smad3 overexpression via gene transfer resulted in increased luminal area, despite enhanced neointimal hyperplasia. Our findings thus demonstrate that TGF-β/Smad3 pathway activation stimulates intimal hyperplasia as well as adaptive remodelling that compensates for that hyperplasia. Results from PCNA immunostaining revealed a significant increase in the number of proliferating cells in the intima, media, and adventitia of AdSmad3-treated arteries. The increased neointimal formation may be related to enhanced SMC proliferation from a direct effect of Smad3 on medial or neointimal SMCs. Moreover, proliferating myofibroblasts from the adventitia may also migrate into and enhance the formation of neointima.
Kobayashi et al
also recently addressed the role of TGF-β/Smad3 in arterial injury. Using a photochemical femoral artery injury model in an Smad3-null mouse, they found that Smad3 gene deficiency, compared with controls, leads to a significantly greater intima:media ratio, luminal loss, and arterial occlusion. These findings would appear to conflict with ours, in that our data suggest that Smad3 overexpression increases intimal hyperplasia. There are several important differences between the two studies.
Kobayashi's group used a different arterial injury model than ours; possibly different injury response mechanisms are elicited by a photochemical vs. the mechanical injury associated with balloon angioplasty. Moreover, the use of an Smad3-null mouse results in a global deficiency of the protein affecting all cells including white blood, endothelial, SMCs, and adventitial cells. Our local gene transfer resulted in the local overexpression of Smad3, limited just to the media. However, overexpression may not always reproduce the true function of an endogenous protein. Future studies employing targeted gene deletion or ‘knock-down’ of Smad3 expression in the arterial wall will be necessary to further clarify the role of endogenous Smad3 in vascular remodelling as well as intimal hyperplasia.
Since gene transfer of Smad3 produced similar effects on arterial remodelling as TGF-β3, as has been previously demonstrated by Kingston et al
it is possible that TGF-β3 actives the Smad3 pathway more profoundly in an injured artery compared with other TGF-β isoforms. However, this hypothesis requires validation.
Our findings, furthermore, offer persuasive evidence that TGF-β/Smad3-induced adaptive remodelling is mediated by medial SMC production of CTGF. Support for this includes the following: (i) both in vitro and in vivo, Smad3 overexpression in medial SMCs leads to increased CTGF production; (ii) blocking CTGF synthesis with siRNA attenuates the crosstalk between SMCs and adventitial fibroblasts; (iii) application of recombinant CTGF to the injured arterial adventitia causes adaptive remodelling and mimics the effect of Smad3 gene transfer.
It has previously been shown that TGF-β stimulates CTGF production via Smad3.34
Moreover, an Smad-binding element has been identified in the CTGF promoter.34
TGF-β plays a profibrotic role in a number of disease processes in which CTGF mediates TGF-β's effects.24,34,35
A strong relationship between TGF-β, CTGF, and human disease is therefore pre-existent. Fibroblasts have been shown to mediate these pathological effects of TGF-β and CTGF.36
CTGF has also been shown to stimulate myofibroblast transformation, proliferation, and matrix protein in skin, lung, and kidney fibroblasts.24,32,35
In the in vitro fibroblast study, medium conditioned by AdNull-infected TGF-β-treated SMCs stimulated both α-actin expression and proliferation of fibroblasts compared with medium conditioned by non-TGF-β-treated SMCs. This stimulatory effect of conditioned medium is potentially mediated by the endogenous Smad3 pathway within SMCs. Consistently, Adnull-infected SMCs responded to TGF-β with enhanced production of CTGF, presumably mediated by endogenous Smad3. Alternatively, TGF-β that used to stimulate SMCs would have remained in the conditioned media as it was too small to be eliminated by filtering. The residual TGF-β could directly act on fibroblasts.
Of note, we observed that endogenous CTGF is up-regulated in the intima, media, and adventitia of AdSmad3-treated arteries. Since CTGF can be synthesized by medial SMCs and secreted into the extracellular space, it is not entirely unexpected that we would detect this soluble factor in all three layers of the arteries. It should also be pointed out that our data do not exclude intimal cells or fibroblasts as sources of CTGF synthesis. However, the fact that only medial SMCs were infected with AdSmad3 in this study suggests that Smad3-expressing cells are the major source of CTGF up-regulation in AdSmad3-infected arteries.
Jiang et al
have recently suggested a role for CTGF in the vein graft remodelling. Using a rabbit vein graft model, these authors found that increased intramural wall stress following vein graft implantation resulted in an up-regulation of both TGF-β and CTGF. They also observed that TGF-β and CTGF levels were inversely correlated with the magnitude of outward remodelling of the vein graft. They found, as we did, that CTGF production was associated with myofibroblast transformation and cell migration. Their data and ours confirm an important role for TGF-β/CTGF in vessel wall remodelling. Contrary to our findings, however, these authors have concluded that the effect of CTGF is to limit, rather than enhance, outward vessel remodelling. Admittedly, the models are quite different; these authors have studied an arteriovenous vein graft fistula, and our studies are after arterial injury. They found an association between elevated CTGF and diminished outward remodelling but did not demonstrate a causal relationship; we have shown that direct application of CTGF to the arterial adventitia produces adaptive remodelling. Nevertheless, taken together, both studies suggest that CTGF, released in response to TGF-β, plays a pivotal role in the architectural changes that follow vessel injury.
Both Smad3 gene transfer and CTGF application led to similar cellular changes in the adventitia: increased cell proliferation, SMA expression, and collagen accumulation. We have assumed that it is through these effects that CTGF produces adaptive remodelling. However, the exact mechanisms that lead to adaptive remodelling remain elusive. Although medial gene transfer of Smad3 and adventitial application of CTGF produced very similar effects on the arterial injury response, the link between Smad3 and CTGF that we observed in cell culture remains to be directly tested in vivo. Our future experiments will focus on using CTGF-specific siRNA to test the ability of Smad3 to regulate arterial remodelling in the absence of CTGF induction.
Myofibroblast transformation, proliferation, and collagen production appear to be involved, but precisely how is not clear. Although excess collagen was originally thought to promote constrictive remodelling, there is increasing evidence, including our current data, suggesting that excess collagen may lead to adaptive remodelling. We found an increase in adventitial collagen in AdSmad3- and CTGF-treated arteries, both of which exhibited adaptive remodelling.
Our observations in vivo
regarding increased collagen deposition in the adventitia of arteries exhibiting adaptive remodelling as well as our in vitro
evidence of increased collagen synthesis by fibroblasts imply that collagen turnover is integral to adaptive remodelling. To this end, matrix metalloproteases (MMPs), specifically MMP-2 and MMP-9, which have been implicated by others in the arterial response to injury, may play an important role in this process. In future studies, we will test this hypothesis using this and similar models.38–40
TGF-β has been shown to induce the expression of MMP-9 in human meningeal cells and in a human head and neck squamous cell carcinoma cell line.41,42
Moreover, using human endometrial carcinoma cell lines, Van Themsche et al.43
demonstrated that TGF-β3 (not β1 or β2) increases the invasiveness of tumour cells through production of MMP-9. It is possible that TGF-β3 through Smad3 up-regulates the expression of MMPs in the injured arterial wall which subsequently promotes arterial remodelling. In future studies, we will test this hypothesis in various arterial injury models.
Our finding that CTGF stimulates both intimal hyperplasia and adaptive remodelling is of interest. We speculate that the expanded intimal hyperplasia in CTGF-treated arteries is, in part, caused by myofibroblast migration into the neointima. It is also possible that exogenous CTGF may diffuse from the adventitia to the media and directly affect medial SMCs. CTGF has been reported to directly stimulate vascular SMC proliferation in vitro
Stimuli that produce intimal hyperplasia presumably simultaneously initiate compensatory expansion. In further support of this concept is the observation of significantly increased neointimal hyperplasia in the Smad3-overexpressing arteries displaying adaptive remodelling.
A limitation of the current study is our inability to detect phosphorylated Smad3 in vivo. Phosphorylation of Smad3 by the TGF-β receptor is a key step in the transduction of TGF-β signal. Although it would be ideal to prove in vivo that Smad3 gene transfer leads to an increase in phosphorylated Smad3, for a number of reasons, we feel confident that Smad3 was activated. First, compared with null virus, in vivo Smad3 expression resulted in a significant change in arterial morphology. Smad3 would not be able to produce an effect without being phosphorylated and consequently activated. Also, it is well established that the rat carotid injury model produces high local levels of TGF-β, which is the direct activator of Smad3. Lastly, we have shown, in vitro, that SMCs overexpressing Smad3 and stimulated with TGF-β produce increased levels of phosphorylated Smad3.
In conclusion, we have established that TGF-β, through an Smad3 pathway, can induce adaptive remodelling following arterial injury. We have established a novel mechanism for this effect. TGF-β activates Smad3 in medial SMCs, causing them to secrete CTGF, which in turn stimulates adventitial fibroblasts to migrate, proliferate, produce collagen, and transform into myofibroblasts. It is presumably through these events that changes in the arterial architecture occur. This process is complex, and our data does not indicate an obvious target that can be manipulated to enhance adaptive remodelling. For example, accentuation of Smad3 or CTGF in the arterial wall does produce adaptive remodelling, but this event is accompanied by the synchronous stimulation of intimal hyperplasia. In the case of Smad3, there is a modest increase in luminal diameter, and with CTGF, there is no net change. It is our hope, however, that these studies will open the door for further investigations of the precise mechanisms of this process, with the goal of eventually defining targets that will specifically enhance adaptive remodelling and diminish the deleterious effects of restenosis.