We have found that there is significantly more cell proliferation in restenotic plaque compared to primary atheromata in patients with lower extremity vascular disease. In addition, restenotic plaque is comprised primarily of SMCs whereas atheromata contain significantly more inflammatory cells. These differences in cellular composition and cell proliferation in conjunction with the novel finding that Smad3 is expressed exclusively in restenotic disease suggest that Smad3 expression may play a role in the mechanism underlying restenotic, as opposed to primary atherosclerotic disease.
It was surprising that we found almost no white blood cells (WBCs) in restenotic plaque, since WBCs and inflammation have been shown to play an integral role in the genesis of atherosclerosis. However, our own immunohistochemical analysis of rat carotid arteries after balloon angioplasty also revealed very few neointimal inflammatory cells, as evidenced by immunohistochemical staining for CD45 and CD68. Moreover our observations are supported by similar findings in other animal models of intimal hyperplasia.22, 23
This differential localization of WBCs predominately in primary but not restenotic lesions highlights another distinction between these two processes. Whereas the inflammatory response is critically important in atherosclerosis; inflammation does not appear to be as essential in the development of restenosis.
While there is a wealth of data regarding the characterization of atherectomy specimens derived from coronary arteries,10-16
there has been only a limited evaluation of peripheral lesions and specifically cell proliferation in peripheral lesions. In several studies rates of apoptosis in peripheral versus central restenotic lesions have been described.3, 24
Others have pooled peripheral and central atherectomy specimens for the characterization of restenotic versus primary atherosclersosis.14, 25, 26
Recently studies with drug-coated stents have implied that the pathophysiology of restenotic disease in coronary versus peripheral vascular disease is quite distinct. Stents coated with rapamycin significantly inhibit restenosis in the coronary circulation whereas rapamycin coated stents were not successful in diminishing the rate of restenosis when used in the peripheral circulation.27
These findings underscore the importance of studying the pathophysiology of restenosis specifically in the peripheral arterial system.
SMC proliferation has been identified as an invariable component of intimal hyperplasia in animals, but previous studies have yielded conflicting results regarding the frequency of cell proliferation in human restenotic lesions.10-16
Pickering et al
. reported a significantly higher percentage of proliferating cells in restenotic as opposed to primary atherosclerotic plaque (59% vs. 20%).14
Our findings support those of Pickering in that we identified that almost 70% of cells in restenotic lesions were PCNA positive whereas slightly over 20% of cells in primary atherosclerotic lesions were proliferating. Our data also suggest that SMC proliferation is a more dominant process in peripheral versus coronary restenotic lesions. Skowasch et al
. were unable to identify proliferation in restenotic coronary lesions.13
Moreover, Glover et al
. found proliferation in only one of twenty coronary restenotic samples as measured by in situ
hybridization for histone 3 mRNA expression.11
O’Brien et al
. found PCNA positivity in 26% of restenotic coronary lesions, but in each of these restenotic lesions <1% of cells were actually PCNA+.10
Similarly, Marek et al
. found only 1-3% of cells expressed PCNA in 41% of restenotic carotid lesions.12
Alternatively, we have found a proliferative index of near 70% in restenotic lesions removed from the superficial femoral artery. Schwartz et al
.hypothesized that restenosis is characterized by proliferation only in the early phase of its development; thus lesions derived from human coronary arteries if they are sampled at a later stage of their development, are not typified by proliferation.26
Our data, however, do not support this hypothesis since all of our peripheral restenotic specimens were taken at a point greater than six months following the initial intervention and as previously noted, proliferation in these lesions was still quite robust.
Having identified proliferating SMCs in the restenotic lesion, we next focused on potential signaling mechanisms that might account for increased proliferation. It is well established that TGF-βligands are upregulated following vascular injury.28
We have previously shown in a rat balloon injury model that the TGF-βsignaling molecule Smad3 is also upregulated after vascular injury. Furthermore, overexpression of Smad3 was associated with increased SMC proliferation in our animal model. This is a surprising finding since TGF-βactivity is traditionally thought to be associated with inhibition of SMC proliferation. In fact, our previous work suggests that while TGF-βcan inhibit SMC proliferation through a non-Smad3 pathway, in the presence of high levels of Smad3, which develop after arterial injury, TGF-βappears to have the converse effect of stimulating SMC proliferation. Our new and similar findings in human vessels and cells serve to confirm this hypothesis and make Smad3 a relevant target in the treatment of human restenotic disease.
There is conflicting data regarding the role of Smad3 in intimal hyperplasia. Kobayashi et al
. have previously shown that SMCs derived from a Smad3 knockout mouse (i.e. cells that are lacking Smad3) have increased baseline proliferation compared to wild type cells.18
These findings would imply that Smad3 is involved in endogenous suppression of SMC proliferation. Mimicking this experimental situation we inhibited Smad3 expression with siRNA and found an opposite result: inhibition of SMC proliferation. The explanation for the discrepancy between ours and Kobayashi’s findings is not entirely clear.
In addition to stimulation of cell proliferation, elevated levels of Smad3 may induce other processes that lead to intimal hyperplasia. Extracellular matrix production has been shown to be an important component of intimal hyperplasia, and we have previously shown that Smad3 is essential for vascular SMC production of fibronectin.29
Alternatively, Smad3 may influence SMC apoptosis, however our in vitro
data suggest that the effect of Smad3 may be minimal. Finally, overexpression of Smad3 may stimulate SMC migration, another process that has been shown to play a role in the development of intimal hyperplasia. This would be one area for future in vitro
One of the limitations of current study is the use of human aortic SMCs for in vitro studies. As the aortic bed does not clinically succumb to occlusive lesions as compared to the SFA, it is possible that SMCs from these two different regions may have distinct responses to intracellular levels of Smad3. Another limitation is the small number of patient samples included. However, our findings even with an n of 3 were quite dramatic and all of our findings were statistically different, suggesting that Smad3-mediated SMC proliferation could be an important mechanism unique to restenosis.
The present study confirms that the mechanism underlying the development of primary atherosclerotic plaque in the superficial femoral artery is quite distinct from that of restenotic disease. White blood cells contribute significantly to primary plaque while restenotic lesions are highly cellular, comprised primarily of SMCs; in addition, SMC proliferation is quite robust. Moreover, the TGF-βsignaling protein Smad3 is upregulated in restenotic lesions and cellular localization studies suggest that this might account for the highly proliferative nature of these lesions. A better understanding of the relationship between Smad3 and cell proliferation will provide us with further insight into the mechanisms by which TGF-βinduces intimal hyperplasia as well as enable us to target specific aspects of this signaling mechanism for therapy to prevent restenosis after treatment of peripheral arterial disease.