4. Discussion
The atherosclerotic plaque is a dynamic lesion that undergoes continuous remodelling of the extracellular matrix on which its structural stability depends. Previous studies demonstrate that acute changes within the carotid plaque, such as IPH, fibrous cap rupture or ulceration can lead to the onset of ischemic cardiovascular events [6]. Recent reports suggest that MMP-9 may have a key role in all stages of the atherosclerotic process during which are being secreted by inflammatory cells and VSMC, although there is still lack of evidence whether MMP-9 can affect the incidence of adverse events [7, 8, 34].
The present pilot trial has demonstrated that the prevalence of unstable plaque was higher in symptomatic than asymptomatic patients, although this was not statistically significant (63% vs. 37%, p =0.077). Our main findings were that the expression of MMP-9 in CD68 cells within the carotid plaque was found to be significantly higher in symptomatic as compared to asymptomatic patients (86% vs. 25% with the highest expression, p =0.014). Importantly, the average MVD by immunohistochemical staining of CD34 was found to be higher and lipid core area larger among both symptomatic patients and unstable carotid plaque specimens, although this did not reach statistical significance (p =0.064 and p =0.132, p =0.360 and p =0.569, respectively). Our study demonstrates the ability of MRI to identify patients with high-risk carotid plaque and to differentiate better which patients could benefit from early surgical revascularization. These findings are in line with the results of other studies and help in understanding that MRI can be the most accurate advanced imaging modality in distinguishing vulnerable from stable carotid plaques preoperatively, although further large prospective comparative studies are still required [7, 19, 20, 28, 35, 36]. Importantly, the most recent ESVS guidelines and recommendations of multicentric randomised controlled trials highlight clinical/imaging features in asymptomatic patients associated with an increased risk of stroke despite the best medical treatment that indicate necessity for carotid revascularisation [1, 4, 25-27]. A recent report comparing carotid MRI, ultrasound and histological findings of carotid plaque specimens has demonstrated that the findings of carotid MRI and ultrasound are closely associated with specimen morphology, but ultrasound alone is insufficient to detect the type of lesion accurately.[36] In addition, we have found that the most common type of carotid plaque as per modified AHA classification by MRI was type IV-V (10 patients, 65%), while type VI was the most common (89 out of 252 plaque specimens) in the report by Cai et al.[28]. Similarly, Million et al. have reported the ability of MRI to identify high-risk carotid plaque and to differentiate symptomatic from asymptomatic patients, although their study was performed using a 3-Tesla system [19].
Furthermore, our results showed that MMP-9 may play a key role in remodelling and destabilization of the carotid plaque. As we hypothesized, the expression of MMP-9 in CD68 cells was significantly higher among symptomatic patients. Similarly, it was also higher in SMA cells in symptomatic patients, but this did not reach statistical difference (p =0.143). Interestingly, symptomatic patients had significantly higher preoperative hsCRP which is in accordance with the inflammatory response and infiltration related to the destabilization of the plaque and potential increased release of MMP-9. Similarly, recent reports showed higher expression levels of MMP-9 obtained from symptomatic when compared to asymptomatic patients [8, 37, 38]. These findings highlighted MMP-9 as one of the most important enzymes related to the plaque instability. On the other hand, Baroncini et al. reported higher MMP-9 concentration in normal tissues and in asymptomatic patients suggesting that MMP-9 is a part of the atherosclerotic process although not associated to acute plaque disruption [15, 16]. Still, this finding remains part of debate asHeo et al. have reported that MMP-9 is significantly associated with plaque rupture [38]. However, recent evidence suggests that an increase in proteolytic activity may lead to an overall increase in matrix degradation which could explain our results which demonstrate that the average MVD could be higher and lipid core area larger among both symptomatic patients and unstable carotid plaque specimens.
There are several limitations to our study. Our results showed a higher prevalence of unstable plaque in symptomatic patients, and a higher average MVD and larger lipid core area among both symptomatic patients and unstable carotid plaque specimens. However, the differences did not reach statistical significance probably because of the lack of power of the study and smaller sample size which are limitations of the present pilot study. Cappendijk et al. also failed to demonstrate a significant difference between symptomatic and asymptomatic patients [39]. However, in times of constraints on resources, both additional MRI analysis and detailed histologic work-up of plaques is costly and time-consuming, thus explaining the necessity of conducting a pilot trial. Although we obtained the best possible resolution for carotid MRI, another limitation of the study could be related to the use of only non-contrast MRI and motion artefacts. Further improvement in coil design, use of 3-Tesla system and modification of imaging parameters could be required, while the use of contrast-enhanced MRI would be less justified in terms of routine preoperative diagnostic work-up. Another limitation is that we have analysed our results semiquantitatively by immunohistochemistry, and the results do not allow accurate quantification. Nevertheless, our estimates were performed independently by two investigators. In addition, it is also possible that we have excluded some regions of plaques where inflammation was more advanced.