Background and purpose Flow diverters (FD) can cause rare but devastating delayed aneurysm ruptures in which matrix metalloproteinases (MMPs) have been potentially implicated. Concomitant coiling or anti-inflammatory medications have been proposed to prevent the risk of delayed ruptures. The aim of this study was to evaluate concomitant coiling and ciclosporin in regulating the expression of MMPs in FD-treated aneurysms.
Materials and methods Elastase-induced aneurysms were created in 20 rabbits. Aneurysms were treated with (1) FD alone; (2) FD with concomitant coiling; (3) FD+ ciclosporin; or (4) left untreated as controls. At sacrifice, MMP levels were analyzed by zymography. Kruskal–Wallis one-way non-parametric ANOVA was performed for each enzyme. If significant results were observed for the Kruskal–Wallis test, pairwise group comparisons were performed using Dunn's test with Bonferroni multiple-testing correction.
Results Significant differences were observed among groups for pro-MMP9 (p=0.0337). Pairwise comparison demonstrated higher levels of pro-MMP9 with concomitant coiling compared with untreated aneurysms (p=0.012), with higher though not significantly different levels of pro-MMP9 in FD with concomitant coiling versus FD alone. While not statistically significant, trends were noted regarding differences in active-MMP9 across groups, with a lower level of active-MMP9 with concomitant coiling compared with the other FD groups. No significant differences were observed for pro- or active-MMP2 across groups, or for FD + ciclosporin compared with FD alone.
Conclusions FD implantation increases the level of pro-MMP9 expression in aneurysms. Provocative trends regarding modulation of active-MMP9 expression with concomitant coiling suggest the need for larger confirmatory preclinical studies. Anti-inflammatory treatment with ciclosporin appears to have a minimal biological effect.
Trial registration number R01NS076491
- Flow Diverter
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Flow diverters (FD) are now largely accepted as standard treatment of selected aneurysms due to their high rates of angiographic occlusion and good clinical outcomes.1–3 However, these devices may have rare but severe complications such as postoperative or delayed aneurysm rupture.4–8 Despite the fact that numerous case series and case reports have reported this complication, there continues to be controversy surrounding its origin with poor evidence regarding the risk factors and mechanisms of these hemorrhagic complications.4 ,6 ,8–12 Previous studies have suggested a potential role of intra-aneurysmal thrombus in the pathophysiologic mechanism of aneurysm rupture.7
As previously described in abdominal aortic aneurysms, leukocytes trapped in the intraluminal thrombus are a source of storage, release, and activation of various proteases such as matrix metalloproteinases (MMP2 and MMP9) and serine proteases. These have high proteolytic activity which could participate in the degradation of structural components of the arterial wall and lead to aneurysm rupture.7 ,13–20 MMPs are secreted as inactive proforms (pro-MMP) and are activated by protein cleavage by other proteinases (active-MMP).20 The overexpression of activated type IV collagenases MMP9 and MMP2 in cerebral ruptured aneurysms21 indicates that effective regulation of MMPs may result in improving the clinical prognosis of cerebral aneurysms.
Some studies have already described the implication of MMPs in cerebral vascular diseases and aneurysms.22–29 Previous studies have demonstrated a higher risk of rupture in giant aneurysms2 ,6 ,7 ,12 and have recommended that giant aneurysms are treated with concomitant coiling and FD treatment in order to protect the dome of the aneurysm in an attempt to prevent delayed ruptures.4 ,6 ,11 ,30 ,31
Ciclosporin A is an anti-inflammatory agent32 widely used to prevent organ transplant rejection or to treat autoimmune disorders.33 Ciclosporin has already been tested for its effect on MMP levels in various models and disorders.32–42 In a study of abdominal aortic aneurysms, ciclosporin decreased MMP9 and stabilized expanding aortic arteries.38 However, it has not been tested in intracranial aneurysms to regulate MMP expression.
The aims of our study were to evaluate the effect of associated coiling and ciclosporin to regulate MMP expression in FD-treated aneurysms in a rabbit model.
Aneurysm creation and treatment
The Institutional Animal Care and Use Committee approved all procedures before the initiation of this study. Some of the rabbits used in this study were originally employed as part of other investigations, where we investigated the gene expression between aneurysms treated with microcoils and FD.43 Elastase-induced saccular aneurysms were created in 20 New Zealand White rabbits as previously described.44 Three weeks after aneurysm creation the rabbits were treated either with FD alone (n=5), FD with concomitant coiling (n=6), FD + ciclosporin (n=5), or left untreated (n=4).45 ,46 The rabbits treated with FD and ciclosporin were given 10 mg cyclosporin A/kg body weight by oral gavage once daily for 4 weeks.
The rabbits were sacrificed 4 weeks after the treatment procedure. At the time of sacrifice the animals were deeply anesthetized. The animals were then euthanized with a lethal injection of pentobarbital. Aneurysms were immediately harvested, frozen in liquid nitrogen and kept at −70°C until used.43
Gelatin zymography of MMPs
Frozen samples were pulverized under liquid nitrogen and extracted in ice-cold lysis buffer (10 mmol/L sodium phosphate, pH 7.2, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, and 0.2% sodium azide). After centrifugation at 10 000 g for 20 min at 4°C, the protein concentration of the supernatant was determined (Pierce Biotechnology). Samples were resolved by non-reducing 10% SDS-PAGE through gels containing 0.1% gelatin (Bio-Rad). Gels were washed with 2.5% Triton X-100 for 1 h, then incubated for 24 h at 37°C in developing buffer (50 mmol/L Tris-HCl, pH 8.5, 5 mmol/L CaCl2 and 0.5 mmol/L ZnCl2). MMP2 and MMP9 are collagenases which act on the gelatin (a partial collagen digest) in the gel and produce gelatinolytic zones. Gelatinolytic zones, representing the activities of MMPs, were visualized after staining the gels with 0.5% Coomassie Blue R-250. The gelatinolytic zones of MMP2 and MMP9 were analyzed densitometrically using Image-J software and converted to quantifiable data in the number of pixels.47 ,48 The intensities of the gelatinolytic bands reflect the activity of the corresponding MMP.
Kruskal–Wallis one-way non-parametric ANOVA was performed for each enzyme (pro-MMP2, active-MMP2, pro-MMP9, and active-MMP9) for each of the four treatment groups. Kruskal–Wallis p values were not corrected for multiple testing. Post-hoc pairwise group comparisons were performed using Dunn’s test with Bonferroni multiple-testing correction.49 Statistical analyses were performed in R (V.3.1.1; Vienna, Austria). Dunn’s test was performed using R package dunn.test (V.1.2.4). A value of α=0.05 was selected as the significance threshold.
In all treatment groups the levels of MMPs in treated aneurysms appeared higher than in the untreated group, with the exception of the FD with concomitant coiling group for active-MMP9; however, significant differences between groups were only found for pro-MMP9. MMP2 levels were similar throughout the three different FD treatment groups.
Compared with the untreated aneurysms, aneurysms treated with FD alone had higher levels of pro-MMP9 (2575 (IQR 697–4917) vs 428 (IQR 90–1941)), active-MMP9 (2666 (IQR 1066–5291) vs 483 (IQR 251–1897)), pro-MMP2 (4644 (IQR 2403–7693) vs 3250 (IQR 2950–6684)), and active-MMP2 (3500 (IQR 2684–4476) vs 1294 (IQR 1143–1666)), but these differences did not reach significant levels (table 2).
In the group treated with FD and concomitant coiling the level of active-MMP9 was similar to the level in the untreated group (496 (IQR 420–1734) vs 483 (IQR 251–1897)). The level of pro-MMP9 was significantly higher in the FD with concomitant coiling group than in the untreated aneurysm group (9774 (IQR 7657–13562) vs 428 (IQR 90–1941); p=0.012, table 2).
We did not observe any statistically significant difference or trends in difference when comparing the MMP levels in aneurysms treated with FD + ciclosporin and FD alone.
This study demonstrates that treatment with FD affects MMP levels in intracranial aneurysms. Specifically, aneurysms treated with FD and concomitant coiling have significantly increased levels of pro-MMP9. In this relatively small study we noted a trend toward decreased active-MMP9 with concomitant coiling compared with the other FD groups. Anti-inflammatory medications with ciclosporin did not significantly affect the levels of MMPs. These findings suggest the need for larger preclinical studies focused on MMP9 biology following treatment with FD.
By analogy with abdominal aneurysms, previous experimental and clinical studies have suggested that the intra-aneurysmal thrombus associated with FD could be a site of activation of MMPs and a potential cause of delayed ruptures.7 ,13–19 Furthermore, prior studies have suggested or demonstrated a higher risk of delayed ruptures after FD in giant aneurysms.2 ,6 ,7 ,12 Since giant aneurysms are generally more likely to have a larger intraluminal thrombus, it is probable that larger FD-treated aneurysms have higher levels of MMP expression. Based on our current findings, we believe that the ongoing focus on MMP9 may provide important insights into FD-related complications.
The trend toward decreased active-MMP9 with concomitant coiling compared with the other FD groups is very important. Indeed, despite previous recommendations for concomitant coiling in aneurysms larger than 15 mm,4 ,6 ,11 ,30 ,31 no study has described its effect on MMP levels. Our study shows that the effect of concomitant coiling is not only related to a mechanical effect of the coils to protect the aneurysms dome but, at least in part, is related to a biological effect on MMP expression. It suggests that concomitant coiling may reduce the level of active-MMP9 expression in the FD-treated aneurysms by blocking the activation of pro-MMP9 in its active form with accumulation of its inactive proform, which could be a potential solution to prevent delayed aneurysm rupture after FD.
Anti-inflammatory and immunosuppressive drugs such as ciclosporin A have proved to be beneficial on MMP levels in abdominal aneurysms.38 ,50 ,51 However, in our study, the expression levels of MMP2 and MMP9 in the group treated with FD + ciclosporin were comparable to those in aneurysms treated with FD alone, with higher levels of active-MMP9. While it was hoped that ciclosporin could be used to control MMP levels, it does not appear to have an effect on either MMP9 or MMP2. Further research is needed to explore the effect of other anti-inflammatory medications on MMP expression. Additionally, the role of pro-inflammatory mediators in aneurysm progression and rupture versus healing after treatment remains to be elucidated. Previous studies have demonstrated that MMPs and monocyte chemoattractant protein (MCP)-1 play key roles in formation and rupture, with MCP-1 promoting MMP9, but post-treatment expression also increases possibly due to aneurysm healing.29 ,52 ,53
Our study has several limitations. We used the rabbit elastase model which has histological, morphological, biological, and hemodynamic similarities to humans and is stable in time with no spontaneous thrombosis.54 However, this model is neither a model of spontaneous rupture nor a model of delayed aneurysm rupture after FD, and some biological aspects may differ when considering rupture-prone aneurysms. To explore these mechanisms, it would be of interest to analyze levels of MMPs in new models for active aneurysms with inflamed aneurysm wall or bioactive thrombus.55 ,56 Also, the reported cases of delayed rupture after FD occurred mostly in large or giant aneurysms but the aneurysms used in this study were less than 20 mm. Some of our results did not reach significant differences, but our ability to detect differences between groups was limited by the size of the treatment groups. Further studies should be done with larger groups. For the effect of concomitant coiling, we did not perform any analysis of the impact of the packing density. Perhaps denser coil packing would increase the effect of MMP regulation. Different anti-inflammatory drugs may have different results, but we decided to use ciclosporin because rabbits are known to be extremely sensitive to steroids.57–60 Furthermore, we analyzed only one dose of ciclosporin and its effect may be different with higher doses. Only one time point was studied following treatment of the aneurysm and protein expression may change over time. Finally, only two MMPs were studied and other MMP isoforms may play important roles in delayed aneurysm rupture.61 Transforming growth factor β (TGF-β) is a key factor for MMP downregulation.38 However, we did not measure the level of TGF-β in this study. It is possible that other proteolytic enzymes such as cathepsin or other pathways may lead to delayed aneurysm rupture.
FD implantation increases the level of pro-MMP9 expression in aneurysms. Provocative trends regarding modulation of active-MMP9 expression with concomitant coiling suggests the need for larger confirmatory preclinical studies. Anti-inflammatory treatment with ciclosporin appears to have minimal biological effect.
Contributors All authors participated in drafting the article and revising it critically for important intellectual content. All authors made substantial contributions to conception and design, acquisition of the data, and analysis and interpretation of the data. All authors provided final approval of the version to be published.
Funding This work was supported by NIH grant R01NS076491
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement The authors are willing to share spreadsheets from their data extraction on request.
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