MMP9 Variation After Thrombolysis Is Associated With Hemorrhagic Transformation of Lesion and Death
Background and Purpose—Experimentally, matrix metalloproteinases (MMPs) play a detrimental role related to hemorrhagic transformation and severity of an ischemic brain lesion. Tissue-type plasminogen activator (tPA) enhances such effects. This study aimed to expand clinical evidence in this connection.
Methods—We measured MMPs 1, 2, 3, 7, 8, 9, and tissue inhibitors of metalloproteinases 1, 2, 4 circulating level in blood taken before and 24 hours after tPA from 327 patients (mean age, 68.9±12.1 years; median National Institutes of Health Stroke Scale, 11) with acute ischemic stroke. Delta median values ([24 hours post tPA–pre tPA]/pre tPA) of each MMP or tissue inhibitors of metalloproteinase were analyzed across subgroups of patients undergoing symptomatic intracerebral hemorrhage, 3-month death, or 3-month modified Rankin Scale score 3 to 6.
Results—Adjusting for major clinical determinants, only matrix metalloproteinase-9 variation proved independently associated with death (odds ratio [95% confidence interval], 1.58 [1.11–2.26]; P=0.045) or symptomatic intracerebral hemorrhage (odds ratio [95% confidence interval], 1.40 [1.02–1.92]; P=0.049). Both matrix metalloproteinase-9 and tissue inhibitors of metalloproteinase-4 changes were correlated with baseline, 24 hours, and 7 days National Institutes of Health Stroke Scale (Spearman P from <0.001 to 0.040).
Conclusions—Our clinical evidence corroborates the detrimental role of matrix metalloproteinase-9 during ischemic stroke treated with thrombolysis, and prompts clinical trials testing agents antagonizing its effects.
In experimental models of brain ischemia, matrix metalloproteinases (MMPs) have been shown consistently to play a detrimental role.1,2 Tissue-type plasminogen activator (tPA) may enhance expression and activity of MMPs, particularly matrix metalloproteinase-9 (MMP9).3 MMP antagonists administered to animals treated with tPA lower the risk of hemorrhagic transformation4 and reduce infarct volume.5 MMPs have been poorly explored in the human stroke setting. We investigated the effect of pre- and post-thrombolysis variations of a number of different MMPs and tissue inhibitors of metalloproteinases (TIMPs) on main adverse outcomes after thrombolysis.
Materials and Methods
Eligible were patients admitted for thrombolysis in 14 Italian centers and were registered in the Safe Implementation of Thrombolysis in Stroke-International Stroke Thrombolysis Register (SITS-ISTR), according to SITS-Monitoring Study criteria.6 Study protocol was approved from each ethical committee, and all patients gave informed consent.
Blood samples were taken before and 24 hours after tPA. Outcomes were defined as follows: (1) symptomatic intracerebral hemorrhage (SICH), using the National Institute of Neurological Disorders and Stroke criteria;7 (2) 3-month death; (3) modified Rankin disability score, dichotomized into good (modified Rankin scale, 0–2) or poor (modified Rankin scale, 3–6) outcome. As a main explanatory variable, we used single patient’s relative pre- and post-thrombolysis variation (Δ median value) of both MMPs and TIMPs levels, calculated according to the formula: (24-hour post-tPA MMP or TIMP–pre-tPA MMP or TIMP)/pre-tPA MMP or TIMP. Differences in these Δ values were analyzed in relation to demographic and clinical features and across subgroups of patients with different outcomes. Methods details are in the online-only Data Supplement.
Between October 2008 and June 2011, 534 patients were registered in the SITS-ISTR, of whom 327 (mean age, 68.9±12.1 years; 58% males) were investigated. The remaining 207 were not studied because of not fulfilling SITS-MOST criteria, not giving consent, blood samples not taken or not stored appropriately, outcomes not assessed, and other protocol violations. Except for onset-to-treatment time, the prevalence of major outcomes determinants did not differ significantly between the 2 groups (demographics and clinical characteristics of both groups are in Table I in the online-only Data Supplement).
Absolute TIMPs or MMPs measures taken before thrombolysis and 24 hours after are reported in the Table II in the online-only Data Supplement.
The Figure shows pre- and post-thrombolysis changes of each MMP and TIMP level measured in patients with and without SICH, in patients who died and in those who survived, and in patients scoring 3 to 6 or 0 to 2 on the 3-month modified Rankin scale. MMP9, TIMP4, and MMP2 level changes showed a tendency toward the association with SICH. Variations of MMP8, MMP9, and TIMP1 levels were significantly associated with death, whereas variations of MMP8, MMP9, and TIMP4 levels were associated with scoring 3 to 6 on the 3-month modified Rankin scale. Grading of hemorrhage severity (according to the European Cooperative Acute Stroke Study II criteria) in the 27 patients with SICH is shown in Table III in the online-only Data Supplement. The association of MMP9 level variation with hemorrhage remained significant (P=0.036) after excluding the patients with less severe transformation (hemorrhagic infarction type 1).
MMP9 level changes were significantly correlated with prethrombolysis, 24-hour, and 7-day National Institutes of Health Stroke Scale score (Spearman P=0.012, 0.040, and <0.001, respectively).
A similar correlation was found for TIMP4 variations (Spearman P<0.001, <0.001, and 0.007, respectively). Adjusting for clinical determinants, among all MMPs and TIMPs examined, only MMP9 changes remained significantly associated with SICH, whereas only MMP9 and TIMP1 level changes remained associated with death (Table).
In the largest series hitherto investigated of tPA-treated patients with ischemic stroke, in whom a number of different MMPs and TIMPs were measured before and after thrombolysis, MMP9 circulating level variation proved associated, independent of major clinical determinants, with SICH or death. The unfavorable role of MMP9 was further supported by the correlation between MMP9 changes and National Institutes of Health Stroke Scale ratings before and after thrombolysis. Our findings are consistent with experimental data demonstrating the detrimental role of MMP9 in the processes aggravating tissue damage in tPA-treated cerebral ischemia.1,2
In the setting of human ischemic stroke treated with thrombolysis, previous smaller studies8 have focused on the relationship between circulating MMPs level variations and either hemorrhagic transformation or clinical outcomes. The different timing of blood sampling limits comparisons across these studies.
One limitation of our study is the lack of a control group of patients with stroke not treated with tPA. A further limitation rests on methods used for measuring MMPs and TIMPs. Actually, we measured the antigen levels of MMPs and TIMPs, not their activities. Conversely, the zymographic assay procedure has poor standardization and high interlaboratory variability.
In conclusion, our study extends experimental data, suggesting a distinct role of MMP9 related to the course of ischemic stroke treated with thrombolysis, and may encourage randomized trials testing MMP9 antagonists administered together with tPA.
We thank the hospital staff for data collection: M. Acampa, Siena; M. Bacigaluppi, Milano; A. Chiti, Pisa; A. De Boni, Vicenza; M.L. De Lodovici, Varese; F. Galati, Vibo Valentia; N. Marcello, Reggio Emilia; N. Micheletti, Verona; F. Muscia, Como; E. Paolino, Ferrara; P. Palumbo, Prato; P. Tosi, Rozzano; E. Mossello, Firenze; M. Torri, Firenze. We thank Maria Elena Della Santa for assistance in preparing the article.
Sources of Funding
Biological Markers Associated with Acute Ischemic Stroke (MAGIC) Study was funded by grants from Italian Ministry of Health, 2006 Finalized Research Programmes (RFPS-2006-1-336520) and Ente Cassa di Risparmio di Firenze (2010.06.03).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.002274/-/DC1.
- Received May 30, 2013.
- Accepted June 20, 2013.
- © 2013 American Heart Association, Inc.
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