Circulating TIMP-1 is associated with hematoma volume in patients with spontaneous intracranial hemorrhage

Matrix metalloproteinases (MMPs) are proteolytic zinc-endopeptidases regulated by tissue Inhibitors of matrix metalloproteinases (TIMPs). We evaluated the potential of MMPs and TIMPs as clinical tools for Intracranial Haemorrhage (ICH). Spontaneous non‐traumatic ICH patients were recruited from two hospitals: Complejo Hospitalario de Navarra (CHN = 29) and Vall d´Hebron (VdH = 76). Plasmatic levels of MMP-1, −2, −7, −9, −10 and TIMP-1 and their relationship with clinical, radiological and functional variables were evaluated. We further studied the effect of TIMP-1 (0.05–0.2 mg/Kg) in an experimental tail-bleeding model. In CHN, TIMP-1 was associated with admission-hematoma volume and MMP-7 was elevated in patients with deep when compared to lobar hematoma. In VdH, admission-hematoma volume was associated with TIMP-1 and MMP-7. When data from both hospitals were combined, we observed that an increase in 1 ng/ml in TIMP-1 was associated with an increase of 0.14 ml in haemorrhage (combined β = 0.14, 95% CI = 0.08–0.21). Likewise, mice receiving TIMP-1 (0.2 mg/Kg) showed a shorter bleeding time (p < 0.01). Therefore, the association of TIMP-1 with hematoma volume in two independent ICH cohorts suggests its potential as ICH biomarker. Moreover, increased TIMP-1 might not be sufficient to counterbalance MMPs upregulation indicating that TIMP-1 administration might be a beneficial strategy for ICH.

www.nature.com/scientificreports www.nature.com/scientificreports/ Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with proteolytic activity that contribute to ECM degradation, tissue remodeling and regeneration 10 . The catalytic activity of MMPs is regulated by tissue inhibitors of matrix metalloproteinases (TIMPs) 11 . The four members of the TIMP family (TIMP 1-4) are broad spectrum inhibitors that directly bind to the catalytic domain of MMPs 12 .
After brain injury, the role of MMPs and their inhibitors may be critical, since both participate in early acute injury along with tissue recovering during later stages 13 . Clinical studies have observed increased MMP-2, −3, −9 and TIMP-1 levels in blood and cerebrospinal fluid of ICH patients when compared to normal population 14,15 , suggesting that MMPs/TIMPs are likely to be involved in brain injury after spontaneous ICH and may be associated with patients' outcome 16 . In the last years our group has studied the role of MMPs in different neurovascular diseases. We showed that TIMP-1 levels were associated with mortality in patients with traumatic brain injury and malignant middle cerebral artery occlusion [17][18][19] , while MMP-2 expression might be implicated in the development of amyloid angiopathy in ICH patients 20 . Moreover, MMPs have been related to hematoma expansion, edema growth and neuronal loss in extensive pre-clinical studies 16 . However, their potential as therapeutic or diagnostic clinical tools has not yet been fully identified.
In this study, we explored the plasmatic levels of several MMPs and TIMP-1 and their relationship with hematoma volume and location as well as with other clinical, radiological and functional variables in two independent cohorts of ICH patients. In addition, we further studied the effect of TIMP-1 in an experimental tail-bleeding model.
In the VdH cohort (

Association of MMPs and TIMP-1 with clinical and radiological variables.
We performed a univariate analysis to assess the relationship between plasma MMPs and TIMP-1 and clinical and radiological variables (Tables 3 and 4).
First, in the CHN cohort, the multivariate analysis showed a significant association of TIMP-1 and hemorrhage volume after adjusting for confounding factors in all tested models (models 1 to 3, Table 5). This association was further confirmed in the VdH cohort in both, the univariate and the multivariate analysis (Table 6).
When we combined the results from the two cohorts, we observed a statistically significant association between TIMP-1 and hematoma volume (Overall β = 0.14, 95% CI = 0.08-0.21, Fig. 1). These results suggest that TIMP-1 might be a potential biomarker for ICH.

TIMP-1 effects in experimental haemorrhage. After finding an association between hematoma volume
and TIMP-1, we further explored the effects of TIMP-1 in an experimental model of tPA-induced haemorrhage. Our results showed that an experimental dose of TIMP-1 (0.05 mg/Kg) did not decrease the maximum bleeding time allowed (30 minutes). However, mice injected with the highest dose of TIMP-1 (0.2 mg/Kg), showed a much shorter bleeding time (1.8 ± 0.7 vs. 28.3 ± 3.3 min, p < 0.01, Fig. 2a) and blood lost (15.3 ± 4.7 vs. 42.8 ± 14.9 μL, p < 0.05, Fig. 2b). These results suggest that TIMP-1 might be able to control experimental haemorrhage.

Discussion
The main objective of this study was to explore the relationship of MMPs and TIMP-1 with clinical, radiological and functional variables in two independent cohorts of ICH patients. Hematoma volume and expansion have been associated with poor prognosis, neurological detriment and disability after ICH 21 . We reported for the first time that circulating levels of TIMP-1 at admission are associated with hematoma volume independently of hypertension, NIHSS and hematoma location in two independent cohorts of ICH patients.
TIMP-1 is an endogenous inhibitor of MMPs present under normal conditions in tissues to regulate MMPs activity 22 . Studies addressing the role of TIMP1 in ICH are scarce. In this study, we report that TIMP-1 is significantly associated with hematoma volume in two independent cohorts, and after a combined meta-analysis, suggesting that TIMP-1 might be potential critical biomarker in ICH. These results are in agreement with other studies showing increased concentrations of TIMP-1 during the acute phase of ICH, that remained highly sustained during the first week after stroke onset 23 , and showing TIMP-1 association with early mortality rates 24 , supporting a potential role of TIMP-1 as severity biomarker in ICH. The elevated secretion of TIMP-1 might be induced to lessen MMPs activity and thus, to prevent further brain damage 25 . However, it might not be enough to counterbalance the concomitant upregulation of MMPs. Likewise, it has been reported a relative increase in MMP-9/TIMP-1 ratio associated with symptomatic ICH 26 and Subarachnoid Haemorrhage (SAH) 27 supporting the hypothesis that perturbations in MMPs/TIMPs levels may contribute to the matrix disruption associated with cerebral aneurysms 28 .
Additionally, we found that administration of TIMP-1 (approximately 10-fold circulating levels) is able to control experimental acute bleeding in mice. These findings are consistent with the overall idea that in conditions of brain haemorrhage, damage, and inflammation, there may be an imbalance in the dynamic equilibrium between MMPs and TIMPs, so that any way of enhancing the inhibitor side of the equation might be beneficial 29,30 .  www.nature.com/scientificreports www.nature.com/scientificreports/ Supporting this hypothesis, previous data in TIMP-1 knockout mice showed exacerbated BBB disruption and brain injury 31 , while adenoviral TIMP-1 overexpression in mice, reduced brain lesion volume, neuronal damage and BBB leakage after ischemia 32 . Moreover, MMP inhibitors significantly decreased bleeding and brain damage in experimental models of haemorrhage, including ICH 33,34 .
One of the main factors that influences ICH outcome is the hematoma location; deep and lobar predominantly 35 that usually results in different hematoma volumes. Despite the relevance of this parameter for ICH outcome, there are few clinical data regarding the association of MMPs with hematoma location. Abilleira et al. reported higher MMP-9 plasmatic levels in patients with deep-associated ICH 36 . Moreover, in cerebral amyloid ICH, typically located in lobar regions, MMP-2 and -9 were up-regulated in perihematomal area of the brains 20,37 . In our study, CHN patients with deep hematoma location showed elevated plasmatic MMP-7 levels compared to patients with lobar hematoma. MMP-7/matrilysin is a small, secreted metalloproteinase capable of digesting a large series of proteins of the extracellular matrix 38 . A recent population-based cohort study followed for more than 10 years, reported that high serum levels of MMP-7 were independently associated with increased risk of incident spontaneous SAH suggesting its possible use as a predictive marker for SAH 39 . Likewise, several studies with MMP-7 null mice indicated that the robust expression of MMP-7 during severe inflammation lead to irreparable tissue damage 40 . Furthermore, we observed an association of this metalloprotease with hematoma volume in the VdH cohort. Therefore, our results suggest that MMP-7 might be involved in the pathophysiology of deep-ICH although it may be partly dependent on risk factors, therefore, further studies must be performed to confirm these data.
Other associations found in this study, such as MMP-10 with age and diabetes and MMP-2 with age, hypertension and 90-days mortality, were only significant in VdH possibly explained by larger sample size and statistical power of this cohort. However, these associations were lost after adjusting for risk factors of ICH. Previous data about the association of high MMP-10 levels with microvascular complications in diabetes type 1 in clinical and  www.nature.com/scientificreports www.nature.com/scientificreports/ experimental studies 41,42 could explain the association found in ICH patients. Regarding MMP-2, several studies support the association of MMP-2 with hypertension and mortality in diabetic patients and in patients with heart failure 43,44 . However, concerning spontaneous ICH patients, elevated levels of MMP-2 were only found at admission decreasing during the first 24 hours of hospitalization 23 and, specifically at tissue level in β-amyloid-damaged vessels located far from the acute ICH 20 . Therefore, future studies with larger sample size might confirm these data.
Our work has several limitations: first the small sample sizes of our series make our results to be interpreted carefully. Therefore, data from this study should be confirmed in larger cohorts before definitive clinical conclusions can be drawn. The observational study design did not allow us to establish a cause-effect relationship between TIMP-1 and hematoma volume, although the effect of TIMP-1 reducing bleeding time in an experimental model of tail bleeding supports the hypothesis that TIMP-1 could be a promising strategy to control hematoma volume in ICH. However, further experimental models of ICH using deficient mice might be performed.

Conclusion
We report for the first time that circulating TIMP-1 is associated with hematoma volume in two independent ICH cohorts and when we combined the results from both cohorts suggesting the potential of TIMP-1 as biomarker for ICH. Additionally, TIMP-1 administration significantly reduces experimental bleeding. Therefore, it could be considered that after ICH increased TIMP-1 levels might not be sufficient to counterbalance the upregulation of MMPs, and that its exogenous administration could be a beneficial strategy for ICH in order to reduce MMPs activity and restore the MMPs/TIMPs balance.

Materials and methods
Study populations. Spontaneous non-traumatic ICH patients were recruited from two different Spanish tertiary hospitals (Tables 1 and 2 respectively. All patients or their "legally authorised representative" provided written informed consent according to Helsinki declaration. The study included only patients with spontaneous nontraumatic supratentorial intracerebral hemorrhage (ICH), diagnosed by experienced neurologists of the Stroke Unit by initial cranial non-contrast computed tomography (CT) at admission in the emergency department. Patients with brainstem/infratentorial ICH and/or secondary ICH (vascular malformation, aneurysm, tumors, ischemic stroke with haemorrhagic transformation) were excluded. We excluded other causes of intracranial haemorrhages, as subdural hematoma, epidural bleeds, and subarachnoid hemorrhage. However, we included patients who had suffered subarachnoid and intraventricular haemorrhage (IVH), although always associated with concurrent parenchymal hematoma.   www.nature.com/scientificreports www.nature.com/scientificreports/ Systemic cancer and inflammatory systemic condition patients were also excluded from the study. ICH was classified by its location by experienced neurologist as previously reported by Aguilar I et al. 45 . Demographics and previous antiplatelet and/or anticoagulation therapy were registered. Reversal of anticoagulation was indicated in some patients according to current guidelines.
Radiologic measurements. The primary radiologic outcomes were hemorrhage volume (admission volume and after the following 24-48 hours), hematoma location, hematoma expansion (more than 6 mL or 33% growth during the first 24 hours) and IVH. Hematoma volume was quantified by using the ABC/2 method 46,47 . This well-validated method, consist on measuring the greatest axial dimension of the hematoma (A), a perpendicular diameter on the same slice (B), and the cranio-caudal dimension (C) estimated by counting the number of slices where the hematoma is present and multiplying that number by the slice thickness. The hematoma volume is estimated by multiplying these three dimensions together and dividing by 2, which estimates the simplified volume of an ellipse.
Neurological and functional outcomes. Clinical severity was assessed at admission using the National Institute of Health Stroke Scale (NIHSS) 48 and initial prognosis was evaluated by ICH score 49 . Functional outcome was defined by modified Rankin Scale (mRS) 50 and mortality rates at 90 days after onset to evaluate the mid-term outcome of patients. Previous functional situation evaluated by mRS was registered at admission. Bleeding assay. Bleeding assay was performed in 5-week-old male C57/BL6J mice (n = 12, Envigo, Spain).
All experiments in this study were conducted according to the European Community guidelines for the ethical animal care and use of laboratory animals (2010/63/EU) and approved by the University of Navarra Animal Research Review Committee (ref. 46,12 ). The experimental protocol is shown in Supplemental Fig. 1. Briefly, animals were anesthetized with 2.5% isoflurane, placed in prone position and tPA (0.05 mg/Kg) was injected through the ocular plexus. Five minutes later, TIMP-1 (0.05 and 0.2 mg/Kg) or saline were injected through the femoral vein catheter (10% bolus and 90% perfusion during 40 minutes). A distal 5 mm segment of the tail was transected with a scalpel blade 5 minutes after starting the treatments. The tail was immediately immersed in a tube containing pre-warmed saline (37 °C in a water bath). Bleeding time was defined as the time elapsed until bleeding stops for a maximum of 30 min. Blood lost was defined as the amount of blood collected during 30 min and absorbance www.nature.com/scientificreports www.nature.com/scientificreports/ was measured in a spectrophotometer (Tecan Sunrise, Switzerland) and quantified against a standard curve generated with known quantities of blood 33 . Statistical analysis. Descriptive statistics (median, interquartile range, frequency and percentage) were defined for all clinical, radiological and functional variables. Normality was assessed using the Shapiro-Wilk test. Bivariate comparisons of characteristics at admission between ICH location (deep and lobar index ICH) were performed using Student t test or Mann-Whitney U test for continuous variables, and χ2 test or Fisher exact test for categorical variables. All covariates associated with the end point in the univariate analysis, considering a threshold of p < 0.1 in both cohorts, were included in the multivariate linear regression analyses for the target molecules of the study (MMPs and TIMP-1). Due to the small sample size in one of the cohorts, the following baseline models were considered for adjustment: model 1, TIMP-1, location, and hypertension (yes/no); and model 2, TIMP-1, location and NIHSS. Finally, we conducted a fixed effect meta-analysis to combine the data from our two cohorts in a meta-analysis. Statistical analysis of the data from experimental model was done using Mann-Whitney U two-tailed test.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.