Abstract
Sturge–Weber syndrome (SWS) consists of a capillary-venous vascular malformation of the brain, skin and eye. Urine vascular biomarkers have been demonstrated to be abnormal in other vascular anomalies and to correlate with clinical severity and progression. The current study investigated the use of urinary matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) levels to non-invasively monitor the progression of SWS. Fifty-four urine samples were collected from patients seen at the Hunter Nelson Sturge–Weber Center at Kennedy Krieger Institute. Urine was analyzed for MMP-2, MMP-9, VEGF and bFGF levels and correlated with clinical outcome at the time of urine collection (n = 48) and 1 year following urine collection (n = 22). Analysis revealed that MMP-2 (p = 0.033) and MMP-9 (p = 0.010) were significantly more likely to be present in the urine of SWS subjects compared to controls and that bFGF was significantly more likely to be present at abnormal levels (p = 0.005). MMP-2 correlated with a more severe clinical score at the time of urine collection, while both MMP-2 and MMP-9 levels correlated with greater disease severity at time of collection. bFGF levels correlated with improved clinical score 1 year after urine collection. These results suggest that MMP-2 and MMP-9 levels may be useful in assessing SWS progression, as well as indicating which patients might benefit from more aggressive treatment, while bFGF levels may be useful in judging the efficacy of neurologic treatment in SWS.
Keywords: angiogenesis, biological markers, fibroblast growth factor, remodeling, risk assessment, vascular endothelial growth factor-A
Introduction
Sturge–Weber syndrome (SWS) is a rare congenital neurocutaneous disorder. It is associated with vascular malformations of the brain, eye, and skin. Patients often present with a combination of leptomeningeal angiomatosis, usually of the occipital and posterior parietal lobes, and a facial capillary malformation (port-wine birthmark).1 They can also have glaucoma, seizures, strokes or stroke-like episodes, cognitive disability, attention problems, and headaches. The cause of SWS is unknown but is postulated to be a somatic mutation occurring early in development that affects vasculature perfusing of both neural and facial skin tissue.2,3 The molecular pathogenesis that leads to abnormal angiogenesis in SWS is unclear.4
Several studies have been conducted on vascular anomalies, tumors and stroke, investigating changes in the expression of vascular endothelial growth factors (VEGF) and basic fibroblast growth factor (bFGF), as well as matrix metalloproteinases (MMPs). MMPs are involved in the degradation of the extracellular matrix and in central nervous system vascularization.5 In particular, MMP-2 and MMP-9 have been found at elevated levels in the urine of patients with hemangiomas and brain tumors compared to controls.5,6,7 VEGF and bFGF have been shown through in vitro and in vivo, as well as clinical studies to be involved in vascular remodeling in tumors, vascular malformations, and strokes.6,8–11
The goal of this study is to investigate the potential of using urine MMPs, VEGF, and bFGF levels as biomarkers to track the progression of SWS. Since MMPs, VEGF, and bFGF all play a role in vascular remodeling, we hypothesized that the level of these angiogenic factors would be higher in the urine of SWS subjects compared to controls and that their level would correlate with disease severity. Here we report the urinary levels of these angiogenic factors in a cross-sectional group of subjects with SWS, compare the levels to those of a published group of controls from the same laboratory, and correlate the results with measures of SWS disease severity.
Methods
The subjects were patients seen at the Kennedy Krieger Institute Hunter Nelson Sturge–Weber Center, a multidisciplinary clinic dedicated to the care and research of SWS. The Johns Hopkins Institutional Review Board approved this research and all subjects provided informed consent. Fifty-four urine samples (Group A, Table 1) were collected from subjects (ages 0–43 years old) with confirmed SWS brain involvement who were evaluated at the Hunter Nelson Sturge–Weber Center between the years of 2005 and 2011. SWS brain involvement was diagnosed based on unilateral or bilateral leptomeningeal enhancement on neuroimaging. Samples were divided into aliquots and frozen as previously reported and shipped on dry ice for subsequent analysis as previously reported by us.6,12
Table 1.
Demographics.
| Group A (n = 54) | Subgroup 1 (n = 48) | Subgroup 2 (n = 22) | Controls (n = 74) | |
|---|---|---|---|---|
| Male/Female | 33/21 | 29/19 | 16/6 | 39/35 |
| Brain involvement | 50 unilateral; 4 bilateral | 44 unilateral; 4 bilateral | 21 unilateral; 1 bilateral | N/A |
| Median age | 7.0 years | 7.0 years | 8.0 years | 6.5 years |
| Age range | 0–43 years | 0–43 years | 0–28 years | 3.5–10.3 yearsa |
| Race | 3 hispanic, 46 non-hispanic, 5 unknown |
3 hispanic, 41 non-hispanic, 4 unknown |
2 hispanic, 18 non-hispanic, 2 unknown |
Not available |
| Ethnicity | 1 American Indian or Alaska native, 2 Asian, 6 black, 41 white, 4 unknown/other |
1 American Indian or Alaska native, 2 Asian, 6 black, 37 white, 2 unknown/other |
2 Asian, 4 black, 14 white, 2 unknown/other |
Not available |
Interquartile range – data on age range was unavailable.
Group A includes all participants providing a one-time urine sample. Subgroup 1 includes all participants who were assigned a SWS clinical score at the time of urine sample. Subgroup 2 includes all participants who were assigned a SWS clinical score 1 year post-urine sample. See Figure 1 for further explanation.
Substrate gel electrophoresis (zymography) for urinary MMP species was performed as previously described by us.12,13 Thirty microliters of each urine sample was loaded into wells of a 10% SDS-PAGE gel containing 0.1% (wt/vol) gelatin (Bio-Rad Laboratories, Hercules, CA, USA) on a mini-gel apparatus. Gels were run at 200 V for 50 minutes then soaked in 2.5% Triton X-100 with gentle shaking for 30 minutes at an ambient temperature. After incubating overnight at 37°C in substrate buffer (50 mM Tris-HCL buffer pH 8, 5 mM CaCl2, and 0.02% NaN3), gels were stained for 30 minutes in 0.5% Coomassie Blue R-250 in acetic acid, ethanol, and water (1:3:6) and destained for 1 hour. MMP levels were quantified by scoring the band intensity by densitometry of each type of MMP on a scale of zero to six, with zero indicating no detectable MMPs and six indicating strong intensity bands, as previously described.13–16
Enzyme-linked immunosorbent assay (ELISA) (Quantikine Kits; R&D Systems, Inc.) were conducted according to the manufacturer’s instructions and as previously reported to quantify levels of VEGF and bFGF.7 Specimens, standards and reagents were prepared according to the manufacturer’s instructions. Protein concentration was determined via the Bradford method using bovine serum albumin as the standard.
All subject charts were evaluated for age, gender, and side of brain, eye, and skin involvement (unilateral or bilateral). SWS clinical scores and involvement scores were assigned prospectively at each visit for a subgroup of 48 subjects (Subgroup 1, Table 1). The SWS clinical scoring system is based on clinical disease severity with respect to seizures, cognition, hemiparesis, and vision (Appendix 1). This scoring system has been clinically validated with imaging findings and quantitative EEG results documenting brain involvement in SWS subjects.17,18 Involvement scores were assigned as follows: a score of 0 (no involvement), 1 (unilateral involvement), or 2 (bilateral involvement) assigned for brain, eye, and skin of each subject (possible range between 0 and 6: 0 = no involvement, 6 = bilateral brain, skin and eye involvement) (Appendix 2).
Data on the presence or absence of different angiogenic factors (MMPs, VEGF and bFGF) from all 54 subjects were compared to data on 74 control subjects previously published from the same laboratory.6 Differences in the levels of factors between genders within SWS subjects were also examined. For the 48 subjects in Subgroup 1, correlations between factor levels and clinical outcome at the time of sample collection were examined.
A subgroup of 22 subjects (Subgroup 2, Table 1) had SWS clinical scores and involvement scores assigned 10–14 months after sample collection. Correlations between levels of MMPs, VEGF or bFGF, and clinical outcome a year after sample collection for this subgroup were examined as well.
All data were analyzed using Spearman correlations, Fisher’s exact tests, and Mann–Whitney U as appropriate using SPSS (Statistical Package for Social Sciences) Version 19 (SPSS Inc., Chicago, IL, USA). The significance level for all analyses was p < 0.05.
Results
Demographics (Table 1)
The 54 subjects in Group A, 33 males and 21 females, had a median age of 7.0 years (range: 0–43 years). Fifty subjects had unilateral brain involvement and four had bilateral brain involvement. The 74 control subjects, 39 males and 35 females, used as comparison data had a median age of 6.5 years (interquartile range: 3.5–10.3 years). Subgroup 1 (n = 48), consisting of 29 males and 19 females, was used for analysis of clinical outcome at the time of sample collection and had a median age of 7.0 years (range: 0–43 years). Forty-four had unilateral brain involvement and four had bilateral brain involvement. Subgroup 2 (n = 22), consisting of 16 males and six females was used for analysis of clinical outcomes 10–14 months after sample collection. Twenty-one subjects had unilateral brain involvement and one had bilateral brain involvement.
SWS vs control subjects
The presence of MMP-9 and MMP-2 in the urine of all 54 SWS subjects was compared to the previously published data on 74 control subjects.6 Both MMP-2 (p = 0.033) and MMP-9 (p = 0.010) were significantly more likely to be present in the urine of SWS subjects compared to controls by Fisher’s exact test (Table 2).
Table 2.
SWS versus control subjects.
| MMP-2 | Present | Absent | |
|---|---|---|---|
| Control (n = 74) | 32 | 42 | |
| SWS (n = 54) | 34 | 20 | *p = 0.0325 |
| MMP-9 | Present | Absent | |
| Control (n = 74) | 22 | 52 | |
| SWS (n = 54) | 29 | 25 | *p = 0.010 |
| bFGF | Abnormal (> 4000 pg/L) | Normal (< 4000 pg/L) | |
| Control (n = 74) | 6 | 68 | |
| SWS (n = 49) | 14 | 35 | **p = 0.0050 |
| VEGF | Abnormal (> 300 pg/mL) | Normal (< 300 pg/mL) | |
| Control (n = 74) | 7 | 67 | |
| SWS (n = 49) | 2 | 47 | p = 0.3145 |
MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor.
p< 0.05,
p<0.01.
The levels of bFGF and VEGF was measured and labeled as either normal or abnormal (> 4000 pg/L for bFGF; > 300 pg/mL for VEGF). bFGF was significantly more likely to be abnormally high in the urine of SWS subjects compared to published data on controls (p = 0.005) by Fisher’s exact test (Table 2). There was no significant difference in the number of subjects with abnormally high VEGF levels between the two groups.
Gender differences within SWS subjects
The amount of MMPs, quantified as described above in Methods, was compared between the 54 male and female SWS subjects by Mann–Whitney U analysis (Table 3). Female SWS subjects were significantly more likely to have higher levels of MMP-9 (p = 0.003) and its related molecules, MMP-9 dimer (p = 0.004), and the MMP-9/neutrophil gelatinase-associated lipocalin (NGAL) complex (p = 0.002) present in their urine than male SWS subjects. There was no significant difference in age between males and females.
Table 3.
Gender differences in SWS subjects.
| MMP-9 | Median | Range | |
|---|---|---|---|
| Males (n = 33) | 0 | 0–4 | |
| Females (n = 21) | 3 | 0–4 | **p = 0.003 |
| MMP-9 dimer | Median | Range | |
| Males (n = 33) | 0 | 0–5 | |
| Females (n = 21) | 1 | 0–4 | **p = 0.004 |
| MMP-9/NGAL complex | Median | Range | |
| Males (n = 33) | 0 | 0–5 | |
| Females (n = 21) | 2 | 0–4 | **p = 0.002 |
MMP, matrix metalloproteinase; NGAL, neutrophil gelatinase-associated lipocalin.
p<0.01.
Angiogenic factors and clinical outcome correlations
In Subgroup 1, for which clinical scores were assigned at the time of urine collection, MMP-2 levels correlated positively with total Neuroscore in males (n = 29; Spearman’s rho = 0.428; p = 0.021), male children (n = 23; Spearman’s rho = 0.523; p = 0.010), subjects with MMP-2 present (n = 29; Spearman’s rho = 0.386; p = 0.038), and children with MMP-9 present (n = 23; Spearman’s rho = 0.522; p = 0.006). MMP-2 levels correlated positively with cognitive function score in females with MMP-2 present (n = 11; Spearman’s rho = 0.670; p = 0.024) and males (n = 29; Spearman’s rho = 0.371; p = 0.048). In short, when present, correlations between MMP-2 and clinical status were positive (i.e. the greater the amount of urine MMP-2, the worse the SWS clinical score).
Both MMP-9 (Spearman’s rho = 0.401; p = 0.031) and MMP-9 dimer (Spearman’s rho = 0.389; p = 0.037) correlated positively with skin involvement score in males (n = 29). The MMP-9/NGAL complex correlated positively with total involvement score in subjects with MMP-2 present (n = 29; Spearman’s rho = 0.411; p = 0.027) and subjects with MMP-9 present (n = 12; Spearman’s rho = 0.642; p = 0.024). The MMP-9/NGAL complex correlated positively with brain involvement score in subjects with MMP-2 present (n = 29; Spearman’s rho = 0.455; p = 0.013) and with eye involvement score in children with MMP-2 present (n = 24; Spearman’s rho = 0.411; p = 0.027). Overall, when present, MMP-9 and MMP-9/NGAL and its related molecules correlated positively with involvement score (i.e. the greater the amount of MMP-9 and MMP-9/NGAL in the urine, the greater the extent of SWS involvement).
In Subgroup 2, for which clinical scores were assigned 1 year after urine collection, bFGF levels correlated negatively with the hemiparesis score for all subjects (n = 22; Spearman’s rho = −0.597; p = 0.005), males (n = 16; Spearman’s rho = −0.428; p = 0.021), children (n = 19; Spearman’s rho = −0.545; p = 0.024), male children (n = 13; Spearman’s rho = − 0.600; p = 0.039), subjects with MMP-2 present (n = 19; Spearman’s rho = −0.598; p = 0.014), and males with MMP-2 present (n = 14; Spearman’s rho = −0.683; p = 0.021). bFGF levels also correlated negatively with total Neuroscore in males (n = 16; Spearman’s rho = −0.529; p = 0.043). In brief, bFGF levels correlated with improved clinical outcome (specifically hemiparesis) a year after sample collection.
Discussion
In this study, we asked whether angiogenic factors such as MMPs and their related proteins and the angiogenic mitogens VEGF and bFGF, when detected in the urine of SWS patients, could provide useful clinical data for the assessment of disease progression. We found that MMP-2 and MMP-9 were significantly more likely to be present in the urine of SWS subjects compared to controls. In addition, bFGF was significantly more likely to be present at abnormal levels in the urine of SWS subjects compared to controls. When utilized in combination with SWS clinical scoring, elevated MMP-2 correlated with a more severe clinical score at the time of urine collection. Both elevated MMP-2/MMP-9 levels correlated with greater disease severity at the time of urine collection. bFGF levels correlated with improved clinical scores 1 year following urine collection.
Previous animal studies have examined several angiogenic factors, including MMP-2/MMP-9, VEGF, and bFGF, in relation to ischemic periods of vascular remodeling following strokes. These studies have shown that ischemia leads to a period of post-stroke brain plasticity that is very similar to the juvenile critical period in terms of cellular structure and molecular expression.19 In both instances, the need for vascular remodeling and neurogenesis led to an increase in the expression of molecules responsible for both phenomena. In particular, MMP-2 and MMP-9 are upregulated in post-stroke cortex to promote re-vascularization of ischemic areas.20 VEGF and bFGF levels have also been shown to be elevated in post-ischemic recovery in the central nervous system.11
In SWS, stroke-like episodes occur when there is decreased blood flow to an area of the brain, and ictal-SPECT (single-photon emission computed tomography) studies have shown that blood flow can decrease to ischemic levels during seizures in SWS subjects.21 It is likely that some level of vascular remodeling occurs in response to these episodes and neurogenesis in the ischemic brain. Comati et al. found increased expression of VEGF and increased mitotic activity of endothelial cells in SWS brain tissue – indicating that angiogenesis is ongoing and SWS lesions are dynamic rather than static structures.9 Elevated serum bFGF levels have been found in post-stroke adult humans up to 14 days after stroke.11,22,23 The presence of MMPs and high levels of bFGF in the urine of subjects with SWS would be consistent with ongoing vascular remodeling, either as a consequence of the vascular anomaly or in response to the abnormal blood flow and tissue hypoxia.
We have shown that female SWS subjects are more likely to have higher levels of MMP-9 and its related molecules. While this is a novel result in the context of SWS, similar results have been found in control subjects and in subjects with diabetes mellitus.24 Unlike the patient population reported in the Thrailkill et al. study, the majority of our female subjects were premenarcheal, making the gender difference less likely to be estrogen-mediated.24 Further investigation into gender differences in SWS is needed, but this difference in MMP-9 levels might provide insight into SWS pathophysiology and in gender differences in other neurological disorders as well.
The correlations between angiogenic factors and clinical outcomes reported here are also supported by previous studies. In 2005, Marler et al. reported that MMP-2 and MMP-9 correlated with both the extent and progression of a wide variety of vascular anomalies in children.6 In SWS, we report here that MMP-2 correlates positively with worse clinical outcome as measured by the SWS neurologic score assigned at the time of collection, and MMP-9 correlates positively with the extent of SWS involvement in the subject. These results suggest that analysis of urinary MMPs may be clinically useful for selecting subjects for more aggressive treatment or future clinical trials.
Furthermore, the data presented here indicate that bFGF might predict future clinical outcome in SWS subjects. Subjects with higher bFGF levels tended to have less evidence of hemiparesis when assigned SWS clinical scores 10–14 months after sample collection. One possible reason for this correlation is that bFGF has been shown to have a protective and reparative effect on the ischemic and post-ischemic brain.25 In vitro and in vivo studies have demonstrated that bFGF is both a vasodilator that can increase cerebral blood flow to regions of infarct and promote neurogenesis, as well as a protective agent for neurons against a variety of toxins and insults, including free radicals that can cause damage to cells as per the calcium hypothesis of ischemic brain injury.26 Additionally, bFGF has been successfully used as an exogenous drug in several studies to promote functional recovery in middle cerebral artery occluded rats.25,27–30 In addition, it has been shown in post-stroke adults that increased serum bFGF levels correlate positively with improvement of clinical neurological deficits.11 Our results suggest that bFGF urinary levels in subjects with SWS may be useful as a biomarker for evaluating treatment response in these patients.
Limitations
The hypotheses made in this study rely on the assumption that the angiogenic factors measured in this study change as a direct result of the subjects’ SWS severity and are not influenced by medications or other factors. All subjects in this study were on one or more anticonvulsants, typically oxcarbazepine and/or levetiracetam, for their seizures, and many were on other medications as well (such as aspirin or stimulants). The influence of these drugs in the proteins of interest will require additional study.
Additionally, there is a high level of phenotypic variation among Sturge–Weber patients. There are many different symptoms associated with SWS, which can present in isolation or in various combinations. Given the low prevalence of SWS, it is difficult to compile a cohort of patients all experiencing the same manifestation of SWS. While we limited our subjects to those with confirmed brain involvement, and noted subjects’ disease severity, we did not standardize or limit participation on the basis of other SWS characteristics. These phenotypic variations among subjects may create difficulty when interpreting the data.
Conclusions
This study takes the first steps towards demonstrating the efficacy of using angiogenic factors in urine as biomarkers to track the progression of SWS and supports their role in the ongoing vascular remodeling. SWS subjects have higher levels of angiogenic factors in their urine compared to controls, the levels of these factors correlate with clinical outcome at the time of collection and they may have predictive value for disease progression as well.
Collecting and analyzing urine samples for angiogenic factors is non-invasive, and safe, especially when compared to blood draws, tissue biopsies, CT or MRI scans, or other diagnostic measures. Because it is non-invasive and does not require the use of anesthesia, samples can be collected on a frequent basis, allowing for close monitoring of disease progression.
Since the expression of angiogenic factors changes dramatically during ischemia, it is likely that strokes and stroke-like episodes have an important effect on the amount of angiogenic factors found in subjects’ urine. Future studies would benefit from investigations into the specific relationship between the strokes and stroke-like episodes suffered by SWS subjects, the vascular remodeling that takes place as a result, and the change in factor levels.
Figure 1.
Consort diagram for Sturge-Weber Syndrome urine biomarker study.
Acknowledgement
We gratefully acknowledge the support of the families and individuals who participated in this research.
Funding
We gratefully acknowledge funding from Hunter’s Dream for a Cure Foundation and from The Brain Vascular Malformation Consortium (BVMC; U54NS065705), which is a part of the National Institutes of Health (NIH) Rare Disease Clinical Research Network (RDCRN), supported through collaboration between the NIH Office of Rare Diseases Research (ORDR) at the National Center for Advancing Translational Science (NCATS), and the National Institute of Neurological Disorders and Stroke (NINDS).
Appendix 1. Sturge–Weber syndrome clinical scores.
| Seizures | ||||||
|---|---|---|---|---|---|---|
| Score | 0 | 1 | 2 | 3 | 4 | |
| No seizures | 1+, but controlled for last 6 months | Breakthrough seizures during last 6 months but not monthly | Monthly | Weekly + | ||
| Hemiparesis | ||||||
| 0 | 1 | 2 | 3 | 4 | ||
| Normal | Intermittent postures | Fine motor impairment | Fine and gross motor impairment | Severe fine and gross motor impairment, poor helper arm function, walks poorly/not at all | ||
| Visual field cut | ||||||
| 0 | 1 | 2 | ||||
| None | Partial hemi-field cut | Full hemi-field cut | ||||
| Cognitive function | ||||||
| 0 | 1 | 2 | 3 | 4 | 5 | |
| Adult: 18+ years | Normal | Lives and works independently | Works in community with parental support | Significant difficulty maintaining employment or satisfactory social relationships | Trainable (group home, supervised work setting) | Full care |
Appendix 2. Sturge–Weber syndrome involvement scores.
| Score | 0 | 1 | 2 |
|---|---|---|---|
| Brain | No involvement | Unilateral involvement | Bilateral involvement |
| Eye | No involvement | Unilateral involvement | Bilateral involvement |
| Skin | No involvement | Unilateral involvement | Bilateral involvement |
Patients are assigned Involvement Scores of 0–2 for brain, eye and skin resulting in an overall range of 0–6 for total Involvement Score.
Footnotes
Conflict of interest
The author declares that there is no conflict of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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