Sturge-Weber Syndrome (SWS) is a rare neurovascular disorder characterized by facial capillary malformation (port-wine birthmark), abnormal leptomeningeal blood vessels, and abnormal vasculature and glaucoma in the eye. Patients present neurologically with seizures, stroke-like episodes, motor and cognitive impairment. SWS and port-wine birthmarks are caused by a somatic R183 mutation in GNAQ, the gene which codes for the protein Gαq.1,2 Cell sorting experiments of tissue samples suggest that the mutation is enriched in endothelial cells in both port-wine birthmarks and SWS.3,4 The mutation is predicted to result in impaired deactivation of Gαq and hyperactivation of downstream pathways, including the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) and mTOR pathways.5,6 These downstream pathways have been implicated in other vascular anomalies, uveal melanoma and overgrowth syndromes.7 We hypothesized, therefore, that mTOR activity would be greater in the endothelial cells of abnormal SWS leptomeningeal vessels, than in those from epilepsy control brain leptomeningeal vessels. Phosphorylated-S6 (p-S6) has previously been used as a marker for mTOR pathway activity8 and has been detected in skin tissue endothelial cells from venous channels and capillaries in SWS patients.9 We aimed therefore to determine p–S6 expression in SWS and epilepsy control (EC) brain tissue samples.
Materials and methods
Tissue samples were collected with written consent and the study was performed with Institutional Review Board approval at John Hopkins University. Brain tissue samples from SWS subjects (n = 7; 2 females, ages 12 months to 24 years) and epilepsy control samples (n = 10; 8 females, ages 2 months to 23 years; 6 Rasmussens, 3 Migration Disorder, 1 post-traumatic Epilepsy) were obtained. We also matched SWS samples with age and sex matched postmortem control samples; however, none of these samples stained well with either DAPI or antibodies, and therefore were not used in the analysis.
Brain tissues were paraffin processed, and 7 µm sections were used for immunocytochemical staining as previously described. H&E was done on a section for each sample. IHC was performed using a specific antibody that recognizes the phosphorylated form of S6 ribosomal protein, a rabbit monoclonal anti-Phospho-S6 Ribosomal Protein (Ser235/236) (Cell Signaling Technology, Cat. no. 4858T), and costained for DAPI (Prolong Gold Antifade Reagent with DAPI, Cell Signaling Technology, Cat. no. 8961). The paraffin-embedded brain sections were deparaffinized in an oven (60°C) and rehydrated using HemoDe and Ethanol. Antigen retrieval was performed using preheated 1× Sodium Citrate Buffer. The slides were incubated with the secondary antibody Alexa Fluor 594-Goat-anti-Rabbit 1:500 (Invitrogen, Cat. no. A-11037).
H&E sections were visualized and photographed under a light microscope (Leitz Laborlux 12 Microscope). IHC stained sections were visualized using an apotome microscope (Zeiss AxioVision Microscope). Microphotographs were taken of each sample at standardized settings, and focused on the leptomeningeal endothelial layer, leptomeningeal vessels, nonvascular leptomeningeal tissue, and cerebral cortex, as previously described.10 Two observers independently analyzed each image, blinded to sample diagnosis, and noted the presence or absence of Phospho-S6-ribosomal protein expression in the leptomeningeal endothelial cell layer, the perivascular cells, and in the cortex. The percentage of vessels staining for Phospho-S6 in the endothelial cell layer was also calculated from the images taken.
Percentage of SWS and EC brain tissue samples demonstrating p-S6 in the leptomeningeal endothelial, cortical, and perivascular cells were calculated, as were the percentage of imaged leptomeningeal vessels with p-S6 staining. Fisher’s Exact tests were used for the statistical analysis in SPSS (IBM, Version 25) and significance was ascertained at P < 0.05.
Histomorphologic assessment by H&E
An increased number of both thick-walled vessels and dilated thin-walled vessels were seen in SWS leptomeninges. Most had capillary-venous morphology; however, an increase in arterialized, thick-walled vessels with layers morphologically consistent with smooth muscle cells was also seen (Figure 1B). In epilepsy control (EC) samples, the leptomeninges were more diverse with eight out of ten (8/10) having normal appearing leptomeninges and two out of ten (2/10) having abnormal, enlarged vessels, although not to the degree seen in the SWS samples (Figure 1A). Evidence of cortical or subcortical calcification was seen in five out of seven (5/7) SWS samples and none of the epilepsy controls (Figure 1A,B).
Phospho-S6 expression in leptomeningeal endothelial cells
Within the leptomeninges, all SWS sections had Phospho-S6 positivity, whereas only 50% of the EC sections did (7/7 SWS versus 5/10 EC, P < 0.05; Figure 2A–C). Within only the samples that were positive for Phospho-S6 expression in the endothelial cell layer (7/7 SWS and 5/10 EC), the percentage of positive vessels was not significantly different between SWS and EC tissues (SWS mean 53% ± 20.7 versus EC mean 75% ± 35.4; NS).
Phospho-S6 expression in other vascular and perivascular cells
Most SWS samples also had Phospho-S6 staining in the leptomeningeal vessel wall, and in the perivascular meningeal cells of the leptomeninges; however, the percent was not significantly different from the EC brain tissue (vessel wall 6/7 SWS versus 5/10 EC; NS; perivascular meningeal cells 7/7 SWS versus 8/10 EC; NS; Figure 3A–C).
Phospho-S6 expression in cortical cells
Phospho-S6 staining in the cortex was present in only 3 out of 7 SWS samples, compared to 7 out of 10 epilepsy control samples; difference was not statistically significant. Phospho-S6 staining of the cerebral cortex revealed cells with neuronal phenotype and at the pial-cortical interface in the SWS samples, and with neuronal phenotype in the EC samples (Figure 4A–E).
Discussion and conclusion
Our data indicate that cortical brain lesions from SWS subjects are significantly more likely to have Phospho-S6 staining in the leptomeningeal endothelial layer than EC subjects. We recently reported increased expression of phosphorylated extracellular signal-regulated kinase (p-ERK) in the endothelial cells of SWS leptomeningeal vessels.10 ERK activation can increase mTOR activity indirectly, and therefore we hypothesized that the abnormal blood vessels in SWS would also demonstrate increased mTOR activity, and therefore Phospho-S6 expression, in leptomeningeal endothelial cells. The results of this study are consistent with this hypothesis and suggest that mTOR inhibitors may improve endothelial cell function in the vascular malformation of patients with SWS.
Sirolimus (rapamycin) is FDA-approved for organ rejection in kidney transplants and for lymphangioleiomyomatosis, a rare progressive lung disease. Sirolimus, an mTOR inhibitor, in conjunction with laser treatment, was suggested in a randomized, placebo-controlled trial to have benefit over laser alone for the treatment of capillary malformations. Some centers are now offering it as routine clinical care. Low-dose mTOR inhibitors are beneficial for the treatment of tumors and seizures in Tuberous Sclerosis Complex in randomized, placebo-controlled clinical trials.11,12 Sirolimus treatment of children ages 0–31 years with other complicated vascular anomalies (not involving the brain) in a phase II clinical trial13 showed that low-dose sirolimus was safe even in the youngest patients. Clinically, sirolimus is now being widely used in the clinical treatment of complicated vascular anomalies. Recently, data has been published from a pilot clinical drug trial of oral sirolimus, in patients with Sturge-Weber Syndrome suggesting that sirolimus may improve cognitive and neurologic status, particularly in patients with a history of stroke and stroke-like episodes.14 In addition, a case was reported of a child treated presymptomatically with sirolimus and low-dose aspirin.15 We hypothesize that the mTOR inhibitor helps normalize endothelial function in SWS cells with the R183Q GNAQ mutation.
Increased expression of vascular factors has been found in SWS brain tissue: vascular endothelial growth factor (VEGF) expression is increased in cortical neurons and glia underlying the abnormal leptomeningeal vessels, whereas VEGFR-1, VEGFR-2, HIF-1α, and HIF-2α expression are increased in endothelial cells lining the abnormal leptomeningeal vessels.16 Expression of vascular endothelial growth factor receptor 2 (VEGFR-2) in endothelial cells is mediated by activated GNAQ, and likely stimulates molecular changes responsible for the vascular remodeling that has been observed in SWS.16 Both the Ras/RAF/MEK/ERK and PI3K/AKT/mTOR pathways modulate VEGF and HIF-1α expression, and in SWS likely interact in doing so to regulate mutant endothelial cell function.
Obtaining age and sex matched brain tissue suitable for immunohistochemistry is challenging. The matched postmortem control samples we obtained were not of sufficient quality to be able to obtain satisfactory IHC results. Therefore, all of the control brain tissue used in this study are also abnormal and were obtained through epilepsy surgery. These EC samples may also not have “normal” Phospho-S6 expression. The issue of controls can be addressed to some degree by using normal and abnormal (port-wine) skin biopsies as previously reported to study p-S6 expression9; however, this biopsy tissue is also very limited. The other significant limitation in this study is that, to date, no reliable methods exist for identifying which cells within the intact tissue section have the somatic mutation; no specific antibody exists for the R183Q mutant Gαq, laser capture is insufficiently selective or unsuccessful, and other less common methods have also been unsuccessful. Therefore, while we hypothesize that the SWS leptomeningeal endothelial cells with p-S6 expression harbor the somatic mutation, this is not able to be confirmed at this time.
Conclusions and future directions
Our study demonstrates that Sturge-Weber brain samples have Phospho-S6 expression in the endothelial cells of abnormal leptomeningeal vessels. This result, along with prior work indicating Phospho-S6 expression in the endothelial cells lining capillary malformations from port-wine birthmarks of patients with SWS, supports the hypothesis that mTOR activity is involved in the pathogenesis and/or progression of the leptomeningeal vascular malformation. Further study is needed in patients with SWS, to understand the role of mTOR activation in pathophysiology and progression of this syndrome, and to assess the role of mTOR inhibitors in the treatment of SWS. Future studies may analyze port-wine skin biopsy expression of Phospho-S6 and/or Phospho-ERK as potential biomarkers for assessing or predicting treatment response to mTOR or other pathway inhibitors. Approaches for identifying the cells harboring the somatic mutation within intact tissue sections are urgently needed, and efforts are underway to address this important technical issue. In vitro and animal models of the R183Q GNAQ mutation in vascular cells are under development; when validated, these models can also be used to better understand the role of mTOR activity in SWS and port-wine birthmarks.
The authors disclosed receipt of the following financial support for the research, authorship, or publication of this article: this work was supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS) (National Institutes of Health [NIH] U54NS065705) (to Michael Lawton; Sturge-Weber Project to AMC). The Brain Vascular Malformation Consortium (U54NS065705) is a part of the NIH Rare Diseases Clinical Research Network (RDCRN), supported through the collaboration between the NIH Office of Rare Diseases Research (ORDR) at the National Center for Advancing Translational Science (NCATS) and the NINDS. Work was also supported by the Kennedy Krieger Institute IDDRC (U54 HD079123). Brain tissue samples were obtained from the NIH Neuro BioBank and the Johns Hopkins Brain Tissue Bank.
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