In vivo demonstration of blood-brain barrier impairment in Moyamoya disease.

BACKGROUND: Moyamoya disease (MMD) is a cerebrovascular disorder characterized by fragile vascular system. Previous studies suggested that the blood-brain barrier (BBB) destabilizing cytokine angiopoietin-2 plays a critical role in increasing vascular plasticity and endothelial disintegration in MMD. The aim of this study was to assess cerebrovascular integrity in vivo in patients affected by MMD. METHODS: We retrospectively analyzed 11 patients that underwent bypass for MMD (MMD group), 11 patients that underwent bypass for atherosclerotic cerebrovascular disease (ACVD-control group I), and 5 patients that underwent clipping for unruptured aneurysms (non-ischemic-control group II). Sodium fluorescein (NaFL) extravasation was evaluated during videoangiography when checking for bypass patency. A grading system (0, +, ++, +++) was used to define the extent of extravasation. Frequency and intensity of leakage was compared among different groups. RESULTS: NaFL extravasation appeared in 10/11 (91%) patients with MMD and in 8/11 (73%) patients with ACVD during bypass procedures. Extravasation was observed in none of the patients undergoing clipping for unruptured aneurysms. Although both chronic ischemic patient groups showed a comparably high incidence of NaFL extravasation, the MMD group was characterized by a much greater intensity of NaFL extravasation (grade +++ in 82%) than the ACVD group (grade +++ in 27%, p < 0.05). CONCLUSIONS: We demonstrate blood-brain barrier impairment in MMD patients for the first time in vivo. This may be due to mechanisms intrinsic to the unique pathology of MMD, probably explaining the higher association with hemorrhage and post-operative hyperperfusion.

Autocrine release of angiopoietin-2 mediates cerebrovascular disintegration in Moyamoya disease.

Moyamoya disease is a rare steno-occlusive cerebrovascular disorder often resulting in hemorrhagic and ischemic strokes. Although sharing the same ischemic stimulus with atherosclerotic cerebrovascular disease, Moyamoya disease is characterized by a highly instable cerebrovascular system which is prone to rupture due to pathological neovascularization. To understand the molecular mechanisms underlying this instability, angiopoietin-2 gene expression was analyzed in middle cerebral artery lesions obtained from Moyamoya disease and atherosclerotic cerebrovascular disease patients. Angiopoietin-2 was significantly up-regulated in Moyamoya vessels, while serum concentrations of soluble angiopoietins were not changed. For further evaluations, cerebral endothelial cells incubated with serum from these patients in vitro were applied. In contrast to atherosclerotic cerebrovascular disease serum, Moyamoya disease serum induced an angiopoietin-2 overexpression and secretion, accompanied by loss of endothelial integrity. These effects were absent or inverse in endothelial cells of non-brain origin suggesting brain endothelium specificity. The destabilizing effects on brain endothelial cells to Moyamoya disease serum were partially suppressed by the inhibition of angiopoietin-2. Our findings define brain endothelial cells as the potential source of vessel-destabilizing factors inducing the high plasticity state and disintegration in Moyamoya disease in an autocrine manner. We also provide new insights into Moyamoya disease pathophysiology that may be helpful for preventive treatment strategies in future.

Intravascular Inflammation Triggers Intracerebral Activated Microglia and Contributes to Secondary Brain Injury After Experimental Subarachnoid Hemorrhage (eSAH).

Activation of innate immunity contributes to secondary brain injury after experimental subarachnoid hemorrhage (eSAH). Microglia accumulation and activation within the brain has recently been shown to induce neuronal cell death after eSAH. In isolated mouse brain capillaries after eSAH, we show a significantly increased gene expression for intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Hence, we hypothesized that extracerebral intravascular inflammatory processes might initiate the previously reported microglia accumulation within the brain tissue. We therefore induced eSAH in knockout mice for ICAM-1 (ICAM-1-/-) and P-selectin glycoprotein ligand-1 (PSGL-1-/-) to find a significant decrease in neutrophil-endothelial interaction within the first 7 days after the bleeding in a chronic cranial window model. This inhibition of neutrophil recruitment to the endothelium results in significantly ameliorated microglia accumulation and neuronal cell death in knockout animals in comparison to controls. Our results suggest an outside-in activation of the CNS innate immune system at the vessel/brain interface following eSAH. Microglia cells, as part of the brain's innate immune system, are triggered by an inflammatory reaction in the microvasculature after eSAH, thus contributing to neuronal cell death. This finding offers a whole range of new research targets, as well as possible therapy options for patients suffering from eSAH.

Vasculogenic and Angiogenic Pathways in Moyamoya Disease.

BACKGROUND: Moyamoya disease (MMD) is a slowly progressing steno-occlusive cerebrovascular disease. The typical moyamoya vessels, which originate from an initial stenosis of the internal carotid, highlight that increased and/or abnormal angiogenic, vasculogenic and arteriogenic processes are involved in the disease pathophysiology. OBJECTIVE: Herein, we summarize the current knowledge on the most important signaling pathways involved in MMD vessel formation, particularly focusing on the expression of growth factors and function of endothelial progenitor cells (EPCs). METHODS AND RESULTS: Higher plasma concentrations of vascular endothelial growth factor, matrix metalloproteinase, hepatocyte growth factor, and interleukin-1ß were reported in MMD. A specific higher level of basic fibroblast growth factor was also found in the cerebrospinal fluid of these patients. Finally, the number and the functionality of EPCs were found to be increased. In spite of the available data, the approaches and findings reported so far do not give an evident correlation between the expression levels of the aforementioned growth factors and MMD severity. Furthermore, the controversial results provided by studies on EPCs, do not permit to understand the true involvement of these cells in MMD pathophysiology. CONCLUSION: Further studies should thus be implemented to extend our knowledge on processes regulating both the arterial stenosis and the excessive formation of collateral vessels. Moreover, we suggest advances of integrated approaches and functional assays to correlate biological and clinical data, arguing for the development of new therapeutic applications for MMD.

Control of the blood-brain barrier function in cancer cell metastasis.

Cerebral metastases are the most common brain neoplasms seen clinically in the adults and comprise more than half of all brain tumours. Actual treatment options for brain metastases that include surgical resection, radiotherapy and chemotherapy are rarely curative, although palliative treatment improves survival and life quality of patients carrying brain-metastatic tumours. Chemotherapy in particular has also shown limited or no activity in brain metastasis of most tumour types. Many chemotherapeutic agents used systemically do not cross the blood-brain barrier (BBB), whereas others may transiently weaken the BBB and allow extravasation of tumour cells from the circulation into the brain parenchyma. Increasing evidence points out that the interaction between the BBB and tumour cells plays a key role for implantation and growth of brain metastases in the central nervous system. The BBB, as the tightest endothelial barrier, prevents both early detection and treatment by creating a privileged microenvironment. Therefore, as observed in several in vivo studies, precise targetting the BBB by a specific transient opening of the structure making it permeable for therapeutic compounds, might potentially help to overcome this difficult clinical problem. Moreover, a better understanding of the molecular features of the BBB, its interrelation with metastatic tumour cells and the elucidation of cellular mechanisms responsible for establishing cerebral metastasis must be clearly outlined in order to promote treatment modalities that particularly involve chemotherapy. This in turn would substantially expand the survival and quality of life of patients with brain metastasis, and potentially increase the remission rate. Therefore, the focus of this review is to summarise the current knowledge on the role and function of the BBB in cancer metastasis.

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5.

Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 ± 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.

Inhibition of proteasome-mediated glucocorticoid receptor degradation restores nitric oxide bioavailability in myocardial endothelial cells in vitro.

BACKGROUND INFORMATION: Glucocorticoids (GCs), including the synthetic GC derivate dexamethasone, are widely used as immunomodulators. One of the numerous side effects of dexamethasone therapy is hypertension arising from reduced release of the endothelium-derived vasodilator nitric oxide (NO). RESULTS: Herein, we described the role of dexamethasone and its glucocorticoid receptor (GR) in the regulation of NO synthesis in vitro using the mouse myocardial microvascular endothelial cell line, MyEND. GC treatment caused a firm decrease of extracellular NO levels, whereas the expression of endothelial NO synthase (eNOS) was not affected. However, GC application induced an impairment of tetrahydrobiopterin (BH4 ) concentrations as well as GTP cyclohydrolase-1 (GTPCH-1) expression, both essential factors for NO production upstream of eNOS. Moreover, dexamethasone stimulation resulted in a substantially decreased GR gene and protein expression in MyEND cells. Importantly, inhibition of proteasome-mediated proteolysis of the GR or overexpression of an ubiquitination-defective GR construct improved the bioavailability of BH4 and strengthened GTPCH-1 expression and eNOS activity. CONCLUSIONS: Summarising our results, we propose a new mechanism involved in the regulation of NO signalling by GCs in myocardial endothelial cells. We suggest that a sufficient GR protein expression plays a crucial role for the management of GC-induced harmful adverse effects, including deregulations of vasorelaxation arising from disturbed NO biosynthesis.

TWIST1 regulates the activity of ubiquitin proteasome system via the miR-199/214 cluster in human end-stage dilated cardiomyopathy.

BACKGROUND: The transcription factor TWIST1 has been described to regulate the microRNA (miR)-199/214 cluster. Genetic disruption of TWIST1 resulted in a cachectic phenotype and early death of the knock-out mice. This might be connected to the activity of the ubiquitin-proteasome-system (UPS), as miR-199a has been suggested to regulate the ubiquitin E2 ligases Ube2i and Ube2g1. METHODS: Cardiac tissue from explanted hearts of 42 patients with dilated cardiomyopathy and 20 healthy donor hearts were analysed for protein expression of TWIST1 and its inhibitors Id-1, MuRF-1 and MAFbx, the expression of miR-199a, -199b and -214, as well as the activity of the UPS by using specific fluorogenic substrates. RESULTS: TWIST1 was repressed in patients with dilated cardiomyopathy by 43% (p=0.003), while Id1 expression was unchanged. This was paralleled by a reduced expression of miR-199a by 38 ± 9% (p=0.053), miR-199b by 36 ± 13% (p=0.019) and miR-214 by 41 ± 11% (p=0.0158) compared to donor hearts. An increased peptidylglutamyl-peptide-hydrolysing activity (p<0.0001) was observed in the UPS, while the chymotrypsin-like and trypsin-like activities were unchanged. The protein levels of the rate limiting ubiquitin E3-ligases MuRF-1 and MAFbx were up-regulated (p=0.005 and p=0.0156, respectively). Mechanistically silencing of TWIST1 using siRNA in primary rat cardiomyocytes led to a down-regulation of the miR-199/214 cluster and to a subsequent up-regulation of Ube2i. CONCLUSION: The TWIST1/miR-199/214 axis is down-regulated in dilated cardiomyopathy, which is likely to play a role in the increased activity of the UPS. This may contribute to the loss of cardiac mass during dilatation of the heart.

Glucocorticoid insensitivity at the hypoxic blood-brain barrier can be reversed by inhibition of the proteasome.

BACKGROUND AND PURPOSE: Glucocorticoids potently stabilize the blood-brain barrier and ameliorate tissue edema in certain neoplastic and inflammatory disorders of the central nervous system, but they are largely ineffective in patients with acute ischemic stroke. The reasons for this discrepancy are unresolved. METHODS: To address the molecular basis for the paradox unresponsiveness of the blood-brain barrier during hypoxia, we used murine brain microvascular endothelial cells exposed to O(2)/glucose deprivation as an in vitro model. In an in vivo approach, mice were subjected to transient middle cerebral artery occlusion to induce brain infarctions. Blood-brain barrier damage and edema formation were chosen as surrogate markers of glucocorticoid sensitivity in the presence or absence of proteasome inhibitors. RESULTS: O(2)/glucose deprivation reduced the expression of tight junction proteins and transendothelial resistance in murine brain microvascular endothelial cells in vitro. Dexamethasone treatment failed to reverse these effects during hypoxia. Proteasome-dependent degradation of the glucocorticoid receptor impaired glucocorticoid receptor transactivation thereby preventing physiological glucocorticoid activity. Inhibition of the proteasome, however, fully restored the blood-brain barrier stabilizing properties of glucocorticoid during O(2)/glucose deprivation. Importantly, mice treated with the proteasome inhibitor Bortezomib in combination with steroids several hours after stroke developed significantly less brain edema and functional deficits, whereas respective monotherapies were ineffective. CONCLUSIONS: We for the first time show that inhibition of the proteasome can overcome glucocorticoid resistance at the hypoxic blood-brain barrier. Hence, combined treatment strategies may help to combat stroke-induced brain edema formation in the future and prevent secondary clinical deterioration.

Glucocorticoid effects on endothelial barrier function in the murine brain endothelial cell line cEND incubated with sera from patients with multiple sclerosis.

Compromised blood-brain barrier integrity is a major hallmark of active multiple sclerosis (MS). Alterations in brain endothelial tight junction protein and gene expression occur early during neuroinflammation but there is little known about the underlying mechanisms. In this study, we analysed barrier compromising effects of sera from MS patients and barrier restoring effects of glucocorticoids on blood-brain barrier integrity in vitro. cEND murine brain microvascular endothelial cell monolayers were incubated with sera from patients in active phase of disease or in relapse. Data were compared with effects of the glucocorticoid dexamethasone alone or in combination with MS sera on barrier integrity. Tight junction protein levels and gene expression were evaluated concomitant with barrier integrity. We reveal downregulation of claudin-5 and occludin protein and mRNA and an accompanying upregulation in expression of matrix metalloproteinase MMP-9 after incubation with serum from active disease and remission and also a minor reconstitution of barrier functions related to dexamethasone treatment. Moreover, we for the first time describe downregulation of claudin-5 and occludin protein after incubation of cEND cells with sera from patients in remission phase of MS. Our findings reveal direct and differential effects of MS sera on blood-brain barrier integrity.

Glucocorticoids increase VE-cadherin expression and cause cytoskeletal rearrangements in murine brain endothelial cEND cells.

Recent studies have shown the influence of glucocorticoids on the expression of the tight junction protein occludin in the brain capillary endothelial cell line cEND, contributing to improvement in endothelial barrier functions. In this study, we investigated glucocorticoid effects on the expression of the adherens junction proteins VE- (vascular-endothelial) cadherin, alpha-catenin and beta-catenin as well as that of ZO-1, the plaque protein shared by both adherens and tight junctions on stimulation with dexamethasone. We were able to show a positive influence of dexamethasone administration on VE-cadherin protein levels as well as a rearrangement of VE-cadherin protein to the cytoskeleton after dexamethasone treatment. Investigation of transcriptional activation of the VE-cadherin promoter by dexamethasone, however, did not point to direct glucocorticoid-mediated VE-cadherin gene induction but rather suggested indirect steroid effects leading to increased VE-cadherin protein synthesis. Dexamethasone was further shown to induce cellular differentiation into a cobblestone cellular morphology and reinforcement of adherens junctions concomitant with the increased anchorage of VE-cadherin to the actin cytoskeleton. We thus propose that glucocorticoid effects on VE-cadherin protein synthesis and organization are important for the formation of both adherens and tight junction, and for improved barrier properties in microvascular brain endothelial cells.