J. Neurochem. (2012) 121, 730–737.
Homeostasis of the brain is dependent on the blood–brain barrier (BBB). This barrier tightly regulates the exchange of essential nutrients and limits the free flow of immune cells into the CNS. Perturbations of BBB function and the loss of its immune quiescence are hallmarks of a variety of brain diseases, including multiple sclerosis (MS), vascular dementia, and stroke. In particular, diapedesis of monocytes and subsequent trafficking of monocyte-derived macrophages into the brain are key mediators of demyelination and axonal damage in MS. Endothelin-1 (ET-1) is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases. Here, we studied the role of different components of the endothelin system, i.e., ET-1, its type B receptor (ETB) and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis of a human brain endothelial cell barrier. Our pharmacological inhibitory and specific gene knockdown studies point to a regulatory function of these proteins in transendothelial passage of monocytes. Results from this study suggest that the endothelin system is a putative target within the brain for anti-inflammatory treatment in neurological diseases.
- ETA receptor
endothelin type A receptor
- ETB receptor
endothelin type B receptor
normal donkey serum
The BBB endothelial cells play the predominant role in actively transporting nutrients to the brain and limit the entrance of potentially harmful blood components, including cells of the immune system. This is essential for brain homeostasis and reliable functioning of the neuronal environment. A common feature of diverse neurological diseases, including MS, vascular dementia and stroke, is the typical loss of the specialized function of the BBB which leads to unstable brain homeostasis, inflammation, and neuronal damage (de Vries et al. 1997; Zlokovic 2008). In the past, we and others showed that loss of BBB function is an early and typical phenomenon in the neuro-inflammatory disease MS and models thereof (Floris et al. 2004; Vos et al. 2005). In MS, infiltrated monocyte-derived macrophages form the major cell type in perivascular infiltrates and are key mediators of demyelination and axonal damage, two characteristic features of MS (Bruck et al. 1996). To travel into the brain and exert their detrimental effects, monocytes have to cross the BBB. This requires the active participation of brain endothelial cells, and we and others have demonstrated the strong contribution of soluble mediators, extracellular proteases and intracellular signaling events (Walters et al. 2002; Reijerkerk et al. 2006, 2008, 2010; Schreibelt et al. 2007).
Endothelin-1 is considered as a potent pro-inflammatory peptide and has been implicated in the development of cardiovascular diseases (Khimji and Rockey 2010). It is of interest that the expression of adhesion proteins which facilitate the interaction between endothelial cells and leukocytes, including intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 is enhanced by ET-1 in brain endothelial cells (McCarron et al. 1993). Moreover, ET-1 has been reported to increase BBB permeability in vivo (Narushima et al. 2003) and inhibit P-glycoprotein-mediated efflux function in cultured brain endothelial cells (Hembury and Mabondzo 2008) and in isolated cerebral capillaries (Hartz et al. 2004). It is of particular interest that plasma levels of ET-1 are significantly elevated in patients with MS (Haufschild et al. 2001), illustrating its pro-inflammatory function.
Brain endothelial cells like other endothelium produce ET-1 (Yoshimoto et al. 1990; Dehouck et al. 1997) which is generated through cleavage of its precursor preproendothelin by ECE-1. ET-1 exerts its biological effects via two types of ET receptor, ETA and ETB, which are members of G-protein-coupled receptor superfamily (Rubanyi and Polokoff 1994). The ETA receptor is found predominantly in smooth muscle cells and cardiac muscles whereas the ETB receptor is abundantly expressed in endothelial cells (Huggins et al. 1993). There has been controversy regarding the ETA and ETB receptor subtype expression and function in brain endothelial cells (Hagiwara et al. 1993; Kobari et al. 1994; Pagotto et al. 1995; Kawai et al. 1997; Naidoo et al. 2004).
Here, we hypothesized that the endothelin system has an important function in the recruitment of leukocytes at sites of inflammation in the brain. Using both pharmacological and specific gene knockdown approaches, we here show that monocyte diapedesis is dependent on the activity of ET-1, ETB receptor, and ECE-1. The results point to a set of putative therapeutic targets for anti-inflammation treatment in neurological diseases such as MS, vascular dementia, and stroke.
Materials and methods
Materials and cells
The human brain endothelial cell line hCMEC/D3 (Weksler et al. 2005) was grown in endothelial cell basal medium-2 supplemented with human epidermal growth factor, hydrocortisone, GA-1000, vascular endothelial growth factor, basic fibroblast growth factor, R3-insulin-like growth factor-1, ascorbic acid, and 2.5% fetal calf serum (Lonza, Basel, Switzerland) on collagen coated plates. Collagen type I (calf skin) was from Sigma-Aldrich (St. Louis, MO, USA). Phalloidin-rhodamine was from Molecular Probes (Leiden, the Netherlands). Anti-ETB receptor [goat, 4 μg/mL; ETBR (C-20) goat polyclonal IgG, sc-21196], anti-ET-1 [goat, 2 μg/mL; ET-1 (N-8); goat polyclonal IgG; sc-21625] anti-ECE-1 [goat, 2 μg/mL; ECE-1 (F-16); goat polyclonal IgG; sc-27557], and anti-lamin B [goat, 2 μg/mL; Lamin B (C-20); goat polyclonal IgG; sc-6216] were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Transendothelial migration of primary monocytes
Human blood monocytes were isolated from buffy coats of healthy donors by Ficoll gradient and CD14-positive beads (Elkord et al. 2005). Primary monocyte transmigration through a monolayer of hCMEC/D3 was assayed as described previously (Reijerkerk et al. 2008). Briefly, 7.5 × 104 monocytes were added to 96-well plates containing hCMEC/D3 monolayers. Monocytes were allowed to settle and migrate over a 4-h period. To monitor monocyte migration, co-cultures were placed in an inverted phase-contrast microscope (Nikon Eclipse TE300, Lijnden, The Netherlands) housed in a temperature-controlled (37°C), 5% CO2 gassed chamber. A field (220 × 220 μm) was randomly selected and recorded for 10 min at 50 times by using a digital video camera using Cell F imaging software (Olympus, Heidelberg, Germany). Monocyte diapedesis was assessed by enumerating the number of monocytes within the field that had either adhered or migrated through the monolayer. Transmigrated monocytes (phase-dark) could be readily distinguished from those remaining on the cell surface by their highly refractive (phase-bright) morphology. To test the role of ETB and ETA receptor and ECE-1 in monocyte diapedesis, inhibitors including BQ-788, BQ-123, phosphoramidon, or DL-Thiorphan (Sigma-Aldrich) were added simultaneously with the monocytes to the brain endothelial cell monolayers.
The hCMEC/D3 brain endothelial cells were cultured in μSlide VI coated with collagen (Ibidi GmbH, München, Germany), fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min and blocked with 5% normal donkey serum (NDS) in PBS with 0.3% Triton X-100 for 15 min. Anti-ETB receptor, anti-ET-1, and anti-ECE-1 were diluted in PBS containing 0.3% Triton X-100 and 1% NDS and added overnight to detect ETB receptors, ET-1, and ECE-1, respectively. The secondary anti-goat-488 antibody (donkey, 1 : 500 dilution; Alexa fluor 488, IgG, A11055; Invitrogen, Breda, The Netherlands) was diluted in PBS containing 0.3% Triton X-100 and 1% NDS and was added for 60 min. The nucleus was stained with antibodies against lamin B for 5 min. Actin was stained with Rhodamin-labeled phalloidin (1 : 300 dilution; Molecular Probes) for 15 min. Imaging was done with a confocal laser scanning microscope (Leica AOBS SP2; PL APO x63/NA1.30, Rijswijk, The Netherlands).
Lentivirus-mediated delivery of shRNA
To establish knockdown of selected proteins we used a vector-based shRNA technique. Expression plasmids encoding specific shRNAs targeting human ET-1, ETB, and ECE-1 were obtained from Sigma-Aldrich (MISSION® shRNA library). Recombinant lentiviruses were produced by co-transfecting subconfluent human embryonic kidney 293T cells with the shRNA lentivirus expression plasmid and packaging plasmids (pMDLg/pRRE and pRSV-Rev) using calcium phosphate as a transfection reagent. Human embryonic kidney 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, in a 37°C incubator with 5% CO2. Infectious lentiviruses were collected 48 h after transfection. The supernatant was centrifuged to remove cell debris and stored at −80°C. hCMEC/D3 cells were transduced with the lentivirus-containing shRNA. Forty-eight hours after infection of hCMEC/D3 cells with the shRNA-expressing lentiviruses, stable cell lines were selected by puromycin treatment (2 μg/mL). The expression knockdown efficiency was determined by western blotting. Subsequently, stable cell lines were subjected to monocyte transmigration studies as described.
For western blot analysis of cell fractions, the nucleus and cytosolic fraction of hCMEC/D3 cells were separated using the NE-PER cell fractioning kit (Thermo Scientific, Rockford, IL, USA). In short, cells were lysed in buffer containing protease inhibitors (Roche Diagnostics Nederland B.V., Almere, the Netherlands). The lysates were centrifuged and the supernatant which contained the cytosolic extracts was harvested and stored. Nuclei were disrupted in buffer containing protease inhibitor cocktail. The nuclear lysates were centrifuged again and the supernatants containing the nuclear proteins were stored at −20°C.
Cell homogenates were prepared by replacing the culture medium with sodium dodecyl sulfate sample buffer containing 5%β-mercaptoethanol and subsequent heating at 95°C for 5 min. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Membranes were blocked with PBS containing 5% normal rabbit serum and incubated with antibodies against ETB receptor, ECE-1, and ET-1. Binding of these antibodies to their respective antigens was visualized using the Odyssee® Infrared Imaging System after application of IgG labeled with Infrared dye (IRdye) 800 (Rockland Immunochemicals, Gilbertsville, PA, USA). Band intensities were quantified using ImageJ software (National Institutes of Health, USA) and normalized to actin.
Statistical analysis was performed with the Mann–Whitney test (Prism 4.0; GraphPad Software, San Diego, CA, USA), and results were considered significant if p-value was < 0.05.
Endothelin system components are expressed in human brain endothelial cells
First, we determined the expression and localization of ET-1 and its ETB receptor in cultured human brain endothelial cells. Immunofluorescence stainings revealed the abundance of ET-1 and ETB receptor protein, which was scattered throughout the cell but concentrated near the cell nucleus (Fig. 1a). hCMEC/D3 cells did not express ETA receptor (not shown). Although G-protein-coupled receptors, such as ETB are usually expressed at the cell membrane, the stainings show that the majority of ETB co-localized with the nuclear envelope, indicated by lamin B staining. To further investigate this, nuclear and cytoplasmic fractions of the brain endothelial cells were subjected to western blotting analysis. The results show that brain endothelial cells express two isoforms of ETB receptor, one (50 kD) residing in the cytoplasm, the larger form (60 kD) being also present in the nuclear fraction (Fig. 1b).
Role of endothelin and ETB receptor in monocyte transmigration
As a next step, we tested whether the endothelin system has a functional role during the traversal of primary monocytes through a monolayer of human brain endothelial cells. Transendothelial migration of monocytes was studied in the absence or presence of a specific inhibitor of the ET-1 receptor or after knockdown by lentiviral mediated delivery of specific shRNA (Fig. 2). Diapedesis through brain endothelial cells was reduced in the presence of BQ-788, an ET receptor antagonist with high affinity for ETB (IC50 = 1.2 nM; control: 100.0% ± 2.2; 0.2 μM: 90.3% ± 3.4; 2 μM: 71.5 ± 2.4; n = 4; p ≤ 0.05 vs. control, Fig. 2a) and upon shRNA-mediated reduction of ETB (control: 100.0% ± 5.8; ETB shRNA: 51.1% ± 1.6; n = 4; p ≤ 0.05 vs. control; Fig. 2b). Of note, treatment with the ETA receptor antagonist BQ-123 did not affect monocyte transmigration (not shown). Similarly, monocyte diapedesis was significantly reduced in cells with reduced expression of ET-1 (control: 100.0% ± 13.3; ET-1 shRNA: 46.9% ± 4.5; n = 4; p ≤ 0.05 vs. control; Fig. 2c). The reduction of ETB and ET-1 expression in hCMEC/D3 cells was corroborated by western blot analysis (Fig. 2b and c, right panels).
Endothelin-converting enzyme expression and contribution to monocyte transmigration
Endothelin-1 is produced from a precursor, preproendothelin. After the removal of a signal peptide, the precursor is selectively processed by an enzyme (a furin-like peptidase) to yield a biologically inactive intermediate called big-endothelin. Big-endothelin is further converted into active ET by ECE. Remarkably, like ETB receptor, ECE-1 localized to the nucleus of human brain endothelial cells as determined by immunofluorescence and immunoblot analysis (Fig. 3a and b). An approach to inhibit the pro-inflammatory effect of ET-1 is to suppress its synthesis by inhibiting ECE-1. We therefore treated brain endothelial cells with phosphoramidon, a potent inhibitor of ECE-1 (IC50 = 0.68 μM), and analyzed monocyte transmigration. The results show that pharmacological inhibition of ECE-1 significantly reduced monocyte transmigration (control: 102.3% ± 4.2; 10 μM: 72.3% ± 6.2; 50 μM: 61.3% ± 4.6; n = 4; p ≤ 0.05 vs. control). Since phosphoramidon can also inhibit the related neutral endopeptidase (NEP, IC50 = 0.034 μM), we also treated brain endothelial cells with the NEP specific inhibitor DL-Thiorphan (IC50 for NEP is 2.1 nM). The results show that this inhibitor only affects monocyte diapedesis at a relatively high dose (control: 100.0% ± 2.2; 10 μM: 98.1% ± 4.1; 50 μM: 66.9% ± 3.8; n = 4; p = 0.68, p ≤ 0.05 vs. control, respectively). The function of ECE-1 in monocyte diapedesis was further supported by specific shRNA-mediated silencing of ECE-1 in brain endothelial cells. Knockdown of ECE-1, which was verified by western blot analysis, significantly reduced the transmigratory capacity of primary monocytes (control: 100.0% ± 6.5; ECE-1 shRNA: 74.1% ± 5.0; n = 4; p ≤ 0.05 vs. control).
Diapedesis of monocytes into the brain is an important hallmark of a variety of neurological diseases, including MS, vascular dementia, and stroke. We here report that this process involves different components of the endothelin system, including ET-1, ETB receptor, and ECE-1. In short, cultured brain endothelial cells express ET-1, its B-type receptor and the protease needed for generation of active ET-1, ECE-1. In this study, we have applied both synthetic inhibitors and gene-specific knockdown approaches to block ET-1, ECE-1, and ETB receptor activity and provide direct evidence for their role in monocyte diapedesis.
Our results show that ET-1, ETB receptor and ECE-1 contribute to inflammation at the BBB. Originally, ET-1 was isolated and identified from conditioned medium of cultured porcine endothelial cells and designated as a potent vasoconstrictive peptide (Yanagisawa et al. 1988). These studies were followed by other reports which implicate the endothelin system in pathology, in particular its role in cardiovascular diseases, including ischemic heart diseases, chronic heart failure, (pulmonary) hypertension, atherosclerosis, and chronic renal failure (reviewed by Khimji and Rockey 2010). It is of interest that ET-1 also has been associated with neurovascular diseases. ET-1 has been implicated as a mediator of the cerebrovascular responses seen with ischemic strokes and subarachnoid hemorrhages (Masaoka et al. 1989; Kohno et al. 1990; Suzuki et al. 1990; Lampl et al. 1997), HIV-1 infection (Chauhan et al. 2007), Alzheimer’s disease (Palmer and Love 2011), and MS (Speciale et al. 2000; Haufschild et al. 2001). In these diseases, the circulating level of ET-1 is high in many cases. In addition, over-expression of ET-1 in endothelial cells leads to more severe vascular permeability and BBB breakdown after transient middle cerebral artery occlusion (Leung et al. 2009). Therefore, the endothelin system is an attractive target for the treatment of brain disorders which involve a dysfunctional neurovasculature. Other investigators have attempted to reduce subarachnoid hemorrhages in animals by treatment with the ECE-1 inhibitor phosphoramidon (Matsumura et al. 1991; Josko et al. 2000) although clinical outcomes in patients could not be improved (Kramer and Fletcher 2009). The involvement of ET-1, ETB receptor and ECE-1 in the pathogenesis of BBB passage by immune cells indicates the potential for endothelin receptor antagonists and ECE-1 inhibitors in the treatment of diseases which are associated with inflammation in the brain, such as MS.
Our results suggest that the ETB receptor is a potential therapeutic target in brain endothelial cells. Others have reported that administration of ETA antagonists can decrease strole-induced tissue injury and reduces leukocyte infiltration (Feuerstein et al. 1994; Barone et al. 1995; Dawson et al. 1999). The expression of ETA receptors in brain endothelial cells is a matter of debate (Hagiwara et al. 1993; Kobari et al. 1994; Pagotto et al. 1995; Kawai et al. 1997; Naidoo et al. 2004). hCMEC/D3 cells did not express ETA and the ETA antagonist BQ-123 did not affect monocyte diapedesis in our studies. In particular, Naidoo et al. have demonstrated the expression of ETA and ETB receptors in several cell types of the brain. Remarkably, in this study only ETB receptors were visualized in cerebral capillary endothelial cells (Naidoo et al. 2004). It is not excluded that the protective effects of ETA inhibitors are dependent on the expression of ETA receptors in other cells such as neurons and glial cells (Kallakuri et al. 2010).
It is currently unknown which mechanism underlies the function of ETB during the transmigration of monocytes through the brain endothelial cell barrier. McCarron et al. have shown that ET-1 can enhance the expression of cell adhesion molecules involved in monocyte diapedesis, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, in brain endothelial cells through ETA (McCarron et al. 1993). However, the expression of these proteins was not affected by pharmacological treatment with BQ-788 or DL-Thiorphan nor in cells which underwent shRNA-mediated knockdown of ETB and ECE-1 (Fig. S1). Since in our study, ETA inhibition did not affect monocyte transmigration we suggest a distinct and currently unknown mechanism of action by which ETB mediates monocyte transmigration. Others have shown that ET-1 can induce the production of inflammatory mediators such as monocyte chemotactic protein-1 and interleukin-8 by isolated brain endothelial cells (Zidovetzki et al. 1999; Chen et al. 2001). It is of interest that an ETB agonist (Sarafotoxin) caused a time-dependent extracellular signal related kinase 1/2 activation in human aortic smooth muscle cells (Chen et al. 2009). We have recently shown that monocyte diapedesis is dependent on activated extracellular signal related kinase 1/2 (Reijerkerk et al. 2008, 2010). Moreover, others reported that in endothelial cells, ET-1 can activate protein kinase B/Akt through phosphoinositide 3-kinase (Liu et al. 2003), two signal transduction proteins previously reported to mediate brain endothelial cell barrier function and monocyte transmigration (Schreibelt et al. 2007).
This article corroborates previous reports on nuclear localization of ETB receptors and ECE-1 in human endothelial cells (Jacques et al. 2005; Hunter and Turner 2006) and underscores the recent notion that nuclear membrane ET-1 receptors may play an important role in overall ET-1 action (Bkaily et al. 2011). Although we show a strong contribution of ETB receptors and ECE-1 to the diapedesis of monocytes through the brain endothelial cell barrier, our studies do not distinguish between the function of cellular and nuclear membrane bound forms. Until now, it has not been determined whether nuclear ECE-1 is functionally active, although functional ETB receptors on the nuclei of human vascular smooth muscle cells, human vascular endothelial cells, and human endocardial endothelial cells were reported (Bkaily et al. 2011). Nuclear expression of ECE-1 and ETB suggest the possibility of a second mode of action of ET which may be part of a rapid autocrine response. Others have extensively reviewed the function of nuclear ETB receptors and suggested that this would provide a specific pathway to directly affect gene-transcription rather than acting indrectly through a cytoplasmic signal transduction cascade (Jacques et al. 2006; Bkaily et al. 2011).
Our brain endothelial cell fractionation and immunoblot analyses revealed that nuclear ETB receptor was comprised of a larger isoform of ∼ 60 kD. The ETB gene can reveal different mRNA splice variants which encode isoforms 1 and 2 and the larger isoform 3. In contrast to the smaller isoforms 1 and 2, the larger isoform lacks a signal peptide sequence which normally directs a protein to the cell membrane (SignalP 3.0; Bendtsen et al. 2004). Moreover, this isoform of ∼ 60 kD is predicted to localize in the nucleus (PredictProtein; Rost and Liu 2003). Of note, based primarily on functional assays two types of the ETB receptor subtypes were described (Battistini et al. 1993), however, the molecular basis for the existence of these subtypes is still lacking. It is also not clear whether both compartments can be as efficiently inhibited by BQ-788. In our studies, relatively high concentrations of BQ-788 were needed to inhibit monocyte transmigration. To overcome this, we have reduced the expression of ETB receptor using a shRNA approach. It is of interest that this leads to a further reduction of the traversal of monocytes. The presence of endothelin system components in nuclear and cellular membrane compartments may provide a new and specific mechanism of endothelin function but also provides excellent opportunities on the development of novel targeted therapies in neuro-inflammatory diseases.
This work was supported by grants from the Top Institute Pharma (AR, BvhH) and the MS Research Foundation (AR, HEdeV). AR and HEdeV thank Dr. J. de Mey for helpful discussions. The authors have no conflicting financial interests.