Inflammation-induced TRPV4 channels exacerbate blood-brain barrier dysfunction in multiple sclerosis.

BACKGROUND: Blood-brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS. MAIN TEXT: In human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium. CONCLUSION: Collectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Elevated monocyte-specific type I interferon signalling correlates positively with cardiac healing in myocardial infarct patients but interferon alpha application deteriorates myocardial healing in rats.

Monocytes are involved in adverse left ventricular (LV) remodelling following myocardial infarction (MI). To provide therapeutic opportunities we aimed to identify gene transcripts in monocytes that relate to post-MI healing and evaluated intervention with the observed gene activity in a rat MI model. In 51 MI patients treated by primary percutaneous coronary intervention (PCI), the change in LV end-diastolic volume index (EDVi) from baseline to 4-month follow-up was assessed using cardiovascular magnetic resonance imaging (CMR). Circulating monocytes were collected at day 5 (Arterioscler Thromb Vasc Biol 35:1066-1070, 2015; Cell Stem Cell 16:477-487, 2015; Curr Med Chem 13:1877-1893, 2006) after primary PCI for transcriptome analysis. Transcriptional profiling and pathway analysis revealed that patients with a decreased LV EDVi showed an induction of type I interferon (IFN) signalling (type I IFN pathway: P value < 0.001; false discovery rate < 0.001). We subsequently administered 15,000 Units of IFN-α subcutaneously in a rat MI model for three consecutive days following MI. Cardiac function was measured using echocardiography and infarct size/cardiac inflammation using (immuno)-histochemical analysis. We found that IFN-α application deteriorated ventricular dilatation and increased infarct size at day 28 post-MI. Moreover, IFN-α changed the peripheral monocyte subset distribution towards the pro-inflammatory monocyte subset whereas in the myocardium, the presence of the alternative macrophage subset was increased at day 3 post-MI. Our findings suggest that induction of type I IFN signalling in human monocytes coincides with adverse LV remodelling. In rats, however, IFN-α administration deteriorated post-MI healing. These findings underscore important but also contradictory roles for the type I IFN response during cardiac healing following MI.

Vascular Endothelial Growth Factor remains unchanged in cerebrospinal fluid of patients with Alzheimer's disease and vascular dementia.

BACKGROUND: Increasing evidence suggests that cerebral vascular dysfunction is associated with the early stages of Alzheimer's disease (AD). Vascular endothelial growth factor (VEGF) is one of the key players involved in the development and maintenance of the vasculature. Here, we hypothesized that VEGF levels in cerebrospinal fluid (CSF) may be altered in AD patients with vascular involvement, characterized by the presence of microbleeds (MB), and in vascular dementia (VaD) patients compared to controls. METHODS: VEGF levels were determined by electrochemilumiscence Meso Scale Discovery (MULTI-SPOT Assay System) in CSF from age-matched groups of controls with subjective cognitive decline (n = 21), AD without MB (n = 25), AD with MB (n = 25), and VaD (n = 21) patients. RESULTS: The average level of VEGF in the different groups was 2.8 ± 1 pg/ml CSF. Adjusted for age and gender, no significant differences were detected between groups (p > 0.5). However, we detected a significant correlation between the concentration of VEGF in the CSF and age (r = 0.22, p = 0.03). In addition, males (n = 54) revealed higher VEGF levels in their CSF compared to females (n = 38) (males = 3.08 ± 0.769 pg/ml (mean ± SD), females = 2.6 ± 0.59; p = 0.006), indicating a gender-related regulation. CONCLUSION: Our study suggests that VEGF levels in the CSF do not reflect the cerebral vascular alterations in either AD or VaD patients. The observed associations of VEGF with age and gender may indicate that VEGF reflects normal aging and that males and females may differ in their aging process.

Expression of Nitric Oxide-Transporting Aquaporin-1 Is Controlled by KLF2 and Marks Non-Activated Endothelium In Vivo.

The flow-responsive transcription factor Krüppel-like factor 2 (KLF2) maintains an anti-coagulant, anti-inflammatory endothelium with sufficient nitric oxide (NO)-bioavailability. In this study, we aimed to explore, both in vitro and in human vascular tissue, expression of the NO-transporting transmembrane pore aquaporin-1 (AQP1) and its regulation by atheroprotective KLF2 and atherogenic inflammatory stimuli. In silico analysis of gene expression profiles from studies that assessed the effects of KLF2 overexpression in vitro and atherosclerosis in vivo on endothelial cells, identifies AQP1 as KLF2 downstream gene with elevated expression in the plaque-free vessel wall. Biomechanical and pharmaceutical induction of KLF2 in vitro is accompanied by induction of AQP1. Chromosome immunoprecipitation (CHIP) confirms binding of KLF2 to the AQP1 promoter. Inflammatory stimulation of endothelial cells leads to repression of AQP1 transcription, which is restrained by KLF2 overexpression. Immunohistochemistry reveals expression of aquaporin-1 in non-activated endothelium overlying macrophage-poor intimae, irrespective whether these intimae are characterized as being plaque-free or as containing advanced plaque. We conclude that AQP1 expression is subject to KLF2-mediated positive regulation by atheroprotective shear stress and is downregulated under inflammatory conditions both in vitro and in vivo. Thus, endothelial expression of AQP1 characterizes the atheroprotected, non-inflamed vessel wall. Our data provide support for a continuous role of KLF2 in stabilizing the vessel wall via co-temporal expression of eNOS and AQP1 both preceding and during the pathogenesis of atherosclerosis.

Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

Kruppel-like factor 2 improves neovascularization capacity of aged proangiogenic cells.

AIMS: Coronary artery disease (CAD) patients have less circulating proangiogenic cells (PACs), formerly known as endothelial progenitor cells, which exhibit impaired neovascularization properties. Inverse correlations were also found between PAC function and risk factors like age. Krüppel-like factor 2 (KLF2) is expressed by mature endothelial cells (ECs), is induced by both shear stress and statins, and provokes endothelial functional differentiation. The aim of this study is to identify whether KLF2 can reverse negative effects of ageing on PAC function. METHODS AND RESULTS: We describe that progenitor cells in the bone marrow and PACs also express KLF2 at a comparable level to mature ECs and that senescence decreases KLF2 levels. To study the effects of ageing on KLF2 levels, we compared progenitor cells of 4 weeks and 16- to 18-month-old C57BL/6 mice. In addition to the three-fold reduction of circulating Sca1(+)/c-Kit(+)/Lin(-) progenitor cells and the 15% reduction of Sca1(+)/Flk1(+) endothelial-committed progenitor cells, the spleen-derived PACs and bone marrow-derived progenitor cells isolated from aged mice showed a lower level of KLF2 when compared with young mice. Lentiviral overexpression of KLF2 increased human PAC numbers and endothelial nitric oxide synthase expression by 60% during in vitro culture. Endothelial lineage-specific KLF2 overexpression in aged bone marrow-derived mononuclear cells strongly augments neovascularization in vivo in a murine hind-limb ischaemia model. CONCLUSION: These results imply that KLF2 is an attractive novel target to rejuvenate PACs before autologous administration to CAD patients.

KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium.

The shear stress-induced transcription factor Krüppel-like factor 2 (KLF2) confers antiinflammatory properties to endothelial cells through the inhibition of activator protein 1, presumably by interfering with mitogen-activated protein kinase (MAPK) cascades. To gain insight into the regulation of these cascades by KLF2, we used antibody arrays in combination with time-course mRNA microarray analysis. No gross changes in MAPKs were detected; rather, phosphorylation of actin cytoskeleton-associated proteins, including focal adhesion kinase, was markedly repressed by KLF2. Furthermore, we demonstrate that KLF2-mediated inhibition of Jun NH(2)-terminal kinase (JNK) and its downstream targets ATF2/c-Jun is dependent on the cytoskeleton. Specifically, KLF2 directs the formation of typical short basal actin filaments, termed shear fibers by us, which are distinct from thrombin- or tumor necrosis factor-alpha-induced stress fibers. KLF2 is shown to be essential for shear stress-induced cell alignment, concomitant shear fiber assembly, and inhibition of JNK signaling. These findings link the specific effects of shear-induced KLF2 on endothelial morphology to the suppression of JNK MAPK signaling in vascular homeostasis via novel actin shear fibers.

Endothelial CD81 is a marker of early human atherosclerotic plaques and facilitates monocyte adhesion.

AIMS: In a recent report, we established at the genome-wide level those genes that are specifically upregulated in the endothelium of atherosclerotic plaques in human arteries. As the transcriptome data revealed that mRNA for the tetraspanin family member CD81 is significantly and specifically upregulated in the endothelium overlying early atheroma, we set out to validate these results on the protein level, and investigate the functional consequences of CD81 upregulation. METHODS AND RESULTS: Immunohistochemical analysis in an independent set of donor arteries verified in the endothelium of early human atherosclerotic lesions the enhanced expression of CD81, which appears oxidative stress-dependent. Using lentiviral overexpression and silencing in human umbilical endothelial cells, we established in an in vitro flow adhesion assay that elevated endothelial CD81 is associated with increased monocyte adhesion to non-activated CD81-transduced endothelial cells, approaching the levels normally only attained after tumour necrosis factor alpha stimulation. The CD81 effect was dependent on both intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as it was abolished in the presence of a mixture of anti-ICAM-1 and anti-VCAM-1 antibodies. Flow cytometry revealed that increased CD81 levels did not increase total ICAM-1 and VCAM-1 surface expression. Instead, it concentrated the available adhesion molecules into membrane clusters, as indicated by confocal and electron microscopy. CD81 also colocalized with ICAM-1 and VCAM-1 in the adhesion rings around bound monocytes. CONCLUSION: Endothelial CD81 upregulated in early human atheroma has the potential to play a crucial role in the initial stages of atherosclerotic plaque formation by increasing monocyte adhesion prior to the full-blown inflammatory response.

VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis.

Angiogenesis is crucial for the progression of colorectal carcinomas in which the bioavailability of Vascular Endothelial Growth Factor (VEGF) plays a major role. VEGF bioavailability is regulated by proteolytic release or cleavage. In colorectal cancer patients, we observed a significant correlation between circulating VEGF and tumour tissue Matrix Metalloproteinase-9 (MMP-9) levels but not with MMP-2. Therefore, we evaluated the role of MMP-9 in regulating VEGF bioavailability and subsequent angiogenesis in 3-dimensional human cell culture models. MMP-9 treatment released VEGF dose-dependently from HT29 colon carcinoma spheroids, comparable to heparitinase, a known mediator of VEGF release. Conditioned medium from human neutrophils, containing high amounts of active MMP-9, released VEGF comparable to recombinant MMP-9, in contrast to myofibroblast medium. MMP-9 treated spheroids showed decreased extracellular levels of heparan sulphates, required for VEGF binding to the matrix, whereas the levels in the medium were increased. Western blot analysis revealed that VEGF(165) is the major isoform released by MMP-9 treatment. In vitro experiments indicated that MMP-9 is not capable to cleave VEGF(165) into smaller isoforms, like plasmin does. These data suggested that MMP-9 mediates release rather than the cleavage of larger VEGF isoforms. Medium from MMP-9 treated HT29 spheroids induced endothelial cell sprouting in an angiogenesis assay, comparable to the effect of recombinant VEGF(165). Anti-VEGF antibody treatment resulted in a strongly reduced number of sprouts. In conclusion, we have shown that neutrophil-derived MMP-9 is able to release biologically active VEGF(165) from the ECM of colon cancer cells by the cleavage of heparan sulphates.

Distinctive expression of chemokines and transforming growth factor-beta signaling in human arterial endothelium during atherosclerosis.

Knowledge about the in vivo role of endothelium in chronic human atherosclerosis has mostly been derived by insights from mouse models. Therefore, we set out to establish by microarray analyses the gene expression profiles of endothelium from human large arteries, as isolated by laser microbeam microdissection, having focal atherosclerosis of the early or the advanced stage. Within individual arteries, the endothelial transcriptomes of the lesional and unaffected sides were compared pairwise, thus limiting genetic and environmental confounders. Specific endothelial signature gene sets were identified with changed expression levels in either early (n = 718) or advanced atherosclerosis (n = 403), relative to their paired plaque-free controls. Gene set enrichment analysis identified distinct sets of chemokines and differential enrichments of nuclear factor-kappaB-, p53-, and transforming growth factor-beta-related genes in advanced plaques. Immunohistochemistry validated the discriminative value of corresponding endothelial protein expression between early (fractalkine/CX3CL1, IP10/CCL10, TBX18) or advanced (BAX, NFKB2) stages of atherosclerosis and versus their plaque-free controls. The functional involvement of transforming growth factor-beta signaling in directing its downstream gene repertoire was substantiated by a consistent detection of activated SMAD2 in advanced lesions. Thus, we identified truly common, local molecular denominators of pathological changes to vascular endothelium, with a marked distinction of endothelial phenotype between early and advanced plaques.

Spatial segregation of transport and signalling functions between human endothelial caveolae and lipid raft proteomes.

Lipid rafts and caveolae are biochemically similar, specialized domains of the PM (plasma membrane) that cluster specific proteins. However, they are morphologically distinct, implying different, possibly complementary functions. Two-dimensional gel electrophoresis preceding identification of proteins by MS was used to compare the relative abundance of proteins in DRMs (detergent-resistant membranes) isolated from HUVEC (human umbilical-vein endothelial cells), and caveolae immunopurified from DRM fractions. Various signalling and transport proteins were identified and additional cell-surface biotinylation revealed the majority to be exposed, demonstrating their presence at the PM. In resting endothelial cells, the scaffold of immunoisolated caveolae consists of only few resident proteins, related to structure [CAV1 (caveolin-1), vimentin] and transport (V-ATPase), as well as the GPI (glycosylphosphatidylinositol)-linked, surface-exposed protein CD59. Further quantitative characterization by immunoblotting and confocal microscopy of well-known [eNOS (endothelial nitric oxide synthase) and CAV1], less known [SNAP-23 (23 kDa synaptosome-associated protein) and BASP1 (brain acid soluble protein 1)] and novel [C8ORF2 (chromosome 8 open reading frame 2)] proteins showed different subcellular distributions with none of these proteins being exclusive to either caveolae or DRM. However, the DRM-associated fraction of the novel protein C8ORF2 (approximately 5% of total protein) associated with immunoseparated caveolae, in contrast with the raft protein SNAP-23. The segregation of caveolae from lipid rafts was visually confirmed in proliferating cells, where CAV1 was spatially separated from eNOS, SNAP-23 and BASP1. These results provide direct evidence for the previously suggested segregation of transport and signalling functions between specialized domains of the endothelial plasma membrane.

Rosuvastatin reduces atherosclerosis development beyond and independent of its plasma cholesterol-lowering effect in APOE*3-Leiden transgenic mice: evidence for antiinflammatory effects of rosuvastatin.

BACKGROUND: Statins can exert anti-inflammatory antiatherosclerotic effects through an anti-inflammatory action, independent of lowering cholesterol. We addressed the question whether the anti-inflammatory activities of statins can reduce atherosclerosis beyond the reduction achieved by cholesterol lowering per se. METHODS AND RESULTS: Two groups of 20 female APOE*3-Leiden mice received either a high-cholesterol diet (HC) or a high-cholesterol diet supplemented with 0.005% (wt/wt) rosuvastatin (HC+R). The HC diet alone resulted in a plasma cholesterol concentration of 18.9+/-1.4 mmol/L, and administration of rosuvastatin lowered plasma cholesterol to 14.1+/-0.7 mmol/L. In a separate low-cholesterol (LC) control group, the dietary cholesterol intake was reduced, which resulted in plasma cholesterol levels that were comparable to the HC+R group (13.4+/-0.8 mmol/L). Atherosclerosis in the aortic root area was quantified after 24 weeks. As compared with the HC group, the LC group had a 62% (P<0.001) reduction in cross-sectional lesion area. When compared with the LC group, the HC+R group showed a further decrease in cross-sectional lesion area (80%, P<0.001), size of individual lesions (63%, P<0.05), lesion number (58%, P<0.001), monocyte adherence (24%, P<0.05), and macrophage-containing area (60%, P<0.001). Furthermore, rosuvastatin specifically suppressed the expression of the inflammation parameters MCP-1 and TNF-alpha in the vessel wall and lowered plasma concentrations of serum amyloid A and fibrinogen, independent of its cholesterol-lowering effect. CONCLUSIONS: Rosuvastatin reduces atherosclerosis beyond and independent of the reduction achieved by cholesterol lowering alone. This additional beneficial effect of rosuvastatin may be explained, at least partly, by its anti-inflammatory activity.

Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2).

The endothelium expresses a large repertoire of genes under apparent transcriptional control of biomechanical forces, many of which are neither cell-type nor flow specific. We set out to identify genes that are uniquely flow responsive in human vascular endothelial cells. Transcriptional profiling using commercial DNA microarrays identified 12 of 18 000 genes that were modulated at least 5-fold after 24 hours of steady laminar flow (25 dyne/cm(2)). After a 7-day exposure to unidirectional pulsatile flow (19 +/- 12 dyne/cm(2)), only 3 of 12 remained elevated at least 5-fold. A custom microarray of ~300 vascular cell-related gene fragments was constructed, and expression analysis revealed that many flow-induced genes are also induced by at least one of the following agents: tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), transforming growth factor-beta, vascular endothelial growth factor, or thrombin, indicating a more general role in adaptive or stress responses. Most flow-induced genes were also induced by TNF-alpha but not IL-1beta, suggesting the involvement of reactive oxygen species. A limited panel of genes that are unique for flow-exposed cultures was identified, including lung Krüppel-like factor (LKLF/KLF2) and cytochrome P450 1B1 (CYP1B1). In marked contrast, both these genes were substantially repressed by TNF-alpha. LKLF but not CYP1B1 mRNA was detected exclusively in the vascular endothelium of healthy human aorta by in situ hybridization and appeared to be flow regulated. To date LKLF is the first endothelial transcription factor that is uniquely induced by flow and might therefore be at the molecular basis of the physiological healthy, flow-exposed state of the endothelial cell.