IntroductionMicrovessels are responsible for facilitating the exchange of nutrients between blood and tissue while maintaining a barrier against the undesired movement of other molecules. This barrier is most finely tuned and structurally competent at the interface between the blood and the brain, the blood-brain barrier (BBB). The BBB is formed by intercellular junctions that limit paracellular diffusion combined with a low-flux transcellular system that selectively transports molecules either into or out of the brain parenchyma. Increasing evidence implicates chronic BBB permeability in the onset of neurologic and neurovascular diseases (eg, Alzheimer disease, dementia, small-vessel disease, stroke). 1 Although the BBB prevents the flux of harmful substances into the brain, it can also exclude the delivery of therapeutic drugs. Understanding the molecular regulation of the BBB is therefore important for the understanding of disease etiology and treatment.Paracellular diffusion of molecules at the BBB is minimized by endothelial cell-cell (EC-EC) junctions. Chief among these are the adherens junctions and tight junctions, of which vascularendothelial cadherin (VE-cadherin; VEC) and claudin-5 are wellknown members, respectively. Although VEC is endothelial specific, the claudin family of proteins is found in many epithelial tissues. However, claudin-5 is specific to tight junctions of ECs and is required to block paracellular diffusion of small molecules (Ͻ ϳ 800 Da) in the microvasculature of the brain. 2 In particular, claudin-5 is now recognized as the predominant claudin isoform in tight junctions of the BBB. Although claudins 1, 10, 11, and 12 (at least) are also expressed in brain endothelium, claudin-5 mRNA is nearly 600-fold higher than the other claudins expressed in microvascular ECs freshly isolated from the brain. 3 On electron microscopy, plasma membranes of adjacent cells associate close enough to merge without apparent intercellular space at tight junctions. 4 Homocysteine (Hcy) is an intermediate aminothiol derived from methionine catabolism, and its elevation in plasma, known as hyperhomocysteinemia (HHcy), is a chronic condition associated with cerebral small-vessel disease, stroke, and dementia (Alzheimer and vascular). 5,6 The list of Hcy-mediated vasculopathies is still growing but includes endothelial dysfunction, vessel wall malformations, loss of extracellular matrix collagen, and loosening of the BBB in rodents and humans. 5,6 Mounting evidence suggests that an increase in BBB permeability is an important factor in initiating cerebral small-vessel disease and lacunar stroke. 7,8 Hence, identifying the mechanisms by which HHcy opens the BBB is of great clinical interest. We reported that treating human umbilical vein ECs with Hcy increases EC monolayer permeability and decreases expression of claudin-5. 9 However, the mechanism by which Hcy triggers these events has not been elucidated, and the intracellular mechanisms for decreased expression of claudin-5 in HHcy are unknown.Recently, a ...

Endothelial dysfunction is a hallmark of systemic inflammatory response underlying multiple organ failure. Here we report a novel function of DHHC-containing palmitoyl acyltransferases (PATs) in mediating endothelial inflammation. Pharmacological inhibition of PATs attenuates barrier leakage and leucocyte adhesion induced by endothelial junction hyperpermeability and ICAM-1 expression during inflammation. Among 11 DHHCs detected in vascular endothelium, DHHC21 is required for barrier response. Mice with DHHC21 function deficiency (Zdhhc21dep/dep) exhibit marked resistance to injury, characterized by reduced plasma leakage, decreased leucocyte adhesion and ameliorated lung pathology, culminating in improved survival. Endothelial cells from Zdhhc21dep/dep display blunted barrier dysfunction and leucocyte adhesion, whereas leucocytes from these mice did not show altered adhesiveness. Furthermore, inflammation enhances PLCβ1 palmitoylation and signalling activity, effects significantly reduced in Zdhhc21dep/dep and rescued by DHHC21 overexpression. Likewise, overexpression of wild-type, not mutant, PLCβ1 augments barrier dysfunction. Altogether, these data suggest the involvement of DHHC21-mediated PLCβ1 palmitoylation in endothelial inflammation.

Aberrant elevation in the levels of the pro-inflammatory cytokine interleukin-1b (IL-1b) contributes to neuroinflammatory diseases. Blood-brain barrier (BBB) dysfunction is a hallmark phenotype of neuroinflammation. It is known that IL-1b directly induces BBB hyperpermeability but the mechanisms remain unclear. Claudin-5 (Cldn5) is a tight junction protein found at endothelial cell-cell contacts that are crucial for maintaining brain microvascular endothelial cell (BMVEC) integrity. Transcriptional regulation of Cldn5 has been attributed to the transcription factors b-catenin and forkhead box protein O1 (FoxO1), and the signaling molecules regulating their nuclear translocation. Non-muscle myosin light chain kinase (nmMlck, encoded by the Mylk gene) is a key regulator involved in endothelial hyperpermeability, and IL-1b has been shown to mediate nmMlck-dependent barrier dysfunction in epithelia. Considering these factors, we tested the hypothesis that nmMlck modulates IL-1b-mediated downregulation of Cldn5 in BMVECs in a manner that depends on transcriptional repression mediated by b-catenin and FoxO1. We found that treating BMVECs with IL-1b induced barrier dysfunction concomitantly with the nuclear translocation of b-catenin and FoxO1 and the repression of Cldn5. Most importantly, using primary BMVECs isolated from mice null for nmMlck, we identified that Cldn5 repression caused by b-catenin and FoxO1 in IL-1b-mediated barrier dysfunction was dependent on nmMlck.

Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-β-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.

A disintegrin and metalloproteinase15 (ADAM15) has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS). An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3′ UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.