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Several lines of evidence suggest that stress induces the neurovascular dysfunction associated with increased blood–brain barrier (BBB) permeability, which could be an important pathology linking stress and psychiatric disorders, including major depressive disorder (MDD). However, the detailed mechanism resulting in BBB dysfunction associated in the pathophysiology of MDD still remains unclear. Herein, we demonstrate the role of vascular endothelial growth factor (VEGF), a key mediator of vascular angiogenesis and BBB permeability, in stress-induced BBB dysfunction and depressive-like behavior development. We implemented an animal model of depression, chronic restraint stress (RS) in BALB/c mice, and found that the BBB permeability was significantly increased in chronically stressed mice. Immunohistochemical and electron microscopic observations revealed that increased BBB permeability was associated with both paracellular and transcellular barrier alterations in the brain endothelial cells. Pharmacological inhibition of VEGF receptor 2 (VEGFR2) using a specific monoclonal antibody (DC101) prevented chronic RS-induced BBB permeability and anhedonic behavior. Considered together, these results indicate that VEGF/VEGFR2 plays a crucial role in the pathogenesis of depression by increasing the BBB permeability, and suggest that VEGFR2 inhibition could be a potential therapeutic strategy for the MDD subtype associated with BBB dysfunction.

Blood vessel endothelium forms a semi-permeable barrier and its permeability controls the traffics of plasma contents. Here we report an intravital evaluation system for vascular permeability in mice using two-photon microscopy. We used various sizes of fluorescein-conjugated dextran as a tracer and its efflux was quantified by measuring the changes of fluorescent intensity both on the blood vessel area and the interstitial space. Using this system, we demonstrated that skin blood vessels limited the passage of dextran larger than 70 kDa under homeostatic conditions. We evaluated the kinetics of vascular permeability in histamine- or IgE-induced type I allergic models and a hapten-induced type IV allergic model. In such inflammatory conditions, the hyperpermeability was selectively induced in the postcapillary venules and dextran as large as 2000-kDa leaked from the bloods. Taken together, our study provides a convenient method to characterize the skin blood vessels as a traffic barrier in physiological conditions.

Metastasis is the leading cause of death in patients with breast cancer (BC). Primary tumors create a premetastatic niche (PMN) in secondary organs for subsequent metastases. Cancer-associated fibroblasts (CAFs) are a predominant stromal component in the tumor microenvironment and serve as a major contributor to tumor metastasis. However, the function and mechanism of primary CAFs in the premetastatic niche of secondary organs remain unclear in BC. We investigated the expression profiles of lncRNAs in pairs of CAFs and NFs derived from breast tumor tissues using lncRNA microarray. The expression levels of lncSNHG5, ZNF281, IGF2BP2, CCL2 and CCL5 were assessed by qRT-PCR; the protein levels of related genes (e.g., ZNF281, IGF2BP2, CCL2, and CCL5) were analyzed using western blotting and/or ELISA in primary and immortalized CAFs and clinical samples. Tubule formation and three-dimensional sprouting assays and tissue fluorescence staining were conducted to investigate angiogenesis. permeability assays, trans-endothelial invasion assays, permeability assays and tissue fluorescence staining were conducted to examine vascular permeability. The regulatory mechanism of lncSNHG5 was investigated by RNA sequencing, fluorescent hybridization, cellular fractionation assay, mass spectrometry, RNA pull-down, RNA immunoprecipitation, gene-specific m6A assay, chromatin immunoprecipitation, dual luciferase reporter assay and actinomycin D treatment in CAFs and NFs. LncSNHG5 was highly expressed in breast CAFs and played an essential role in premetastatic niche formation by promoting angiogenesis and vascular leakiness through regulation of ZNF281 in CAFs. lncSNHG5 enhanced ZNF281 mRNA stability by binding with the m6A reader IGF2BP2. Enhanced ZNF281 transcriptionally regulated CCL2 and CCL5 expression to activate P38 MAPK signaling in endothelial cells. High CCL2 and CCL5 expression was associated with tumor metastasis and poor prognosis in BC patients. The inhibitors RS102895, marasviroc and cenicriviroc inhibited angiogenesis and vascular permeability in the PMN by blocking the binding of CCL2/CCR2 and CCL5/CCR5. The lncSNHG5-ZNF281-CCL2/CCL5 signaling axis plays an essential role in inducing premetastatic niche formation to promote BC metastasis. Our work demonstrates that lncSNHG5 and its downstream signaling ZNF281-CCL2/CCL5 in CAFs play a crucial role in premetastatic niche formation in breast cancer and may serve as potential targets for the diagnosis and treatment of BC metastasis.

The blood–retinal barrier (BRB) consists of tightly interconnected capillary endothelial cells covered with pericytes and glia, but the role of the pericytes in BRB regulation is not fully understood. Here, we show that platelet-derived growth factor (PDGF)-B/PDGF receptor beta (PDGFRβ) signalling is critical in formation and maturation of BRB through active recruitment of pericytes onto growing retinal vessels. Impaired pericyte recruitment to the vessels shows multiple vascular hallmarks of diabetic retinopathy (DR) due to BRB disruption. However, PDGF-B/PDGFRβ signalling is expendable for maintaining BRB integrity in adult mice. Although selective pericyte loss in stable adult retinal vessels surprisingly does not cause BRB disintegration, it sensitizes retinal vascular endothelial cells (ECs) to VEGF-A, leading to upregulation of angiopoietin-2 (Ang2) in ECs through FOXO1 activation and triggering a positive feedback that resembles the pathogenesis of DR. Accordingly, either blocking Ang2 or activating Tie2 greatly attenuates BRB breakdown, suggesting potential therapeutic approaches to reduce retinal damages upon DR progression. Blood-retinal barrier (BRB) is composed of tightly connected endothelium and supporting pericytes and glia. Here, the authors show that pericytes are crucial for BRB buildup during retinal development and its maintenance in adult retinas in response to VEGF-A-induced endothelial sensitization by regulating the Tie2/FOXO1/Ang2 axis.

Increased microvascular permeability to plasma proteins and neutrophil emigration are hallmarks of innate immunity and key features of numerous inflammatory disorders. Although neutrophils can promote microvascular leakage, the impact of vascular permeability on neutrophil trafficking is unknown. Here, through the application of confocal intravital microscopy, we report that vascular permeability-enhancing stimuli caused a significant frequency of neutrophil reverse transendothelial cell migration (rTEM). Furthermore, mice with a selective defect in microvascular permeability enhancement (VEC-Y685F-ki) showed reduced incidence of neutrophil rTEM. Mechanistically, elevated vascular leakage promoted movement of interstitial chemokines into the bloodstream, a response that supported abluminal-to-luminal neutrophil TEM. Through development of an in vivo cell labeling method we provide direct evidence for the systemic dissemination of rTEM neutrophils, and showed them to exhibit an activated phenotype and be capable of trafficking to the lungs where their presence was aligned with regions of vascular injury. Collectively, we demonstrate that increased microvascular leakage reverses the localization of directional cues across venular walls, thus causing neutrophils engaged in diapedesis to reenter the systemic circulation. This cascade of events offers a mechanism to explain how local tissue inflammation and vascular permeability can induce downstream pathological effects in remote organs, most notably in the lungs.

Background Sepsis-associated encephalopathy (SAE), a diffuse cerebral dysfunction in the absence of direct CNS infection, is associated with increased rates of mortality and morbidity in patients with sepsis. Increased cytokine production and disruption of the blood-brain barrier (BBB) are implicated in the pathogenesis of SAE. The induction of pro-inflammatory mediators is driven, in part, by activation of NF-κΒ. Lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, potently activates NF-κΒ and its downstream targets, including cyclooxygenase-2 (Cox-2). Cox-2 catalyzes prostaglandin synthesis and in the brain prostaglandin, E2 is capable of inducing endothelial permeability. Depletion of polymerase δ-interacting protein 2 (Poldip2) has previously been reported to attenuate BBB disruption, possibly via regulation of NF-κΒ, in response to ischemic stroke. Here we investigated Poldip2 as a novel regulator of NF-κΒ/cyclooxygenase-2 signaling in an LPS model of SAE. Methods Intraperitoneal injections of LPS (18 mg/kg) were used to induce BBB disruption in Poldip2 +/+ and Poldip2 +/− mice. Changes in cerebral vascular permeability and the effect of meloxicam, a selective Cox-2 inhibitor, were assessed by Evans blue dye extravasation. Cerebral cortices of Poldip2 +/+ and Poldip2 +/− mice were further evaluated by immunoblotting and ELISA. To investigate the role of endothelial Poldip2, immunofluorescence microscopy and immunoblotting were performed to study the effect of siPoldip2 on LPS-mediated NF-κΒ subunit p65 translocation and Cox-2 induction in rat brain microvascular endothelial cells. Finally, FITC-dextran transwell assay was used to assess the effect of siPoldip2 on LPS-induced endothelial permeability. Results Heterozygous deletion of Poldip2 conferred protection against LPS-induced BBB permeability. Alterations in Poldip2 +/+ BBB integrity were preceded by induction of Poldip2, p65, and Cox-2, which was not observed in Poldip2 +/− mice. Consistent with these findings, prostaglandin E2 levels were significantly elevated in Poldip2 +/+ cerebral cortices compared to Poldip2 +/− cortices. Treatment with meloxicam attenuated LPS-induced BBB permeability in Poldip2 +/+ mice, while having no significant effect in Poldip2 +/− mice. Moreover, silencing of Poldip2 in vitro blocked LPS-induced p65 nuclear translocation, Cox-2 expression, and endothelial permeability. Conclusions These data suggest Poldip2 mediates LPS-induced BBB disruption by regulating NF-κΒ subunit p65 activation and Cox-2 and prostaglandin E2 induction. Consequently, targeted inhibition of Poldip2 may provide clinical benefit in the prevention of sepsis-induced BBB disruption.

Aims/hypothesisMicrovascular endothelial hyperpermeability, mainly caused by claudin-5 deficiency, is the initial pathological change that occurs in diabetes-associated cardiovascular disease. The ketone body β-hydroxybutyrate (BHB) exerts unique beneficial effects on the cardiovascular system, but the involvement of BHB in promoting the generation of claudin-5 to attenuate cardiac microvascular hyperpermeability in diabetes is poorly understood.MethodsThe effects of BHB on cardiac microvascular endothelial hyperpermeability and claudin-5 generation were evaluated in rats with streptozotocin-induced diabetes and in high glucose (HG)-stimulated human cardiac microvascular endothelial cells (HCMECs). To explore the underlying mechanisms, we also measured β-catenin nuclear translocation, binding of β-catenin, histone deacetylase (HDAC)1, HDAC3 and p300 to the Claudin-5 (also known as CLDN5) promoter, interaction between HDAC3 and β-catenin, and histone acetylation in the Claudin-5 promoter.ResultsWe found that 10 weeks of BHB treatment promoted claudin-5 generation and antagonised cardiac microvascular endothelial hyperpermeability in rat models of diabetes. Meanwhile, BHB promoted claudin-5 generation and inhibited paracellular permeability in HG-stimulated HCMECs. Specifically, BHB (2 mmol/l) inhibited HG-induced HDAC3 from binding to the Claudin-5 promoter, although nuclear translocation or promoter binding of β-catenin did not change with BHB treatment. In addition, BHB prevented the binding and co-localisation of HDAC3 to β-catenin in HG-stimulated HCMECs. Furthermore, using mass spectrometry, acetylated H3K14 (H3K14ac) in the Claudin-5 promoter following BHB treatment was identified, regardless of whether cells were stimulated by HG or not. Although reduced levels of acetylated H3K9 in the Claudin-5 promoter were found following HG stimulation, increased H3K14ac was specifically associated with BHB treatment.Conclusions/interpretationBHB inhibited HDAC3 and caused acetylation of H3K14 in the Claudin-5 promoter, thereby promoting claudin-5 generation and antagonising diabetes-associated cardiac microvascular hyperpermeability.

Key Points Modalities of cell–cell communication of relevance to angiogenesis, inflammation and fibrosis include paracrine signalling, mechanosignalling, direct signal transduction via gap junctions and tunnelling nanotubes, and communication via the release and uptake of exosomes. Intercellular communication might be targeted by altering gap junctions and nanotubes, and exosomes are potential drug delivery vehicles as well as diagnostic markers. Leukocytes interact with microvascular cells to affect angiogenesis. These interactions are potentially druggable processes for the regulation of angiogenesis. Angiogenesis can occur through three different mechanisms — sprouting, intussusception and looping — which should be taken into account when designing new pharmacological treatments to reduce or increase angiongesis. Different classes of leukocytes are potential targets to reduce uncontrolled fibrosis. Inhibited recruitment of inflammatory leukocytes together with on-site reprogramming of macrophages could promote fibrosis resolution. In response to different signals from the environment, such as interferons, Toll-like receptor ligands or interleukins, macrophages undergo classical (M1) or alternative activation, which results in a continuum of diverse phenotypes depending on activation states. The identification of the underlying regulation of macrophage plasticity and polarized activation provides targets for macrophage-centred therapies. Delineation of the endothelial heterogeneity is important to the development of strategies for the targeted delivery of drugs to organ-specific vascular beds to limit adverse effects. Cells within the microvascular compartment, particularly leukocytes, can affect angiogenesis, inflammation and fibrosis. Kreuger and Phillipson discuss how to target these cells therapeutically, focusing on ways to interfere with intracellular communication and reprogramme leukocytes, which could have applications in the design of drugs and their delivery systems. Regulation of vascular permeability, recruitment of leukocytes from blood to tissue and angiogenesis are all processes that occur at the level of the microvasculature during both physiological and pathological conditions. The interplay between microvascular cells and leukocytes during inflammation, together with the emerging roles of leukocytes in the modulation of the angiogenic process, make leukocyte–vascular interactions prime targets for therapeutics to potentially treat a wide range of diseases, including pathological and dysfunctional vessel growth, chronic inflammation and fibrosis. In this Review, we discuss how the different cell types that are present in and around microvessels interact, cooperate and instruct each other, and in this context we highlight drug targets as well as emerging druggable processes that can be exploited to restore tissue homeostasis.

Histamine is a mediator of allergic inflammation released mainly from mast cells. Although histamine strongly increases vascular permeability, its precise mechanism under in vivo situation remains unknown. We here attempted to reveal how histamine induces vascular hyperpermeability focusing on the key regulators of vascular permeability, blood flow and endothelial barrier. Degranulation of mast cells by antigen-stimulation or histamine treatment induced vascular hyperpermeability and tissue swelling in mouse ears. These were abolished by histamine H1 receptor antagonism. Intravital imaging showed that histamine dilated vasculature, increased blood flow, while it induced hyperpermeability in venula. Whole-mount staining showed that histamine disrupted endothelial barrier formation of venula indicated by changes in vascular endothelial cadherin (VE-cadherin) localization at endothelial cell junction. Inhibition of nitric oxide synthesis (NOS) by L-NAME or vasoconstriction by phenylephrine strongly inhibited the histamine-induced blood flow increase and hyperpermeability without changing the VE-cadherin localization. In vitro, measurements of trans-endothelial electrical resistance of human dermal microvascular endothelial cells (HDMECs) showed that histamine disrupted endothelial barrier. Inhibition of protein kinase C (PKC) or Rho-associated protein kinase (ROCK), NOS attenuated the histamine-induced barrier disruption. These observations suggested that histamine increases vascular permeability mainly by nitric oxide (NO)-dependent vascular dilation and subsequent blood flow increase and maybe partially by PKC/ROCK/NO-dependent endothelial barrier disruption.

Background Burn injury is associated with a long-standing inflammatory reaction. The use of albumin solutions for plasma volume support is controversial because of concerns of increased capillary leakage, which could aggravate the commonly seen interstitial oedema. Methods In the present open controlled clinical trial, an intravenous infusion of 20% albumin at 3 mL/kg was given over 30 min to 15 burn patients and 15 healthy volunteers. Blood samples and urine were collected for 5 h. Plasma dilution, plasma albumin and colloid osmotic pressure were compared. Mass balance calculations were used to estimate plasma volume expansion and capillary leakage of fluid and albumin. Results The patients were studied between 4 and 14 (median, 7) days after the burn injury, which spread over 7–48% (median, 15%) of the total body surface area. The albumin solution expanded the plasma volume by almost 15%, equivalent to twice the infused volume, in both groups. The urinary excretion exceeded the infused volume by a factor of 2.5. Capillary leakage of albumin occurred at a rate of 3.4 ± 1.5 g/h in burn patients and 3.7 ± 1.6 g/h in the volunteers ( P  = 0.61), which corresponded to 2.4 ± 1.0% and 2.5 ± 1.2% per hour of the intravascular pool ( P  = 0.85). The median half-life of the plasma volume expansion was 5.9 (25th–75th percentiles 2.7–11.7) h in the burn patients and 6.9 (3.4–8.5) h in the volunteers ( P  = 0.56). Conclusions Albumin 20% was an effective volume expander in patients at 1 week post-burn. No relevant differences were found between burn patients and healthy volunteers. Trial registration EudraCT 2016-000996-26 on May 31, 2016.