Leveraging avidin-biotin interaction to quantify permeability property of microvessels-on-a-chip networks.

Microvessels-on-a-chip have enabled in vitro studies to closely simulate in vivo microvessel environment. However, assessing microvessel permeability, a functional measure of microvascular exchange, has not been attainable in nonpermeable microfluidic platforms. This study developed a new approach that enables permeability coefficients (Ps) to be quantified in microvessels developed in nonpermeable chip platforms by integrating avidin-biotin technology. Microvessels were developed on biotinylated fibronectin-coated microfluidic channels. Solute transport was assessed by perfusing microvessels with fluorescence-labeled avidin. Avidin molecules that crossed endothelium were captured by substrate biotin and recorded with real-time confocal images. The Ps was derived from the rate of avidin-biotin accumulation at the substrate relative to solute concentration difference across microvessel wall. Avidin tracers with different physiochemical properties were used to characterize the barrier properties of the microvessel wall. The measured baseline Ps and inflammatory mediator-induced increases in Ps and endothelial cell (EC) [Ca2+]i resembled those observed in intact microvessels. Importantly, the spatial accumulation of avidin-biotin at substrate defines the transport pathways. Glycocalyx layer is well formed on endothelium and its degradation increased transcellular transport without affecting EC junctions. This study demonstrated that in vitro microvessels developed in this simply designed microfluidics structurally possess in vivo-like glycocalyx layer and EC junctions and functionally recapitulate basal barrier properties and stimuli-induced responses observed in intact microvessels. This new approach overcomes the limitations of nonpermeable microfluidics and provides an easily executed highly reproducible in vitro microvessel model with in vivo microvessel functionality, suitable for a wide range of applications in blood and vascular research and drug development.NEW & NOTEWORTHY Our study developed a novel method that allows permeability coefficient to be measured in microvessels developed in nonpermeable microfluidic platforms using avidin-biotin technology. It overcomes the major limitation of nonpermeable microfluidic system and provides a simply designed easily executed and highly reproducible in vitro microvessel model with permeability accessibility. This model with in vivo-like endothelial junctions, glycocalyx, and permeability properties advances microfluidics in microvascular research, suitable for a wide range of biomedical and clinical applications.

Increased circulating microparticles contribute to severe infection and adverse outcomes of COVID-19 in patients with diabetes.

Patients with diabetes infected with COVID-19 have greater mortality than those without comorbidities, but the underlying mechanisms remain unknown. This study aims to identify the mechanistic interactions between diabetes and severe COVID-19. Microparticles (MPs), the cell membrane-derived vesicles released on cell activation, are largely increased in patients with diabetes. To date, many mechanisms have been postulated for increased severity of COVID-19 in patients with underlying conditions, but the contributions of excessive MPs in patients with diabetes have been overlooked. This study characterizes plasma MPs from normal human subjects and patients with type 2 diabetes in terms of amount, cell origins, surface adhesive properties, ACE2 expression, spike protein binding capacity, and their roles in SARS-CoV-2 infection. Results showed that over 90% of plasma MPs express ACE2 that binds the spike protein of SARS-CoV-2. MPs in patients with diabetes increase 13-fold in quantity and 11-fold in adhesiveness when compared with normal subjects. Perfusion of human plasma with pseudo-typed SARS-CoV-2 virus or spike protein-bound MPs into human endothelial cell-formed microvessels-on-a chip demonstrated that MPs from patients with diabetes, not normal subjects, interact with endothelium and carry SARS-CoV-2 into cells through endocytosis, providing additional virus entry pathways and enhanced infection. Results also showed a large percentage of platelet-derived tissue factor-bearing MPs in diabetic plasma, which could contribute to thrombotic complications with SARS-CoV-2 infection. This study reveals a dual role of diabetic MPs in promoting SARS-CoV-2 entry and propagating vascular inflammation. These findings provide novel mechanistic insight into the high prevalence of COVID-19 in patients with diabetes and their propensity to develop severe vascular complications.NEW & NOTEWORTHY This study provides the first evidence that over 90% of human plasma microparticles express ACE2 that binds SARS-CoV-2 S protein with high affinity. Thus, the highly elevated adhesive circulating microparticles identified in patients with diabetes not only have greater SARS-CoV-2 binding capacity but also enable additional viral entry through virus-bound microparticle-endothelium interactions and enhanced infection. These findings reveal a novel mechanistic insight into the adverse outcomes of COVID-19 in patients with diabetes.

Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

KEY POINTS: Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. Using a diabetic rat model, we identified a >130-fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. Using combined single microvessel perfusion and systemic cross-transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multi-organ microvascular dysfunction. ABSTRACT: In various cardiovascular diseases, microparticles (MPs), the membrane-derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease-induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin-induced diabetic rat model with well-characterized microvascular functions. Our study revealed a >130-fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre-coated with a lipid-binding protein, annexin V, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi-organ vascular dysfunction that commonly occurs in diabetic patients.

Selective knockout of astrocytic Na+ /H+ exchanger isoform 1 reduces astrogliosis, BBB damage, infarction, and improves neurological function after ischemic stroke.

Stimulation of Na+ /H+ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1flox/flox (Nhe1f/f ) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1f/f mice with Gfap-CreERT2 Cre-recombinase mice. Gfap-CreERT2+/- ;Nhe1f/f mice at postnatal day 60-90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post-injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil-vehicle group (control), Tam-treated Gfap-CreERT2+/- ;Nhe1f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP-9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.

Enhanced permeability responses to inflammation in streptozotocin-induced diabetic rat venules: Rho-mediated alterations of actin cytoskeleton and VE-cadherin.

Diabetes is a progressive disease that often leads to microvascular complications. This study investigates the impact of diabetes on microvessel permeability under basal and inflammatory conditions. Streptozotocin-induced diabetic rats were used to mimic type 1 diabetes. Parallel experiments were conducted in intact mesenteric venules in normal rats and diabetic rats experiencing hyperglycemia for 2-3 wk. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). The correlated changes in endothelial intracellular Ca(2+) concentration ([Ca(2+)]i), adherens junctions, and cytoskeleton F-actin were examined with fluorescence imaging. Diabetic vessels showed moderately increased basal Lp, but upon platelet-activating factor (PAF) exposure, these vessels showed an ~10-fold higher Lp increase than the normal vessels. Concomitantly, we observed higher increases in endothelial [Ca(2+)]i, enhanced stress fiber formation, vascular endothelial-cadherin separation, and larger gap formation between endothelial cells than those occurring in normal vessels. PAF receptor staining showed no significant difference between normal and diabetic vessels. The application of Rho kinase inhibitor Y27632 did not affect PAF-induced increases in endothelial [Ca(2+)]i but significantly reduced PAF-induced Lp increases by 90% in diabetic vessels. The application of both Y27632 and nitric oxide (NO) synthase inhibitor attenuated PAF-induced Lp increases more than using one inhibitor alone. Our studies indicate that diabetic conditions prime endothelial cells into a phenotype with increased susceptibility to inflammation without altering receptor expression and that the increased Rho activation and NO production play important roles in exaggerated permeability increases when diabetic vessels were exposed to inflammatory mediators, which may account for the exacerbated vascular dysfunction when diabetic patients are exposed to additional inflammation.

H2O2-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules.

Although elevated levels of H(2)O(2) have been implicated to play important roles in the pathogenesis of various cardiovascular diseases, the underlying mechanisms remain unclear. This study aims to examine the effect of H(2)O(2) on endothelial nitric oxide (NO) production in intact venules, and elucidate the role and mechanisms of NO in H(2)O(2)-induced increases in microvessel permeability. Experiments were conducted on individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial [Ca(2+)](i) was measured on fura-2-loaded vessels. Perfusion of H(2)O(2) (10 µM) caused a delayed and progressively increased endothelial [Ca(2+)](i) and Lp, a pattern different from inflammatory mediator-induced immediate and transient response. Under the same experimental conditions, measuring endothelial NO via DAF-2 and the spatial detection of cell apoptosis by fluorescent markers revealed that H(2)O(2) induced two phases of NO production followed by caspase activation, intracellular Ca(2+) accumulation, and vascular cell apoptosis. The initial NO production was correlated with increased endothelial NO synthase (eNOS) Ser(1177) phosphorylation in the absence of elevated endothelial [Ca(2+)](i), whereas the second phase of NO depended on increased [Ca(2+)](i) and was associated with Thr(495) dephosphorylation without increased Ser(1177) phosphorylation. Inhibition of NOS prevented H(2)O(2)-induced caspase activation, cell apoptosis, and increases in endothelial [Ca(2+)](i) and Lp. Our results indicate that H(2)O(2) at micromolar concentration is able to induce a large magnitude of NO in intact venules, causing caspase activation-mediated endothelial Ca(2+) accumulation, cell apoptosis, and increases in permeability. The mechanisms revealed from intact microvessels may contribute to the pathogenesis of oxidant-related cardiovascular diseases.

Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules.

Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.

Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca²âº]i, nitric oxide, and gap formation in intact venules.

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca(2+)](i), NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca(2+)](i) was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca(2+)](i), NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca(2+)](i) preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca(2+)](i) in each cell, suggesting that the initial levels of endothelial [Ca(2+)](i) determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca(2+) response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.

Improved measurements of intracellular nitric oxide in intact microvessels using 4,5-diaminofluorescein diacetate.

4,5-Diaminofluorescein diacetate (DAF-2 DA) has been widely used for the measurement of nitric oxide (NO) in living cells and tissues. We previously established a method that demonstrated platelet activating factor (PAF)-induced endothelial NO production in intact venules using DAF-2 DA. In previous applications, the loading dye was removed from the extracellular space before NO measurements. However, in high permeability vessels, endothelial cells quickly released the accumulated intracellular DAF-2 after the washout, which compromises the NO measurement. The objective of this study was to investigate if the presence of DAF-2 DA during NO measurements could overcome the dye retention problem and enhance the sensitivity of NO detection. Experiments were conducted in individually perfused rat venules, and endothelial NO was measured using fluorescence imaging under basal and stimulated conditions with continuous perfusion of DAF-2 DA. Continuous dye perfusion was found to promote a relatively constant endothelial dye concentration in both normal and high permeability vessels throughout the experiment. With the use of this method, the basal and stimulated NO was quantified after endothelial DAF-2 concentrations reached a steady state. Our results showed enhanced sensitivity of detecting PAF-stimulated NO compared with a previous method. We also found that the hydrolyzed intracellular DAF-2, the precursor of DAF-2 triazole, contributed significantly to the measured fluorescence and that an appropriate subtraction of non-NO-dependent intracellular DAF-2 fluorescence is critical for the assessment of NO in living tissues. This method overcame the dye leakage problem, enhanced the sensitivity of NO detection, and improved NO quantification, demonstrating significant advantages over existing methodologies using DAF-2.

Activation of endothelial Wnt/ß-catenin signaling by protective astrocytes repairs BBB damage in ischemic stroke.

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na+/H+ exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ∼177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/ß-catenin inhibitor, XAV-939, blocked the reparative effects of Nhe1-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to "protective" by inducing Wnt production to promote BBB repair after ischemic stroke.

Activating Sphingosine-1-phospahte signaling in endothelial cells increases myosin light chain phosphorylation to decrease endothelial permeability thereby inhibiting cancer metastasis.

Targeting the metastatic process to prevent disease dissemination in cancer remains challenging. One step in the metastatic cascade involves cancer cells transiting through the vascular endothelium after inflammation has increased the permeability of this cellular layer. Reducing inflammation-mediated gaps in the vascular endothelium could potentially be used to retard metastasis. This study describes the development of a novel ASR396-containing nanoparticle designed to activate the Sphingosine-1-Phosphate Receptor 1 (S1PR1) in order to tighten the junctions between the endothelial cells lining the vascular endothelium thereby inhibiting metastasis. ASR396 was derived from the S1PR1 agonist SEW2871 through chemical modification enabling the new compound to be loaded into a nanoliposome. ASR396 retained S1PR1 binding activity and the nanoliposomal formulation (nanoASR396) made it systemically bioavailable upon intravenous injection. Studies conducted in microvessels demonstrated that nanoASR396 significantly attenuated inflammatory mediator-induced permeability increase through the S1PR1 activation. Similarly, nanoASR396 inhibited gap formation mediated by inflammatory agents on an endothelial cell monolayer by decreasing levels of phosphorylated myosin light chain protein thereby inhibiting cellular contractility. In animal models, nanoASR396 inhibited lung metastasis by up to 80%, indicating its potential for retarding melanoma metastasis. Thus, a novel bioavailable nanoparticle-based S1PR1 agonist has been developed to negate the effects of inflammatory mediators on the vascular endothelium in order to reduce the metastatic dissemination of cancer cells.

Sphingosine-1-phosphate prevents permeability increases via activation of endothelial sphingosine-1-phosphate receptor 1 in rat venules.

Sphingosine-1-phosphate (S1P) has been demonstrated to enhance endothelial barrier function in vivo and in vitro. However, different S1P receptor subtypes have been indicated to play different or even opposing roles in the regulation of vascular barrier function. This study aims to differentiate the roles of endogenous endothelial S1P subtype receptors in the regulation of permeability in intact microvessels using specific receptor agonist and antagonists. Microvessel permeability was measured with hydraulic conductivity (L(p)) in individually perfused rat mesenteric venules. S1P-mediated changes in endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura-2-loaded venules. Confocal images of fluorescent immunostaining illustrated the spatial expressions of three S1P subtype receptors (S1P(R1-3)) in rat venules. The application of S1P (1 µM) in the presence of S1P(R1-3) inhibited platelet-activating factor- or bradykinin-induced permeability increase. This S1P effect was reversed only with a selective S1P(R1) antagonist, W-146, and was not affected by S1P(R2) or S1P(R3) antagonists JTE-013 and CAY-10444, respectively. S1P(R1) was also identified as the sole receptor responsible for S1P-mediated increases in endothelial [Ca(2+)](i). S1P(R2) or S1P(R3) antagonist alone affected neither basal L(p) nor platelet-activating factor-induced permeability increase. The selective S1P(R1) agonist, SEW-2871, showed similar [Ca(2+)](i) and permeability effect to that of S1P. These results indicate that, despite the presence of S1P(R1-3) in the intact venules, only the activation of endothelial S1P(R1) is responsible for the protective action of S1P on microvessel permeability and that endogenous S1P(R2) or S1P(R3) did not exhibit functional roles in the regulation of permeability under basal or acutely stimulated conditions.

Oxidative Stress and Microvessel Barrier Dysfunction.

Clinical and experimental evidence indicate that increased vascular permeability contributes to many disease-associated vascular complications. Oxidative stress with increased production of reactive oxygen species (ROS) has been implicated in a wide variety of pathological conditions, including inflammation and many cardiovascular diseases. It is thus important to identify the role of ROS and their mechanistic significance in microvessel barrier dysfunction under pathological conditions. The role of specific ROS and their cross talk in pathological processes is complex. The mechanisms of ROS-induced increases in vascular permeability remain poorly understood. The sources of ROS in diseases have been extensively reviewed at enzyme levels. This review will instead focus on the underlying mechanisms of ROS release by leukocytes, the differentiate effects and signaling mechanisms of individual ROS on endothelial cells, pericytes and microvessel barrier function, as well as the interplay of reactive oxygen species, nitric oxide, and nitrogen species in ROS-mediated vascular barrier dysfunction. As a counter balance of excessive ROS, nuclear factor erythroid 2 related factor 2 (Nrf2), a redox-sensitive cell-protective transcription factor, will be highlighted as a potential therapeutic target for antioxidant defenses. The advantages and limitations of different experimental approaches used for the study of ROS-induced endothelial barrier function are also discussed. This article will outline the advances emerged mainly from in vivo and ex vivo studies and attempt to consolidate some of the opposing views in the field, and hence provide a better understanding of ROS-mediated microvessel barrier dysfunction and benefit the development of therapeutic strategies.

Gut-resident CX3CR1hi macrophages induce tertiary lymphoid structures and IgA response in situ.

Intestinal mononuclear phagocytes (MPs) are composed of heterogeneous dendritic cell (DC) and macrophage subsets necessary for the initiation of immune response and control of inflammation. Although MPs in the normal intestine have been extensively studied, the heterogeneity and function of inflammatory MPs remain poorly defined. We performed phenotypical, transcriptional, and functional analyses of inflammatory MPs in infectious Salmonella colitis and identified CX3CR1+ MPs as the most prevalent inflammatory cell type. CX3CR1+ MPs were further divided into three distinct populations, namely, Nos2 +CX3CR1lo, Ccr7 +CX3CR1int (lymph migratory), and Cxcl13 +CX3CR1hi (mucosa resident), all of which were transcriptionally aligned with macrophages and derived from monocytes. In follow-up experiments in vivo, intestinal CX3CR1+ macrophages were superior to conventional DC1 (cDC1) and cDC2 in inducing Salmonella-specific mucosal IgA. We next examined spatial organization of the immune response induced by CX3CR1+ macrophage subsets and identified mucosa-resident Cxcl13 +CX3CR1hi macrophages as the antigen-presenting cells responsible for recruitment and activation of CD4+ T and B cells to the sites of Salmonella invasion, followed by tertiary lymphoid structure formation and the local pathogen-specific IgA response. Using mice we developed with a floxed Ccr7 allele, we showed that this local IgA response developed independently of migration of the Ccr7 +CX3CR1int population to the mesenteric lymph nodes and contributed to the total mucosal IgA response to infection. The differential activity of intestinal macrophage subsets in promoting mucosal IgA responses should be considered in the development of vaccines to prevent Salmonella infection and in the design of anti-inflammatory therapies aimed at modulating macrophage function in inflammatory bowel disease.

H2 O2 -induced microvessel barrier dysfunction: the interplay between reactive oxygen species, nitric oxide, and peroxynitrite.

Elevated H2 O2 is implicated in many cardiovascular diseases. We previously demonstrated that H2 O2 -induced endothelial nitric oxide synthase (eNOS) activation and excessive NO production contribute to vascular cell injury and increases in microvessel permeability. However, the mechanisms of excessive NO-mediated vascular injury and hyperpermeability remain unknown. This study aims to examine the functional role of NO-derived peroxynitrite (ONOO- ) in H2 O2 -induced vascular barrier dysfunction by elucidating the interrelationships between H2 O2 -induced NO, superoxide, ONOO- , and changes in endothelial [Ca2+ ]i and microvessel permeability. Experiments were conducted on intact rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+ ]i , NO, and O2- were assessed with fluorescence imaging. Perfusion of vessels with H2 O2 (10 µmol/L) induced marked productions of NO and O2- , resulting in extensive protein tyrosine nitration, a biomarker of ONOO- . The formation of ONOO- was abolished by inhibition of NOS with NG -Methyl-L-arginine. Blocking NO production or scavenging ONOO- by uric acid prevented H2 O2 -induced increases in endothelial [Ca2+ ]i and Lp. Additionally, the application of exogenous ONOO- to microvessels induced delayed and progressive increases in endothelial [Ca2+ ]i and microvessel Lp, a pattern similar to that observed in H2 O2 -perfused vessels. Importantly, ONOO- caused further activation of eNOS with amplified NO production. We conclude that the augmentation of NO-derived ONOO- is essential for H2 O2 -induced endothelial Ca2+ overload and progressively increased microvessel permeability, which is achieved by self-promoted amplifications of NO-dependent signaling cascades. This novel mechanism provides new insight into the reactive oxygen and/or reactive nitrogen species-mediated vascular dysfunction in cardiovascular diseases.

New insights into shear stress-induced endothelial signalling and barrier function: cell-free fluid versus blood flow.

AIMS: Fluid shear stress (SS) is known to regulate endothelial cell (EC) function. Most of the studies, however, focused on the effects of cell-free fluid-generated wall SS on ECs. The objective of this study was to investigate how changes in blood flow altered EC signalling and endothelial function directly through wall SS and indirectly through SS effects on red blood cells (RBCs). METHODS AND RESULTS: Experiments were conducted in individually perfused rat venules. We experimentally induced changes in SS that were quantified by measured flow velocity and fluid viscosity. The concomitant changes in EC [Ca2+]i and nitric oxide (NO) were measured with fluorescent markers, and EC barrier function was assessed by fluorescent microsphere accumulation at EC junctions using confocal imaging. EC eNOS activation was evaluated by immunostaining. In response to changes in SS, increases in EC [Ca2+]i and gap formation occurred only in blood or RBC solution perfused vessels, whereas SS-dependent NO production and eNOS-Ser1177 phosphorylation occurred in both plasma and blood perfused vessels. A bioluminescent assay detected SS-dependent ATP release from RBCs. Pharmacological inhibition and genetic modification of pannexin-1 channels on RBCs abolished SS-dependent ATP release and SS-induced increases in EC [Ca2+]i and gap formation. CONCLUSIONS: SS-induced EC NO production occurs in both cell free fluid and blood perfused vessels, whereas SS-induced increases in EC [Ca2+]i and EC gap formation require the presence of RBCs, attributing to SS-induced pannexin-1 channel dependent release of ATP from RBCs. Thus, changes in blood flow alter vascular EC function through both wall SS and SS exerted on RBCs, and RBC released ATP contributes to SS-induced changes in EC barrier function.

Transendothelial movement of adiponectin is restricted by glucocorticoids.

Altered permeability of the endothelial barrier in a variety of tissues has implications both in disease pathogenesis and treatment. Glucocorticoids are potent mediators of endothelial permeability, and this forms the basis for their heavily prescribed use as medications to treat ocular disease. However, the effect of glucocorticoids on endothelial barriers elsewhere in the body is less well studied. Here, we investigated glucocorticoid-mediated changes in endothelial flux of Adiponectin (Ad), a hormone with a critical role in diabetes. First, we used monolayers of endothelial cells in vitro and found that the glucocorticoid dexamethasone increased transendothelial electrical resistance and reduced permeability of polyethylene glycol (PEG, molecular weight 4000 Da). Dexamethasone reduced flux of Ad from the apical to basolateral side, measured both by ELISA and Western blotting. We then examined a diabetic rat model induced by treatment with exogenous corticosterone, which was characterized by glucose intolerance and hyperinsulinemia. There was no change in circulating Ad but less Ad protein in skeletal muscle homogenates, despite slightly higher mRNA levels, in diabetic vs control muscles. Dexamethasone-induced changes in Ad flux across endothelial monolayers were associated with alterations in the abundance of select claudin tight junction (TJ) proteins. shRNA-mediated knockdown of one such gene, claudin-7, in HUVEC resulted in decreased TEER and increased adiponectin flux, confirming the functional significance of Dex-induced changes in its expression. In conclusion, our study identifies glucocorticoid-mediated reductions in flux of Ad across endothelial monolayers in vivo and in vitro This suggests that impaired Ad action in target tissues, as a consequence of reduced transendothelial flux, may contribute to the glucocorticoid-induced diabetic phenotype.

Reduction of Endothelial Nitric Oxide Increases the Adhesiveness of Constitutive Endothelial Membrane ICAM-1 through Src-Mediated Phosphorylation.

Nitric oxide (NO) is a known anti-adhesive molecule that prevents platelet aggregation and leukocyte adhesion to endothelial cells (ECs). The mechanism has been attributed to its role in the regulation of adhesion molecules on leukocytes and the adhesive properties of platelets. Our previous study conducted in rat venules found that reduction of EC basal NO synthesis caused EC ICAM-1-mediated firm adhesion of leukocytes within 10-30 min. This quick response occurred in the absence of alterations of adhesion molecules on leukocytes and also opposes the classical pattern of ICAM-1-mediated leukocyte adhesion that requires protein synthesis and occurs hours after stimulation. The objective of this study is to investigate the underlying mechanisms of reduced basal NO-induced EC-mediated rapid leukocyte adhesion observed in intact microvessels. The relative levels of ICAM-1 at different cell regions and their activation status were determined with cellular fractionation and western blot using cultured human umbilical vein ECs. ICAM-1 adhesiveness was determined by immunoprecipitation in non-denatured proteins to assess the changes in ICAM-1 binding to its inhibitory antibody, mAb1A29, and antibody against total ICAM-1 with and without NO reduction. The adhesion strength of EC ICAM-1 was assessed by atomic force microscopy (AFM) on live cells. Results showed that reduction of EC basal NO caused by the application of caveolin-1 scaffolding domain (AP-CAV) or NOS inhibitor, L-NMMA, for 30 min significantly increased phosphorylated ICAM-1 and its binding to mAb1A29 in the absence of altered ICAM-1 expression and its distribution at subcellular regions. The Src inhibitor, PP1, inhibited NO reduction-induced increases in ICAM-1 phosphorylation and adhesive binding. AFM detected significant increases in the binding force between AP-CAV-treated ECs and mAb1A29-coated probes. These results demonstrated that reduced EC basal NO lead to a rapid increase in ICAM-1 adhesive binding via Src-mediated phosphorylation without de novo protein synthesis and translocation. This study suggests that a NO-dependent conformational change of constitutive EC membrane ICAM-1 might be the mechanism of rapid ICAM-1 dependent leukocyte adhesion observed in vivo. This new mechanistic insight provides a better understanding of EC/leukocyte interaction-mediated vascular inflammation under many disease conditions that encounter reduced basal NO in the circulation system.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca(2+)]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca(2+)]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Insulin decreases atherosclerosis by inducing endothelin receptor B expression.

Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe-/- mice (Irs1/Apoe-/-) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE-/- mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin's enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE-/- mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr-/- and Irs1/Ldlr-/- mice decreased NO production and accelerated atherosclerosis, compared with Ldlr-/- mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production.