Increased Expression Of Adhesion Molecules Research Articles

Oxidative Stress and Thrombophilia: Focus on Pregnancy-Related Pathophysiological Mechanisms

Abstract Thrombophilia, characterized by an increased tendency to form blood clots, presents a substantial risk during pregnancy, potentially impacting maternal and fetal outcomes. This review explores the intricate relationship between thrombophilia and oxidative stress, shedding light on their combined influence on blood clot formation. Specifically, endothelial dysfunction, driven by oxidative stress, emerges as a pivotal factor in thrombophilia, setting the stage for increased platelet activation and altered coagulation factors. Factors like Factor V Leiden mutation, Prothrombin G20210A mutation, and deficiencies in antithrombin, protein C, and protein S contribute to the pro-thrombotic state observed in thrombophilia. Furthermore, inflammation, closely intertwined with oxidative stress, exacerbates the risk of blood clot formation. Inflammatory responses lead to endothelial activation, altered endothelial function, and increased adhesion molecules expression, disrupting the delicate balance between pro- and anti-coagulant factors. Chronic inflammatory conditions, such as autoimmune disorders, potentiate a persistent state of heightened clotting risk. Additionally, impaired antioxidant defenses compound the prothrombotic state by allowing the accumulation of reactive oxygen species, thereby contributing to oxidative stress-induced endothelial dysfunction. Understanding the interplay between these factors is crucial for tailored thrombophilia management, particularly in pregnancy. Treatment strategies encompass a multifaceted approach, including anticoagulant medications, lifestyle modifications, and targeted interventions to improve endothelial health. The complex nature of thrombophilia underscores the need for a collaborative healthcare approach, involving hematologists and high-risk pregnancy specialists. Early detection, vigilant monitoring, and timely intervention are paramount in mitigating risks for both mother and baby.

Kupffer Cell Inactivation Alters Endothelial Cell Adhesion Molecules in Cecal Ligation and Puncture-Induced Sepsis.

The activation of Kupffer cells, resident macrophages in the liver, is closely associated with the inflammatory response during sepsis, which leads to multiple-organ failure. However, how Kupffer cell activation affects adhesion molecules (ICAM-1 and VCAM-1) in sepsis has not been determined. This study investigated Kupffer cell inactivation's (by gadolinium chloride; GdCl3) effects on adhesion molecule expression in CLP-induced sepsis. The induction of sepsis resulted in increased expression of liver and lung ICAM-1 and VCAM-1. GdCl3 pretreatment significantly decreased liver ICAM-1 expression but had no effect on VCAM-1 expression. In contrast, GdCl3 pretreatment had no effect on sepsis-induced increased adhesion molecule expression in the lungs. Similarly, the immunoreactivity of ICAM-1 was decreased in liver sinusoidal endothelial cells but increased in pulmonary endothelial cells in septic mice pretreated with GdCl3. Further, GdCl3 pretreatment had no effect on the immunoreactivity of VCAM-1 in endothelial cells of the liver and lungs. Hence, the findings of this study demonstrate the differential effects of Kupffer cell inactivation on liver and lung adhesion molecules and suggest the complexity of their involvement in the pathophysiology of sepsis.

Calprotectin blockade inhibits long-term vascular pathology following peritoneal dialysis-associated bacterial infection.

Bacterial infections and the concurrent inflammation have been associated with increased long-term cardiovascular (CV) risk. In patients receiving peritoneal dialysis (PD), bacterial peritonitis is a common occurrence, and each episode further increases late CV mortality risk. However, the underlying mechanism(s) remains to be elucidated before safe and efficient anti-inflammatory interventions can be developed. Damage-Associated Molecular Patterns (DAMPs) have been shown to contribute to the acute inflammatory response to infections, but a potential role for DAMPs in mediating long-term vascular inflammation and CV risk following infection resolution in PD, has not been investigated. We found that bacterial peritonitis in mice that resolved within 24h led to CV disease-promoting systemic and vascular immune-mediated inflammatory responses that were maintained up to 28 days. These included higher blood proportions of inflammatory leukocytes displaying increased adhesion molecule expression, higher plasma cytokines levels, and increased aortic inflammatory and atherosclerosis-associated gene expression. These effects were also observed in infected nephropathic mice and amplified in mice routinely exposed to PD fluids. A peritonitis episode resulted in elevated plasma levels of the DAMP Calprotectin, both in PD patients and mice, here the increase was maintained up to 28 days. In vitro, the ability of culture supernatants from infected cells to promote key inflammatory and atherosclerosis-associated cellular responses, such as monocyte chemotaxis, and foam cell formation, was Calprotectin-dependent. In vivo, Calprotectin blockade robustly inhibited the short and long-term peripheral and vascular consequences of peritonitis, thereby demonstrating that targeting of the DAMP Calprotectin is a promising therapeutic strategy to reduce the long-lasting vascular inflammatory aftermath of an infection, notably PD-associated peritonitis, ultimately lowering CV risk.

Loss Of Nicotinamide Nucleotide Transhydrogenase Promotes Redox Dependent Endothelial Activation In Atherosclerosis

Heart disease is the leading cause of death in the United States and atherosclerosis accounts for nearly 75% of all deaths from heart disease. Atherosclerosis is a chronic inflammatory disease and mitochondrial reactive oxygen species (mtROS) are critical contributors to disease development. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that maintains mitochondrial NADPH pools and supports the enzymatic degradation of mtROS. Preliminary data indicates that NNT expression is decreased in the endothelium of human atherosclerotic plaques and we sought to test the hypothesis that the loss of NNT increases mtROS and promotes atherosclerosis by enhancing endothelial and vascular dysfunction. Previous we have demonstrated that the loss of NNT was associated with increased vascular ROS production, plaque formation, and plaque size in response to treatment with PCSK9 and high fat diet (HFD). We now show that the loss of NNT in endothelial specific NNT knockout mice promotes enhanced endothelial VCAM-1 expression in response to HFD and disturbed flow. Similarly, in vitro, the loss of NNT promoted both VCAM-1 and ICAM-1 expression in response to oxLDL. Increased adhesion molecule expression associated with the loss of NNT also augmented monocyte adhesion in response to oxLDL consistent with vascular inflammation observed in early atherosclerosis. Additionally, macrophages isolated from NNT knockout mice display enhanced M1 polarization, decreased lipid uptake, and impaired lipid utilization. Co- treatment with the mitochondria targeted antioxidant MitoEbselen is able to normalize both endothelial and macrophage function, underscoring a critical role for mtROS in promoting an inflammatory phenotype in these cells. In both global and endothelial cell specific NNT knockout mice the loss of NNT was associated with increased necrotic core formation in response to PCSK9 treatment and 16 weeks of high fat diet. Taken together, these data indicate that decreased NNT expression promotes endothelial activation, increased inflammatory cell trafficking, macrophage metabolic dysfunction and M1 polarization that could contribute to an acceleration of necrotic core development and severe atherosclerotic disease. NIH P20GM121307. NIH 1 R01 HL145171-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Current Nanomedicine for Targeted Vascular Disease Treatment: Trends and Perspectives.

Nanotechnology has been developed to deliver cargos effectively to the vascular system. Nanomedicine is a novel and effective approach for targeted vascular disease treatment including atherosclerosis, coronary artery disease, strokes, peripheral arterial disease, and cancer. It has been well known for some time that vascular disease patients have a higher cancer risk than the general population. During atherogenesis, the endothelial cells are activated to increase the expression of adhesion molecules such as Intercellular Adhesion Molecule 1 (ICAM-1), Vascular cell adhesion protein 1 (VCAM-1), E-selectin, and P-selectin. This biological activation of endothelial cells gives a targetability clue for nanoparticle strategies. Nanoparticle formation has a passive targeting pathway due to the increased adhesion molecule expression on the cell surface as well as increased cell activation. In addition, the VCAM-1-targeting peptide has been widely used to target the inflamed endothelial cells. Biomimetic nanoparticles using platelet and leukocyte membrane fragment strategies have been promising techniques for targeted vascular disease treatment. Cyclodextrin, a natural oligosaccharide with a hydrophobic cavity, increase the solubility of cholesterol crystals at the atherosclerotic plaque site and has been used to deliver the hydrophobic drug statin as a therapeutic in a targeted manner. In summary, nanoparticles decorated with various targeting molecules will be an effective and promising strategy for targeted vascular disease treatment.

Effect of cytokine-induced alterations in extracellular matrix composition on diabetic retinopathy-relevant endothelial cell behaviors

Retinal vascular basement membrane (BM) thickening is an early structural abnormality of diabetic retinopathy (DR). Recent studies suggest that BM thickening contributes to the DR pathological cascade; however, much remains to be elucidated about the exact mechanisms by which BM thickening develops and subsequently drives other pathogenic events in DR. Therefore, we undertook a systematic analysis to understand how human retinal microvascular endothelial cells (hRMEC) and human retinal pericytes (hRP) change their expression of key extracellular matrix (ECM) constituents when treated with diabetes-relevant stimuli designed to model the three major insults of the diabetic environment: hyperglycemia, dyslipidemia, and inflammation. TNFα and IL-1β caused the most potent and consistent changes in ECM expression in both hRMEC and hRP. We also demonstrate that conditioned media from IL-1β-treated human Müller cells caused dose-dependent, significant increases in collagen IV and agrin expression in hRMEC. After narrowing our focus to inflammation-induced changes, we sought to understand how ECM deposited by hRMEC and hRP under inflammatory conditions affects the behavior of naïve hRMEC. Our data demonstrated that diabetes-relevant alterations in ECM composition alone cause both increased adhesion molecule expression by and increased peripheral blood mononuclear cell (PBMC) adhesion to naïve hRMEC. Taken together, these data demonstrate novel roles for inflammation and pericytes in driving BM pathology and suggest that inflammation-induced ECM alterations may advance other pathogenic behaviors in DR, including leukostasis.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft SFB1123 European Research Council AdG_692511 Background Genome wide association studies revealed a strong association between the CXCL12 gene locus and cardiovascular diseases, highlighting its receptors CXCR4 and atypical chemokine receptor-3 (ACKR3) as targets for CVD research. Although vascular CXCR4 is well studied in atherosclerosis, the exact role of ACKR3 remains elusive. Therefore, the aim of this project is to decipher the role of vascular ACKR3 in atherosclerosis. Methods Vascular endothelium (Bmx-cre) specific Ackr3 deficient mice on Apoe-/- background were fed with a cholesterol-rich diet. Lesions in the aortic roots and arches were analyzed via H&E staining. The phenotype was further characterized employing ex-vivo perfusion and intravital microscopy for cell adhesion and Evans-blue injection for endothelial permeability. Signaling studies are performed in human coronary artery endothelial cells (HCAECs). Results Endothelial Ackr3 deficiency resulted in significantly smaller atherosclerotic lesions, accompanied by a more stable plaque phenotypes (more lesional collagen and smooth muscle cell content, smaller necrotic cores). Endothelial-Ackr3-/- mice revealed a reduction in plaque macrophage content and ICAM positive endothelial cells in atherosclerotic lesions. Moreover, immune cell adhesion on carotid arteries lacking endothelial ACKR3 was decreased whilst endothelial permeability was not affected. ACKR3 silencing in TNF-stimulated HCAECs decreased adhesion molecule expression and downregulated MAPK signaling and NF-kB p65 phosphorylation. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in Ackr3 deficient mice. Conclusion Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways resulting in increased adhesion molecule expression.

Intrinsic blood-brain barrier dysfunction contributes to multiple sclerosis pathogenesis.

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the CNS are early hallmarks of multiple sclerosis (MS). The mechanisms leading to BBB dysfunction are incompletely understood and generally thought to be a consequence of neuroinflammation. Here, we have challenged this view and asked if intrinsic alterations in the BBB of MS patients contribute to MS pathogenesis. To this end, we made use of human induced pluripotent stem cells derived from healthy controls and MS patients and differentiated them into brain microvascular endothelial cell (BMEC)-like cells as in vitro model of the BBB. MS-derived BMEC-like cells showed impaired junctional integrity, barrier properties and efflux pump activity when compared to healthy controls. Also, MS-derived BMEC-like cells displayed an inflammatory phenotype with increased adhesion molecule expression and immune cell interactions. Activation of Wnt/β-catenin signalling in MS-derived endothelial progenitor cells enhanced barrier characteristics and reduced the inflammatory phenotype. Our study provides evidence for an intrinsic impairment of BBB function in MS patients that can be modelled in vitro. Human iPSC-derived BMEC-like cells are thus suitable to explore the molecular underpinnings of BBB dysfunction in MS and will assist in the identification of potential novel therapeutic targets for BBB stabilization.

Endothelial Intracellular ANG (Angiogenin) Protects Against Atherosclerosis by Decreasing Endoplasmic Reticulum Stress.

ANG (angiogenin) is essential for cellular adaptation to endoplasmic reticulum (ER) stress, a process closely associated with cardiovascular diseases, including atherosclerosis. We aimed to investigate the role of ANG in the progression of atherosclerosis and elucidate its underlying molecular mechanisms. We constructed adenoassociated virus 9 ANG overexpression vectors and endothelial ANG- and ApoE (apolipoprotein E)-deficient mice to determine the effects of ANG on ER stress and atherosclerotic lesions. RNA sequencing of endothelial ANG- and ApoE-deficient mice identified ANG-dependent downregulation of ST3GAL5 (ST3 beta-galactoside alpha-2,3-sialyltransferase 5) expression, and the direct regulation of ST3GAL5 by ANG was verified by chromatin immunoprecipitation sequencing and luciferase reporter assay results. Reanalysis of expression profiling datasets indicated decreased ANG levels in patients' atherosclerotic lesions, and these data were validated in aortas from ApoE-/- mice. ER stress marker and adhesion molecule levels, aortic root lesions and macrophage deposition were substantially reduced in ApoE-/- mice injected with an adenoassociated virus 9 ANG without signal peptide (ANG-ΔSP) overexpression vector compared with empty and full-length ANG overexpression vectors. Endothelial ANG deficiency significantly elevated ER stress and increased adhesion molecule expression, which aggravated atherosclerotic lesions and enhanced THP-1 monocyte adhesion to endothelial cells in vivo and in vitro, respectively. Furthermore, ANG-ΔSP overexpression significantly attenuated oxidized low-density lipoprotein-induced ER stress and THP-1 monocyte adhesion to endothelial cells, which were reversed by ST3GAL5 inhibition. These results suggest that endothelial intracellular ANG is a novel therapeutic against atherosclerosis and exerts atheroprotective effects via ST3GAL5-mediated ER stress suppression.

Abstract P157: Loss Of Nicotinamide Nucleotide Transhydrogenase Promotes Endothelial Activation In Atherosclerosis

Heart disease is the leading cause of death in the United States and atherosclerosis accounts for nearly 75% of all deaths from heart disease. Atherosclerosis is a chronic inflammatory disease and mitochondrial reactive oxygen species (mtROS) are critical contributors to disease development. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that maintains mitochondrial NADPH pools and supports the enzymatic degradation of mtROS. Preliminary data indicates that NNT expression is decreased in the endothelium of human atherosclerotic plaques and we sought to test the hypothesis that the loss of NNT increases mtROS and promotes atherosclerosis by enhancing endothelial and vascular dysfunction. Previous we have demonstrated that the loss of NNT was associated with increased vascular ROS production, plaque formation, and plaque size in response to treatment with PCSK9 and high fat diet (HFD). We now show that the loss of NNT in endothelial specific NNT knockout mice promotes enhanced endothelial VCAM-1 expression in response to HFD and disturbed flow. Similarly, in vitro , the loss of NNT promoted both VCAM-1 and ICAM-1 expression in response to oxLDL. Increased adhesion molecule expression associated with the loss of NNT also augmented monocyte adhesion in response to oxLDL consistent with vascular inflammation observed in early atherosclerosis. Additionally, macrophages isolated from NNT knockout mice display enhanced M1 polarization, decreased lipid uptake, and impaired lipid utilization. Co-treatment with the mitochondria targeted antioxidant MitoEbselen is able to normalize both endothelial and macrophage function, underscoring a critical role for mtROS in promoting an inflammatory phenotype in these cells. In both global and endothelial cell specific NNT knockout mice the loss of NNT was associated with increased necrotic core formation in response to PCSK9 treatment and 16 weeks of high fat diet. Taken together, these data indicate that decreased NNT expression promotes endothelial activation, increased inflammatory cell trafficking, macrophage metabolic dysfunction and M1 polarization that could contribute to an acceleration of necrotic core development and severe atherosclerotic disease.

Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis.

Aims Mental stress substantially contributes to the initiation and progression of human disease, including cardiovascular conditions. We aim to investigate the underlying mechanisms of these contributions since they remain largely unclear.Methods and results Here, we show in humans and mice that leucocytes deplete rapidly from the blood after a single episode of acute mental stress. Using cell-tracking experiments in animal models of acute mental stress, we found that stress exposure leads to prompt uptake of inflammatory leucocytes from the blood to distinct tissues including heart, lung, skin, and, if present, atherosclerotic plaques. Mechanistically, we found that acute stress enhances leucocyte influx into mouse atherosclerotic plaques by modulating endothelial cells. Specifically, acute stress increases adhesion molecule expression and chemokine release through locally derived norepinephrine. Either chemical or surgical disruption of norepinephrine signalling diminished stress-induced leucocyte migration into mouse atherosclerotic plaques.Conclusion Our data show that acute mental stress rapidly amplifies inflammatory leucocyte expansion inside mouse atherosclerotic lesions and promotes plaque vulnerability.

Hypoxia acts as an environmental cue for the human tissue-resident memory T cell differentiation program.

Tissue-resident memory T cells (TRM) provide frontline defense against infectious diseases and contribute to antitumor immunity; however, aside from the necessity of TGF-β, knowledge regarding TRM-inductive cues remains incomplete, particularly for human cells. Oxygen tension is an environmental cue that distinguishes peripheral tissues from the circulation, and here, we demonstrate that differentiation of human CD8+ T cells in the presence of hypoxia and TGF-β1 led to the development of a TRM phenotype, characterized by a greater than 5-fold increase in CD69+CD103+ cells expressing human TRM hallmarks and enrichment for endogenous human TRM gene signatures, including increased adhesion molecule expression and decreased expression of genes involved in recirculation. Hypoxia and TGF-β1 synergized to produce a significantly larger population of TRM phenotype cells than either condition alone, and comparison of these cells from the individual and combination conditions revealed distinct phenotypic and transcriptional profiles, indicating a programming response to milieu rather than a mere expansion. Our findings identify a likely previously unreported cue for the TRM differentiation program and can enable facile generation of human TRM phenotype cells in vitro for basic studies and translational applications such as adoptive cellular therapy.

TRPV4 channel activation induces the transition of venous and arterial endothelial cells toward a pro-inflammatory phenotype.

The Transient Receptor Potential Vanilloid 4 (TRPV4) of endothelial cells contributes to many important functions including the regulation of Ca2+ homeostasis, cell volume, endothelial barrier permeability, and smooth muscle tone. However, its role in the transition of endothelial cells toward a pro‐inflammatory phenotype has not been studied so far. Using both arterial and venous endothelial cells, we first show that the pharmacological activation of TRPV4 channels with GSK1016790A, a potent TRPV4 agonist, triggers robust and sustained Ca2+ increases, which are blocked by both TRPV4 antagonists HC067047 and RN9893. TRPV4 activation also triggers the actin cytoskeleton and adherens junction (VE‐Cadherin) rearrangement in both arterial and venous endothelial cells and leads to rapid decreases of trans‐endothelial electrical resistance. In addition to its effect on endothelial barrier integrity, TRPV4 activation selectively increases ICAM‐1 surface expression in arterial and venous endothelial cells, due to the stimulation of ICAM‐1 gene expression through the NF‐κB transcription factor. TRPV4 channel activation also induced apoptosis of venous and arterial endothelial cells, while TRPV4 blockade reduced apoptosis, even in the absence of TRPV4 activation. As altered barrier integrity, increased adhesion molecule expression and apoptosis are hallmarks of the pro‐inflammatory state of endothelial cells, our results indicate that TRPV4 channel activity can induce the transition of both venous and arterial endothelial cells toward a pro‐inflammatory phenotype.

Methyl-β-cyclodextrin suppresses the monocyte-endothelial adhesion triggered by lipopolysaccharide (LPS) or oxidized low-density lipoprotein (oxLDL)

Context Recent studies demonstrated the anti-atherosclerotic efficacy of cyclodextrin. However, it remains unclear whether cyclodextrin exerts the anti-atherosclerotic effect via regulating monocyte-endothelial adhesion. Objective To answer that question by recruiting methyl-β-cyclodextrin (MβCD) as a cyclodextrin representative. Materials and methods Human umbilical vein endothelial cells (HUVECs) were not treated, or treated with 1 µg/mL liposaccharide (LPS) or 50 µg/mL oxidized low-density lipoprotein (oxLDL) for 12 h, 5 mM MβCD for 1 h, and LPS/oxLDL (1 and 50 µg/mL, respectively for 12 h) plus MβCD (5 mM for 1 h), respectively. The effects of MβCD on LPS/oxLDL-triggered monocyte-endothelial adhesion and related molecules in signalling pathways were evaluated via confocal microscopy, flow cytometry, RT-PCR, western blotting, and cell adhesion assay. Results MβCD with an IC50 of 27.66 mM (1 h treatment) exerted no significant cytotoxicity at ≤5 mM for ≤2 h. Compared with the control, both LPS and oxLDL induced an ∼2–3-fold increase in adhesion molecule expression (ICAM-1 and VCAM-1 at protein and mRNA levels) and NF-κB phosphorylation (p-NF-κB/pP65), an increase in IκB kinase (IKK), and a decrease in phosphorylated protein kinase B (p-Akt), respectively. Moreover, more monocytes (2-fold higher for LPS and 15% higher for oxLDL) were attached on LPS/oxLDL-stimulated HUVECs. 5 mM MβCD reversed the LPS/oxLDL-induced changes back to the control levels. Conclusions MβCD significantly suppresses the LPS/oxLDL-triggered monocyte-endothelial adhesion by downregulating adhesion molecule expression probably via LPS-IKK-NF-κB or oxLDL-Akt-NF-κB pathway. This study demonstrates a potential mechanism of the anti-atherosclerotic efficacy of cyclodextrin from the angle of monocyte-endothelial adhesion.

Double-Hit-Induced Leukocyte Extravasation Driven by Endothelial Adherens Junction Destabilization.

During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.

OroxylinA reverses lipopolysaccharide-induced adhesion molecule expression and endothelial barrier disruption in the rat aorta

Vascular dysfunction plays a critical role in the pathogenesis of sepsis. We elucidated the mechanisms underlying the amelioration of lipopolysaccharide (LPS)-induced vascular inflammation by oroxylin A (OroA) post-treatment in rats. The animals were intraperitoneally injected with LPS (10 mg/kg) to induce systemic inflammation and intravenously (iv) administered OroA (15 mg/kg) 6 h after the LPS treatment. The assessments included biochemical changes in peripheral blood, vascular reactivity which was evaluated by blood-vessel myography, morphological/histological assessment of inflammation, toll-like receptor (TLR)-4-mediated interleukin-1-receptor-associated-kinase (IRAK)-4 activation, changes in adhesion molecule expression, and endothelial junctional stability in the aorta. LPS significantly enhanced the proinflammatory cytokine release, increased vascular cell adhesion molecule (VCAM)-1 expression, disrupted endothelial tight junction, reduced vascular endothelial barrier stability, and increased macrophage infiltration and accumulation in the aorta. All observed pathological changes and vascular inflammation were significantly reversed by the OroA post-treatment. Importantly, OroA suppressed the increased adhesion molecule expression and the endothelial barrier disruption by inhibiting LPS-activated IRAK-4-targeted inhibitory nuclear factor kappa B kinase (IKK) α/β complex phosphorylation, without directly affecting the interaction between LPS and TLR-4. Moreover, the iNOS activity induced by the LPS challenge was inhibited by the OroA pretreatment of the isolated aortic rings. These results suggest that OroA regulates the vascular tone by inhibiting vascular hyporeactivity caused by NO overproduction and reverses the endothelial barrier dysfunction and inflammation by inhibiting the IRAK-4-mediated IKKα/β phosphorylation. Overall, these findings suggest OroA administration as a potentially useful therapeutic approach for clinical interventions in septic shock.

Resveratrol decreases TNFα-induced ICAM-1 expression and release by Sirt-1-independent mechanism in intestinal myofibroblasts

Up-regulation of intercellular adhesion molecule-1 (ICAM-1) and its soluble form are involved in the chronic inflammation. For the first time, we demonstrated that resveratrol (RE), a natural polyphenol with antioxidant and anti-inflammatory properties, reduces the increase of expression and release of ICAM-1, due to TNFα-induced oxidative stress, in a myofibroblast cell line derived from human colonic (18Co cells). RE is scavenger of radical oxygen species (ROS) and modulates signaling pathways in which Sirt-1 and NF-κB are involved. Effectively, in TNFα-stimulated 18Co cells RE decreases ROS production and increases Sirt-1 expression and activity, but it reduces TNFα-induced ICAM-1 up-regulation by a Sirt-1-independent mechanism, as demonstrated by EX527 and Sirt-1 siRNA treatments. RE inhibits TNFα-induced activation of NF-κB by reducing both ROS and the degradation of IκB-α, an endogenous inhibitor of NF-κB, with consequent decrease of NF-κB nuclear translocation. This study also shows that NF-κB is not the only factor involved in the TNFα-induced ICAM-1 up-regulation and confirms our previous evidence according to which TNFα increases ICAM-1 levels by redox- and non-redox-regulated mechanisms. RE can represent good and useful support in therapies for intestinal inflammatory diseases in which TNFα plays a crucial role in the increase of adhesion molecule expression.

Nucleotide-binding oligomerization domain 1 (NOD1) modulates liver ischemia reperfusion through the expression adhesion molecules

In liver transplantation, organ shortage leads to the use of marginal grafts that are more susceptible to ischemia-reperfusion (IR) injury. We identified nucleotide-binding oligomerization domain 1 (NOD1) as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in IR. Herein, we aimed to elucidate the role of NOD1 in IR injury, particularly focusing on its effects on the endothelium and hepatocytes. Nod1 WT and KO mice were treated with NOD1 agonists and subjected to liver IR. Expression of adhesion molecules was analyzed in total liver, isolated hepatocytes and endothelial cells. Interactions between PMNs and hepatocytes were studied in an ex vivo co-culture model using electron microscopy and lactate dehydrogenase levels. We generated NOD1 antagonist-loaded nanoparticles (np ALINO). NOD1 agonist treatment increased liver injury, PMN tissue infiltration and upregulated ICAM-1 and VCAM-1 expression 20 hours after reperfusion. NOD1 agonist treatment without IR increased expression of adhesion molecules (ICAM-1, VCAM-1) in total liver and more particularly in WT hepatocytes, but not in Nod1 KO hepatocytes. This induction is dependent of p38 and ERK signaling pathways. Compared to untreated hepatocytes, a NOD1 agonist markedly increased hepatocyte lysis in co-culture with PMNs as shown by the increase of lactate dehydrogenase in supernatants. Interaction between hepatocytes and PMNs was confirmed by electron microscopy. In a mouse model of liver IR, treatment with np ALINO significantly reduced the area of necrosis, aminotransferase levels and ICAM-1 expression. NOD1 regulates liver IR injury through induction of adhesion molecules and modulation of hepatocyte-PMN interactions. NOD1 antagonist-loaded nanoparticles reduced liver IR injury and provide a potential approach to prevent IR, especially in the context of liver transplantation. Nucleotide-binding oligomerization domain 1 (NOD1) is as an important modulator of polymorphonuclear neutrophil (PMN)-induced liver injury, which occurs in ischemia-reperfusion. Here, we show that the NOD1 pathway targets liver adhesion molecule expression on the endothelium and on hepatocytes through p38 and ERK signaling pathways. The early increase of adhesion molecule expression after reperfusion emphasizes the importance of adhesion molecules in liver injury. In this study we generated nanoparticles loaded with NOD1 antagonist. These nanoparticles reduced liver necrosis by reducing PMN liver infiltration and adhesion molecule expression.

Vascular Targeting to Increase the Efficiency of Immune Checkpoint Blockade in Cancer.

Boosting natural immunity against malignant cells has had a major breakthrough in clinical cancer therapy. This is mainly due to the successful development of immune checkpoint blocking antibodies, which release a break on cytolytic anti-tumor-directed T-lymphocytes. However, immune checkpoint blockade is only effective for a proportion of cancer patients, and a major challenge in the field is to understand and overcome treatment resistance. Immune checkpoint blockade relies on successful trafficking of tumor-targeted T-lymphocytes from the secondary lymphoid organs, through the blood stream and into the tumor tissue. Resistance to therapy is often associated with a low density of T-lymphocytes residing within the tumor tissue prior to treatment. The recruitment of leukocytes to the tumor tissue relies on up-regulation of adhesion molecules and chemokines by the tumor vasculature, which is denoted as endothelial activation. Tumor vessels are often poorly activated due to constitutive pro-angiogenic signaling in the tumor microenvironment, and therefore constitute barriers to efficient leukocyte recruitment. An emerging possibility to enhance the efficiency of cancer immunotherapy is to combine pro-inflammatory drugs with anti-angiogenic therapy, which can enable tumor-targeted T-lymphocytes to access the tumor tissue by relieving endothelial anergy and increasing adhesion molecule expression. This would pave the way for efficient immune checkpoint blockade. Here, we review the current understanding of the biological basis of endothelial anergy within the tumor microenvironment, and discuss the challenges and opportunities of combining vascular targeting with immunotherapeutic drugs as suggested by data from key pre-clinical and clinical studies.

High glucose-induced endothelial microparticles increase adhesion molecule expression on endothelial cells.

The experimental aim of this study was to determine, in vitro, the effects of glucose-induced EMPs on endothelial cell expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 and platelet cell adhesion molecule-1 (PECAM-1). Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 105 cells/condition. HUVECs were incubated with media containing either 25mM d-glucose (concentration representing a hyperglycemic state) or 5mM d-glucose (normoglycemic condition) for 48h to generate EMPs. EMP identification (CD144+) and concentration were determined by flow cytometry. HUVECs (3 × 106 cells/condition) were treated with either high glucose-derived EMPs (hgEMPs) or normal glucose-derived (ngEMPs) for 24h and surface expression of E-selectin (CD62E-PE), ICAM-1 (CD54-FITC), VCAM-1 (CD106-APC) and PECAM-1 (CD31-BV) was assessed by flow cytometry and reported as mean fluorescent intensity (MFI). Hyperglycemic-derived EMPs induced significantly higher surface expression of E-selectin (2614 ± 132 vs. 2010 ± 204MFI), ICAM-1 (2110 ± 81 vs. 1688 ± 152MFI), VCAM-1 (3589 ± 431 vs. 2134 ± 386) and PECAM-1 (4237 ± 395 vs. 2525 ± 269MFI) on endothelial cells than EMPs from normoglycemic conditions. Microparticle-induced cell adhesion molecule expression provides potential novel mechanistic insight regarding the accelerated risk of atherosclerotic vascular disease associated with hyperglycemia.