Leukocyte Adhesion Molecules Research Articles

Molecular mechanisms of coronary artery disease risk at the PDGFD locus

Genome wide association studies for coronary artery disease (CAD) have identified a risk locus at 11q22.3. Here, we verify with mechanistic studies that rs2019090 and PDGFD represent the functional variant and gene at this locus. Further, FOXC1/C2 transcription factor binding at rs2019090 is shown to promote PDGFD transcription through the CAD promoting allele. With single cell transcriptomic and histology studies with Pdgfd knockdown in an SMC lineage tracing male atherosclerosis mouse model we find that Pdgfd promotes expansion, migration, and transition of SMC lineage cells to the chondromyocyte phenotype. Pdgfd also increases adventitial fibroblast and pericyte expression of chemokines and leukocyte adhesion molecules, which is linked to plaque macrophage recruitment. Despite these changes there is no effect of Pdgfd deletion on overall plaque burden. These findings suggest that PDGFD mediates CAD risk by promoting deleterious phenotypic changes in SMC, along with an inflammatory response that is primarily focused in the adventitia.

Transcriptomic landscapes of effective and failed liver regeneration in humans

Although extensive experimental evidence on the process of liver regeneration exists, in humans, validation is largely missing. However, liver regeneration is critically affected by underlying liver disease. Within this project, we aimed to systematically assess early transcriptional changes during liver regeneration in humans and further assess how these processes differ in people with dysfunctional liver regeneration. Blood samples of 154 patients and intraoperative tissue samples of 46 patients undergoing liver resection were collected and classified with regard to dysfunctional postoperative liver regeneration. Of those, a matched cohort of 21 patients were used for RNA sequencing. Samples were assessed for circulating cytokines, gene expression dynamics, intrahepatic neutrophil accumulation, and spatial transcriptomics. Individuals with dysfunctional liver regeneration demonstrated an aggravated transcriptional inflammatory response with higher intracellular adhesion molecule-1 induction. Increased induction of this critical leukocyte adhesion molecule was associated with increased intrahepatic neutrophil accumulation and activation upon induction of liver regeneration in individuals with dysfunctional liver regeneration. Comparing baseline gene expression profiles in individuals with and without dysfunctional liver regeneration, we found that dual-specificity phosphatase 4 (DUSP4) expression, a known critical regulator of intracellular adhesion molecule-1 expression in endothelial cells, was markedly reduced in patients with dysfunctional liver regeneration. Mimicking clinical risk factors for dysfunctional liver regeneration, we found liver sinusoidal endothelial cells of two liver disease models to have significantly reduced baseline levels of DUSP4. Exploring the landscape of early transcriptional changes of human liver regeneration, we observed that people with dysfunctional regeneration experience overwhelming intrahepatic inflammation. Subclinical liver disease might account for DUSP4 reduction in liver sinusoidal endothelial cells, which ultimately primes the liver for an aggravated inflammatory response. Using a unique human biorepository, focused on liver regeneration (LR), we explored the landscape of circulating and tissue-level alterations associated with both functional and dysfunctional LR. In contrast to experimental animal models, people with dysfunctional LR demonstrated an aggravated transcriptional inflammatory response, higher intracellular adhesion molecule-1 (ICAM-1) induction, intrahepatic neutrophil accumulation and activation upon induction of LR. Although inflammatory responses appear rapidly after liver resection, people with dysfunctional LR have exaggerated inflammatory responses that appear to be related to decreased levels of LSEC DUSP4, challenging existing concepts of post-resectional LR.

Fuzapladib reduces postsurgical inflammation in the intestinal muscularis externa.

Postoperative ileus (POI) is a surgical complication that induces emesis and anorexia. Fuzapladib (FUZ), an inhibitor of leukocyte-function-associated antigen type 1 (LFA-1) activation, a leukocyte adhesion molecule, exerts anti-inflammatory effects by inhibiting leukocyte migration into the inflammatory site. In this study, we examined the prophylactic impact of FUZ on POI in a mouse model. POI model mice were generated by intestinal manipulation, and the effect of FUZ on intestinal transit and the infiltration of inflammatory cells into the ileal muscularis externa was assessed. The increased number of macrophages was significantly suppressed by FUZ, whereas the infiltration of neutrophils into the ileal muscularis externa was not sufficiently inhibited in the POI model mice. Additionally, FUZ did not ameliorate delayed gastrointestinal transit in POI model mice. In conclusion, our results suggest that FUZ does not improve delayed gastrointestinal transit but partially inhibits inflammation in the ileal muscularis externa in POI model mice. FUZ may be a potential anti-inflammatory agent for the management of post-surgical inflammation.

MyD88-TLR4-dependent choroid plexus activation precedes perilesional inflammation and secondary brain edema in a mouse model of intracerebral hemorrhage.

The functional neurological outcome of patients with intracerebral hemorrhage (ICH) strongly relates to the degree of secondary brain injury (ICH-SBI) evolving within days after the initial bleeding. Different mechanisms including the incitement of inflammatory pathways, dysfunction of the blood-brain barrier (BBB), activation of resident microglia, and an influx of blood-borne immune cells, have been hypothesized to contribute to ICH-SBI. Yet, the spatiotemporal interplay of specific inflammatory processes within different brain compartments has not been sufficiently characterized, limiting potential therapeutic interventions to prevent and treat ICH-SBI. We used a whole-blood injection model in mice, to systematically characterized the spatial and temporal dynamics of inflammatory processes after ICH using 7-Tesla magnetic resonance imaging (MRI), spatial RNA sequencing (spRNAseq), functional BBB assessment, and immunofluorescence average-intensity-mapping. We identified a pronounced early response of the choroid plexus (CP) peaking at 12-24h that was characterized by inflammatory cytokine expression, epithelial and endothelial expression of leukocyte adhesion molecules, and the accumulation of leukocytes. In contrast, we observed a delayed secondary reaction pattern at the injection site (striatum) peaking at 96h, defined by gene expression corresponding to perilesional leukocyte infiltration and correlating to the delayed signal alteration seen on MRI. Pathway analysis revealed a dependence of the early inflammatory reaction in the CP on toll-like receptor 4 (TLR4) signaling via myeloid differentiation factor 88 (MyD88). TLR4 and MyD88 knockout mice corroborated this observation, lacking the early upregulation of adhesion molecules and leukocyte infiltration within the CP 24h after whole-blood injection. We report a biphasic brain reaction pattern after ICH with a MyD88-TLR4-dependent early inflammatory response of the CP, preceding inflammation, edema and leukocyte infiltration at the lesion site. Pharmacological targeting of the early CP activation might harbor the potential to modulate the development of ICH-SBI.

Emerging vascular cell-specific roles for mineralocorticoid receptor: implications for understanding sex differences in cardiovascular disease.

As growing evidence implicates extrarenal mineralocorticoid receptor (MR) in cardiovascular disease (CVD), recent studies have defined both cell- and sex-specific roles. MR is expressed in vascular smooth muscle (SMC) and endothelial cells (ECs). This review integrates published data from the past 5 years to identify novel roles for vascular MR in CVD, with a focus on understanding sex differences. Four areas are reviewed in which there is recently expanded understanding of the cell type- or sex-specific role of MR in 1) obesity-induced microvascular endothelial dysfunction, 2) vascular inflammation in atherosclerosis, 3) pulmonary hypertension, and 4) chronic kidney disease (CKD)-related CVD. The review focuses on preclinical data on each topic describing new mechanistic paradigms, cell type-specific mechanisms, sexual dimorphism if addressed, and clinical implications are then considered. New data support that MR drives vascular dysfunction induced by cardiovascular risk factors via sexually dimorphic mechanisms. In females, EC-MR contributes to obesity-induced endothelial dysfunction by regulating epithelial sodium channel expression and by inhibiting estrogen-induced nitric oxide production. In males with hyperlipidemia, EC-MR promotes large vessel inflammation by genomic regulation of leukocyte adhesion molecules, which is inhibited by the estrogen receptor. In pulmonary hypertension models, MRs in EC and SMC contribute to distinct components of disease pathologies including pulmonary vessel remodeling and RV dysfunction. Despite a female predominance in pulmonary hypertension, sex-specific roles for MR have not been explored. Vascular MR has also been directly implicated in CKD-related vascular dysfunction, independent of blood pressure. Despite these advances, sex differences in MR function remain understudied.

ALCAM Deficiency Alleviates LPS-Induced Acute Lung Injury by Inhibiting Inflammatory Response.

We investigated the effects and underlying mechanisms of activated leukocyte adhesion molecule (ALCAM) on acute lung injury (ALI) by using lipopolysaccharide (LPS)-induced ALI animal model and LPS-induced inflammation in vitro. In LPS-stimulated mice, ALCAM deficiency relieved lung injury, which manifested as reduced pathological changes in the lung tissue, reduced pulmonary edema, and reduced vascular permeability. Furthermore, we demonstrated that ALCAM deficiency reduced the infiltration of inflammatory cells, including neutrophil, eosinophil, and macrophages; the release of inflammatory cytokines, including IL-1β, IL-6, TNF-α, and COX2; and reduced the protein level of TLR4/NF-κB pathway (TLR4, MyD88, p-IkBɑ, and p-NF-κB p65). We also demonstrated that ALCAM deficiency reduced the expression of oxidative stress-related proteins (Nrf-2, HO-1, and NQO-1) and endoplasmic reticulum stress-related proteins (CHOP, GRP78, ATF-6, and p-eIF2ɑ). In addition, in LPS-induced inflammation in vitro, ALCAM overexpression promoted inflammatory response, oxidative stress, and ER stress. We established that ALCAM deficiency can suppress the ALI process by reducing inflammatory response, oxidative stress, and endoplasmic reticulum stress.

Interleukin-1 in Coronary Artery Disease.

Cardiovascular disease is the leading cause of mortality worldwide. Inflammation has long been established as a key component in the pathophysiology of coronary artery disease. The interleukin-1 family consists of 11 members that regulate the inflammatory response through both pro- and anti-inflammatory properties with the Nod-like receptor (NLR) family pyrin domain containing 3 inflammasome having a pivotal role in the process of converting interleukin-1 beta and interleukin- 18, two key inflammatory mediators, into their mature forms. Interleukin-1 affects various cell types that participate in the pathogenesis of atherosclerosis as it enhances the expression of leukocyte adhesion molecules on the surface of endothelial cells and augments the permeability of the endothelial cell barrier, attracting monocytes and macrophages into the vessel wall and aids the migration of smooth muscle cells toward atheroma. It also enhances the aggregation of low-density lipoprotein particles in endothelium and smooth muscle cells and exhibits procoagulant activity by inducing synthesis, cell-surface expression and release of tissue factor in endothelial cells, promoting platelet adhesion. The value of interleukin-1 as a diagnostic biomarker is currently limited, but interleukin-1 beta, interleukin-18 and interleukin-37 have shown promising data regarding their prognostic value in coronary artery disease. Importantly, target anti-inflammatory treatments have shown promising results regarding atherosclerosis progression and cardiovascular events. In this review article, we focus on the immense role of interleukin-1 in atherosclerosis progression, inflammation cascade and in the clinical application of target anti-inflammatory treatments.

Membrane attack complexes, endothelial cell activation, and direct allorecognition.

Endothelial cells (ECs) form a critical immune interface regulating both the activation and trafficking of alloreactive T cells. In the setting of solid organ transplantation, donor-derived ECs represent sites where alloreactive T cells encounter major and minor tissue-derived alloantigens. During this initial encounter, ECs may formatively modulate effector responses of these T cells through expression of inflammatory mediators. Direct allorecognition is a process whereby recipient T cells recognize alloantigen in the context of donor EC-derived HLA molecules. Direct alloresponses are strongly modulated by human ECs and are galvanized by EC-derived inflammatory mediators.Complement are immune proteins that mark damaged or foreign surfaces for immune cell activation. Following labeling by natural IgM during ischemia reperfusion injury (IRI) or IgG during antibody-mediated rejection (ABMR), the complement cascade is terminally activated in the vicinity of donor-derived ECs to locally generate the solid-phase inflammatory mediator, the membrane attack complex (MAC). Via upregulation of leukocyte adhesion molecules, costimulatory molecules, and cytokine trans-presentation, MAC strengthen EC:T cell direct alloresponses and qualitatively shape the alloimmune T cell response. These processes together promote T cell-mediated inflammation during solid organ transplant rejection.In this review we describe molecular pathways downstream of IgM- and IgG-mediated MAC assembly on ECs in the setting of IRI and ABMR of tissue allografts, respectively. We describe work demonstrating that MAC deposition on ECs generates ‘signaling endosomes’ that sequester and post-translationally enhance the stability of inflammatory signaling molecules to promote EC activation, a process potentiating EC-mediated direct allorecognition. Additionally, with consideration to first-in-human xenotransplantation procedures, we describe clinical therapeutics based on inhibition of the complement pathway. The complement cascade critically mediates EC activation and improved understanding of relevant effector pathways will uncover druggable targets to obviate dysregulated alloimmune T cell infiltration into tissue allografts.

Colchicine Impacts Leukocyte Trafficking in Atherosclerosis and Reduces Vascular Inflammation.

BackgroundInflammation strongly contributes to atherosclerosis initiation and progression. Consequently, recent clinical trials pharmacologically targeted vascular inflammation to decrease the incidence of atherosclerosis-related complications. Colchicine, a microtubule inhibitor with anti-inflammatory properties, reduced cardiovascular events in patients with recent acute coronary syndrome and chronic coronary disease. However, the biological basis of these observations remains elusive. We sought to explore the mechanism by which colchicine beneficially alters the course of atherosclerosis.Methods and ResultsIn mice with early atherosclerosis (Apoe-/- mice on a high cholesterol diet for 8 weeks), we found that colchicine treatment (0.25 mg/kg bodyweight once daily over four weeks) reduced numbers of neutrophils, inflammatory monocytes and macrophages inside atherosclerotic aortas using flow cytometry and immunohistochemistry. Consequently, colchicine treatment resulted in a less inflammatory plaque composition and reduced plaque size. We next investigated how colchicine prevented plaque leukocyte expansion and found that colchicine treatment mitigated recruitment of blood neutrophils and inflammatory monocytes to plaques as revealed by adoptive transfer experiments. Causally, we found that colchicine reduced levels of both leukocyte adhesion molecules and receptors for leukocyte chemoattractants on blood neutrophils and monocytes. Further experiments showed that colchicine treatment reduced vascular inflammation also in post-myocardial infarction accelerated atherosclerosis through similar mechanisms as documented in early atherosclerosis. When we examined whether colchicine also decreased numbers of macrophages inside atherosclerotic plaques by impacting monocyte/macrophage transitioning or in-situ proliferation of macrophages, we report that colchicine treatment did not influence macrophage precursor differentiation or macrophage proliferation using cell culture experiments with bone marrow derived macrophages.ConclusionsOur data reveal that colchicine prevents expansion of plaque inflammatory leukocytes through lowering recruitment of blood myeloid cells to plaques. These data provide novel mechanistic clues on the beneficial effects of colchicine in the treatment of atherosclerosis and may inform future anti-inflammatory interventions in patients at risk.

EFFECTS OF RESVERATROL ON HUMAN HEALTH

Grape is a perennial plant and its fruit and leaves are consumed as food. Its homeland is thought to be the Middle East. There is evidence that the cultivation of this plant began 7-8 thousand years ago. When we look at the content of ripe grapes, 100 grams of grapes contain 69 kilos of energy, 81% of them are water, 18% are carbohydrates, 1% is protein, and contain very little fat, vitamin K and vitamin E. Resveratrol (RSV) was first produced in the early 1930s by the medicinal plant Veratrum grandifolium Loes. fil., many studies have shown that it will be useful in clinical use due to the flavonoids and polyphenolic compounds found in grapes. RSV is a natural non-flavonoid polyphenol belonging to the stilbenoid group, especially found in the skin of black grapes. Besides grapes, it is also found in various plants such as strawberries, blueberries, pomegranates, mulberries, pistachios and peanuts. It is thought that RSV, which is found in dietary components, may have many effects such as antioxidant, anti-inflammatory, anticarcinogenic, neuroprotective, cardioprotective, lipid regulating, antidiabetic and life-prolonging. The potential benefits of RSV have been widely studied in some animal experiments, with positive results. Clinical trials in humans, on the other hand, are less numerous and the results are not yet conclusive. Considering the scientific studies done so far, it has been determined that RSV has tumor-suppressive properties in many stages of cancer. It is a powerful antioxidant that can prevent cell damage caused by free radicals. Radiotherapy or chemotherapy causes inflammation in neighboring cells of cancer cells, while RSV stimulates leukocytes to eliminate these lysed cells, thereby accelerating their apoptosis. RSV improves lipid profile by reducing hepatic 3-hydroxy-3-methyl-glutaryl-CoA reductase in mRNA expression and potentially leading to reverse cholesterol transport. It has been shown that it can lower blood pressure by increasing endothelial nitric oxide (NO) production, reducing oxidative damage and calcium influx. While RSV does not cause any changes in blood pressure acutely, when used for a long time, it improves endothelial function, especially in hypertensive women with higher LDL cholesterol levels. Because polyphenols reduce atherogenesis by preventing low-density lipoprotein oxidation, fat accumulation and platelet aggregation. Down-regulation of inflammatory pathways and regular signaling, decrease in IL-6, TNF alpha, interferon-gamma, Herp, IL-8 and leptin levels, down-regulation of leukocyte adhesion molecules, changes in mRNAs involved in inflammatory modulation, anti-inflammatory effect through an increase in IL-10 plasma levels shows. Recent studies have shown that RSV has a curative effect on Alzheimer's disease and prevents neurological damage in case of cerebral ischemia. It has a protective effect against chronic obstructive pulmonary disease. Some recent studies on RSV have determined that it prolongs lifespan in some yeasts, threadworms, fruit flies, fish, and obese mice. Keywords: Grape seeds, antioxidant, effects of resveratrol.

P1016: POLYCYTHEMIA VERA AND IMMUNE THROMBOGENESIS

Background: Polycythemia Vera (PV) is a Ph1-negative myeloproliferative neoplasm characterized by vascular thrombosis and poor survival. The polycythemic thrombogenesis is associated with erythrocytosis and red cell adhesiveness angiopathy. Recent studies reported a JAK/STAT-mediated reduction of the regulatory T cells (Tregs) (CD4+, CD25high, CD127low, FoxP3+) and loss of self-tolerance. Aims: We investigated Tregs, anti-endothelial cell antibodies (AECA), and endothelial, platelet and coagulation activation, in Polycythemia Vera (PV) and thrombosis. Methods: We enrolled 60WHO-defined PV patients (30 men, 30 women; mean age 45±10 years) without cardiovascular risk factors, autoimmune disease or thrombotic history. Of PV patients, 40/60 had thromosis includingmyocardial infarction (MI) (10/60) according to the WHO critera, deep vein thrombosis (20/60) and pulmonary embolism (10/60) on lower-limb ultrasonography and computed tomography angiography, respectively. All patients were evaluated for JAK2V617F allele burden, Tregs, AECA,Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1), Intercellular Adhesion Molecule-1 (ICAM-1),prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and D-dimer (DD). JAK2V617F allele burden were analyzed by Polymerase chain reaction, Tregs were measured by flow cytometry,AECA, ELAM-1 and ICAM-1 by ELISA, PT and APTT by coagulometric test, Fib using Clauss method, and DD using ELISA.Complete blood hemostasis was studied by PFA-100 on Collagen/ADP (CT-ADP) and Collagen/Epinephrine (CT-EPI) cartridges and Thromboelastometry method on Clotting Time (CT), Clotting Formation Time (CFT), Maximum Clot Firmness (MCF), and clot lysis at 30 minutes (LY-30). Results: The patients with thrombosis had JAK2V616F allele burden higher (> 50%) compared to patients without thrombosis (< 50Kroll%), lower Tregs (1,5±0,5% vs 3.5±1%),higher AECA (200±20% vs 110±10%), ELAM-1 (80±10 ng/ml vs 45±5 ng/ml), and ICAM (170 ng/mL±10 vs 110±10 ng/mL), longer PT (30±10 s vs 20±2 s) and PTT (60±10 s vs 38±5 s), lower Fib (90±20 mg/dl vs 120±20 mg/dl), higher DD (650±100 mg/l vs 300±50 mg/l) and shorter C/ADP and C/EPI (C/ADP, n.v. 68-121 s (40±10 s vs 55±20 s) and C/EPI n.v. 84-160 s (35±5 s vs 60±10 s). The patients with thrombosis had shorter CT (INTEM 35±20 s vs 70±20 s, EXTEM 20±10 s vs 30±5 s), shorter CFT (INTEM 15±10 s vs 25±5 s EXTEM 18±10 s vs 28±5 s), longer MCF (INTEM 130±10 mm vs 90±10 mm, EXTEM 120±10 mm vs 82±10 mm), and lower LY-30 (INTEM 0.9% vs 15%, EXTEM 0.8% vs 15%). A positive correlation there was between Tregs and AECA, ELAM-1 and ICAM-1 and thrombosis. Summary/Conclusion: These findings shed new light on thrombotic pathogenesis in patients with PV.

Abstract 6382: Targeting mast cells restores T cell infiltration and sensitizes sarcomas to PD-L1 inhibition

Abstract Despite recent advances in cancer research, developments in sarcoma treatment have been slow. The efficacy of chemotherapy in sarcoma is limited by the pathophysiology of surrounding tumor stroma. Abnormalities in the tumor microenvironment (TME) result in stiffening of the tumor and accumulation of mechanical stresses which in turn cause the extreme deformation and collapse of intratumoral vessels. Strategies to induce TME reprogramming with the aim to restore mechanical properties of the tissue as well as tumor blood vessel functionality involve the use of “mechanotherapeutics”. Herein, we exploited the potential of the antihistamine drug and mast cell stabilizer, ketotifen, to induce mechanotherapeutic effects in murine models of soft tissue and bone sarcoma. Mast cells are the first population of immune cells drawn to the tumorigenic nidus where they interact with multiple components of the TME. Yet, their role in tumor stroma remains enigmatic as they either promote or inhibit tumor development depending on the conditions. In agreement with other mechanotherapeutics, we found that ketotifen induces vascular normalization in a dose dependent manner as indicated by the increase in perfusion, higher open lumen fraction and pericyte coverage of the vessels. Moreover, in vivo ultrasound shear wave elastography confirmed the mechanotherapeutic properties of ketotifen in reducing tissue stiffness, while further ex vivo validation indicated that TME reprogramming is mediated by a significant reduction in cancer associated fibroblast and inhibition of collagen synthesis as well as of that of other extracellular matrix components. Furthermore, we found that ketotifen modulates the immune microenvironment to promote immunostimulation. Immune stimulation was mediated by both intratumoral T cell accumulation and higher cytotoxic to regulatory T cell ratio concomitantly with a pronounced reduction in tissue hypoxia and upregulation of leukocyte adhesion molecules on endothelium. As a result of ketotifen-induced TME reprogramming, the addition of ketotifen to doxorubicin-aPDL1 combination treatment exhibited therapeutic superiority as opposed to doxorubicin-aPDL1, offering a durable tumor remission and immunological memory following tumor rechallenge experiments. Taken together, our findings demonstrate that ketotifen has a dual role in shaping the TME: i) reducing intratumoral physical forces to improve perfusion and ii) establishing favorable immunogenic conditions. Therefore, incorporating such agents in clinical practice holds great promise in the treatment of refractory, advanced sarcomas. Citation Format: Myrofora Panagi, Fotios Mpekris, Chrysovalantis Voutouri, Christina Michael, Anastasia Constantinidou, John D. Martin, Triantafyllos Stylianopoulos. Targeting mast cells restores T cell infiltration and sensitizes sarcomas to PD-L1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6382.

Dehydroabietic Acid Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice and VCAM-1 Expressions in-vitro

Dehydroabietic acid (DHA) is an analog of abietic acid (AA) for cardiovascular disease prevention, known to act pharmacologically against aging, inflammation, bacterial infections and cancer. The current research investigated the molecular mechanisms of DHA on the adhesiveness of endothelial leukocytes and activation of NF-κB in TNF-α treated HAEC and ApoE−/− mice in experimental atherosclerosis. The HAECs were tested for toxicity using MTT assay at various DHA concentrations. The cell-surface expression of CAM against endothelial leukocyte adhesion molecule-1, ICAM -1, or human VCAM-1 was determined by the ELISA test, followed by western blot analysis. Endothelial cell-leukocyte adhesion assay involving U937 cells was carried out followed by the determination of NF-κB p65 expression. ApoE−/− mice were fed with a high cholesterol diet every day followed by oral administration of DHA (10 and 20 mg/kg/day). The DHA (5 and 10 mM) substantially reduced (p &lt; 0.05) human (U937) cell lines binding to TNF-α activated human endothelial aortic cells. The assays involving the 32P-labelled NF-kB as a probe demonstrated that DHA pre-treatment decreased the shifted bands density following the stimulation of TNF-α. Nuclear extracts immune blot assessment and immune fluorescence staining showed a significant decrease (p &lt; 0.05) in the NF-κB p65 concentration in the nuclei with DHA treated human endothelial aortic cells. Together these findings indicate that DHA inhibits nuclear trans localization of TNF-α-induced NF-κB p65, and thus considerably suppresses (p &lt; 0.05) the VCAM-1 expression, which contributes to lower leukocyte adherence suggesting that DHA helps in preventing inflammatory atherosclerosis in vivo.

Impact of Anti-Endothelial Cell Antibodies (AECAs) in Patients with Polycythemia Vera and Thrombosis.

Polycythemia vera (PV) causes thrombosis. Erythrocytosis and cell adhesiveness are responsible for thrombosis. JAK2V617F causes inflammation and autoimmunity; however, whether or not autoimmunity or inflammation causes thrombosis has yet to be proven. In 60 PV patients, we analyzed JAK2V671F and its allele burden, autoimmune Th17 cells, interleukin-17 (IL-17), anti-endothelial cell antibodies (AECAs), endothelial leukocyte adhesion molecule-1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and von Willebrand factor antigen (VWF:Ag). Fifty blood donors were used as the controls. All patients were on phlebotomy-maintaining hematocrit <45% and aspirin. Of the 60 patients, 40 had thrombosis. Those patients with thrombosis had a higher JAK2V617F allele burden than those without thrombosis, andTh17 cells and IL-17 were also higher in patients with thrombosis. Interestingly, we observed a high AECA IgG ELISA ratio (ER) in patients with thrombosis, which was normal in patients without thrombosis. We found high ELAM-1 and ICAM-1 as well as high VWF:Ag in patients with thrombosis compared to patients without thrombosis. AECA-positive sera from patients with thrombosis showed enhanced binding to cytokine-treated HUVEC and a positive antibody-dependent cellular cytotoxicity, suggesting that AECA may contribute to vascular injury. A positive correlation between AECAs, allele burden, and thrombosis was found. These results suggest that autoimmunity may be an additional mechanism in PV thrombogenesis.

Pericyte Loss Leads to Capillary Stalling Through Increased Leukocyte-Endothelial Cell Interaction in the Brain.

The neurovascular unit is a functional unit composed of neurons, glial cells, pericytes, and endothelial cells which sustain brain activity. While pericyte is a key component of the neurovascular unit, its role in cerebral blood flow regulation remains elusive. Recently, capillary stalling, which means the transient interruption of microcirculation in capillaries, has been shown to have an outsized impact on microcirculatory changes in several neurological diseases. In this study, we investigated capillary stalling and its possible causes, such as the cerebral endothelial glycocalyx and leukocyte adhesion molecules after depleting pericytes postnatally in mice. Moreover, we investigated hypoxia and gliosis as consequences of capillary stalling. Although there were no differences in the capillary structure and RBC flow, longitudinal optical coherence tomography angiography showed an increased number of stalled segments in capillaries after pericyte loss. Furthermore, the extent of the cerebral endothelial glycocalyx was decreased with increased expression of leukocyte adhesion molecules, suggesting enhanced interaction between leukocytes and endothelial cells. Finally, pericyte loss induced cerebral hypoxia and gliosis. Cumulatively, the results suggest that pericyte loss induces capillary stalling through increased interaction between leukocytes and endothelial cells in the brain.

Current Insights on the Role of Irisin in Endothelial Dysfunction.

Endothelial dysfunction is a crucial physiopathological mechanism for cardiovascular diseases that results from the harmful impact of metabolic disorders. Irisin, a recently discovered adipomyokine, has been shown to exert beneficial metabolic effects by increasing energy consumption, improving insulin sensitivity, and reducing the proinflammatory milieu. Multiple preclinical models have assessed irisin's possible role in the development of endothelial dysfunction, displaying that treatment with exogenous irisin can decrease the production of oxidative stress mediators by up-regulating Akt/mTOR/Nrf2 pathway, promote endothelial-dependent vasodilatation through the activation of AMPK-PI3K-AkteNOS pathway, and increase the endothelial cell viability by activation of ERK proliferation pathway and downregulation of Bad/Bax/Caspase 3 pro-apoptotic pathway. However, there is scarce evidence of these mechanisms in clinical studies, and available results are controversial. Some have shown negative correlations of irisin levels with the burden of coronary atherosclerosis and leukocyte adhesion molecules' expression. Others have demonstrated associations between irisin levels and increased atherosclerosis risk and higher carotid intima-media thickness. Since the role of irisin in endothelial damage remains unclear, in this review, we compare, contrast, and integrate the current knowledge from preclinical and clinical studies to elucidate the potential preventive role and the underlying mechanisms and pathways of irisin in endothelial dysfunction. This review also comprises original figures to illustrate these mechanisms.

Does AGE-RAGE Stress Play a Role in the Development of Coronary Artery Disease in Obesity?

This article deals with the role of AGE (advanced glycation end products)-RAGE (receptor for AGE) stress (AGE/sRAGE) in the development of coronary artery disease (CAD) in obesity. CAD is due to atherosclerosis in coronary artery. The serum/plasma levels of AGE and sRAGE are reduced, while AGE-RAGE stress and expression of RAGE are elevated in obese individuals. However, the levels of AGE are elevated in obese individuals with more than one metabolic syndrome. The increases in the AGE-RAGE stress would elevate the expression and production of atherogenic factors, including reactive oxygen species, nuclear factor-kappa B, cytokines, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, endothelial leukocyte adhesion molecules, monocyte chemoattractant protein-1, granulocyte-macrophage colony-stimulating factor, and growth factors. Low levels of sRAGE would also increase the atherogenic factors. The increases in the AGE-RAGE stress and decreases in the levels of sRAGE would induce development of atherosclerosis, leading to CAD. The therapeutic regimen for AGE-RAGE stress-induced CAD in obesity would include lowering of AGE intake, prevention of AGE formation, degradation of AGE in vivo, suppression of RAGE expression, blockade of AGE-RAGE interaction, downregulation of sRAGE expression, and use of antioxidants. In conclusion, the data suggest that AGE-RAGE stress is involved in the development of CAD in obesity, and the therapeutic interventions to reduce AGE-RAGE would be helpful in preventing, regressing, and slowing the progression of CAD in obesity.

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.

POS-406 Mac-1 (CD11b/CD18) deficiency ameliorates AKI to CKD progression via promoting alternative activation of macrophages

Acute kidney injury (AKI), even if followed by renal recovery, is a risk factor for the future development of chronic kidney disease (CKD). Persistent chronic inflammation has been proposed to contribute to the progression of AKI-to-CKD. Macrophages are a heterogeneous cell group implicated in injury, repair, and fibrosis during AKI-to-CKD process. However, the underlying mechanism of how macrophages participate in the progress of disease is largely unclear. Mac-1 (CD11b/CD18), a leukocyte adhesion molecule, expressed on neutrophils, eosinophils and macrophages has been shown to mediate several adhesion-dependent processes.

The Role of Ferroptosis in Blood-Brain Barrier Injury.

The blood-brain barrier (BBB) is an important barrier that maintains homeostasis within the central nervous system. Brain microvascular endothelial cells are arranged to form vessel walls and express tight junctional complexes that limit the paracellular pathways of the BBB and therefore play a crucial role in ensuring brain function. These vessel walls tightly regulate the movement of ions, molecules, and cells between the blood and the brain, which protect the neural tissue from toxins and pathogens. Primary damage caused by BBB dysfunction can disrupt the expression of tight junctions, transport proteins and leukocyte adhesion molecules, leading to brain edema, disturbances in ion homeostasis, altered signaling and immune infiltration, which can lead to neuronal cell death. Various neurological diseases are known to cause BBB dysfunction, but the mechanism that causes this disorder is not clear. Recently, ferroptosis has been found to play an important role in BBB dysfunction. Ferroptosis is a new form of regulatory cell death, which is caused by the excessive accumulation of lipid peroxides and iron-dependent reactive oxygen species. This review summarizes the role of ferroptosis in BBB dysfunction and the latest progress of ferroptosis mechanism, and further discusses the influence of various factors of ferroptosis on the severity and prognosis of BBB dysfunction, which may provide better therapeutic targets for BBB dysfunction.