Leukocyte-endothelial Cell Interactions Research Articles

Effect of Non-Surgical Periodontal Treatment on Oxidative Stress Markers in Leukocytes and Their Interaction with the Endothelium in Obese Subjects with Periodontitis: A Pilot Study.

Aim: The primary objective of this pilot study was to evaluate the effect of non-surgical periodontal treatment. The secondary aim was to evaluate the effect of dietary therapy on both parameters of oxidative stress in leukocytes and leukocyte-endothelial cell interactions in an obese population. Methods: This was a pilot study with a before-and-after design. Forty-nine obese subjects with periodontitis were randomized by means of the minimization method and assigned to one of two groups, one of which underwent dietary therapy while the other did not. All the subjects underwent non-surgical periodontal treatment. We determined periodontal, inflammatory and oxidative stress parameters—total reactive oxygen species (ROS), superoxide production, intracellular Ca2+, mitochondrial membrane potential and superoxide dismutase (SOD) activity. We also evaluated interactions between leukocytes and endothelium cells—velocity, rolling flux and adhesion—at baseline and 12 weeks after intervention. Results: Periodontal treatment improved the periodontal health of all the patients, with a reduction in serum retinol-binding protein 4 (RBP4), total superoxide production and cytosolic Ca2+ in leukocytes. In the patients undergoing dietary therapy, there were less leukocyte adhesion to the endothelium, an effect that was accompanied by a decrease in TNFα, P-selectin and total ROS and an increase in SOD activity. Conclusions: Whereas non-surgical periodontal treatment induces an improvement in leukocyte homeostasis, dietary therapy as an adjuvant reduces systemic inflammation and increases antioxidant status which, in turn, modulates leukocyte-endothelium dynamics.

Fibrinogen concentrations in ischaemic stroke patients with metabolic disorders.

Hyperfibrinogenemia plays a crucial role in the coagulation cascade leading to the formation of clots. It is involved in the process of platelet aggregation, primary haemostasis, and leukocyte-endothelial cell interactions. The aim of our study was to assess the correlations between fibrinogen concentration and particular risk factors for vascular diseases and atherosclerotic changes in stroke patients. The study group consisted with 94 patients with acute ischaemic stroke with normo- or hyperglycaemia and normoor hyperlipidemia. 21 healthy subjects served as a control group. Fibrinogen level, HbA1c, and lipid profile were measured in all patients. Using a flow cytometer, we assessed CD61 positive microparticles which were defined as platelet-derived microparticles (PDMPs). The level of sP-selectin in serum was measured using the ELISA method. A significant positive correlation was observed between fibrinogen concentration and sP-selectin (p = 0.001), HbA1c (p < 0.05) level, and percentage of PDMPs (p < 0.05) in the study patients. Furthermore, we noticed a significant negative correlation between fibrinogen concentration and the level of HDL (p < 0.05). No correlation was observed between fibrinogen and TC, LDL and TG levels. Our findings suggest that an elevated fibrinogen level may represent a marker of prothrombotic condition exacerbated in the state of hyperglycaemia and activation of platelets and endothelial cells. This suggests an important role played by fibrinogen in the process of thrombogenesis.

Experimental Approaches to Evaluate Leukocyte-Endothelial Cell Interactions in Sepsis and Inflammation.

Sepsis is a life-threatening syndrome of organ dysfunction caused by a dysregulated host response to infection characterized by excessive neutrophil infiltration into vital organs. In sepsis, patients often die of organ failure and therapies directed against endothelial cell dysfunction and tissue damage are important targets for treatment of this disease. Novel approaches are required to understand the underlying pathophysiology of neutrophil dysregulation and neutrophil-endothelial cell interactions that play a critical role in the early course of organ damage and disruption of endothelial protective barrier. Here, we review methodologies that our laboratories have employed to study neutrophil-endothelial interaction and endothelial barrier function in in vivo and in vitro models of sepsis. We will focus on in vivo rodent models of sepsis and in vitro tools that use human cell culture models under static conditions and the more physiologically relevant biomimetic microfluidic assays. This Methods paper is based on our presentation in the Master Class Symposium at the 41st Annual Conference on Shock 2018.

Electronic cigarette exposure disrupts blood-brain barrier integrity and promotes neuroinflammation

Electronic cigarette (e-cigarette) use has grown substantially since inception, particularly among adolescents and combustible tobacco users. Several cigarette smoke constituents with known neurovascular effect are present in e-cigarette liquids or formed during the vapor generation. The present study establishes inhaled models of cigarette and e-cigarette use with normalized nicotine delivery, then characterizes the impact on blood-brain barrier (BBB) function. Sequencing of microvessel RNA following exposure revealed downregulation of several genes with critical roles in BBB function. Reduced protein expression of Occludin and Glut1 is also observed at the tight junction in all groups following exposure. Pro-inflammatory changes in leukocyte-endothelial cell interaction are also noted, and mice exposed to nicotine-free e-cigarettes have impaired novel object recognition performance. On this basis, it is concluded that long term e-cigarette use may adversely impact neurovascular health. The observed effects are noted to be partly independent of nicotine content and nicotine may even serve to moderate the effects of non-nicotinic components on the blood-brain barrier.

In Vitro Approaches for Investigating the Influence of MIF on Leukocyte-Endothelial Cell Interactions Under Flow Conditions.

The exit of leukocytes from the bloodstream into inflamed sites involves a sequence of interactions with vascular endothelial cells, in which leukocytes, moving rapidly in flowing blood, first tether and roll on the endothelial surface before arresting and then transmigrating across the endothelial barrier. Examining the mechanisms of these interactions in human systems has involved the use of in vitro flow chamber assays, using a variety of cells and immobilized molecules as adhesive substrata. Here we describe how to perform these assays using human umbilical vein endothelial cells and human leukocytes.

Molecular and Pathophysiological Mechanisms of Diabetic Retinopathy in Relation to Adhesion Molecules.

Diabetic Retinopathy (DR) is considered as a most common microvascular complication of diabetes affected by one in three people who are suffered for diabetes. Several pathophysiological mechanisms and adhesion molecules may play an etiologic role in the development of diabetes and its complications. The adhesion molecules located on both leucocytes and endothelial cells and considered as important molecules which can assessed the endothelial function. The functions of adhesion molecules involved in the cellular margination, slow rolling and transmigration of leukocytes. Hyperglycemia and its immediate biochemical sequelae or the low-grade inflammation directly alter endothelial function or influence endothelial cell functioning indirectly by induce oxidative stress and activates leukocytosis and leukocyte-endothelial cell interactions by the increased expression of adhesion molecules, growth factors, inflammatory factors, chemokines etc. and results DR. This review summarized the several pathophysiological mechanisms and role of adhesion molecules in disruption of homeostasis of vasculature by leukocytes in the development of diabetic retinopathy.

SGLT-2 (Sodium-Glucose Cotransporter 2) Inhibition Reduces Ang II (Angiotensin II)-Induced Dissecting Abdominal Aortic Aneurysm in ApoE (Apolipoprotein E) Knockout Mice.

Abdominal aortic aneurysm (AAA) is a pathological condition of permanent vessel dilatation that predisposes to the potentially fatal consequence of aortic rupture. SGLT-2 (sodium-glucose cotransporter 2) inhibitors have emerged as powerful pharmacological tools for type 2 diabetes mellitus treatment. Beyond their glucose-lowering effects, recent studies have shown that SGLT-2 inhibitors reduce cardiovascular events and have beneficial effects on several vascular diseases such as atherosclerosis; however, the potential effects of SGLT-2 inhibition on AAA remain unknown. This study evaluates the effect of oral chronic treatment with empagliflozin-an SGLT-2 inhibitor-on dissecting AAA induced by Ang II (angiotensin II) infusion in apoE (apolipoprotein E)-/- mice. Approach and Results: Empagliflozin treatment significantly reduced the Ang II-induced increase in maximal suprarenal aortic diameter in apoE-/- mice independently of blood pressure effects. Immunohistochemistry analysis revealed that empagliflozin diminished Ang II-induced elastin degradation, neovessel formation, and macrophage infiltration at the AAA lesion. Furthermore, Ang II infusion resulted in a marked increase in the expression of chemokines (CCL-2 [chemokine (C-C motif) ligand 2] and CCL-5 [chemokine (C-C motif) ligand 5]), VEGF (vascular endothelial growth factor), and MMP (matrix metalloproteinase)-2 and MMP-9 in suprarenal aortic walls of apoE-/- mice, and all were reduced by empagliflozin cotreatment. Western blot analysis revealed that p38 MAPK (p38 mitogen-activated protein kinase) and NF-κB (nuclear factor-κB) activation was also reduced in the suprarenal aortas of apoE-/- mice cotreated with empagliflozin. Finally, in vitro studies in human aortic endothelial cells and macrophages showed that empagliflozin inhibited leukocyte-endothelial cell interactions and release of proinflammatory chemokines. Pharmacological inhibition of SGLT-2 by empagliflozin inhibits AAA formation. SGLT-2 inhibition might represent a novel promising therapeutic strategy to prevent AAA progression.

The dorsal skinfold chamber: A valuable model for the in vivo evaluation of topical formulations

In this study, we introduce the mouse dorsal skinfold chamber model as a valuable approach for the in vivo evaluation of topical formulations. For this purpose, dorsal skinfold chambers were implanted into BALB/c mice. Tumor necrosis factor (TNF)-α was administered to the chamber tissue for the local induction of inflammation followed by the application of diclofenac-containing or diclofenac-free (control) gel onto the skin of the chamber backside. Intravital fluorescence microscopy was repetitively performed throughout an observation period of 24hours to study macromolecular leakage, leucocyte-endothelial cell interactions and microhaemodynamic parameters. In addition, infiltration of the inflamed tissue with different immune cell subtypes was assessed by immunohistochemistry. In a second set of experiments, the effect of dermal application of a diclofenac-containing gel on photochemically induced thrombus formation was analysed. It was observed that macromolecular leakage, numbers of adherent leucocytes and tissue infiltrating myeloperoxidase (MPO)-positive neutrophilic granulocytes and CD68-positive macrophages were significantly reduced in dorsal skinfold chambers treated with diclofenac-containing gel when compared to controls. Moreover, the diclofenac-containing gel exerted an anti-thrombotic activity, as indicated by a significantly prolonged complete vessel occlusion time. These findings demonstrate that the mouse dorsal skinfold chamber represents a valid and versatile tool to evaluate the effects of topical formulationsin vivo.

273-LB: Leukocyte-Endothelial Interaction Is Reduced by Insulin-Induced Downregulation of Endothelial CXCR4

An activated, pro-inflammatory endothelium is a key feature of obesity and type 2 diabetes and may contribute to adipose tissue inflammation, atherosclerosis and tumor formation. We have previously published a striking 4-fold increase in leukocyte adhesion to endothelium in mice with endothelial cell knockout of the insulin receptor. Here, we searched for insulin-regulated genes involved in leukocyte-endothelial cell interaction by performing RNA sequencing in primary human endothelial cells. Among genes with the largest change in expression after insulin treatment was C-X-C motif chemokine receptor 4 (CXCR4), which was downregulated by 54%. This reduction was completely prevented by pretreatment with wortmannin but not by U0126, an inhibitor of MAPK signaling. In CD31+ cells isolated by fluorescence-activated cell sorting of enzymatically dissociated tissue, CXCR4 mRNA in was 77% higher in mice with diet-induced obesity compared to lean controls and 37% higher in db/db mice compared to db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. Stromal-derived factor-1 (SDF-1), the ligand for CXCR4, increased leukocyte adhesion to cultured endothelial cells. Insulin treatment of endothelial cells prevented 80% of this increase which in turn was abrogated by plerixafor, a clinically approved CXCR4 antagonist, or deletion of CXCR4 by gene editing. To study gain of endothelial cell insulin signaling in vivo, we examined transgenic mice with endothelial overexpression of insulin receptor substrate-1 (IRS-1). In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling and adhesion after intravenous injection of SDF-1 in wildtype mice which was reduced by 83% in transgenic mice. We conclude that endothelial cell insulin signaling limits leukocyte-endothelial cell interaction through downregulation of CXCR4 and that targeting endothelial CXCR4 may protect against inflammation in insulin resistant states. Disclosure B. Kunkemoeller: None. T. Rathjen: Employee; Self; Bayer AG. J.C. Patti: None. Q. Li: None. K. Park: None. G.L. King: Research Support; Self; Sanofi. C. Rask-Madsen: None.

Role and Molecular Mechanisms of Pericytes in Regulation of Leukocyte Diapedesis in Inflamed Tissues.

Leukocyte recruitment is a hallmark of the inflammatory response. Migrating leukocytes breach the endothelium along with the vascular basement membrane and associated pericytes. While much is known about leukocyte-endothelial cell interactions, the mechanisms and role of pericytes in extravasation are poorly understood and the classical paradigm of leukocyte recruitment in the microvasculature seldom adequately discusses the involvement of pericytes. Emerging evidence shows that pericytes are essential players in the regulation of leukocyte extravasation in addition to their functions in blood vessel formation and blood-brain barrier maintenance. Junctions between venular endothelial cells are closely aligned with extracellular matrix protein low expression regions (LERs) in the basement membrane, which in turn are aligned with gaps between pericytes. This forms preferential paths for leukocyte extravasation. Breaching of the layer formed by pericytes and the basement membrane entails remodelling of LERs, leukocyte-pericyte adhesion, crawling of leukocytes on pericyte processes, and enlargement of gaps between pericytes to form channels for migrating leukocytes. Furthermore, inflamed arteriolar and capillary pericytes induce chemotactic migration of leukocytes that exit postcapillary venules, and through direct pericyte-leukocyte contact, they induce efficient interstitial migration to enhance the immunosurveillance capacity of leukocytes. Given their role as regulators of leukocyte extravasation, proper pericyte function is imperative in inflammatory disease contexts such as diabetic retinopathy and sepsis. This review summarizes research on the molecular mechanisms by which pericytes mediate leukocyte diapedesis in inflamed tissues.

Narciclasine exerts anti-inflammatory actions by blocking leukocyte-endothelial cell interactions and down-regulation of the endothelial TNF receptor 1.

The alkaloid narciclasine has been characterized extensively as an anticancer compound. Accumulating evidence suggests that narciclasine has anti-inflammatory potential; however, the underlying mechanism remains poorly understood. We hypothesized that narciclasine affects the activation of endothelial cells (ECs), a hallmark of inflammatory processes, which is a prerequisite for leukocyte-EC interaction. Thus, we aimed to investigate narciclasine's action on this process in vivo and to analyze the underlying mechanisms in vitro. In a murine peritonitis model, narciclasine reduced leukocyte infiltration, proinflammatory cytokine expression, and inflammation-associated abdominal pain. Moreover, narciclasine decreased rolling and blocked adhesion and transmigration of leukocytes in vivo. In cultured ECs, narciclasine inhibited the expression of cell adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin and blocked crucial steps of the NF-κB activation cascade: NF-κB promotor activity, p65 nuclear translocation, inhibitor of κB α (IκBα) phosphorylation and degradation, and IκBα kinase β and TGF-β-activated kinase 1 phosphorylation. Interestingly, these effects were based on the narciclasine-triggered loss of TNF receptor 1 (TNFR1). Our study highlights narciclasine as an interesting anti-inflammatory compound that effectively inhibits the interaction of leukocytes with ECs by blocking endothelial activation processes. Most importantly, we showed that the observed inhibitory action of narciclasine on TNF-triggered signaling pathways is based on the loss of TNFR1.-Stark, A., Schwenk, R., Wack, G., Zuchtriegel, G., Hatemler, M. G., Bräutigam, J., Schmidtko, A., Reichel, C. A., Bischoff, I., Fürst, R. Narciclasine exerts anti-inflammatory actions by blocking leukocyte-endothelial cell interactions and down-regulation of the endothelial TNF receptor 1.

The cyclooxygenase-1/mPGES-1/endothelial prostaglandin EP4 receptor pathway constrains myocardial ischemia-reperfusion injury

The use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX)-1 and COX-2, increases heart failure risk. It is unknown whether microsomal (m) prostaglandin (PG) E synthase (S)-1, a target downstream of COX, regulates myocardial (M) ischemia/reperfusion (I/R) injury, a key determinant of heart failure. Here we report that COX-1 and mPGES-1 mediate production of substantial amounts of PGE2 and confer cardiac protection in MI/R. Deletion of mPges-1 impairs cardiac microvascular perfusion and increases inflammatory cell infiltration in mouse MI/R. Consistently, mPges-1 deletion depresses the arteriolar dilatory response to I/R in vivo and to acetylcholine ex vivo, and enhances leukocyte-endothelial cell interaction, which is mediated via PGE receptor-4 (EP4). Furthermore, endothelium-restricted Ep4 deletion impairs microcirculation, and exacerbates MI/R injury, irrespective of EP4 agonism. Treatment with misoprostol, a clinically available PGE analogue, improves microcirculation and reduces MI/R injury. Thus, mPGES-1, a key microcirculation protector, constrains MI/R injury and this beneficial effect is partially mediated via endothelial EP4.

In vitro and in vivo testing of a novel local nicardipine delivery system to the brain: a preclinical study.

The management of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains a highly demanding challenge in critical care medicine. Despite all efforts, the calcium channel antagonist nimodipine remains the only drug approved for improving outcomes after aSAH. However, in its current form of application, it provides less than optimal efficacy and causes dose-limiting hypotension in a substantial number of patients. Here, the authors tested in vitro the release dynamics of a novel formulation of the calcium channel blocker nicardipine and in vivo local tolerance and tissue reaction using a chronic cranial window model in mice. To characterize the release kinetics in vitro, dissolution experiments were performed using artificial cerebrospinal fluid over a time period of 21 days. The excipients used in this formulation (NicaPlant) for sustained nicardipine release are a mixture of two completely degradable polymers. A chronic cranial window in C57BL/6 mice was prepared, and NicaPlant slices were placed in proximity to the exposed cerebral vasculature. Epifluorescence video microscopy was performed right after implantation and on days 3 and 7 after surgery. Vessel diameter of the arteries and veins, vessel permeability, vessel configuration, and leukocyte-endothelial cell interaction were quantified by computer-assisted analysis. Immunofluorescence staining was performed to analyze inflammatory reactions and neuronal alterations. In vitro the nicardipine release profile showed an almost linear curve with about 80% release at day 15 and full release at day 21. In vivo epifluorescence video microscopy showed a significantly higher arterial vessel diameter in the NicaPlant group due to vessel dilatation (21.6 ± 2.6 µm vs 17.8 ± 1.5 µm in controls, p < 0.01) confirming vasoactivity of the implant, whereas the venous diameter was not affected. Vessel dilatation did not have any influence on the vessel permeability measured by contrast extravasation of the fluorescent dye in epifluorescence microscopy. Further, an increased leukocyte-endothelial cell interaction due to the implant could not be detected. Histological analysis did not show any microglial activation or accumulation. No structural neuronal changes were observed. NicaPlant provides continuous in vitro release of nicardipine over a 3-week observation period. In vivo testing confirmed vasoactivity and lack of toxicity. The local application of this novel nicardipine delivery system to the subarachnoid space is a promising tool to improve patient outcomes while avoiding systemic side effects.

Distinct contributions of hyperglycemia and high-fat feeding in metabolic syndrome-induced neuroinflammation

BackgroundHigh-fat feeding and hyperglycemia, key risk factors for the development of metabolic syndrome (MetS), are emerging to associate with increased risk of developing dementia and cognitive decline. Despite this, clinical and experimental studies have yet to elucidate the specific contributions of either high-fat feeding or hyperglycemia to potential neuroinflammatory components. In this study, we delineate these individual components of MetS in the development of neuroinflammation.MethodsMale C57Bl/6 J adult mice were treated with either citrate vehicle (CIT) or streptozotocin (STZ; 55 mg/kg) 3, 5 and 7 days before commencement of either a normal or high-fat diet for 9 or 18 weeks. By creating separate models of high-fat feeding, STZ-induced hyperglycemia, as well as in combination, we were able to delineate the specific effects of a high-fat diet and hyperglycemia on the brain. Throughout the feeding regime, we measured the animals’ body weight and fasting blood glucose levels. At the experimental endpoint, we assessed plasma levels of insulin, glycated haemoglobin and performed glucose tolerance testing. In addition, we examined the effect of high fat-feeding and hyperglycemia on the levels of systemic inflammatory cytokines, gliosis in the hippocampus and immune infiltration in cerebral hemispheric tissue. Furthermore, we used intravital multiphoton microscopy to assess leukocyte-endothelial cell interactions in the cerebral vasculature of mice in vivo.ResultsWe showed that acute hyperglycemia induces regional-specific effects on the brain by elevating microglial numbers and promotes astrocytosis in the hippocampus. In addition, we demonstrated that chronic hyperglycemia supported the recruitment of peripheral GR1+ granulocytes to the cerebral microvasculature in vivo. Moreover, we provided evidence that these changes were independent of the systemic inflammation associated with high-fat feeding.ConclusionsHyperglycemia alone preferentially induces microglial numbers and astrocytosis in the hippocampus and is associated with the peripheral recruitment of leukocytes to the cerebrovasculature, but not systemic inflammation. High-fat feeding alone, and in combination with hyperglycemia, increases the systemic pro-inflammatory cytokine milieu but does not result in brain-specific immune gliosis. These results shed light on the specific contributions of high-fat feeding and hyperglycemia as key factors of MetS in the development of neuroinflammation.

Krüppel-like factor 14, a coronary artery disease associated transcription factor, inhibits endothelial inflammation via NF-κB signaling pathway

Background and aimsHuman genetic studies indicated that variations near the transcription factor Krüppel-like factor 14 (KLF14) gene locus are highly associated with coronary artery disease. Activation of endothelial cells (ECs) by pro-inflammatory molecules and pathways is a primary step in atherosclerosis development. We aimed to investigate the effects and mechanism of KLF14 on inflammatory responses in ECs. MethodsAdenovirus-mediated overexpression of human KLF14 and EC specific Klf14 knockout mice were applied to study the role of KLF14 in EC inflammation. Intravital microscopy was used to examine leukocyte-endothelial cell interactions in vivo. ResultsThe expression of Klf14 was markedly decreased in mouse aortic ECs in both acute and chronic inflammatory conditions. Overexpression of KLF14 inhibited inflammatory activation of human ECs stimulated by interleukin 1β and tumor necrosis factor α. Primary pulmonary ECs from Klf14 knockout mice showed increased expression of adhesion molecules under IL-1β stimuli. Mechanistically, KLF14 inhibited NF-κB signaling pathway by transcriptionally suppressing the expression of p65, resulting in significantly decreased leukocyte adhesion to activated ECs. Using intravital microscopy, an increased leukocyte-endothelial cell interaction was observed in endothelial specific Klf14 knockout mice compared to wild type control mice. Additionally, perhexiline, a KLF14 activator, induces KLF14 expression in ECs and reduced leukocyte-endothelial cell interactions in vitro and in vivo. ConclusionsThe data revealed that KLF14 inhibited the inflammatory response in ECs and the protective effects were mediated by transcriptional inhibition of NF-κB signaling pathway. Endothelial KLF14 could be a potential therapeutic target for cardiovascular diseases.

Obesity impairs leukocyte-endothelium cell interactions and oxidative stress in humans.

To evaluate the relationship between leukocyte-endothelial cell interactions and oxidative stress parameters in non-diabetic patients with different grades of obesity. For this cross-sectional study, 225 subjects were recruited from January 1, 2014 to December 31, 2016 and divided into groups according to BMI (<30kg/m2 , 30-40kg/m2 and >40kg/m²). We determined clinical parameters, systemic inflammatory markers, soluble cellular adhesion molecules, leukocyte-endothelium cell interactions-rolling flux, velocity and adhesion-, oxidative stress parameters-total ROS, total superoxide, glutathione-and mitochondrial membrane potential in leukocytes. We verified that HOMA-IR and hsCRP increased progressively as obesity developed, whereas A1c, IL6 and TNFα were augmented in the BMI>40kg/m² group. The cellular adhesion molecule sP-selectin was increased in patients with obesity, while sICAM, total ROS, total superoxide and mitochondrial membrane potential were selectively higher in the BMI>40kg/m² group. Obesity induced a progressive decrease in rolling velocity and an enhancement of rolling flux and leukocyte adhesion. Our findings reveal that endothelial dysfunction markers are altered in human obesity and are associated with proinflammatory cytokines and increased oxidative stress parameters.

Insulin Action on Endothelial Cells Limits Leukocyte-Endothelial Interaction through CXCR4 and Counteracts Intestinal Tumor Formation in Obesity

An activated, pro-inflammatory endothelium is a key feature of obesity and type 2 diabetes but little is known about its contribution to the increased cancer risk in these conditions. We recently published that insulin resistance in endothelial cells increases VCAM-1 expression, increases recruitment of tumor-associated neutrophils and promotes intestinal tumor formation. Here, we report that tumor-prone Apc(Min/+) mice have 2.1-fold more intestinal tumors during high-fat feeding. However, gain of insulin signaling specifically in endothelial cells by overexpression of IRS1 prevented 44% of tumors in diet-induced obesity. RNA sequencing in primary human endothelial cells identified 17 genes changed by insulin treatment with a log(2) change of at least ±0.5, among them C-X-C motif chemokine receptor 4 (CXCR4), which was downregulated by 54%. Downregulation of CXCR4 after insulin treatment was completely prevented by pretreatment with wortmannin but not by U0126, an inhibitor of MAPK signaling. Using FACS of enzymatically dissociated tissue, CXCR4 mRNA in CD31+ cells was 77% higher in mice with diet-induced obesity compared to lean controls and 37% higher in db/db mice compared to db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. CXCL12, the ligand for CXCR4, increased leukocyte adhesion to cultured endothelial cells and this was completely prevented by plerixafor, a clinically approved CXCR4 antagonist. In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling and adhesion after intravenous injection of CXCL12 and this change was abrogated in transgenic mice with endothelial overexpression of IRS1. We conclude that endothelial cell insulin signaling limit leukocyte-endothelial cell interaction through downregulation of CXCR4 and that improving insulin signaling in endothelial cells may protect against tumor development in obesity. Disclosure T. Rathjen: Employee; Self; Novo Nordisk A/S, Bayer AG. Q. Li: None. K. Park: None. G. Povlsen: Employee; Self; Novo Nordisk A/S. G.S. Olsen: Employee; Self; Novo Nordisk A/S. G.L. King: Research Support; Self; Sanofi-Aventis. C. Rask-Madsen: Research Support; Self; Novo Nordisk A/S.

Targeting the Microcirculation by Indole-3-carbinol and Its Main Derivate 3,3,'-diindolylmethane: Effects on Angiogenesis, Thrombosis and Inflammation

The pharmacological targeting of microcirculatory dysregulations is a therapeutic strategy for the treatment of numerous pathological conditions, such as cancer, thrombosis and inflammation. A promising candidate for this purpose is indole-3-carbinol (I3C), a phytochemical compound of cruciferous vegetables, and its main derivate 3,3,'-diindolylmethane (DIM). As summarized in this review, I3C and DIM affect multiple molecular and cellular processes within the microcirculation due to their pleiotropic action profile. These include angiogenesis, leukocyte-endothelial cell interaction, cytokine and reactive oxygen species (ROS) production, thrombus formation and microvascular leakage. Hence, I3C may serve as a lead compound for the future chemical synthesis of novel drugs that exert comparable beneficial effects while exhibiting an improved bioavailability.

Ronald W. Busuttil, MD, PhD: William P. Longmire, Jr, Chair of Surgery, Chief of Liver and Pancreas Transplantation, Director, UCLA-Dumont Transplant & Liver Cancer Center.

Ronald W. Busuttil, MD, PhD: William P. Longmire, Jr, Chair of Surgery, Chief of Liver and Pancreas Transplantation, Director, UCLA-Dumont Transplant & Liver Cancer Center.

Leukocyte-endothelial cell interaction is enhanced in podocalyxin-deficient mice.

The highly sialoglycosylated extracellular domain of podocalyxin (Podxl) is a constituent of the endothelial glycocalyx of most blood vessels but it is unknown if Podxl plays a prominent role in the function of the glycocalyx as a regulator of leukocyte-endothelial adhesion. We have recently found that mice lacking Podxl in the vascular endothelium develop histological lesions compatible with severe vasculitis resulting in organ failure and premature death. In this work, we show that these mice have an increased quantity of resident leukocytes within the peritoneal cavity in both basal and inflammatory conditions. Adhesion of macrophagic cells to lung endothelial cells from Podxl-deficient mice was increased under inflammatory stimuli. Both, chemokine binding and chemokine-mediated adhesion of immune cells were significantly higher in Podxl-deficient endothelial cells. Moreover, glycocalyx function assessed by measuring the anticoagulant capacity of endothelial cell monolayers to inactivate thrombin was significantly altered in the absence of Podxl. Overall, the results suggest that Podxl is an essential component of the glycocalyx and has an important so far unknown role in preventing leukocyte-endothelial cell adhesion under resting and inflammatory conditions.