Apical Surface Of Endothelial Cells Research Articles

Mechanisms of Formation of Antibodies against Blood Group Antigens That Do Not Exist in the Body.

The system of the four different human blood groups is based on the oligosaccharide antigens A or B, which are located on the surface of blood cells and other cells including endothelial cells, attached to the membrane proteins or lipids. After transfusion, the presence of these antigens on the apical surface of endothelial cells could induce an immunological reaction against the host. The final oligosaccharide sequence of AgA consists of Gal-GlcNAc-Gal (GalNAc)-Fuc. AgB contains Gal-GlcNAc-Gal (Gal)-Fuc. These antigens are synthesised in the Golgi complex (GC) using unique Golgi glycosylation enzymes (GGEs). People with AgA also synthesise antibodies against AgB (group A [II]). People with AgB synthesise antibodies against AgA (group B [III]). People expressing AgA together with AgB (group AB [IV]) do not have these antibodies, while people who do not express these antigens (group O [0; I]) synthesise antibodies against both antigens. Consequently, the antibodies are synthesised against antigens that apparently do not exist in the body. Here, we compared the prediction power of the main hypotheses explaining the formation of these antibodies, namely, the concept of natural antibodies, the gut bacteria-derived antibody hypothesis, and the antibodies formed as a result of glycosylation mistakes or de-sialylation of polysaccharide chains. We assume that when the GC is overloaded with lipids, other less specialised GGEs could make mistakes and synthesise the antigens of these blood groups. Alternatively, under these conditions, the chylomicrons formed in the enterocytes may, under this overload, linger in the post-Golgi compartment, which is temporarily connected to the endosomes. These compartments contain neuraminidases that can cleave off sialic acid, unmasking these blood antigens located below the acid and inducing the production of antibodies.

Endothelial Glycocalyx Preservation-Impact of Nutrition and Lifestyle.

The endothelial glycocalyx (eGC) is a dynamic hair-like layer expressed on the apical surface of endothelial cells throughout the vascular system. This layer serves as an endothelial cell gatekeeper by controlling the permeability and adhesion properties of endothelial cells, as well as by controlling vascular resistance through the mediation of vasodilation. Pathogenic destruction of the eGC could be linked to impaired vascular function, as well as several acute and chronic cardiovascular conditions. Defining the precise functions and mechanisms of the eGC is perhaps the limiting factor of the missing link in finding novel treatments for lifestyle-related diseases such as atherosclerosis, type 2 diabetes, hypertension, and metabolic syndrome. However, the relationship between diet, lifestyle, and the preservation of the eGC is an unexplored territory. This article provides an overview of the eGC's importance for health and disease and describes perspectives of nutritional therapy for the prevention of the eGC's pathogenic destruction. It is concluded that vitamin D and omega-3 fatty acid supplementation, as well as healthy dietary patterns such as the Mediterranean diet and the time management of eating, might show promise for preserving eGC health and, thus, the health of the cardiovascular system.

Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1–2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability.

Evaluation of endothelial glycocalyx in healthy volunteers - An observational study.

Endothelial glycocalyx (EG) is a carbohydrate-rich gel-like mesh covering the apical surface of endothelial cells. It has been linked to the microvascular pathophysiology and tissue metabolism. However, little is known about its condition in young healthy adults. We aimed to describe the condition of EG in young healthy adults by in vivo EG imaging and measurement of syndecan-1, a plasma marker of EG integrity in order to obtain reference values. For in vivo EG studies we used Side-stream Dark Field imaging of the sublingual microcirculation. Recordings were analysed automatically by GlycoCheck software providing the Perfused Boundary Region (PBR) as a marker of EG thickness. Levels of syndecan-1 were analysed in plasma samples by ELISA. 21 volunteers were included in the study. Median of the PBR value was 1.82 μm (interquartile range 1.69-2.01, 95% CI 1.79-1.97). Median concentration of syndecan-1 was 0.3 ng/ml (interquartile range 0.23-0.39, 95% CI 0.27-0.49). This study provides a comparison for cohorts of patients with a particular disease where the EG is presumably damaged. Our findings do not entirely comply with already published data in healthy individuals.

Abstract P087: Mitochondrial Reactive Oxygen Species Mediate Endothelial Glycocalyx Damage And Vascular Dysfunction In Hemorrhagic Shock And Resuscitation

The endothelial glycocalyx forms an anti-thrombotic layer on the apical surface of endothelial cells and maintains the selective permeability barrier of blood vessels. Ischemia reperfusion injury like hemorrhagic shock is shown to cause glycocalyx damage. We have previously shown that mitochondrial reactive oxygen species (mitoROS) mediate glycocalyx damage in cultured endothelial cells. Of note, angiotensin II elevates endothelial mitoROS, suggesting a possible exacerbation of glycocalyx damage in hypertensive patients. It is unknown, however, whether mitoROS mediate glycocalyx damage in vivo . We hypothesize that mitoROS mediate glycocalyx damage in a rat model of hemorrhagic shock.We investigated the effect of mitochondrial ROS on the endothelial glycocalyx in vivo , in a rat model of hemorrhagic shock. In anesthetized rats, mean arterial pressure was reduced to 40 mmHg by withdrawing blood from the femoral artery and kept at 40 mmHg for 30 minutes. The rats were then resuscitated with IV (jugular vein) Ringer’s lactate solution for 30 minutes more. Sham rats received the vascular lines only. Syndecan-1 in the plasma was increased after 30 minutes of resuscitation with LR in hemorrhage/resuscitation (H/R) rats (p=0.02) but was not elevated in Sham rats (p=0.52). Resuscitation with IV mitoROS scavenger mitoTEMPOL significantly blunted this increase (5.9 pg/ml vs. 8.7 pg/ml, p=0.03). At the organ level, the glycocalyx was decreased in the endothelium of muscle (p=0.0444) and intestine (P=5.47 * 10 -7 ) vascular beds in H/R rats vs. Sham rats. Our findings show that mitoROS mediate the glycocalyx damage after H/R. This mechanism suggests possible therapies that target mitoROS generation. Future work will investigate whether preexisting hypertension increases glycocalyx damage during H/R due to exacerbated mitoROS levels.

Effect of short-term administration of lipid emulsion on endothelial glycocalyx integrity in ICU patients - A microvascular and biochemical pilot study.

Endothelial glycocalyx (EG) is a carbohydrate-rich vascular lining of the apical surface of endothelial cells. It has been proved to have an essential role in vascular homeostasis. Lipid emulsions as part of parenteral nutrition (PN) are widely used in patients in the setting of critical care and perioperative medicine. Due to their structure, lipids may potentially interact with EG. The aim of the study was to evaluate the effect of lipid emulsion on EG. To assess the influence of lipid emulsion on EG integrity in ICU patients using a videomicroscopic and biochemical methods. Patients in surgical ICU after major abdominal surgery or cardio surgery and in general ICU were assessed for eligibility for this pilot observational study in University Hospital. The study was performed during the first day of adding lipids as a part of their PN. The patients were given the SMOFlipid 20% for 6 hours in prescribed dose of approx. 1 g/kg of body weight. EG integrity was measured indirectly by automated sublingual videomicroscopy calculating a parameter PBR which describes the amount of lateral deviation of red blood cells from the central column and by levels of syndecan-1 and syndecan-4 in plasma as EG degradational products. Measurements were performed before lipid administration (T0) and 30 minutes after (T6) the infusion of lipid emulsion was completed. The statistical analysis was performed at the level of significance p < 0.05, data are expressed as mean ± standard deviation (SD) and for PBR as median and interquartile range (IQR). Fifteen patients were studied, from them 9 included in final analysis. PBR (expressed in μm) increased after the lipid infusion with no statistical significance (T0 = 2.10; 1.97-2.33 vs. 2.28; 2.11-2.45, p = 0.13). At T6 both syndecans showed statistically significant decrease in their particular levels. Syndecan-1 at T0 = 2580±1013 ng/l, resp. at T6 = 2365±1077 ng/l, p = 0.02; syndecan-4 at T0 = 134±29 ng/l, resp. at T6 = 123±43 ng/l, p = 0.04. In our study, we showed that six hours long SMOFlipid 20% infusion had no detrimental effect on the EG integrity as assessed by PBR value and by syndecan-1 and syndecan-4 plasmatic levels. Observed decrease of syndecans shortly after lipid infusion allows us to hypothesize even possibly protecting effect of lipids on EG.

Hyaluronidase2 (Hyal2) modulates low shear stress-induced glycocalyx impairment via the LKB1/AMPK/NADPH oxidase-dependent pathway.

The endothelium glycocalyx layer (ECL), presents on the apical surface of endothelial cells, creates a barrier between circulating blood and the vessel wall. Low shear stress (LSS) may accelerate the degradation of the glycocalyx via hyaluronidase2 (Hyal2) and then alter the cell polarity. Yet the liver kinase B1 (LKB1) signaling pathway plays an important role in regulating cell polarity. However, the relationship between LKB1 and glycocalyx during LSS is not clear. In the current study, we demonstrate that LSS attenuates LKB1 and AMP-activated protein kinase activation as well as activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47phox ) and Hyal2 in the human umbilical vein endothelial cell (HUVEC). Pretreatment with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside(AICAR), or diphenyleneiodonium(DPI chloride) and transfection with LKB1 overexpression vector and p47phox small interfering RNA downregulated LSS-induced Hyal2 activation. By coimmunoprecipitation, we discovered the existence of p47phox /Hyal2 complex. LSS induced the dissociation of p47phox /Hyal2 complex, which was inhibited by LKB1 overexpression and AICAR. Furthermore, knockdown of Hyal2 performed a positive feedback on LKB1 activity. In addition, we also show that LSS enhanced LKB1 translocation from the cytosol to the nucleus. Taken together, these data indicate that Hyal2 regulates LSS-induced injury of the glycocalyx via LKB1/AMPK/NADPH oxidase signaling cascades.

Alpha-1 Antitrypsin Transcytosis and Secretion.

Protective levels of Alpha-1 antitrypsin (A1AT) are achieved in the lung through the uptake of the pulmonary vasculature of hepatocyte-secreted A1AT. The anti-inflammatory, anti-apoptotic, and anti-protease properties of A1AT are critical toward maintaining the function of pulmonary endothelial and epithelial cells and for the structural integrity of the pulmonary interstitium. To perform these functions A1AT must cross the pulmonary-endothelial barrier. Using transwell inserts, we have demonstrated that the endocytosis of A1AT at the apical surface of endothelial cells, followed by the transcytosis and secretion at the basolateral surface, is a mechanism through which A1AT is transported into the lung epithelium and interstitium.

Similarities and differences in the regulation of leukocyte extravasation and vascular permeability

Leukocyte extravasation is regulated and mediated by a multitude of adhesion and signaling molecules. Many of them enable the capturing and docking of leukocytes to the vessel wall. Others allow leukocytes to crawl on the apical surface of endothelial cells to appropriate sites of exit. While these steps are well understood and the adhesion molecules mediating these interactions are largely identified, a still growing number of adhesion receptors mediate the diapedesis process, the actual migration of leukocytes through the endothelial cell layer, and the underlying basement membrane. In most cases, it is not known which molecular processes they actually mediate, whether they enable the migration of leukocytes through the endothelial cell layer or whether they are involved in the destabilization of endothelial junctions. In addition, leukocytes are able to circumvent junctions and transcytose directly through the body of endothelial cells. While this latter route indeed exists, recent work has highlighted in vivo the junctional pathway as the prevalent way of leukocyte exit in various inflamed tissues. Recent work elucidating molecular mechanisms that regulate endothelial junctions and thereby leukocyte extravasation and vascular permeability will be discussed.

Targeting Thrombomodulin to Endothelial ICAM-1 Rather Than PECAM-1 Allows Partnering with the Endothelial Protein C Receptor and Enhances Protection of Endothelial Barrier Function

Abstract 3351The thrombomodulin-protein C pathway has important antithrombotic and anti-inflammatory roles in a variety of disease models and human illnesses, including severe sepsis, acute lung injury, and focal ischemia & reperfusion. We recently reported that anchoring recombinant thrombomodulin (TM) to PECAM-1 on the luminal surface of the vascular endothelium is protective in a mouse model of inflammatory lung injury. To this point, it has been unclear whether targeted thrombomodulin is able to partner with the endothelial protein C receptor (EPCR) in the same way as its endogenous counterpart. Here we demonstrate that anchoring TM to endothelial ICAM-1, rather than PECAM-1, results in approximately 10-fold greater activation of protein C. Furthermore, blocking protein C binding to EPCR results in marked reduction of protein C activation when TM is targeted to ICAM-1, whereas protein C activation is largely independent of EPCR when PECAM-1 is used as the target ligand. Consistent with this in vitro observation, anti-ICAM/TM fusion protein provides greater in vivo protection in a mouse model of inflammatory lung injury than its PECAM-targeted analogue, more potently blocking expression of pro-inflammatory cytokines and more effectively stabilizing endothelial barrier function. Since ICAM, endogenous TM, and EPCR are all thought to localize to microdomains on the apical surface of endothelial cells, we hypothesize that ICAM-targeting more effectively mimics the natural configuration and brings TM within sufficient proximity to allow access to its membrane co-factor. These observations could have profound implications for the therapeutic efficacy of a whole series of endothelial-targeted proto-drugs and their potential for translational success. Disclosures:No relevant conflicts of interest to declare.

Hemodynamic shear stress characteristic of atherosclerosis-resistant regions promotes glycocalyx formation in cultured endothelial cells

The endothelial glycocalyx, a glycosaminoglycan layer located on the apical surface of vascular endothelial cells, has been shown to be important for several endothelial functions. Previous studies have documented that the glycocalyx is highly abundant in the mouse common carotid region, where the endothelium is exposed to laminar shear stress, and it is resistant to atherosclerosis. In contrast, the glycocalyx is scarce or absent in the mouse internal carotid sinus region, an area exposed to nonlaminar shear stress and highly susceptible to atherosclerosis. On the basis of these observations, we hypothesized that the expression of components of the endothelial glycocalyx is differentially regulated by distinct hemodynamic environments. To test this hypothesis, human endothelial cells were exposed to shear stress waveforms characteristic of atherosclerosis-resistant or atherosclerosis-susceptible regions of the human carotid, and the expression of several components of the glycocalyx was assessed. These experiments revealed that expression of several components of the endothelial glycocalyx is differentially regulated by distinct shear stress waveforms. Interestingly, we found that heparan sulfate expression is increased and evenly distributed on the apical surface of endothelial cells exposed to the atheroprotective waveform and is irregularly present in cells exposed to the atheroprone waveform. Furthermore, expression of a heparan sulfate proteoglycan, syndecan-1, is also differentially regulated by the two waveforms, and its suppression mutes the atheroprotective flow-induced cell surface expression of heparan sulfate. Collectively, these data link distinct hemodynamic environments to the differential expression of critical components of the endothelial glycocalyx.

Assessing the Role of the Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) Three-finger Domain in Binding Lipoprotein Lipase

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial cell protein that transports lipoprotein lipase (LPL) from the subendothelial spaces to the capillary lumen. GPIHBP1 contains two main structural motifs, an amino-terminal acidic domain enriched in aspartates and glutamates and a lymphocyte antigen 6 (Ly6) motif containing 10 cysteines. All of the cysteines in the Ly6 domain are disulfide-bonded, causing the protein to assume a three-fingered structure. The acidic domain of GPIHBP1 is known to be important for LPL binding, but the involvement of the Ly6 domain in LPL binding requires further study. To assess the importance of the Ly6 domain, we created a series of GPIHBP1 mutants in which each residue of the Ly6 domain was changed to alanine. The mutant proteins were expressed in Chinese hamster ovary (CHO) cells, and their expression level on the cell surface and their ability to bind LPL were assessed with an immunofluorescence microscopy assay and a Western blot assay. We identified 12 amino acids within GPIHBP1, aside from the conserved cysteines, that are important for LPL binding; nine of those were clustered in finger 2 of the GPIHBP1 three-fingered motif. The defective GPIHBP1 proteins also lacked the ability to transport LPL from the basolateral to the apical surface of endothelial cells. Our studies demonstrate that the Ly6 domain of GPIHBP1 is important for the ability of GPIHBP1 to bind and transport LPL.

Cyclic Force Applied to FAs Induces Actin Recruitment Depending on the Dynamic Loading Pattern

Mechanical forces acting on focal adhesions (FAs) are believed to be an important determinant for cytoskeletal reorganization. However, the effect of the temporal pattern of forces on cellular responses has not been elucidated. In the present study, we examined the responses of FAs to locally-applied cyclic forces. Magnetic micro beads coated with fibronectin were attached to the apical surface of endothelial cells and continuous or cyclic forces at frequencies of 0.1-10 Hz with duty cycles of 0-100% were applied to the beads using a newly developed electromagnetic tweezer. A significant increase in actin recruitment around the beads was observed when cyclic forces at 1-2 Hz and 25-50% duty cycles were applied. This tendency disappeared upon modification of myosin activity. These results indicate that the sensitivity to temporal patterns of forces is detemined by the viscoelastic properes of FAs and depends on myosin activity.

Inside-out regulation of ICAM-1 dynamics in TNF-alpha-activated endothelium.

BackgroundDuring transendothelial migration, leukocytes use adhesion molecules, such as ICAM-1, to adhere to the endothelium. ICAM-1 is a dynamic molecule that is localized in the apical membrane of the endothelium and clusters upon binding to leukocytes. However, not much is known about the regulation of ICAM-1 clustering and whether membrane dynamics are linked to the ability of ICAM-1 to cluster and bind leukocyte integrins. Therefore, we studied the dynamics of endothelial ICAM-1 under non-clustered and clustered conditions.Principal FindingsDetailed scanning electron and fluorescent microscopy showed that the apical surface of endothelial cells constitutively forms small filopodia-like protrusions that are positive for ICAM-1 and freely move within the lateral plane of the membrane. Clustering of ICAM-1, using anti-ICAM-1 antibody-coated beads, efficiently and rapidly recruits ICAM-1. Using fluorescence recovery after photo-bleaching (FRAP), we found that clustering increased the immobile fraction of ICAM-1, compared to non-clustered ICAM-1. This shift required the intracellular portion of ICAM-1. Moreover, biochemical assays showed that ICAM-1 clustering recruited beta-actin and filamin. Cytochalasin B, which interferes with actin polymerization, delayed the clustering of ICAM-1. In addition, we could show that cytochalasin B decreased the immobile fraction of clustered ICAM-1-GFP, but had no effect on non-clustered ICAM-1. Also, the motor protein myosin-II is recruited to ICAM-1 adhesion sites and its inhibition increased the immobile fraction of both non-clustered and clustered ICAM-1. Finally, blocking Rac1 activation, the formation of lipid rafts, myosin-II activity or actin polymerization, but not Src, reduced the adhesive function of ICAM-1, tested under physiological flow conditions.ConclusionsTogether, these findings indicate that ICAM-1 clustering is regulated in an inside-out fashion through the actin cytoskeleton. Overall, these data indicate that signaling events within the endothelium are required for efficient ICAM-1-mediated leukocyte adhesion.

Measurements of strain on single stress fibers in living endothelial cells induced by fluid shear stress

Fluid shear stress (FSS) acting on the apical surface of endothelial cells (ECs) can be sensed by mechano-sensors in adhesive protein complexes found in focal adhesions and intercellular junctions. This sensing occurs via force transmission through cytoskeletal networks. This study quantitatively evaluated the force transmitted through cytoskeletons to the mechano-sensors by measuring the FSS-induced strain on SFs using live-cell imaging for actin stress fibers (SFs). FSS-induced bending of SFs caused the SFs to align perpendicular to the direction of the flow. In addition, the displacement vectors of the SFs were detected using image correlation and the FSS-induced axial strain of the SFs was calculated. The results indicated that FSS-induced strain on SFs spanned the range 0.01–0.1% at FSSs ranging from 2 to 10 Pa. Together with the tensile property of SFs reported in a previous study, the force exerted on SFs was estimated to range from several to several tens of pN.

Role of stress fibers and focal adhesions as a mediator for mechano-signal transduction in endothelial cells in situ.

Fluid shear stress is the mechanical force generated by the blood flow which is applied over the apical surface of endothelial cells in situ. The findings of a recent study suggest that stress fibers and its associated focal adhesions play roles in mechano-signal transduction mechanism. Stress fibers are present along the apical and the basal portion of the endothelial cells. Endothelial cells respond to fluid shear stress and change their morphological characteristics in both their cell shape and cytoskeletal organization. Atherosclerosis is a common disease of the arteries and it occurs in areas around the branching site of blood vessels where the cells are exposed to low fluid shear stress. The organization of stress fibers and focal adhesions are strongly influenced by shear stress, and therefore the generation of atherosclerotic lesions seem to be associated with the cytoskeletal components of endothelial cells. This review describes the possible role of the cytoskeleton as a mechano-transducer in endothelial cells in situ.

Erratum

Immunological ReviewsVolume 219, Issue 1 p. 205-205 Free Access Erratum This article corrects the following: Functional insights on the polarized redistribution of leukocyte integrins and their ligands during leukocyte migration and immune interactions Olga Barreiro, Hortensia De La Fuente, María Mittelbrunn, Francisco Sánchez-Madrid, Volume 218Issue 1Immunological Reviews pages: 147-164 First Published online: July 9, 2007 First published: 10 September 2007 https://doi.org/10.1111/j.1600-065X.2007.00569.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Barreiro O, de la Fuente H, Mittelbrunn M, Sánchez-Madrid F. Functional insights on the polarized redistribution of leukocyte integrins and their ligands during leukocyte migration and immune interactions. Immunol Rev 2007;218:147–164. In [1] a typographical error occurred. The sentence on page 152, column 2, lines 8 – 14 should have read: The existence is feasible of tetraspaninenriched adhesive platforms at the apical surface of endothelial cells, containing VCAM-1 and ICAM-1, and a variable proportion of most of the receptors involved in the adhesion and transmigration of leukocytes, including CD44 and plateletendothelial cell adhesion molecule-1 (PECAM-1) (O. Barreiro et al., unpublished data). We apologize for this error. Reference 1 Barreiro O, De La Fuente H, Mittelbrunn M, Sánchez-Madrid F. Functional insights on the polarized redistribution of leukocyte integrins and their ligands during leukocyte migration and immune interactions. Immunol Rev 2007; 218: 147– 164. Volume219, Issue1October 2007Pages 205-205 ReferencesRelatedInformation

Stanniocalcin-1 regulates endothelial gene expression and modulates transendothelial migration of leukocytes

The mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) inhibits monocyte chemotactic protein-1- and stromal-derived factor-1alpha (SDF-1alpha)-mediated chemotaxis and diminishes chemokinesis in macrophage-like RAW264.7 and U937 cells in a manner that may involve attenuation of the intracellular calcium signal. STC1 is strongly induced in the kidney following obstructive injury. We hypothesized that STC1 may serve to attenuate the influx of inflammatory cells to the site of tissue injury. In this study, we examined the effect of STC1 on the migration of freshly isolated human macrophages, neutrophils, and T and B lymphocytes through quiescent or IL-1beta-treated human umbilical vein endothelial cell (HUVEC) monolayers. STC1 inhibited transmigration of macrophages and T lymphocytes through quiescent or IL-1beta-activated HUVECs but did not attenuate the transmigration of neutrophils and B lymphocytes. STC1 regulates gene expression in cultured endothelial cells and is detected on the apical surface of endothelial cells in vivo. The data suggest that STC1 plays a critical role in transendothelial migration of inflammatory cells and is involved in the regulation of numerous aspects of endothelial function.

Endothelial inflammation: the role of differential expression of N-deacetylase/N-sulphotransferase enzymes in alteration of the immunological properties of heparan sulphate.

Heparan sulphate N-deacetylase/N-sulphotransferase (NDST) enzymes catalyse the reaction that initiates sulphation and subsequent modification of the oligosaccharide, heparan sulphate (HS). The extent and distribution of sulphate substitution on HS plays a vital role in regulation of the binding of a range of proteins, including IFN-gamma, several interleukins and most chemokines. In this study, the expression of NDST transcripts was found to be non-uniform between a range of cell types, suggesting that different cells produce characteristic HS species. It was found that stimulation of the HMEC-1 microvascular endothelial cell line with the pro-inflammatory cytokines IFN-gamma and TNF-alpha caused a transient decrease in the level of NDST-1 and -2 transcripts after 4 hours (P < 0.05 and P < 0.01 respectively), but the expression of NDST-1 increased above control levels after 16 hours (P < 0.01). The change in NDST expression was concurrent with an increase in the abundance of sulphated HS epitopes on the cell surface; this was not caused by variation in the expression of proteoglycans or by changes in the rate of GAG turnover. Cytokine-stimulated endothelial cells also showed an increase in their potential to bind RANTES (CCL5); this was abrogated by chlorate blockade of sulphotransferase activity or by heparitinase cleavage of cell surface HS. Monolayers of cytokine-stimulated HMEC-1 also supported an enhanced leukocyte chemotactic response towards RANTES. This study demonstrated that pro-inflammatory cytokines can increase NDST expression leading to increased sulphation of HS and a corresponding increase in sequestration of functional RANTES at the apical surface of endothelial cells. This may enhance leukocyte extravasation at sites of inflammation.

Regulation of endothelial cell and platelet receptor-ligand binding by the 12- and 15-lipoxygenase monohydroxides, 12-, 15-HETE and 13-HODE

In previous studies, we reported that vascular wall cells such as endothelial cells metabolize linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) via the 15-lipoxygenase pathway. Endothelial cell 13-HODE levels vary inversely with endothelial cell reactivity to platelets, which, in turn, varies directly with the expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells. We and others have also found that tumour cell adhesivity is dependent, in part, upon the relative amounts of intracellular 13-HODE and the arachidonic acid monohydroxide(s), 12- and/or 15-hydroxyeicosatetraenoic acids (12-, 15-HETE). In addition, we and others have found that platelet adhesivity is dependent upon the intraplatelet level of its major lipoxygenase metabolite, 12-HETE. Finally, we have demonstrated that 13-HODE and VnR co-localize in nonadhesive endothelial cells but dissociate following endothelial cell injury, at which time, the VnR relocates on the endothelial cell apical surface. These data suggest to us that lipoxygenase-derived monohydroxides regulate the ability of various receptors to recognize their specific ligands. The latter data also suggest that these monohydroxides act directly by a physiochemical mechanism. The present study supports this possibility. Thus, we demonstrate that 13-HODE downregulates VnR binding with vitronectin (Vn) > fibronectin (Fn) > fibrinogen (Fgn), whereas 12- and 15-HETE upregulate specific VnR/ligand binding, using purified VnR/liposomes and purified ligands in an adhesion assay; and that 12- and 15-HETE upregulate GPIIb/IIIa:liposome binding of Fgn > Fn > Vn. We conclude that cell-specific monohydroxides influence cell-specific receptor-ligand binding directly through a physiochemical mechanism.