Luminal Endothelial Surface Research Articles

CXCL1 promotes arteriogenesis through enhanced monocyte recruitment into the peri-collateral space.

The mechanisms of monocyte recruitment to arteriogenic collaterals are largely unknown. We investigated the role of chemokine (C-X-C-motif) ligand 1 (CXCL1) and its cognate receptor, chemokine (C-X-C-motif) receptor 2 (CXCR2) in arteriogenesis. After femoral artery ligation in Sprague-Dawley rats, either native collaterals were harvested or placebo, CXCL1 or CXCR2 blocker was administered via an osmopump. Perfusion recovery was measured with Laser Doppler, leukocyte populations were analyzed by fluorescence-activated cell sorting, and hind limb sections were stained for macrophage marker cluster of differentiation 68 (CD68). In vitro, fluorescent CXCL1 or human acute monocytic leukemia cell line (THP-1) monocytic cells were flown over shear-stressed endothelium. CXCL1 mRNA expression in collaterals was dramatically upregulated already 1 h after ligation (ratio ligated/sham 5.73). CD68 mRNA was upregulated from 12 h until 3 days after ligation (peak ratio ligated/sham 2.65). CXCL1 treatment augmented perfusion recovery at 3 and 7 days (p < 0.05) after ligation, and a significant increase in the number of peri-collateral macrophages was evident concomitantly (p < 0.05). Conversely, CXCR2 antagonist treatment caused a decrease in perfusion recovery both at 7 and 10 days postligation (p = 0.01) and also significantly reduced the number of peri-collateral macrophages (p < 0.05). In vitro, CXCL1 tethered to and was taken up by endothelial cells under shear stress conditions and enhanced THP-1 adherence compared to control (p < 0.05). In contrast, CXCR2 antagonist compromised THP-1 adherence to endothelial cells (p < 0.05). CXCL1 presented on the luminal endothelial surface leads to an increase in the number of peri-collateral macrophages, thus improving the arteriogenic response after arterial ligation.

Nanoparticles for imaging: top or flop?

Nanoparticles are frequently suggested as diagnostic agents. However, except for iron oxide nanoparticles, diagnostic nanoparticles have been barely incorporated into clinical use so far. This is predominantly due to difficulties in achieving acceptable pharmacokinetic properties and reproducible particle uniformity as well as to concerns about toxicity, biodegradation, and elimination. Reasonable indications for the clinical utilization of nanoparticles should consider their biologic behavior. For example, many nanoparticles are taken up by macrophages and accumulate in macrophage-rich tissues. Thus, they can be used to provide contrast in liver, spleen, lymph nodes, and inflammatory lesions (eg, atherosclerotic plaques). Furthermore, cells can be efficiently labeled with nanoparticles, enabling the localization of implanted (stem) cells and tissue-engineered grafts as well as in vivo migration studies of cells. The potential of using nanoparticles for molecular imaging is compromised because their pharmacokinetic properties are difficult to control. Ideal targets for nanoparticles are localized on the endothelial luminal surface, whereas targeted nanoparticle delivery to extravascular structures is often limited and difficult to separate from an underlying enhanced permeability and retention (EPR) effect. The majority of clinically used nanoparticle-based drug delivery systems are based on the EPR effect, and, for their more personalized use, imaging markers can be incorporated to monitor biodistribution, target site accumulation, drug release, and treatment efficacy. In conclusion, although nanoparticles are not always the right choice for molecular imaging (because smaller or larger molecules might provide more specific information), there are other diagnostic and theranostic applications for which nanoparticles hold substantial clinical potential.

Thrombomodulin regulation in human brain microvascular endothelial cells in vitro: Role of cytokines and shear stress

Thrombomodulin (TM), an important determinant of blood vessel homeostasis, is expressed on the luminal vascular endothelial cell surface and is released into serum in response to circulatory signals. This includes the cerebrovascular endothelium, where the anti-coagulant and anti-inflammatory properties of TM are thought to be critical to the brain microcirculation and blood–brain barrier (BBB) integrity. Much is still unknown however about how circulatory stimuli may regulate TM activity within the brain microvasculature. To address this, the current short paper investigated the effects of opposing regulatory signals, namely cytokines (TNF-α, IL-6) and laminar shear stress, on the cellular levels and release of TM in cultured human brain microvascular endothelial cells (HBMvECs). Treatment of confluent HBMvECs with either TNF-α or IL-6 (100ng/ml, 18h) reduced TM protein levels by up to 70%, whilst inducing TM release into media by up to 4.4 and 5.5 fold, respectively. The effects of either cytokine (0–100ng/ml) on TM protein levels (6 or 18h) and release (0–18h) were also found to be concentration- and time-dependent. Either cytokine (100ng/ml, 24–72h) also reduced TM mRNA levels by >50%. When exposed to laminar shear stress for 24h at 8dyn/cm2 (SI unit equivalent=0.8Pa), TM protein levels were upregulated by 65% in parallel with a 2-fold increase in TM mRNA levels. Shear stress also proved to be a much more potent stimulus for TM release from HBMvECs, yielding media TM levels of 1000pg/105 cells, when compared to 175 and 210pg/105 cells for TNF-α and IL-6, respectively, after parallel 18h treatments. Finally, shear-conditioned media was found to completely block thrombin-induced permeabilization of HBMvECs, confirming the functional efficacy of released TM. In summary, our data indicate that TM is differentially regulated within cultured HBMvECs by humoral and biomechanical signals.

Medical significance of endothelial glycocalyx. Part 2: Its role in vascular diseases and in diabetic complications

Endothelial glycocalyx is a layer composed by glycosaminoglycans, proteoglycans and glycoproteins attached to the vascular endothelial luminal surface. Shredding of glycocalyx appears as an essential initial step in the pathophysiology of atherosclerosis and microangiopathic complications of diabetes mellitus, as well as in chronic venous disease. Atherosclerosis risk factors, as hypercholesterolemia (LDL), hyperglycemia, inflammation, salt excess and altered shear stress can damage glycocalyx. This lead to endothelial dysfunction and allows LDL and leukocytes to filtrate to the subendothelial space initiating atheroma plaque formation. Degradation of glycocalyx in diabetes mellitus is mainly due to oxidative stress and enables protein filtration (albuminuria) and endothelial disorder of microangiopathy. Chronic venous hypertension brings to altered shears stress which results in shredded glycocalyx, this allows leukocytes to migrate into venous wall and initiate inflammation leading to morphologic and functional venous changes of the chronic venous disease. Treatment with glycosaminoglycans (sulodexide) prevents or recovers the damaged glycocalyx and several of its consequences. This drug improves chronic venous disease and promotes healing of chronic venous ulcers. It has also been useful in peripheral arterial obstructive disease and in diabetic nephropathy with albuminuria.

EphrinB-mediated reverse signalling controls junctional integrity and pro-inflammatory differentiation of endothelial cells

The EphB/ephrinB receptor-ligand system is pivotal for the development of the embryonic vasculature and for angiogenesis in the adult organism. We observed that (i) the expression of ephrinB2 and ephrinB1 is up-regulated in capillaries during inflammation, that (ii) these ligands are localised on the luminal endothelial surface, and that (iii) they interact with the ephrinB-receptor EphB2 on monocyte/macrophages. This study delineates the impact of ephrinB-mediated reverse signalling on the integrity and proinflammatory differentiation of the endothelium. To this end, in vitro analyses with human cultured endothelial cells reveal that knockdown of ephrinB2 or ephrinB1 impairs monocyte transmigration through the endothelium. While ephrinB2 but not ephrinB1 interacts with PECAM-1 (CD31) in this context, reverse signalling by ephrinB1 but not ephrinB2 elicits a c-JunN-terminal kinase (JNK)-dependent up-regulation of E-selectin expression. Furthermore, treatment of endothelial cells with soluble EphB2 receptor bodies or EphB2-overexpressing mouse myeloma cells links ephrinB2 to PECAM-1 and induces its Src-dependent phosphorylation while diminishing Src homology phosphotyrosyl phosphatase-2 (SHP-2) activity and increasing endothelial cell permeability. We conclude that extravasation of EphB2 positive leukocyte populations is facilitated by lowering the integrity of endothelial cell junctions and enhancing the pro-inflammatory phenotype of the endothelium through activation of ephrinB ligands.

G protein-coupled receptors mediate coronary flow- and agonist-induced responses via lectin-oligosaccharide interactions

Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the following. 1) Two-dimensional SDS-PAGE (2D-SDS-PAGE) of the LESL lectinic fraction revealed at least 167 Ls. 2) Among these Ls, we identified three selectins and the GPCRs: angiotensin II, bradykinin (B2-R), adenosine A1 and A2, prolactin, endothelin, α1-adrenergic (α1A-R), thromboxane A2, β1-adrenergic, β3-adrenergic, and insulin receptors; the first six GPCRs are known to be flow sensitive. 3) The amplitude of receptor-induced vascular responses by α1A-R and B2-R activation (phenylephrine or bradykinin, respectively) was a function of flow and O (hyaluronidate). Our results support a novel mechanism of GPCR-mediated responses to flow via L·O interaction.

Nanomechanics of the Endothelial Glycocalyx in Experimental Sepsis

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal side of the endothelium, regulates vascular adhesiveness and permeability. Although central to the pathophysiology of vascular barrier dysfunction in sepsis, glycocalyx damage has been generally understudied, in part because of the aberrancy of in vitro preparations and its degradation during tissue handling. The aim of this study was to analyze inflammation-induced damage of the eGC on living endothelial cells by atomic-force microscopy (AFM) nanoindentation technique. AFM revealed the existence of a mature eGC on the luminal endothelial surface of freshly isolated rodent aorta preparations ex vivo, as well as on cultured human pulmonary microvascular endothelial cells (HPMEC) in vitro. AFM detected a marked reduction in glycocalyx thickness (266 ± 12 vs. 137 ± 17 nm, P<0.0001) and stiffness (0.34 ± 0.03 vs. 0.21 ± 0.01 pN/mn, P<0.0001) in septic mice (1 mg E. coli lipopolysaccharides (LPS)/kg BW i.p.) compared to controls. Corresponding in vitro experiments revealed that sepsis-associated mediators, such as thrombin, LPS or Tumor Necrosis Factor-α alone were sufficient to rapidly decrease eGC thickness (-50%, all P<0.0001) and stiffness (-20% P<0.0001) on HPMEC. In summary, AFM nanoindentation is a promising novel approach to uncover mechanisms involved in deterioration and refurbishment of the eGC in sepsis.

Importancia médica del glucocáliz endotelial

Endothelial glycocalyx is a layer composed by glycosaminoglycans, proteoglycans and glycoproteins attached to the vascular endothelial luminal surface. It has several physiological roles: shear stress mechanotransduction to the endothelial cells, regulation of fluids and macromolecules vascular permeability, of coagulation cascade activation and fibrinolysis, and protects the endothelium from platelets and leukocytes adhesion. In general, glycocalyx protects vascular wall against pathogenic insults. The glycocalyx may be damaged by abnormal shear stress, reactive oxygen species, hypernatremia, hyperglycemia, hypercholesterolemia and inflammatory molecules, resulting in endothelial dysfunction, enhanced vascular permeability, lipoproteins leakage to subendothelial space, activation of plasma coagulation, and increased adherence of platelets and leukocytes to the endothelial cells. Shredding of glycocalyx appears as an important initial step in the pathophysiology of vascular diseases.

Ca2+‐activated K+ channels of small and intermediate conductance control the activation of endothelial cell connexin‐ and pannexin‐formed hemichannels

Nitric Oxide (NO), Ca2+‐activated K+ channels (KCa) of small (SKCa) and intermediate (IKCa) conductance and hemichannels formed by connexins (Cx‐HC) or pannexins (Panx‐HC) are involved in the control of vasomotor tone. However, the mechanisms of Cx‐ and Panx‐based hemichannel activation in endothelial cells are unknown. In mesenteric vessels, we evaluated the expression and activity of Cx‐HC and Panx‐HC by immunofluorescence, biotinylation of membrane proteins of endothelial cell luminal surface and ethidium uptake. Cx37, Cx40, Cx43 and Panx‐1 were detected in endothelial cells. Stimulation with acetylcholine (ACh) or the NO‐donor S‐nitroso‐N‐acetyl‐D‐penicillamine (SNAP) increased ethidium uptake in endothelial cells, which was abolished by the NO synthase inhibitor NG‐nitro‐L‐arginine (L‐NA) or by the Panx‐HC blockers probenecid and Panx10 and attenuated by the Cx‐HC blocker La3+. In addition, ACh‐activated ethidium uptake was also abolished by blockade of KCa with tetraethylammonium (TEA) or IKCa with charybdotoxin (ChTX) or triarylmethane‐34(TRAM‐34), but only attenuated by the SKCa blocker apamin. TEA did not affect the SNAP‐activated ethidium uptake. Treatment with TEA, ChTX or apamin also inhibits the ACh‐induced NO production. These results indicate that SKCa and IKCa regulate the activity of Cx‐HC and Panx‐HC through the modulation of NO production.FONDECYT 1100850, ANILLOS ACT‐71

Correlative evaluation of variable pressure-scanning electron microscopy to histology methods for vascular tissues

For pathological investigation, vascular tissues implanted with biomaterials or medical devices require microscopic histology and scanning electron microscopy (SEM) assessment to evaluate biocompatibility with the vascular luminal surface. However, these valuable specimens are often susceptible to SEM and histology tissue preparation artifacts. Correlative variable pressure-scanning electron microscopy (VP-SEM) and histology methods were developed and evaluated, using current SEM technology, allowing direct imaging of hydrated samples without lengthy tissue processing, labor, equipment, and their sequela artifacts. Unstained and silver-stained, fixed swine carotid arteries were assessed using a cooling stage and an environmental secondary electron detector. After VP-SEM imaging, arteries were processed for histology evaluation. Assessment of luminal endothelial surface SEM images and tissue histology of the underlying mural wall was made to determine if low vacuum, cryo-freezing, electron beam exposure, and/or staining affected tissue morphology. Conventional SEM requires lengthy tissue processing with dedicated equipment for solvent dehydration, critical point drying, and metallic sputter coating in order to image under high vacuum for secondary electron imaging. This process causes notable artifacts when samples are later processed for microscopic histology. In contrast, silver staining of swine carotid arteries, followed by aldehyde fixation, facilitated VP-SEM imaging and histology preparation with results that were comparable to conventional SEM and had acceptable tissue morphology. VP-SEM with a cooling stage and an environmental secondary electron detector allows efficient SEM imaging and correlative histological assessment of vascular tissue samples. This correlative method may be further developed and implemented for research applications with biomaterial and medical device implanted tissues.

Duffy antigen receptor for chemokines and its involvement in patterning and control of inflammatory chemokines.

Leukocyte functions are linked to their migratory responses, which, in turn, are largely determined by the expression profile of classical chemokine receptors. Upon binding their cognate chemokines, these G-protein-coupled receptors (GPCRs) initiate signaling cascades and downstream molecular and cellular responses, including integrin activation and cell locomotion. Chemokines also bind to an alternative subset of chemokine receptors, which have serpentine structure characteristic for GPCRs but lack DRYLAIV consensus motive required for coupling to G-proteins. Duffy antigen receptor for chemokines (DARC) is a member of this atypical receptor subfamily. DARC binds a broad range of inflammatory CXC and CC chemokines and is expressed by erythrocytes, venular endothelial cells, and cerebellar neurons. Erythrocyte DARC serves as blood reservoir of cognate chemokines but also as a chemokine sink, buffering potential surges in plasma chemokine levels. Endothelial cell DARC internalizes chemokines on the basolateral cell surface resulting in subsequent transcytosis of chemokines and their immobilization on the tips of apical microvilli. These DARC-mediated endothelial cell interactions allow chemokines produced in the extravascular tissues to optimally function as arrest chemokines on the luminal endothelial cell surface.

Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization

Changes in blood flow regulate gene expression and protein synthesis in vascular endothelial cells, and this regulation is involved in the development of atherosclerosis. How mechanical stimuli are transmitted from the endothelial luminal surface to the nucleus is incompletely understood. The linker of nucleus and cytoskeleton (LINC) complexes have been proposed as part of a continuous physical link between the plasma membrane and subnuclear structures. LINC proteins nesprin-1, -2, and -4 have been shown to mediate nuclear positioning via microtubule motors and actin. Although nesprin-3 connects intermediate filaments to the nucleus, no functional consequences of nesprin-3 mutations on cellular processes have been described. Here we show that nesprin-3 is robustly expressed in human aortic endothelial cells (HAECs) and localizes to the nuclear envelope. Nesprin-3 regulates HAEC morpho-logy, with nesprin-3 knockdown inducing prominent cellular elongation. Nesprin-3 also organizes perinuclear cytoskeletal organization and is required to attach the centrosome to the nuclear envelope. Finally, nesprin-3 is required for flow-induced polarization of the centrosome and flow-induced migration in HAECs. These results represent the most complete description to date of nesprin-3 function and suggest that nesprin-3 regulates vascular endothelial cell shape, perinuclear cytoskeletal architecture, and important aspects of flow-mediated mechanotransduction.

Bioengineering of a Semiautologous Arterial Vessels with Reconstructed Media and Intima, Longtime Tested In Vivo

AbstractGoal was to engineer biological, arterial grafts with antithrombotic, autologous endothelial luminal surface combined with extraluminal smooth vascular muscle layer (VSMC) and to test in vivo. (1) Different decellularisation methods described in literature were compared to identify the most suitable one with focus on the preservation of extracellular fibre matrix. (2) Endothelial precursor cells (EPC), isolated from bone marrow and VSMC from small venous segments of donor animals were cultivated. Cells were seeded sandwich‐like on homologous decellularized venous scaffolds and conditioned under pulsatile circulation in a bioreactor. (3) The semiautologous grafts were implanted in carotidal position on both sides in five Beagle dogs (n = 10; group 2) as interposition. A group of five animals, receiving only acellular grafts in both carotid positions (n = 10; group 1) served as controls. Comparison of four in literature described decellularisation methods showed different preservation of elastic and collagen fibres compared with native veins, whereas decellularity was similar in all methods. This forced us to choose a decellularization protocol with the best preservation of the extracellular matrix. The in vivo experiments showed in group 1 (control) already after one week a complete thrombotic occlusion of the decellularized implants, whereas in group 2 9/10 semiautologous grafts were patent after 98 ± 4 days in ultrasound, angiography and histology (p = 0.0001). A complete incorporation of semiautologous grafts in the surrounding tissue could be shown. The seeding with two different cell types preserved an aneurysmatic degeneration under arterial conditions with patency without anticoagulation.

Salt overload damages the glycocalyx sodium barrier of vascular endothelium

Sodium overload stiffens vascular endothelial cells in vitro and promotes arterial hypertension in vivo. The hypothesis was tested that the endothelial glycocalyx (eGC), a mesh of anionic biopolymers covering the surface of the endothelium, participates in the stiffening process. By using a mechanical nanosensor, mounted on an atomic force microscope, height (∼400 nm) and stiffness (∼0.25 pN/nm) of the eGC on the luminal endothelial surface of split-open human umbilical arteries were quantified. In presence of aldosterone, the increase of extracellular sodium concentration from 135 to 150 mM over 5 days (sodium overload) led the eGC shrink by ∼50% and stiffening by ∼130%. Quantitative eGC analyses reveal that sodium overload caused a reduction of heparan sulphate residues by 68% which lead to destabilization and collapse of the eGC. Sodium overload transformed the endothelial cells from a sodium release into a sodium-absorbing state. Spironolactone, a specific aldosterone antagonist, prevented these changes. We conclude that the endothelial glycocalyx serves as an effective buffer barrier for sodium. Damaged eGC facilitates sodium entry into the endothelial cells. This could explain endothelial dysfunction and arterial hypertension observed in sodium abuse.

Crawling Phagocytes Recruited in the Brain Vasculature after Pertussis Toxin Exposure through IL6, ICAM1 and ITGαM

The cerebral vasculature is constantly patrolled by rod-shaped leukocytes crawling on the luminal endothelial surface. These cells are recruited in greater numbers after exposure to bacterial lipopolysaccharide (LPS) by a mechanism involving tumor necrosis factor (TNF), interleukin-1β (IL1β) and angiopoietin-2 (Angpt2). Here, we report that the population of crawling leukocytes, consisting mainly of granulocytes, is also increased in the brains of mice suffering from experimental autoimmune encephalomyelitis (EAE) or injected with pertussis toxin (PTX), which is commonly used to induce EAE. However, this recruitment occurs through an alternative mechanism, independent of Angpt2. In a series of experiments using DNA microarrays, knockout mice and neutralizing antibodies, we found that PTX acts indirectly on the endothelium in part through IL6, which is essential for the post-transcriptional upregulation of intercellular adhesion molecule 1 (ICAM1) in response to PTX but not to LPS. We also found that phagocytes adhere to brain capillaries through the interaction of integrin αM (ITGαM) with ICAM1 and an unidentified ligand. In conclusion, this study supports the concept that PTX promotes EAE, at least in part, by inducing vascular changes necessary for the recruitment of patrolling leukocytes.

Expression of Follicle-Stimulating Hormone Receptor in Tumor Blood Vessels

In adult humans, the follicle-stimulating hormone (FSH) receptor is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. It is minimally expressed by the endothelial cells of gonadal blood vessels. We used immunohistochemical and immunoblotting techniques involving four separate FSH-receptor-specific monoclonal antibodies that recognize different FSH receptor epitopes and in situ hybridization to detect FSH receptor in tissue samples from patients with a wide range of tumors. Immunoelectron microscopy was used to detect FSH receptor in mouse tumors. In all 1336 patients examined, FSH receptor was expressed by endothelial cells in tumors of all grades, including early T1 tumors. The tumors were located in the prostate, breast, colon, pancreas, urinary bladder, kidney, lung, liver, stomach, testis, and ovary. In specimens obtained during surgery performed to remove tumors, the FSH receptor was not expressed in the normal tissues located more than 10 mm from the tumors. The tumor lymphatic vessels did not express FSH receptor. The endothelial cells that expressed FSH receptor were located at the periphery of the tumors in a layer that was approximately 10 mm thick; this layer extended both into and outside of the tumor. Immunoelectron microscopy in mice with xenograft tumors, after perfusion with anti–FSH-receptor antibodies coupled to colloidal gold, showed that the FSH receptor is exposed on the luminal endothelial surface and can bind and internalize circulating ligands. FSH receptor is selectively expressed on the surface of the blood vessels of a wide range of tumors. (Funded by INSERM.).

Microanatomy of lymphocyte-endothelial interactions at the high endothelial venules of lymph nodes.

Lymphocyte trafficking into lymph nodes and Peyer's patches is mediated primarily by specifically differentiated venules, called high endothelial venules (HEVs), located in the tissue parenchyma. HEVs have a unique morphology and phenotype, which enables them to interact with circulating lymphocytes efficiently. That is, the HEV endothelial cells have a tall and plump appearance, and constitutively express multiple adhesion molecules and chemokines on their surface. These molecules can interact with cognate receptors on circulating lymphocytes, thereby mediating the stepwise and sequential lymphocyte adhesion and transendothelial migration (TEM) at the HEV endothelial luminal surface. This review summarizes the fine morphological aspects of the unique HEV endothelial cells, with special reference to the spatial distribution of the adhesion molecules and chemokines that regulate lymphocyte migration.

CCR5 Signaling Suppresses Inflammation and Reduces Adverse Remodeling of the Infarcted Heart, Mediating Recruitment of Regulatory T Cells

Myocardial infarction triggers an inflammatory reaction that is involved in cardiac remodeling. Cardiac repair is dependent on regulatory mechanisms that suppress inflammation and prevent excessive matrix degradation. Chemokine induction in the infarcted heart mediates recruitment of leukocyte subsets with distinct properties. We demonstrate that signaling through the CC chemokine receptor 5 (CCR5) prevents uncontrolled postinfarction inflammation and protects from adverse remodeling by recruiting suppressive mononuclear cells. CCR5 and its ligands macrophage inflammatory protein (MIP)−1α and MIP-1β were markedly induced in the infarcted mouse myocardium. In addition, almost 40% of the mononuclear cells infiltrating the infarct expressed CCR5. CCR5−/− mice exhibited marked upregulation of proinflammatory cytokine and chemokine expression in the infarct. In wild-type infarcts CCR5+ mononuclear cells had anti-inflammatory properties, expressing higher levels of IL-10 than CCR5− cells. In contrast, mononuclear cells isolated from CCR5−/− infarcts had reduced IL-10 expression. Moreover, enhanced inflammation in the absence of CCR5 was associated with impaired recruitment of CD4+/foxp3+ regulatory T cells (Tregs). The CCR5+ Treg subset exhibited increased IL-10 expression, reflecting potent anti-inflammatory activity. Accentuated inflammation in CCR5−/− infarcts was associated with increased matrix metalloproteinase (MMP) expression, reduced TIMP levels, and enhanced MMP-2 and MMP-9 activity, resulting in worse cardiac dilation. These results suggest that CCR5-mediated Treg recruitment may restrain postinfarction inflammation, preventing excessive matrix degradation and attenuating adverse remodeling.

Chemokine patterning by glycosaminoglycans and interceptors

Chemokines mediate leukocyte emigration from blood into tissues. This process is triggered by chemokines binding and signaling through their cognate G-protein-coupled receptors on leukocytes and requires the involvement of leukocyte and endothelial cell adhesion molecules. Additionally, in vivo chemokine activity depends on their interaction with "auxiliary" molecules expressed by the vascular endothelial cells. Secreted chemokines can be immobilized on the luminal and abluminal endothelial cell surfaces by glycosaminoglycans. In order to be targeted to their presentation sites on the luminal endothelial cell surface, the tissue-derived chemokines have to cross the endothelial cell barrier. For inflammatory chemokines this is accomplished by active transport involving Duffy antigen, an 'interceptor' expressed by venular endothelial cells. Other chemokine interceptors, D6 in particular, may act as scavenging decoys and are involved in clearance of chemokines. The interceptor-mediated transport or elimination of chemokines, together with their immobilization by glycosaminoglycans, lead to chemokine patterning at the blood-tissue interface and within tissues. The resulting chemokine gradients induce leukocyte emigration from blood and may also be necessary for directed leukocyte migration within tissues.

Phosphatidylethanolamine at the Luminal Endothelial Surface—Implications for Hemostasis and Thrombotic Autoimmunity

Accumulating evidence suggests that phosphatidylethanolamine (PE) is physically present at the luminal endothelial surface, where it tentatively functions as a critical anticoagulant. The goal of the current investigation was 3-fold: to characterize the distribution profile of PE at the luminal endothelial surface; to examine the immunoreactivity to the vascular endothelium by anti-PE (aPE) sera from patients presenting with thrombosis; and to discuss the potential mechanism of PE upregulation by endothelial cells. The rat aortic arch was selected as major conduit vessel under significant hemodynamic burden. The presence of PE and the antigenic profile of aPE sera at the luminal endothelial surface were examined using duramycin as a PEbinding probe and immunohistochemistry. Phosphatidylethanolamine upregulation at endothelial cell surface was investigated using cultured monolayer subject to laminar shear stress or thrombin treatment. High levels of PE were detected at the luminal endothelial surface of aortic flow dividers, the ascending aorta, and the outer curvature of the aortic arch. The antigenic profiles of aPE sera, which are highly associated with elevated thrombotic risks in patients, are consistent with PE distribution along the endothelial surface. Finally, PE is upregulated at the surface of cultured endothelial cells in response to luminal shear stress but not thrombin. The current data describe the physical distribution of vascular PE at the blood-endothelium interface. The luminal PE presents a vulnerability to anti-PE autoimmunity and is consistent with the association between aPE and elevated risk for idiopathic thrombosis.