Current Views on Structure and Function of Endothelial Adhesion Molecules

The consecutive events that occur in a living body following injury are commonly referred to as inflammation (inflammare: to set on fire). In the first century A.D. observations of clinical patients had allowed Cornelius Celsus to formulate his famous “cardinal signs” of inflammation: calor, rubor, tumor and dolor. These still holds true today. The four characteristics of inflammation are redness and swelling with heat and pain. From these early studies it was obvious that blood vessels played an important role in the development of an inflammatory process, which coincided with plasma leakage and accumulation of leucocytes in extravascular tissue.     

骨桥蛋白13肽抑制球囊内皮剥脱术后血管狭窄的实验研究

目的：利用含有骨桥蛋白（OPN）多种功能位点的13肽（Gly^158-Lys^170）,从细胞和整体水平观察其对VSMC和单核巨噬细胞黏附、浸润以及内膜增生的影响,并初步探讨其作用机制。方法：用不同浓度OPN13肽（0,100,200,300mg／L）检测其对体外培养的平滑肌细胞（VSMc）与OPN黏附的抑制作用;以不合黏附序列的6肽分子为对照组,用以确定13肽抑制黏附特异性。用球囊内皮剥脱法建立大鼠内膜增生模型。实验动物分为4组：治疗组大鼠自术前1h及术后静脉滴注13肽,连续给药7d;对照组大鼠给予非特异性对照6肽分子;模型组大鼠给予相同剂量的生理盐水;正常对照组大鼠施假手术。并利用免疫组织化学染色和Western印迹分析方法,检测血管壁中OPN、FAK、ILK的表达变化。结果：OPN13肽能特异性的及浓度依赖性的抑制VSMC与OPN的相互作用,血管内膜剥脱后给予13肽治疗组血管壁单核／巨噬细胞浸润减少,OPN及其下游信号分子ILK,FAK表达下调,内膜增生被明显抑制。结论：含有OPN多功能位点的13肽可通过阻断OPN与膜受体的相互作用而抑制血管炎症的进展和内膜增生。     

Effect of selenium supplementation in asthmatic subjects on the expression of endothelial cell adhesion molecules in culture

Endothelial cells play a major role in immunologic reactions, in which cellular adhesion molecules P-selectin, ICAM-1, VCAM-1, and ELAM-1 are important mediators in the recruitment of leukocytes in pulmonary inflammation. Selenium (Se) is known to modulate immunological mechanisms of asthma. The aim of our investigation was to examine whether Se supplementation in cortico-dependent asthmatic patients may modulate adhesion molecule expression in cultured endothelium. Our findings indicated that P-selectin, VCAM-1, and ELAM-1 expression on human umbilical vein endothelial cells stimulated with peripheral blood mononuclear cells obtained from asthmatics before supplementation with Se was significantly higher than from healthy donors (p < 0.05). The production of ICAM-1 showed only slight augmentation. The levels of VCAM-1 and ELAM-1 expression were significantly decreased after 3 mo of Se supplementation (p < 0.05). After 6 mo of intervention period the intensity of P-selectin and ICAM-1 expression was also significantly reduced (p < 0.05 andp < 0.01, respectively). The inhibitory effect of Se on the adhesion molecule expression was studied in cultured endothelial cells after interferon-γ stimulation. Our data suggest that Se affects the expression of P-selectin, ICAM-1, VCAM-1, and ELAM-1 in a dosedependent manner and the half-maximal inhibitory concentrations were 3.4, 0.5, 4, and 3.8 μg/mL, respectively. The maximal inhibitions (greater than 80%) were observed in vitro with 10 μg/mL Se (p < 0.01). Regulation of adhesion molecule expression may be an important mechanism through which the inflammation may be controlled.     

The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

The Role of Cellular and Extracellular Matrix Adhesion Proteins in Organ Transplantation

The specific adhesion of cells to other cells or to particular tissue microenvirorvments is a basic function of cell migration and recognition, and underlines many biologic processes including embryogenesis, repair and immunity. Leukocytes express an array of surface receptors broadly known as “accessory adhesion molecules.” which mediate most cell -cell interactions, direct lymphocyte traffic between anatomical compartments, and facilitate cellular adhesion to the inflammation or alloantigenic sites (Springer 1990). In addition, adhesion molecules are involved in the process of antigen recognition, and may costimulate cell activation and transformation. These proteins are thought to affect the very early antigen independent events between host leukocytes and vascular endothelium. Because of these activities, the subject of adhesion molecules is gaining interest in the field of organ transplantation, in both conceptualization and development of novel therapeutic strategies (de Sousa et al. 1991, Kupiec-Weglinski et al. 1993a, Heemann et al. 1993).     

Forging the endothelium during inflammation: pushing at a half-open door?

During an inflammatory response, changes in the adhesive properties of the endothelium occur that enable normally non-adherent blood-borne leukocytes to adhere and subsequently to traverse the endothelium through small gaps at inter-cellular junctions. This review concentrates on the role played by inter-endothelial adhesion molecules during transmigration and the way in which their expression may be regulated during inflammation. We show that the final "open" signals that lead to the formation of clefts between adjacent endothelial cells may be derived from inflamed tissue underlying the endothelium and from activated leukocytes.     

Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx

An ultrasound-based molecular imaging technique capable of detecting endothelial cell markers of inflammation may allow early, non-invasive assessment of vascular disease. Clinical application of targeted, acoustically-active microbubbles requires optimization of microbubble-endothelial adhesion strength to maximize image signal-to-noise ratio, as well as the ability to discern the degree of inflammation along a continuum of dysfunction. Accordingly, we hypothesized that adhesion of intercellular adhesion molecule-1 (ICAM-1)-targeted microbubbles is dependent on the degree of endothelial inflammation, and that microbubbles multi-targeted to both ICAM-1 (via anti-ICAM-1 antibodies) and selectins (via sialyl Lewisx) demonstrate greater adhesion strength than microbubbles targeted to either inflammatory marker alone. In a radial flow chamber, microbubbles were perfused across endothelial cells activated with interleukin-1beta to four different levels of inflammation, as assessed by quantitative ICAM-1 expression. ICAM-1-targeted microbubble adhesion strength increased with increasing degree of inflammation, with a relationship that was both positive and linear (r > 0.99). Microbubble adhesion strength was significantly higher for the multi-targeted microbubbles than either of the single-targeted microbubbles. These data thus demonstrate that multi-targeting of contrast microbubbles may offer improved adhesion characteristics, allowing for greater sensitivity to inflammation. Furthermore, the adhesion strength of targeted microbubbles is linearly dependent on the degree of inflammation, suggesting that targeted ultrasound imaging may offer differentiation between various degrees of endothelial dysfunction, and thus detect not only the presence, but also the severity of inflammatory disease processes.     

Soluble intercellular adhesion molecules, soluble vascular cell adhesion molecules, and risk of coronary heart disease

Objective: We examined the association of circulating levels of soluble intercellular adhesion molecules (sICAM‐1) and soluble vascular cell adhesion molecules (sVCAM‐1) with coronary heart disease (CHD) risk factors and whether the adhesion molecules alone, and in combination, can serve as predictors of coronary CHD. Research Methods and Procedures: Among 18,225 men from the Health Professional Follow‐up Study who provided blood in 1994, we documented 266 incidents of non‐fatal myocardial infarction or fatal CHD during 6 years of follow‐up. The cases were matched 1:2 with non‐cases on age, smoking, and month of blood draw. We found both adhesion molecules directly associated with BMI, inflammatory biomarkers, and triglycerides and inversely associated with high‐density lipoprotein and alcohol intake (p < 0.05). After adjustment for C‐reactive protein, cholesterol‐to‐high‐density lipoprotein ratio, age, smoking, BMI, physical activity, alcohol intake, history of diabetes, parental history of CHD, aspirin use, antihypertensive drug use, and fasting status, the relative risk of CHD was 1.69 [95% confidence interval (CI), 1.14 to 2.51] for sICAM‐1 and 1.34 (95% CI, 0.91 to 1.96) for sVCAM‐1, when comparing the top quintile with the lower four quintiles. Control for other inflammatory or lipid biomarkers did not appreciably attenuate the associations. When we cross‐classified participants based on their sICAM‐1 and sVCAM‐1 levels, only the men in the top quintile of both biomarkers [relative risk = 2.39 (95% CI, 1.45 to 3.91)] had a significantly elevated risk of CHD (P interaction = 0.01, multivariate model). Discussion: sICAM‐1 and sVCAM‐1 are directly associated with obesity and other CHD risk factors. The combination of high levels of both adhesion molecules might be associated with the development of CHD, independent of other CHD risk factors.     

Neutralization of TNF by the Antibody cA2 Reveals Differential Regulation of Adhesion Molecule Expression on TNF-Activated Endothelial Cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

Low glucose availability alters the expression of genes involved in initial adhesion of human glioblastoma cancer cell line SF767

Studying the metabolic pathways of cancer cells is considered as a key to control cancer malignancies and open windows for effective drug discovery against cancer. Of all the properties of a tumor, metastasis potential is a defining characteristic. Metastasis is controlled by a variety of factors that directly control the expression of cell adhesion proteins. In this study we have investigated the expression of cell to cell and cell to matrix adhesion protein genes during the initial phases of attachment of human glioblastoma cancer cell line SF767 (66Y old human female: UCSF Neurosurgery Tissue Bank) to the attachment surface under (Cell culture treated polystyrene plate bottom) glucose-rich and glucose-starved conditions. The aim was to imitate the natural microenvironment of glucose availability to cancer cells inside a tumor that triggers epithelial to mesenchymal transition (EMT). In this study, we have observed the gene expression of epithelial and mesenchymal isoforms of cadherin (E-CAD and N-CAD) and Ig like cell adhesion molecules (E-CAM and N-CAM) along with Integrin family subunits for the initial attachment of cancer cells. We observed that high glucose environments promoted cell survival and cell adhesion, whereas low glucose accelerated EMT by downregulating the expression level of integrin, E-CAD, and N-CAD, and upregulation of N-CAM during early period of cell adhesion. Low glucose availability also downregulated variety of structural and regulatory genes, such as zinc finger E-box binding home box 1A), cytokeratin, Snail, and β catenin, and upregulation of hypoxia-inducible factor 1, matrix metalloprotease 13/Collagenase 3, vimentim, p120, and fructose 1,6 bisphosphatase. Glucose conditions are more efficient for cancer studies in this case glioblastoma cells.     

Somatostatin regulates intracellular signaling in human carotid endothelial cells

Somatostatin (somatotropin release inhibitory factor; SRIF) is an endogenous peptide produced at sites of inflammation, making the SRIF a candidate in regulating vascular inflammation. We have used primary human coronary artery endothelial cells (hCAEC) as a model to study SRIF's vascular actions. RT-PCR analysis of hCAEC total mRNA demonstrated the presence of the sst(4) receptor subtype, providing a target for SRIF intracellular signaling. Western blotting with phospho-specific ERK1/2 antibodies showed that SRIF-14 acutely inhibited basal phosphorylation of the extracellular regulated kinases (ERK1/2) by 80%. In addition, SRIF-14 treated hCAEC cell lysates showed a 2.6-fold increase in phosphatase activity, which was inhibited by sodium vanadate. Furthermore, SRIF-14 appeared to be anti-inflammatory in hCAEC as IL-1beta-induced adhesion molecule expression was reduced by 50%. Together, these results show that the coronary artery endothelium is a direct target of SRIF action.     

Characterization of the solution structure of a neuroligin/β-neurexin complex

Neuroligins are post-synaptic cell adhesion molecules that promote synaptic maturation and stabilization upon binding with pre-synaptic partners, the α- and β-neurexins. Using a combination of analytical ultracentrifugation, small angle X-ray, and neutron scattering, we have characterized the low-resolution three-dimensional structure of the extracellular domain of the neuroligins, free in solution, and in complex with β-neurexin. The globular extracellular domain of the neuroligins forms stable homodimers through a four-helix bundle typical of the cholinesterases and other members of the α/β-hydrolase fold family. The presence of the stalk region adds to the extracellular domain of neuroligin-1 an elongated structure, suggesting a rod-like nature of the stalk domain. Sedimentation equilibrium coupled with solution scattering data of the β-neurexin/neuroligin-1 complex indicated a 2:2 stoichiometry where two β-neurexin molecules bind to a neuroligin-1 dimer. Deuteration of neurexin allowed us to collect neutron scattering data that, in combination with other biochemical techniques, provide a basis for optimizing the positioning of each component in a detailed computational model of the neuroligin/neurexin complex. As several mutations of both neurexin and neuroligin genes have been linked to autism spectrum disorders and mental retardation, these new structures provide an important framework for the study of altered structure and function of these synaptic proteins.     

Targeted delivery of antibody conjugated liposomal drug carriers to rat myocardial infarction

Immunoliposome (IL) targeting to areas of inflammation after an acute myocardial infarction (MI) could provide the means by which pro-angiogenic compounds can be selectively targeted to the infarcted region. The adhesion of model drug carriers and ILs coated with an antibody to P-selectin was quantified in a rat model of MI following left coronary artery ligation. Anti-P-selectin coated model drug carriers showed a 140% and 180% increase in adhesion in the border zone of the MI 1 and 4 h post-MI, respectively. Radiolabeled anti-P-selectin ILs injected immediately post-MI and allowed to circulate 24 h showed an 83% increase in targeting to infarcted myocardium when compared to adjacent non-infarcted myocardium. Radiolabeled anti-P-selectin ILs injected 4 h post-MI and allowed to circulate for 24 h showed a 92% increase in accumulation in infarcted myocardium when compared to adjacent non-infarcted myocardium. Targeting to upregulated adhesion molecules on the endothelium provides a promising strategy for selectively delivering compounds to the infarct region of the myocardium using our liposomal-based drug delivery vehicle.     

The Role of Selectins and CD18 in Leukotriene B4-Mediated White Blood Cell Emigration in Human Skin Grafts Transplanted on SCID Mice

The purpose of this study was to examine the role of selectins and CD18 cell adhesion molecules (CAMs) in inflammation induced by injection of leukotriene B₄(LTB₄) into human skin. To accomplish this, the expression of CAMs and the ability of specific antibodies against CAMs to block white blood cell (WBC) transmigration were studied in an in vivo model consisting of human skin transplanted onto mice with the severe combined immune deficiency (SCID) mutation. The results indicate that LTB₄-induced WBC transmigration in the human/SCID model is rapid and pronounced; however, it is not accompanied by a significant upregulation of the baseline expression of endothelial P-selectin, E-selectin, ICAM-1 or VCAM-1. An anti-murine CD18 mAb markedly inhibited white cell infiltration (89% inhibition) confirming the importance of β₂integrins in the process. The role of selectins was also examined. MEL-14, a bioactive antibody against murine L-selectin inhibited transmigration by 66%. A significant, but smaller, effect (39% inhibition) was observed by blocking E-selectin function. These results indicate that LTB₄-induced inflammation does not require upregulation of endothelial CAM expression and, in contrast to TNFα-induced transmigration, is only partially blocked by anti-E-selectin antibodies.     

C-reactive protein induced expression of adhesion molecules in cultured cerebral microvascular endothelial cells

Ischemic stroke can trigger an acute phase response resulting in a rise of plasma concentration of C-reactive protein (CRP). Clinical data about the relationship between CRP and prognosis suggest that CRP might be involved in the pathogenesis of cerebral ischemia. In the present work, a significant increase of circulating level of CRP was observed in an vivo rat brain ischemia model of middle cerebral artery occlusion. To determine the possible effects of CRP on brain microvessel endothelium, we performed a dose-dependent experiment in mouse brain microvascular endothelial cells (bEnd.3 cells) with emphasis on its relation to cell adhesions molecules. Incubation with CRP (1-75 mg/L) for 24 h significantly increased Lactate dehydrogenase (LDH) leakage from bEnd.3 cells (P<0.01) in a dose-dependent manner, and induced significant up-regulations of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions analyzed by Western blotting (P<0.01). In contrast to earlier report, CRP also induced significant increase in ICAM-1 expression in the absence of serum (P<0.01). In conclusion, the present results suggest that CRP may be involved directly in the development of inflammation in response to cerebral ischemia.     

Adenosine in inflammatory joint diseases

Inflammatory joint diseases are a group of heterogeneous disorders with a variety of different etiologies and disease manifestations. However, there are features that are common to all of them: first, the recruitment of various inflammatory cell types that are attracted to involved tissues over the course of the disease process. Second, the treatments used in many of these diseases are commonly medications that suppress or alter immune function. The demonstration that adenosine has endogenous anti-inflammatory functions and that some of the most commonly used anti-rheumatic medications exert their therapeutic effects through stimulation of adenosine release suggest an important role for purinergic signaling in inflammatory rheumatic disorders.