Active Site Formation, Not Bond Kinetics, Limits Adhesion Rate between Human Neutrophils and Immobilized Vascular Cell Adhesion Molecule 1

The formation of receptor ligand bonds at the interface between different cells and between cells and substrates is a widespread phenomenon in biological systems. Physical measurements of bond formation rates between cells and substrates have been exploited to increase our understanding of the biophysical mechanisms that regulate bond formation at interfaces. Heretofore, these measurements have been interpreted in terms of simple bimolecular reaction kinetics. Discrepancies between this simple framework and the behavior of neutrophils adhering to surfaces expressing vascular cell adhesion molecule 1 (VCAM-1) motivated the development of a new kinetic framework in which the explicit formation of active bond formation sites (reaction zones) are a prerequisite for bond formation to occur. Measurements of cells interacting with surfaces having a wide range of VCAM-1 concentrations, and for different durations of contact, enabled the determination of novel kinetic rate constants for the formation of reaction zones and for the intrinsic bond kinetics. Comparison of these rates with rates determined previously for other receptor-ligand pairs points to a predominant role of extrinsic factors such as surface topography and accessibility of active molecules to regions of close contact in determining forward rates of bond formation at cell interfaces.     

Endothelial Cell Adhesion Molecule Expression and Lymphocyte Adhesion to Endothelial Cells: Effect of Nitric Oxide

Interactions between circulating leukocytes and vascular endothelial cells are of fundamental importance in controlling normal recirculation and migration of cells into sites of inflammation. Nitric oxide (NO), which is synthesized by vascular endothelial cells, has been reported to decrease the binding of platelets, monocytes, macrophages, and neutrophils to endothelial cells. Using NO donors and inhibitors of the enzyme NO synthase, we found no evidence that physiologically relevant levels of NO alter adhesion of purified lymphocytes to an endothelial cell line derived from human umbilical vein endothelial cells (SGHEC-7). In addition, NO donors did not alter the cell surface expression of VCAM-1, ICAM-1, or E-selectin on SGHEC-7 cells.     

A Single Immunoglobulin-like Domain of the Human Neural Cell Adhesion Molecule L1 Supports Adhesion by Multiple Vascular and Platelet Integrins

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃. The interaction between L1-Ig6 and α_IIbβ₃ was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with α_vβ₃ and α_vβ₁ supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via α_vβ₃, α_vβ₁, α₅β₁, and α_IIbβ₃. Whereas β₃ integrin binding to L1-Ig6 was evident in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, a corresponding interaction with the β₁ integrins was only observed in the presence of Mn²⁺. Furthermore, such Mn²⁺-dependent binding by α₅β₁ and α_vβ₁ was significantly inhibited by exogenous Ca²⁺. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that α_vβ₃ or active α_IIbβ₃ > α_vβ₁ > α₅β₁. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.     

Endothelial Cell E- and P-Selectin and Vascular Cell Adhesion Molecule-1 Function as Signaling Receptors

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca²⁺]_i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca²⁺]_i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca²⁺]_i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca²⁺]_i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca²⁺]_i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca²⁺]_i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLe^x), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca²⁺]_i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLe^x and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca²⁺]_i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca²⁺]_i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca²⁺]_i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.     

Elevated Concentrations of Circulating Intercellular Adhesion Molecule 1 (ICAM-1) and of Vascular Cell Adhesion Molecule 1 (VCAM-1) in HIV-1 Infection

The aim of this study was to determine whether elevated levels of circulating forms of the soluble adhesion molecules, Intercellular Adhesion Molecule-1 (cICAM-1), Vascular Cell Adhesion Molecule-1 (cVCAM-1) and E-Selectin (cE-Selectin) are observed in the sera of HIV-1 infected individuals as compared to healthy HIV seronegative adults and whether these elevated levels can be correlated with disease progression. Significantly elevated levels of cICAM-1—ranging from 184 to 1116 ng/ml with a mean of 617 ng/ml—and cVCAM-1—ranging from 653 to 3456 ng/ml with a mean of 1500 ng/ml—were observed in the sera of 29 HIV-1 infected individuals as compared to controls-ranging from 152 to 354 ng/ml with a mean of 248 ng/ml for cICAM-1 and from 328 to 792 ng/ml with a mean of 560 ng/ml for cVCAM-1 (P < 0.001). The serum concentrations of cE-Selectin of the HIV-1 infected individuals did not differ from those of the healthy controls. The elevated levels of cICAM-1, cVCAM-1 did not correlate with the CD4 count or the serum concentration of C-reactive protein. However, a significant correlation was observed between the serum concentrations of cVCAM-1 and those of neopterin. Since cICAM-1 as well as cV-CAM-1 can interfere with adhesion events leading to immunological functions, it can be suggested that the high amounts of these circulating forms of adhesion molecules, when present in the sera of HIV-1 positive individuals, can further disturb the immune system of these patients. In addition, the present study also suggests that the seric concentrations of cVCAM-1 can be used as pronostic indicators.     

Novel Cytokine-independent Induction of Endothelial Adhesion Molecules Regulated by Platelet/Endothelial Cell Adhesion Molecule (CD31)

Tumor necrosis factor–α, interleukin-1, and endotoxin stimulate the expression of vascular endothelial cell (EC) adhesion molecules. Here we describe a novel pathway of adhesion molecule induction that is independent of exogenous factors, but which is dependent on integrin signaling and cell–cell interactions. Cells plated onto gelatin, fibronectin, collagen or fibrinogen, or anti-integrin antibodies, expressed increased amounts of E-selectin, vascular cell adhesion molecule–1, and intercellular adhesion molecule–1. In contrast, ECs failed to express E-selectin when plated on poly-l-lysine or when plated on fibrinogen in the presence of attachment-inhibiting, cyclic Arg-Gly-Asp peptides. The duration and magnitude of adhesion molecule expression was dependent on EC density. Induction of E-selectin on ECs plated at confluent density was transient and returned to basal levels by 15 h after plating when only 7 ± 2% (n = 5) of cells were positive. In contrast, cells plated at low density displayed a 17-fold greater expression of E-selectin than did high density ECs with 57 ± 4% (n = 5) positive for E-selectin expression 15 h after plating, and significant expression still evident 72 h after plating. The confluency-dependent inhibition of expression of E-selectin was at least partly mediated through the cell junctional protein, platelet/endothelial cell adhesion molecule–1 (PECAM-1). Antibodies against PECAM-1, but not against VE-cadherin, increased E-selectin expression on confluent ECs. Co– culture of subconfluent ECs with PECAM-1– coated beads or with L cells transfected with full-length PECAM-1 or with a cytoplasmic truncation PECAM-1 mutant, inhibited E-selectin expression. In contrast, untransfected L cells or L cells transfected with an adhesion-defective domain 2 deletion PECAM-1 mutant failed to regulate E-selectin expression. In an in vitro model of wounding the wound front displayed an increase in the number of E-selectin–expressing cells, and also an increase in the intensity of expression of E-selectin positive cells compared to the nonwounded monolayer. Thus we propose that the EC junction, and in particular, the junctional molecule PECAM-1, is a powerful regulator of endothelial adhesiveness.The endothelial lining of the vascular system normally displays a nonactivated, nonadhesive phenotype. Stimulation with agents such as tumor necrosis factor-α (TNF-α)¹, interleukin-1 (IL-1), or lipopolysaccharide (LPS) are known to induce the expression of proteins on the endothelial surface that mediate coagulation (Bevilacqua et al., 1986), leukocyte adhesion (Bevilacqua et al., 1985; Gamble et al., 1985; Pober et al., 1986b ; Doherty et al., 1989), and leukocyte transendothelial migration (Furie et al., 1989; Moser et al., 1989). The endothelial antigens that are important for the adhesion of leukocytes are members of the selectin family, E- and P-selectin, and the immunoglobulin gene superfamily, vascular cell adhesion molecule–1 (VCAM-1) and intercellular adhesion molecule–1 (ICAM-1) (Carlos and Harlan, 1994; Litwin et al., 1995).The induction of E-selectin expression on endothelial cells (ECs) in vitro after cytokine stimulation is transient and independent of the continued presence of the stimulant (Pober et al., 1986a ). Previous studies have shown that E-selectin mRNA and protein levels peak between 2 and 4 h, respectively, after treatment with an agonist, returning to near basal levels by 24 h (Bevilacqua et al., 1989; Read et al., 1994). VCAM-1 (Osborn et al., 1989) and ICAM-1 (Pober et al., 1986b ) are maximal 6 and 12 h, respectively, after stimulation.In contrast to the transiency of E-selectin and VCAM expression demonstrated by the in vitro data, these antigens have been detected on venular endothelium in chronic inflammatory lesions, such as the synovium in rheumatoid arthritis (Koch et al., 1991), and the skin in psoriasis (Petzelbauer et al., 1994). E-selectin expression is also detected on angiogenic vessels in human hemangiomas, a noninflammatory angiogenic disease (Kraling et al., 1996). Moreover, the architecture and anatomic localization of capillary loops influence the pattern of endothelial expression of E-selectin and VCAM-1, independently of the availability of cytokines (Petzelbauer et al., 1994). Thus it is likely that alternate control mechanisms exist to allow prolonged, locality-based expression of adhesion molecules on the endothelium. At least one of these alternate mechanisms may be flow, since increased shear stress has been shown to selectively modulate adhesion molecule expression, upregulating ICAM-1 but not E-selectin or VCAM-1 (Nagel et al., 1994).Since sites of inflammation are often associated with morphological changes including cell retraction of the endothelium (Schumacher, 1973), we hypothesized that cell contacts may be important in the regulation of endothelial phenotype. We describe here the central role of the junctional protein, platelet/endothelial cell adhesion molecule–1 (PECAM-1), through the formation of cell–cell interactions, in the maintenance of the functional integrity of the endothelial monolayer. Furthermore, we demonstrate a novel pathway for the induction of adhesion molecules on endothelial cells that is independent of exogenous addition of cytokines, but is related to integrin- and cell shape–associated signaling events.     

剪切流动法研究表面固定化配基与目标分子的相互作用力

基于剪切流动腔技术,以微球作为受力载体,设计了一套可用于研究表面固定化配基与目标分子特异性相互作用力的实验和分析方法,并以人免疫球蛋白 G (human IgG) 和羊抗人免疫球蛋白 G (goat anti-human IgG) 分别作为模型配基和模型目标分子进行了研究 . 基于平面 Poiseuille 层流模型设计了流场参数,以数值计算结果验证了设计的合理性 . 使用牛血清白蛋白 (BSA) 作为非特异性对照,判断微球与基片表面的结合力来自配基和目标分子的生物特异性相互作用,并由进一步的目标分子灭活对比实验确认了这一结论 . 实验观察到微球与基片表面的结合力受到配基面密度的影响,说明发生结合的是多对而非单对蛋白质分子 . 将 95 ％的微球被剥离时对应的壁面剪切率设定为临界剪切率,由大量实验结果拟合得到了临界剪切率与配基面密度间的定量关系 . 在受力分析模型中,考虑到多分子的结合,以及分子键位置不同造成的力臂长度的差异,最终计算得到单对配基与目标分子的平均结合力约为 342pN.     

Podocalyxin Regulates Murine Lung Vascular Permeability by Altering Endothelial Cell Adhesion

Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin and endoglycan) as vascular endothelial cell markers, there is remarkably little known of their vascular function. Podocalyxin (gene name Podxl), in particular, has been difficult to study in adult vasculature as germ-line deletion of podocalyxin in mice leads to kidney malformations and perinatal death. We generated mice that conditionally delete podocalyxin in vascular endothelial cells (Podxl^ΔEC mice) to study the homeostatic role of podocalyxin in adult mouse vessels. Although Podxl^ΔEC adult mice are viable, their lungs display increased lung volume and changes to the matrix composition. Intriguingly, this was associated with increased basal and inflammation-induced pulmonary vascular permeability. To further investigate the etiology of these defects, we isolated mouse pulmonary endothelial cells. Podxl^ΔEC endothelial cells display mildly enhanced static adhesion to fibronectin but spread normally when plated on fibronectin-coated transwells. In contrast, Podxl^ΔEC endothelial cells exhibit a severely impaired ability to spread on laminin and, to a lesser extent, collagen I coated transwells. The data suggest that, in endothelial cells, podocalyxin plays a previously unrecognized role in maintaining vascular integrity, likely through orchestrating interactions with extracellular matrix components and basement membranes, and that this influences downstream epithelial architecture.     

Interleukin-1α Released from Epithelial Cells after Adenovirus Type 37 Infection Activates Intercellular Adhesion Molecule 1 Expression on Human Vascular Endothelial Cells

A key event in virus-induced inflammation (leukocyte extravasation through the endothelium) is the local activation of endothelial cells, as indicated by the expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin. In order to identify triggers of inflammation in adenovirus infection, we inoculated respiratory and ocular epithelial cells with adenovirus type 37 (Ad37), a human pathogen associated with keratoconjunctivitis as well as urogenital and respiratory infections. Fluids from virus-infected epithelial cells activated ICAM-1 (and to a lesser extent, VCAM-1) expression on cultured human umbilical vein endothelial cells. Blocking studies with anticytokine antibodies implicated interleukin-1alpha (IL-1alpha) as the epithelial cell-derived factor which activated endothelial cell ICAM-1 expression. The results thus identify epithelial cell-derived IL-1alpha as a potentially important activator of endothelial cells in Ad37-induced inflammation.     

野生型p53基因对内皮细胞细胞间粘附分子-1表达的影响

细胞间粘附分子－１（ＩＣＡＭ－１）是介导白细胞与内皮细胞粘附的重要粘附分子．为研究野生型ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响,分别采用流式细胞术和ＲＴ－ＰＣＲ／ＨＰＬＣ方法测定ＩＣＡＭ－１蛋白及ｍＲＮＡ水平．静息状态的内皮细胞表面结构性地表达有少量的ＩＣＡＭ－１,在肿瘤坏死因子α（ＴＮＦα,１０～１０００Ｕ／ｍｌ）诱导下,其表达呈剂量依赖性增加．将ｐ５３基因导入内皮细胞,则显著抑制ＴＮＦα诱导的内皮细胞表面ＩＣＡＭ－１的表达．ｐ５３基因的导入对静息状态内皮细胞表面结构性表达的ＩＣＡＭ－１影响较小．ｐ５３基因主要通过降低ＩＣＡＭ－１的ｍＲＮＡ水平而抑制内皮细胞表面ＩＣＡＭ－１的表达,但对蛋白的抑制程度小于对ｍＲＮＡ的抑制程度．提示：ｐ５３基因对内皮细胞ＩＣＡＭ－１表达的影响除转录水平控制外,还存在转录后水平的调控     

血管内皮细胞衰老与血管疾病

细胞衰老是指细胞在各种应激条件下出现周期阻滞,不可逆地丧失增殖能力,其形态、基因表达和功能都发生特定变化的过程。研究表明,血管内皮细胞衰老可以通过削弱血管功能,促进衰老相关血管疾病的发生发展。然而,有关内皮细胞衰老的发生机制以及内皮细胞衰老影响血管功能及衰老相关血管疾病的潜在机制尚待挖掘。本文从血管内皮细胞衰老相关的信号通路,以及血管内皮细胞衰老与血管功能和血管相关疾病（动脉粥样硬化、高血压和糖尿病血管并发症）的最新研究进展进行综述,为进一步认识血管疾病的发病机制,延缓血管衰老提供新的思路。     

凝血酶与血管内皮细胞

凝血酶不仅在凝血过程中起主导作用,还可以引发多种凝血酶受体介导的分子和细胞间相互作用,在动脉粥样硬化和再狭窄形成中起着重要的作用。现就凝血酶及其受体的特性,以及对血管内皮细胞的作用,包括通透性改变、内皮生长因子、基质金属蛋白酶、黏附分子表达作一综述。     

同型半胱氨酸诱导血管内皮细胞凋亡的研究

观察不同浓度同型半胱氨酸(homocysteine,HCY)在Cu2 介导下,能否诱导培养的人脐静脉内皮细胞凋亡,以揭示HCY致血管内皮损伤的机制。采用细胞计数板检测脱落细胞量;比色法分别测定乳酸脱氢酶释放率、细胞内丙二醛含量和超氧化物歧化酶、谷胱甘肽过氧化物酶活力的改变;Hoechst 33258染色观察凋亡细胞核形态变化及流式细胞术定量测定细胞凋亡。结果表明HCY在生理浓度Cu2 的介导下,可能通过氧化应激损伤的机制而导致血管内皮细胞凋亡,这提示在体内可能通过此途径诱发动脉粥样硬化。     

Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Promotes Neuritogenesis and Cell Survival

The cell adhesion molecule L1 is a Lewis^x-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewis^x-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg⁶⁸⁷ yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system.