Mechanisms regulating endothelial cell barrier function

Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.     

Mechanisms by which soluble endothelial cell protein C receptor modulates protein C and activated protein C function

The endothelial cell protein C receptor (EPCR) functions as an important regulator of the protein C anticoagulant pathway by binding protein C and enhancing activation by the thrombin-thrombomodulin complex. EPCR binds to both protein C and activated protein C (APC) with high affinity. A soluble form of EPCR (sEPCR) circulates in plasma and inhibits APC anticoagulant activity. In this study, we investigate the mechanisms by which sEPCR modulates APC function. Soluble EPCR inhibited the inactivation of factor Va by APC only in the presence of phospholipid vesicles. By using flow cytometric analysis in the presence of 3 mM CaCl(2) and 0. 6 mM MgCl(2), sEPCR inhibited the binding of protein C and APC to phospholipid vesicles (K(i) = 40 +/- 7 and 33 +/- 4 nM, respectively). Without MgCl(2), the K(i) values increased approximately 4-fold. Double label flow cytometric analysis using fluorescein-APC and Texas Red-sEPCR indicated that the APC.sEPCR complex does not interact with phospholipid vesicles. By using surface plasmon resonance, we found that sEPCR also inhibited binding of protein C to phospholipid in a dose-dependent fashion (K(i) = 32 nM). To explore the possibility that sEPCR evokes structural changes in APC, fluorescence spectroscopy studies were performed to monitor sEPCR/Fl-APC interactions. sEPCR binds saturably to Fl-APC (K(d) = 27 +/- 13 nM) with a maximum decrease in Fl-APC fluorescence of 10.8 +/- 0.6%. sEPCR also stimulated the amidolytic activity of APC toward synthetic substrates. We conclude that sEPCR binding to APC blocks phospholipid interaction and alters the active site of APC.     

Exercise metaboreflex activation and endothelial function impairment in atrial fibrillation

Exercising muscle hypoperfusion stimulates afferents (metaboreceptors) involved in the regulation of ventilation. Atrial fibrillation (AF), particularly when combined with diseases causing endothelial (ED) impairment, such as hypertension (HP) and diabetes mellitus (DM), depresses the ED activity and enhances exercise hyperventilation. The relationship between these two functions and the underlying mechanisms have not been explored previously. In lone AF or AF associated with HP or DM (12 subjects in each cohort), we investigated the brachial artery flow-mediated dilatation (ED function) and ventilation during the recovery phase of handgrip (metaboreflex) exercise for subjects receiving placebo or oral vitamin C (double-blind crossover), both before and after cardioversion (CV) to sinus rhythm. Baseline ED impairment was increasingly more severe and the ergoreflex activity more pronounced in AF + HP and AF + DM compared with lone AF. Vitamin C and CV significantly improved both flow-mediated dilatation and metaboreflex activity in lone AF and AF + HP, and vitamin C did not produce any additive effect when administered after CV. In AF + DM, neither vitamin C nor CV was effective. This study provides the following information: AF generates oxidative injury, which is less when the arrhythmia is lone AF and greater when the arrhythmia is associated with HP. In DM, the oxidative injury generated by AF is refractory to a rather weak antioxidant, like vitamin C, or the baseline damage is such as to prevent any additive influence of AF. In AF, a cause-effect link exists between ED dysfunction and metaboreflex activity. Ventilatory advantages of CV seem to be inversely related with the extension of the underlying ED oxidative impairment.     

Circulating vascular endothelial growth factor and its soluble receptors in patients with liver cirrhosis: possible association with hepatic function impairment

Recent studies provided in vivo evidences of an increased angiogenesis in animal model of portal hypertension and cirrhosis which was linked to increased expression of vascular endothelial growth factor. The aim of study was to evaluate the plasma concentration of VEGF and its receptors in liver cirrhosis and the possible association with the degree of liver insufficiency. Methods. Vascular endothelial growth factor (VEGF) and its soluble receptors: sVEGF-R1, sVEGF-R2 were measured in plasma of 78 patients with liver cirrhosis by ELISA. Results. The significant increase of plasma VEGF and sVEGF-R1 was observed in liver cirrhosis compared to healthy individuals (153.1+/-51.9 vs. 46.8+/-4.1pg/mL, P<0.05; 279.8+/-34.3 vs. 105.1+/-5.9pg/mL, P<0.001, respectively). Plasma VEGF and foremost sVEGF R1 showed significant associations with biochemical indices of liver function. Among clinical parameters, only ascites revealed significant association with plasma VEGR and sVEGF-R1. VEGF and sVEGF-R1 were increased respectively to the degree of liver insufficiency. It was demonstrated through a significant positive correlation with Child-Pugh score and MELD classification. In conclusion, our study suggests that serum VEGF and VEGF-R1 may reflect the hepatic function impairment in liver cirrhosis and seems to be associated with portal hypertension symptoms.     

Structural studies of soluble oligomers of the Alzheimer beta-amyloid peptide

Recent studies have suggested that non-fibrillar soluble forms of Abeta peptides possess neurotoxic properties and may therefore play a role in the molecular pathogenesis of Alzheimer's disease. We have identified solution conditions under which two types of soluble oligomers of Abeta40 could be trapped and stabilized for an extended period of time. The first type of oligomers comprises a mixture of dimers/tetramers which are stable at neutral pH and low micromolar concentration, for a period of at least four weeks. The second type of oligomer comprises a narrow distribution of particles that are spherical when examined by electron microscopy and atomic force microscopy. The number average molecular mass of this distribution of particles is 0.94 MDa, and they are are stable at pH 3 for at least four weeks. Circular dichroism studies indicate that the dimers/tetramers possess irregular secondary structure that is not alpha-helix or beta-structure, while the 0.94 MDa particles contain beta-structure. Fluorescence resonance energy transfer experiments indicate that Abeta40 moieties in amyloid fibrils or protofibrils are more similar in structure to those in the 0.94 MDa particles than those in the dimers/tetramers. These findings indicate that soluble oligomeric forms of Abeta peptides can be trapped for extended periods of time, enabling their study by high resolution techniques that would not otherwise be possible.     

Detection of soluble forms of the beta-amyloid precursor protein in human plasma

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.     

Immunization with soluble murine CD4 induces an anti-self antibody response without causing impairment of immune function

The T cell surface molecule CD4 (L3T4 in mouse) is important in the T lymphocyte response to Ag presented in association with MHC class II molecules. To examine the role of CD4 in immune function, we expressed a soluble form of murine CD4 by deleting the transmembrane and cytoplasmic regions of the L3T4 gene and transfecting the altered gene into Chinese hamster ovary cells. The recombinant soluble mouse CD4 (smCD4) retained the native conformation of the external portion, as indicated by the binding of L3T4 mAb. In vitro, smCD4 did not inhibit class II-dependent, Ag-specific, T cell proliferation or MLR, even at concentrations 300-fold greater, on a molar basis, than that of anti-CD4 mAb. Immunization of mice with smCD4 induced a strong anti-CD4 response. These antibodies showed some binding to native cell surface CD4, indicating that immunization with smCD4 generated an anti-self response. Analysis of lymphoid cells from spleen, lymph node, and thymus of smCD4-treated mice revealed no alteration in subset phenotypes. Also, Th cell function, as measured by response to soluble Ag, was not compromised. Thus, smCD4 did not inhibit T cell activity in vitro, and the autoimmune response arising from immunization with smCD4 had no apparent consequences for normal immune function.     

Mechanisms of bacterial lipopolysaccharide-induced endothelial apoptosis

Gram-negative bacterial sepsis remains a common, life-threatening event. The prognosis for patients who develop sepsis-related complications, including the development of acute respiratory distress syndrome (ARDS), remains poor. A common finding among patients and experimental animals with sepsis and ARDS is endothelial injury and/or dysfunction. A component of the outer membrane of gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin, has been implicated in the pathogenesis of much of the endothelial cell injury and/or dysfunction associated with these disease states. LPS is a highly proinflammatory molecule that elicits a wide array of endothelial responses, including the upregulation of cytokines, adhesion molecules, and tissue factor. In addition to activation, LPS induces endothelial cell death that is apoptotic in nature. This review summarizes the evidence for LPS-induced vascular endothelial injury and examines the molecular signaling pathways that activate and inhibit LPS-induced endothelial apoptosis. Furthermore, the role of apoptotic signaling molecules in mediating LPS-induced activation of endothelial cells will be considered.     

Production of soluble Neprilysin by endothelial cells

A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC₅₀ values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.     

Mechanisms regulating the sorting of soluble lysosomal proteins

Lysosomes are key regulators of many fundamental cellular processes such as metabolism, autophagy, immune response, cell signalling and plasma membrane repair. These highly dynamic organelles are composed of various membrane and soluble proteins, which are essential for their proper functioning. The soluble proteins include numerous proteases, glycosidases and other hydrolases, along with activators, required for catabolism. The correct sorting of soluble lysosomal proteins is crucial to ensure the proper functioning of lysosomes and is achieved through the coordinated effort of many sorting receptors, resident ER and Golgi proteins, and several cytosolic components. Mutations in a number of proteins involved in sorting soluble proteins to lysosomes result in human disease. These can range from rare diseases such as lysosome storage disorders, to more prevalent ones, such as Alzheimer’s disease, Parkinson’s disease and others, including rare neurodegenerative diseases that affect children. In this review, we discuss the mechanisms that regulate the sorting of soluble proteins to lysosomes and highlight the effects of mutations in this pathway that cause human disease. More precisely, we will review the route taken by soluble lysosomal proteins from their translation into the ER, their maturation along the Golgi apparatus, and sorting at the trans-Golgi network. We will also highlight the effects of mutations in this pathway that cause human disease.     

Mechanisms of impairment of precise movements during long-term hypokinesia

Antiorthostaic hypokinesia (AOHK) was found to entail a considerable diminishing of the control system's precision abilities. In the first 14-30 days of the AOHK (the 1st stage), variability of interimpike intervals (ISI) was sharply increased as well as the degree of synchronisation of the motor units' (MU) activity; starting from the 30th day (the 2nd stage), a regular diminishing of the ISI occurred and the MU synchronisation disappeared. The data obtained suggest the different nature of the precision disorders during the 1st and the 2nd stages of the AOHK: a reflex responses to support unloading and the atrophic processes in the muscles, respectively.     

Dissociation of ERK and Akt signaling in endothelial cell angiogenic responses to beta-amyloid

Cerebrovascular deposits of beta-amyloid (Abeta) peptides are found in Alzheimer's disease and cerebral amyloid angiopathy with stroke or dementia. Dysregulations of angiogenesis, the blood-brain barrier and other critical endothelial cell (EC) functions have been implicated in aggravating chronic hypoperfusion in AD brain. We have used cultured ECs to model the effects of beta-amyloid on the activated phosphorylation states of multifunctional serine/threonine kinases since these are differentially involved in the survival, proliferation and migration aspects of angiogenesis. Serum-starved EC cultures containing amyloid-beta peptides underwent a 2- to 3-fold increase in nuclear pyknosis. Under growth conditions with sublethal doses of beta-amyloid, loss of cell membrane integrity and inhibition of cell proliferation were observed. By contrast, cell migration was the most sensitive to Abeta since inhibition was significant already at 1 muM (P = 0.01, migration vs. proliferation). In previous work, intracellular Abeta accumulation was shown toxic to ECs and Akt function. Here, extracellular Abeta peptides do not alter Akt activation, resulting instead in proportionate decreases in the phosphorylations of the MAPKs: ERK1/2 and p38 (starting at 1 microM). This inhibitory action occurs proximal to MEK1/2 activation, possibly through interference with growth factor receptor coupling. Levels of phospho-JNK remained unchanged. Addition of PD98059, but not LY294002, resulted in a similar decrease in activated ERK1/2 levels and inhibition of EC migration. Transfection of ERK1/2 into Abeta-poisoned ECs functionally rescued migration. The marked effect of extracellular Abeta on the migration component of angiogenesis is associated with inhibition of MAPK signaling, while Akt-dependent cell survival appears more affected by cellular Abeta.     

血管内皮型一氧化氮合酶的调节机制

血管内皮型一氧化氮合酶(eNOS)的调控机制可分为基因表达水平调节和蛋白水平调节两个方面。其中,eNOS的基因表达水平调节主要包含启动子的调节和mRNA的稳定性调节两方面。而eNOS的蛋白水平调节又可分为三个方面:eNOS细胞内转位的调节机制;eNOS复合体形成的调节机制;eNOS氨基酸残基磷酸化的调节机制。eNOS的分子调控机制与临床疾病的发生、发展及其治疗有着密切的关系,故对eNOS分子调控机制的进一步了解有着非常重要的意义。     

Mechanisms of glucocorticoid-induced growth retardation: impairment of cartilage mineralization

Immature female mice were treated with daily doses of triamcinolone diacetate. Using histochemical and fluorescent methods, the mineralization pattern of the proximal epiphyseal plate of the humerus was studied at various intervals. Following 1 injection, a noticeable decrease was noted in the oxytetracycline incorporation at the mineralizing zone of the growth plate. By the 10th injection, the mineralization process was almost totally arrested. Marked accumulation of phospholipids appeared in experimental plates concomitantly with a lack of acidic glycosaminoglycans' synthesis as well as degradation. Chondroclastic activity was also inhibited resulting in an increased number of hypertrophic chondrocytes at the mineralization zone. It is suggested that the hormone's antianabolic effect led to an impairment in the activity of the chondrocytes' hydrolytic enzymes and thereby interfered with the maturation of the matrix, a prerequisite phase for normal mineralization.     

The expression of p75 neurotrophin receptor protects against the neurotoxicity of soluble oligomers of beta-amyloid

In this paper, evidence is provided that p75 neurotrophin receptor (p75NTR) exerts an opposite role on the cytotoxic function of β-amyloid (Aβ) depending on the different state of the peptide, fibrillar or oligomeric soluble form. Previous work in our laboratory has shown that the expression of p75NTR is required for cell death in vitro by Aβ peptides in fibrillar form (G. Perini, V. Della-Bianca, V. Politi, G. Della Valle, I. Dal-Pra, F. Rossi, U. Armato. Role of p75 neurotrophin receptor in the neurotoxicity by beta-amyloid peptides and synergistic effect of inflammatory cytokines. J. Exp. Med. 195 (2002) 907-918). In the present study, performed by using the same cell clones and procedures as in previous paper, we show that: (a) soluble oligomers of Aβ(1-42) exert a cytotoxic activity independent of p75NTR, (b) the expression of p75NTR exerts a protective role against the toxic activity of soluble oligomers, (c) this role is due to an active function of the juxtamembrane sequence of the cytoplasmic region of p75NTR and (d) the protective function is mediated by phosphatidylinositide 3-kinase (PI3K) activity.     

Production of soluble P-selectin by platelets and endothelial cells

The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for approximately 10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A2 analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up approximately 10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.