Ras guanyl nucleotide releasing protein 2 affects cell viability and cell–matrix adhesion in ECV304 endothelial cells

Ras guanyl nucleotide releasing proteins (RasGRPs) are guanine nucleotide exchange factors that activate Ras and Rap. We recently reported that xrasgrp2, which is a homolog of the human rasgrp2, plays a role in vasculogenesis and/or angiogenesis during early development of Xenopus embryos. However, the function of RasGRP2 in human vascular endothelium remains unknown. Therefore we aimed to analyze the function of human RasGRP2 in vascular endothelial cells. RasGRP2 overexpression did not increase Ras activation. However, it slightly increased Ras expression and increased proliferation in ECV304 cells. Furthermore, RasGRP2 overexpression increased Rap1 activation and cell–matrix adhesion in ECV304 cells. These data demonstrate that RasGRP2 increases cell viability and cell–matrix adhesion through increased Ras expression and Rap1 activation, respectively, in endothelial cells.     

Localization and activity of calmodulin is involved in cell–cell adhesion of tumor cells and endothelial cells in response to hypoxic stress

Adhesion of tumor cells to endothelial cells is known to be involved in the hematogenous metastasis of cancer, which is regulated by hypoxia. Hypoxia is able to induce a significant increase in free intracellular Ca²⁺ levels in both tumor cells and endothelial cells. Here, we investigate the regulatory effects of calmodulin (CaM), an intracellular calcium mediator, on tumor cell–endothelial cell adhesion under hypoxic conditions. Hypoxia facilitates HeLa cell–ECV304 endothelial cell adhesion, and results in actin cytoskeleton rearrangement in both endothelial cells and tumor cells. Suppression of CaM activation by CaM inhibitor W-7 disrupts actin cytoskeleton organization and CaM distribution in the cell–cell contact region, and thus inhibits cell–cell adhesion. CaM inhibitor also downregulates hypoxia-induced HIF-1-dependent gene expression. These results suggest that the Ca²⁺-CaM signaling pathway might be involved in tumor cell-endothelial cell adhesion, and that co-localization of CaM and actin at cell–cell contact regions might be essential for this process under hypoxic stress. W.-G. Shen and W.-X. Peng Contributed to this paper equally     

Binding Modes of Phthalocyanines to Amyloid β Peptide and Their Effects on Amyloid Fibril Formation

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer’s and Parkinson’s diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid β-peptide (Aβ) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aβ₄₀ revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aβ₄₀-PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aβ₄₀-PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aβ₄₀ and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of pathological aggregates.     

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β₂-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.     

A hot water extract of Curcuma longa inhibits adhesion molecule protein expression and monocyte adhesion to TNF-α-stimulated human endothelial cells

The recruitment of arterial leukocytes to endothelial cells is an important step in the progression of various inflammatory diseases. Therefore, its modulation is thought to be a prospective target for the prevention or treatment of such diseases. Adhesion molecules on endothelial cells are induced by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), and contribute to the recruitment of leukocytes. In the present study, we investigated the effect of hot water extract of Curcuma longa (WEC) on the protein expression of adhesion molecules, monocyte adhesion induced by TNF-α in human umbilical vascular endothelial cells (HUVECs). Treatment of HUVECs with WEC significantly suppressed both TNF-α-induced protein expression of adhesion molecules and monocyte adhesion. WEC also suppressed phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) induced by TNF-α in HUVECs, suggesting that WEC inhibits the NF-κB signaling pathway.     

Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-κB-dependent pathway, leading to monocyte adhesion to endothelial cells

Enterotoxigenic Bacteroides fragilis (ETBF) produces a ～ 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in mucosal inflammation. Although a variety of inflammatory cells is found at ETBF-infected sites, little is known about leukocyte adhesion in response to BFT stimulation. We investigated whether BFT affected the expression of ICAM-1 and monocytic adhesion to endothelial cells (ECs). Stimulation of HUVECs and rat aortic ECs with BFT resulted in the induction of ICAM-1 expression. Upregulation of ICAM-1 was dependent on the activation of IκB kinase (IKK) and NF-κB signaling. In contrast, suppression of AP-1 did not affect ICAM-1 expression in BFT-stimulated cells. Suppression of NF-κB activity in HUVECs significantly reduced monocytic adhesion, indicating that ICAM-1 expression is indispensable for BFT-induced adhesion of monocytes to the endothelium. Inhibition of JNK resulted in a significant attenuation of BFT-induced ICAM-1 expression in ECs. Moreover, inhibition of aldose reductase significantly reduced JNK-dependent IKK/NF-κB activation, ICAM-1 expression, and adhesion of monocytes to HUVECs. These results suggest that a signaling pathway involving aldose reductase, JNK, IKK, and NF-κB is required for ICAM-1 induction in ECs exposed to BFT, and may be involved in the leukocyte-adhesion cascade following infection with ETBF.     

IFN-γ-induced BST2 mediates monocyte adhesion to human endothelial cells

BST2 is a type II transmembrane protein that had been initially identified as a surface molecule expressed on terminally differentiated B cells. Here, we characterize the expression of BST2 in human endothelial cells, HUVECs. IFN-γ, among various inflammatory stimuli, dramatically upregulates BST2 expression in HUVECs. We also address a novel putative role of BST2 in IFN-γ-stimulated HUVECs as an intercellular adhesion-related molecule. We show that purified extracellular domain of BST2 protein specifically and significantly decreased the adhesion of human monocytes to HUVECs, which suggests that IFN-γ-induced BST2 expression may be involved in monocyte migration from blood through the endothelium to the inflammation site. Furthermore, we show that the monocytic cell line U937 can directly adhere to BST2 extracellular domain-coated tissue culture wells. These results provide experimental evidence to support a novel role for BST2 in the interaction between human monocyte and IFN-γ-stimulated endothelium.     

Amyloid β protein and Alzheimer disease

Amyloid beta protein is predominant in senile plaques, the neuropathologic hallmarks of Alzheimer disease. Researchers in Winnipeg have shown that this protein can overstimulate certain hydrolytic enzymes to break down the phospholipid building blocks of the brain-cell wall. They speculate that the abnormal destruction of phospholipids gradually drains the energy resources a neuron uses to rebuild its membrane. As neurons "burn out," the brain loses its ability to function normally. In view of evidence that NSAID therapy may interfere with the hydrolysis of phospholipids, the researchers will focus on finding an NSAID-related compound effective against Alzheimer disease.     

Interactions of Amyloid β Peptide 1–40 and Cerebrosterol

Amyloid β peptides appear to play a role in physiological processes; however, they are also involved in the pathogenesis of Alzheimer disease. Their actions under normal conditions are probably mediated by soluble monomeric l-isoforms at low concentrations, perhaps via highly specific interactions. On the contrary, toxic effects of aggregated natural l-isoforms/synthetic d-isoforms on membranes are very similar, but synthetic reverse/random l-isoforms without pronounced aggregation properties are not toxic. Our previous work reported interactions of non-aggregated/aggregated l-isoforms of amyloid β peptides 1–40/1–42 with racemic 24-hydroxycholesterol. In this study, stereospecificity in the interactions of natural 24(S)hydroxycholesterol (cerebrosterol) or synthetic 24(R)hydroxycholesterol with soluble fragment 1–40 was evaluated by means of an in vitro test based on increased vulnerability of the hemicholinium-3 sensitive high-affinity choline uptake system in rat hippocampal cholesterol-depleted synaptosomes to the actions of amyloid β; computational simulations were also performed. Our results suggest that: (1) 24(S)hydroxycholesterol interacts with l-peptide 1–40 but not with the reverse l-peptide 40–1, (2) 24(R)hydroxycholesterol does not interact with l-peptide 1–40 or reverse 40–1, and (3) both enantiomers can probably interact with d-peptide 1–40. Therefore, the binding of 24(S)hydroxycholesterol is not fully stereospecific and the interaction could not reflect a physiological mechanism. Data from the computational simulation indicate that the hydrophobic core of the amyloid β molecule interacts with the hydrophobic part of 24(S)hydroxycholesterol, but no hydrogen bonds with high stability were found. Using this procedure, globular amyloid β could retain 24(S)hydroxycholesterol and thus contribute to its pathological accumulation in the brains of patients with Alzheimer disease.     

Sarpogrelate inhibits the expression of ICAM-1 and monocyte–endothelial adhesion induced by high glucose in human endothelial cells

Hyperglycemia is the major cause of diabetic angiopathy. Sarpogrelate hydrochloride is an antiplatelet drug, and expected to be useful in the treatment of chronic arterial occlusive diseases. The aim of our study was to evaluate the possible effects of sarpogrelate hydrochloride on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. Intercellular adhesion molecule-1 (ICAM-1) expression and superoxide dismutase (SOD) activity were determined after endothelial cells were exposed to high glucose in the absence and presence of sarpogrelate hydrochloride. Coincubation of endothelial cells with high glucose for 24 h resulted in a significant increase of monocyte–endothelial cell adhesion and the expression of ICAM-1 (P < 0.01). These effects were abolished by sarpogrelate hydrochloride and sarpogrelate hydrochloride significantly increased SOD activities (40 ± 8 vs. 47 ± 7, n = 8, P < 0.01). The low dose sarpogrelate group (0.1 μM) had significantly higher monocyte–endothelial cell adhesion and the expression of ICAM-1 than medium dose sarpogrelate group (1.0 μM) and high dose sarpogrelate group (10.0 μM) (P < 0.05 for comparison among three groups and P < 0.01 for difference between low and high dose sarpogrelate groups). These findings suggested that sarpogrelate hydrochloride was able to protect vascular endothelium from dysfunction induced by high glucose.     

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a "tet-on" inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that (1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; (2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; (3) angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization.     

Trophinin-mediated cell adhesion induces apoptosis of human endometrial epithelial cells through PKC-δ

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-δ protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-δ associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-δ became tyrosine phosphorylated, dissociated from trophinin and entered the nucleus. In PKC-δ knockdown endometrial cells, GWRQ did not induce apoptosis. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch to induce apoptosis through the PKC-δ pathway in endometrial epithelial cells. Thus, trophinin-mediated induction of apoptosis of endometrial epithelial cells, which function as a barrier to embryo invasion, allows trophoblast invasion of maternal tissue and embryo implantation in humans.Key words: blastocyst, embryo implantation, apoptosis, cell adhesion, signal transduction     

Ultrastructural responses of pancreatic β cells to metabolic alkalosis

Summary The ultrastructural changes in pancreatic cells were studied following glucose-induced insulin secretion in vitro, at two different extracellular pH (7.4 and 7.8). The pancreata perfused at pH 7.4 exhibited a biphasic insulin response to glucose challenge together with signs of increased emiocytotic activity and numerous microtubules in the cells. Conversely, the pancreata perfused at pH 7.8 showed a significant decrease in insulin secretion, and their cells revealed scarce emiocytotic images and a marked increase of intracellular granulolysis. These results represent the ultrastructural correlate of the reduced insulin secretion produced by metabolic alkalosis in the perfused rat pancreas.The authors wish to thank Mrs. Elma P. de Gagliardino and Mrs. Susana Rivas for excellent technical assistance.This research was partially supported by funds from CONICET and CIC, Pcia de Bs.As. C.L. Gómez Dumm, O.R. Rebolledo and J.J. Gagliardino are members of Carrera del Investigador del CONICET (Argentina)     

Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells

Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury.This process is primarily regulate...     

Histidine-rich glycoprotein blocks collagen-binding integrins and adhesion of endothelial cells through low-affinity interaction with α2 integrin

The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2β1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.