Constitutive activation of peroxisome proliferator-activated receptor-gamma suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated nuclear receptor that has an essential role in adipogenesis and glucose homeostasis. PPAR-gamma is expressed in vascular tissues including endothelial cells (ECs). PPAR-gamma activity can be regulated by many pathophysiological and pharmacological agonists. However, the role of PPAR-gamma activation in ECs remains unclear. In this study, we examined the effect of the constitutive activation of PPAR-gamma on the phenotypic modulation of ECs. Adenovirus-mediated expression of a constitutively active mutant of PPAR-gamma resulted in significant ligand-independent activation of PPAR-gamma and specific induction of the PPAR-gamma target genes. However, PPAR-gamma activation significantly suppressed the expression of vascular adhesion molecules in ECs and the ensuing leukocyte recruitment. Furthermore, constitutive activation of PPAR-gamma resulted in simultaneous repression of AP-1 and NF-kappaB activity, which suggests that PPAR-gamma may reduce pro-inflammatory phenotypes via, at least in part, suppression of the AP-1 and NF-kappaB pathways. Therefore, using a gain-of-function approach, our study provides novel evidence showing that constitutive activation of PPAR-gamma is sufficient to prevent ECs from converting into a pro-inflammatory phenotype. These results also suggest that, in addition to pharmacological agonists, the genetic modification of the PPAR-gamma activity in ECs may be a potential approach for therapeutic intervention in various inflammatory disorders.     

Biochemical properties of vascular endothelial cells

Present knowledge in the field of vascular endothelial cells is reviewed. The role of endothelial cells in the synthesis of matrix proteins and glycosaminoglycans is described. Endothelial cells play a considerable role in the processes of coagulation and fibrinolysis. They also interact with neurotransmitters and vasomotoric substances, and participate in inflammation and immunological responses. They procuce several different growth factors. Their role in lipoprotein metabolism is of special importance to research into atherosclerosis.     

血管内皮细胞内质网应激

内质网是调控细胞内膜型/分泌型蛋白质合成、钙稳态和细胞凋亡的重要细胞器,多种因素影响内质网稳态、触发内质网应激。适当的内质网应激通过激活未折叠蛋白反应促进内质网紊乱的恢复,但过度内质网应激触发内质网相关凋亡途径,参与多种疾病的发生。血管内皮细胞具有高度发达的内质网,对内质网应激非常敏感,本文综述血管内皮细胞内质网应激反应及其在血管损伤相关疾病中的作用。     

血管内皮细胞容量激活的氯通道

氯通道是血管内皮细胞上主要的离子通道,容量激活的氯通道是其中一种主要类型并广为研究。已经主宰容量激活的氯通道在维持静息膜电位,调节细胞内钙、ｐＨ值,影响细胞增殖和分化中起着重要的作用。本文综述了血管内皮细胞容量激活氯通道的基本电生理及分子生物学特性,并初步探讨该通道的调节机制。     

Chained vesicles in vascular endothelial cells.

There is extensive ultrastructural evidence in endothelium for the presence of chained vesicles or clusters of attached vesicles, and they are considered to be involved in specific transport mechanisms, such as the formation of trans-endothelial channels. However, few details are known about their mechanical characteristics. In this study, the formation mechanism and mechanical aspects of vascular endothelial chained vesicles are investigated theoretically, based on membrane bending strain energy analysis. The shape of the axisymmetric vesicles was computed on the assumption that the cytoplasmic side of the vesicle has a molecular layer or cytoskeleton attached to the lipid bilayer, which induces a spontaneous curvature in the resting state. The bending strain energy is the only elasticity involved, while the shear elasticity is assumed to be negligible. The surface area of the membrane is assumed to be constant due to constant lipid bilayer thickness. Mechanically stable shapes of chained vesicles are revealed, in addition to a cylindrical tube shape. Unfolding of vesicles into a more flattened shape is associated with increase in bending energy without a significant increase in membrane tension. These results provide insights into the formation mechanism and mechanics of the chained vesicle.     

Estrogen induces the Akt-dependent activation of endothelial nitric-oxide synthase in vascular endothelial cells

Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells (HUVECs) and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), 17beta-estradiol (E2), but not 17alpha-E2, caused acute activation of eNOS that was unaffected by actinomycin D and was specifically blocked by the pure estrogen receptor antagonist ICI-182,780. Treatment of both TRLECs and HUVECs with 17beta-E2 stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the 17beta-E2-induced activation of Akt. 17beta-E2-induced Akt activation was also inhibited by ICI-182,780, but not by actinomycin D. Either treatment with wortmannin or exogenous expression of a dominant negative Akt in TRLECs decreased the 17beta-E2-induced eNOS activation. Moreover, 17beta-E2-induced Akt activation actually enhances the phosphorylation of eNOS. 17beta-E2-induced Akt activation was dependent on both extracellular and intracellular Ca(2+). We further examined the 17beta-E2-induced Akt activity in Chinese hamster ovary (CHO) cells transiently transfected with cDNAs for estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta). 17beta-E2 stimulated the activation of Akt in CHO cells expressing ERalpha but not in CHO cells expressing ERbeta. Our findings suggest that 17beta-E2 induced eNOS activation through an Akt-dependent mechanism, which is mediated by ERalpha via a nongenomic mechanism.     

Receptor-regulated dynamic S-nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells

The endothelial isoform of nitric-oxide synthase (eNOS) is regulated by a complex pattern of post-translational modifications. In these studies, we show that eNOS is dynamically regulated by S-nitrosylation, the covalent adduction of nitric oxide (NO)-derived nitrosyl groups to the cysteine thiols of proteins. We report that eNOS is tonically S-nitrosylated in resting bovine aortic endothelial cells and that the enzyme undergoes rapid transient denitrosylation after addition of the eNOS agonist, vascular endothelial growth factor. eNOS is thereafter progressively renitrosylated to basal levels. The receptor-mediated decrease in eNOS S-nitrosylation is inversely related to enzyme phosphorylation at Ser(1179), a site associated with eNOS activation. We also document that targeting of eNOS to the cell membrane is required for eNOS S-nitrosylation. Acylation-deficient mutant eNOS, which is targeted to the cytosol, does not undergo S-nitrosylation. Using purified eNOS, we show that eNOS S-nitrosylation by exogenous NO donors inhibits enzyme activity and that eNOS inhibition is reversed by denitrosylation. We determine that the cysteines of the zinc-tetrathiolate that comprise the eNOS dimer interface are the targets of S-nitrosylation. Mutation of the zinc-tetrathiolate cysteines eliminates eNOS S-nitrosylation but does not eliminate NO synthase activity, arguing strongly that disruption of the zinc-tetrathiolate does not necessarily lead to eNOS monomerization in vivo. Taken together, these studies suggest that eNOS S-nitrosylation may represent an important mechanism for regulation of NO signaling pathways in the vascular wall.     

Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.     

Cytotoxicity of water-soluble fullerene in vascular endothelial cells

Nanoscale materials are presently under development for diagnostic (nanomedicine) and electronic purposes. In contrast to the potential benefits of nanotechnology, the effects of nanomaterials on human health are poorly understood. Nanomaterials are known to translocate into the circulation and could thus directly affect vascular endothelial cells (ECs), causing vascular injury that might be responsible for the development of atherosclerosis. To explore the direct effects of nanomaterials on endothelial toxicity, human umbilical vein ECs were treated with 1–100 µg/ml hydroxyl fullerene [C₆₀(OH)₂₄; mean diameter, 7.1 ± 2.4 nm] for 24 h. C₆₀(OH)₂₄ induced cytotoxic morphological changes such as cytosolic vacuole formation and decreased cell density in a dose-dependent manner. Lactate dehydrogenase assay revealed that a maximal dose of C₆₀(OH)₂₄ (100 µg/ml) induced cytotoxic injury. Proliferation assay also showed that a maximal dose of C₆₀(OH)₂₄ inhibited EC growth. C₆₀(OH)₂₄ did not seem to induce apoptosis but caused the accumulation of polyubiquitinated proteins and facilitated autophagic cell death. Formation of autophagosomes was confirmed on the basis of Western blot analysis using a specific marker, light chain 3 antibody, and electron microscopy. Chronic treatment with low-dose C₆₀(OH)₂₄ (10 µg/ml for 8 days) inhibited cell attachment and delayed EC growth. In the present study, we have examined, for the first time, the toxicity of water-soluble fullerenes to ECs. Although fullerenes changed morphology in a dose-dependent manner, only maximal doses of fullerenes caused cytotoxic injury and/or death and inhibited cell growth. EC death seemed to be caused by activation of ubiquitin-autophagy cell death pathways. Although exposure to nanomaterials appears to represent a risk for cardiovascular disorders, further in vivo validations are necessary. nanomaterials; ubiquitin proteasome; autophagy; atherosclerosis     

Role of vascular endothelial cells in bone biology

Bone development and remodeling depend on complex interactions between bone-forming osteoblasts, bone-degrading osteoclasts, and other cells present within the bone microenvironment. Balanced control of bone formative and degradative processes is normally carefully maintained in the adult skeleton but becomes uncoupled in the course of aging or in various pathological disease states. Systemic regulators of bone metabolism and local mediators, including matrix molecules, cytokines, prostaglandins, leukotrienes, and other autocrine or paracrine factors, regulate the recruitment, differentiation, and function of cells participating in bone formation and turnover. Although some of these interactions are now understood, many yet remain to be elucidated. Recent studies have begun exploring in detail how vascular endothelial cells and their products function in bone physiology. The findings are revealing that bone vascular endothelial cells may be members of a complex communication network in bone which operates between endothelial cells, osteoblasts, osteoclasts, macrophages, stromal cells, and perhaps other cell types found in bone as well. Therefore, multiple systemic and locally produced signals may be received, transduced, and integrated by individual cells and then propagated by the release from these cells of further signals targeted to other members of the bone cell network. In this manner, bone cell activities may be continuously coordinated to afford concerted actions and rapid responses to physiological changes. The bone microvasculature may play a pivotal role in these processes, both in linking circulatory and local signals with cells of the bone microenvironment and in actively contributing itself to the regulation of bone cell physiology. Thus, skeletal homeostasis and the coupling observed between bone resorption and bone formation during normal bone remodeling may be manifestations of this dynamic interactive communication network, operating via diverse signals not only between osteoblasts and osteoclasts but between many cell types residing within bone. © 1994 Wiley-Liss, Inc.     

Phenotypic heterogeneity of vascular endothelial cells in the human kidney

Summary To clarify the structural base of immune response occurring in the kidney, we investigated the antigenic and functional properties of vascular endothelial cells. Peritubular capillary endothelial cells exhibited the same immuno-histochemical characteristics (OKM5-positive, HLA-DR-positive, Factor VIII/von Willebrand factor antigen-negative, Interleukin 1-positive) as a peripheral blood macrophage subset capable of presenting soluble antigens and triggering the autologous mixed lymphocyte reaction. On the other hand, endothelial cells of glomerular capillary loops, considered to be involved in blood coagulation, were OKM5-negative, HLA-DR-positive, Factor VIII/von Willebrand factor antigen-positive, Interleukin 1-positive. Thus the results of this study suggest that vascular endothelial cells in different anatomic compartments of the kidney express surface antigens heterogenously and may play different roles in the immune reaction.     

Heterotypic and homotypic cell-cell adhesion molecules in endothelial cells

Sickle red blood cells display an abnormal propensity to adhere to cultured bovine aortic endothelial cells when compared to normal red blood cells. The adherence was potentiated three-fold by endothelial cell derived conditioned medium, enriched in multimers of von Willebrand factor. Such adherence was ablated by 80% by either the synthetic peptide (RGDS) or antibody to GPIIb/IIIa, indicating the presence of RGD peptide recognition domain/receptor in either endothelial cells or sickle cells or both. The adherence was also inhibited by 70% by phosphatidylserine, but not by other phospholipids, indicating the presence of putative receptors for this phospholipid in endothelial cells. The labeling of cultured bovine aortic endothelial cells with monoclonal antibodies revealed the localization of MAB D2 to regions of cell-cell contact. The antigen on endothelial cells which cross-reacts with this antibody has a Mr of 130,000. The addition of such an antibody during the plating of endothelial cells disrupted monolayer formation. It appears that a 130-kDa polypeptide antigen in endothelial cells which is recognized by MAB D2, may be a cell-cell adhesion molecule.     

Biochemical properties of vascular endothelial cells.

Present knowledge in the field of vascular endothelial cells is reviewed. The role of endothelial cells in the synthesis of matrix proteins and glycosaminoglycans is described. Endothelial cells play a considerable role in the processes of coagulation and fibrinolysis. They also interact with neurotransmitters and vasomotoric substances, and participate in inflammation and immunological responses. They produce several different growth factors. Their role in lipoprotein metabolism is of special importance to research into atherosclerosis.     

Conversion of vascular endothelial cells into multipotent stem-like cells

Mesenchymal stem cells can give rise to several cell types, but varying results depending on isolation methods and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an activin-like kinase-2 (ALK2) receptor-dependent mechanism. In lesions from individuals with fibrodysplasia ossificans progressiva (FOP), a disease in which heterotopic ossification occurs as a result of activating ALK2 mutations, or from transgenic mice expressing constitutively active ALK2, chondrocytes and osteoblasts expressed endothelial markers. Lineage tracing of heterotopic ossification in mice using a Tie2-Cre construct also suggested an endothelial origin of these cell types. Expression of constitutively active ALK2 in endothelial cells caused endothelial-to-mesenchymal transition and acquisition of a stem cell-like phenotype. Similar results were obtained by treatment of untransfected endothelial cells with the ligands transforming growth factor-β2 (TGF-β2) or bone morphogenetic protein-4 (BMP4) in an ALK2-dependent manner. These stem-like cells could be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. We suggest that conversion of endothelial cells to stem-like cells may provide a new approach to tissue engineering.     

Cation transport in vascular endothelial cells and aging

Summary To understand the generation and maintenance of Na and K gradients in cultured vascular endothelial cells, net Na and K movements were studied. Ouabain-sensitive (OS) net Na gain and K loss were estimated as the difference between the cation content in the presence of ouabain and that in the control. Ouabain-and furosemide-resistant (OFR) fluxes were determined in the presence of the two inhibitors. When the normal medium bicarbonate and phosphate buffers were replaced by N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid both the OS ans OFR fluxes decreased more than 50%. Ouabain-sensitive and ouabain-and furosemide-resistant fluxes decreased with increasing cellular age (passage number) an effect not observed when the cation movements were studied in the absence of bicarbonate and phosphate. These results suggest that cultured vascular endothelial cells possess bicarbonate-and phosphate-dependent Na and K pathways which account for a significant portion of their passive movements. Furthermore, the behavior of cation permeabilities with passage number suggests that these modulations may be related to the cellular aging process.     

AlphaB-crystallin inhibits glucose-induced apoptosis in vascular endothelial cells

Recent studies implicate hyperglycemia as a cause of vascular complications in diabetes. Our study confirmed that high concentration of glucose (30 mM) induces apoptosis in cultures of human umbilical vein endothelial cells. After 5 days of culture TUNEL positive cells in high concentration of glucose were nearly 63% higher when compared to normal concentration of glucose (5 mM). Transfection of pcDNA3-rat alphaB-crystallin into these cells inhibited high glucose-induced apoptosis by approximately 36%, such an effect was not observed when cells were transfected with an empty vector. AlphaB-crystallin transfection inhibited by about 35% of high glucose induced activation of caspase-3. High concentration of glucose enhanced formation of reactive oxygen species (ROS) in these cells but this was significantly (p < 0.001) curtailed by transfection of alphaB-crystallin. Results of our study indicate that alphaB-crystallin effectively inhibits both ROS formation and apoptosis in cultured vascular endothelial cells and provide a basis for future therapeutic interventions in diabetic vascular complications.