Cytoplasm Of Endothelial Cells Research Articles

Ultrastructure of endothelium in ovules ofPenstemon gentianoidesPoir. (Scrophulariaceae) at mature embryo sac phase

In this study ultrastructural differences between endothelial cells of different location in Penstemon gentianoides have been examined with electron microscope at mature embryo sac phase. Embryo sac is of the Polygonum type and surrounded by endothelium except the micropylar region. The cuticle is located primarily around the chalazal three-fourths of the embryo sac. Endothelium cells around the chalaza and toward the micropylar region are rich in cytoplasmic organelles. The cytoplasm of endothelial cells near the central cell has large vacuoles and few organelles. There are also plasmodesmas on the anticlinal walls of endothelial cells. The endothelium and the micropylar integumentary cells play a role in transport of metabolites into the embryo sac.

Molecular Anatomy of the Brain Endothelial Barrier: An Overview of the Distributional Features

The blood-brain barrier (BBB) impedes the influx of intravascular compounds from the blood to the brain. The elements composing the BBB are endothelial cells, pericytes and the end-feet of astrocytes. Among them, the endothelial cell barrier line is the most critical for preventing toxic substances from entering the brain. In this review, we focus on the ultrastructural distribution of important components in the intracellular junction and cytoplasm of brain endothelial cells. The ultrastructural distribution of tight junction-specific integral membrane proteins such as occludin, junctional adhesion molecules, claudin, peripheral zonula occludens protein-1 (ZO-1), adherens junction-specific transmembrane protein cadherin, and adherens junction-associated peripheral proteins alpha-catenin, beta-catenin, and p120 catenin is reviewed. P-glycoprotein and some other transporters recently discovered in endothelial cells prevent several compounds from entering the brain parenchyma. It is likely that the transient inhibition of P-glycoprotein by antidepressants enables other medicines to enter the brain. Vesicular transport with clathrin-mediated or adsorptive endocytosis through endothelial cells is also critical for transportation of blood-born substances from the bloodstream to the brain. How medicines pass the BBB to reach the brain parenchyma is discussed.

The Ets-1 Transcription Factor is Involved in Pterygial Angiogenesis

Pterygial pathology is characterized by abnormal corneal epithelial proliferation, stromal modulation, matrix degradation and a strong tendency for otherwise absent corneal vascularization. As the proto-oncogene Ets-1 is known to play a key role in angiogenesis and matrix degradation in other tissues, its involvement in corneal vascularization was investigated. Fifteen pterygia representing two groups were studied. Group 1 consisted of five clinically active pterygia, and group 2 consisted of 10 samples of clinically non-active pterygia. (35)S-labelled ets-1 antisense and sense riboprobes were used for in-situ hybridization of Ets-1 transcription factor in all pterygia. The cytoplasm of blood vessel endothelial cells showed strong expression of ets-1 mRNA in all group 1 pterygia. In contrast, no expression of ets-1 was found in group 2 pterygia. Proto-oncogene ets-1 expression has been shown for the first time in the metaplastic pterygium, an eye tissue of unknown pathogenesis.

The expression and localization of extracellular matrix-degrading proteases in the mammary gland during development, function and involution in the cow

Extracellular matrix (ECM) degrading proteases, like matrix metalloproteases (MMP) and the plasminogen activator (PA) system may play a critical role in extensive remodelling that occurs in the bovine mammary gland during development, lactation and involution. Therefore, the aim of our study was to investigate the mRNA expression of MMP-1, MMP-2, MMP-14, MMP-19, tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, urokinase type (u)PA, tissue type (t)PA, uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) by quantitative real-time PCR (Rotor-Gene) and to localize with immunohistochemistry MMP-1, MMP-2, MMP-14, and TIMP-2 proteins in bovine mammary gland during pubertal mammogenesis, lactogenesis, galactopoiesis and involution. Expression of mRNA for each of the studied factors was relatively lower during galactopoiesis and early involution but was markedly increased during mammogenesis and late involution, two stages where tissue remodeling is especially pronounced. The localization of proteins for MMP-1, MMP-14 and TIMP-2 showed a similar trend with strong staining intensity in cytoplasm of mammary duct and alveolar epithelial cells during pubertal mammogenesis and late involution. Interestingly, MMP-2 protein was only localized in cytoplasm of endothelial cells during late involution. Our study demonstrated clearly that expression of extracellular matrix-degrading proteases coincides with a concomitant expression of their inhibitors. High expression levels of MMP, TIMP and PA family members seem to be a typical feature of non-lactating mammary gland.

The Blood-Brain Barrier and Cancer: Transporters, Treatment, and Trojan Horses

Despite scientific advances in understanding the causes and treatment of human malignancy, a persistent challenge facing basic and clinical investigators is how to adequately treat primary and metastatic brain tumors. The blood-brain barrier is a physiologic obstruction to the delivery of systemic chemotherapy to the brain parenchyma and central nervous system (CNS). A number of physiologic properties make the endothelium in the CNS distinct from the vasculature found in the periphery. Recent evidence has shown that a critical aspect of this barrier is composed of xenobiotic transporters which extrude substrates from the brain into the cerebrospinal fluid and systemic circulation. These transporters also extrude drugs and toxins if they gain entry into the cytoplasm of brain endothelial cells before they enter the brain. This review highlights the properties of the blood-brain barrier, including the location, function, and relative importance of the drug transporters that maintain this barrier. Primary and metastatic brain malignancy can compromise this barrier, allowing some access of chemotherapy treatment to reach the tumor. The responsiveness of brain tumors to systemic treatment found in past clinical research is discussed, as are possible explanations as to why CNS tumors are nonetheless able to evade therapy. Finally, strategies to overcome this barrier and better deliver chemotherapy into CNS tumors are presented.

Expression and Localization of Extracellular Matrix-Degrading Proteinases and Their Inhibitors in the Bovine Mammary Gland During Development, Function, and Involution

In degrading the extracellular matrix, matrix metalloproteinases (MMP) and the plasminogen activator (PA) system may play a critical role in extensive remodeling that occurs in the bovine mammary gland during development, lactation, and involution. Therefore, the aim of our study was to investigate the mRNA expression of MMP-1, MMP-2, MMP-14, MMP-19, tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, urokinase-type PA, tissue-type PA, urokinase-type PA receptor, and PA inhibitor-1 by quantitative PCR and to localize with immunohistochemistry MMP-1, MMP-2, MMP-14, and TIMP-2 proteins in the bovine mammary gland during pubertal mammogenesis, lactogenesis, galactopoiesis, and involution. Expression of mRNA for each of the studied factors was relatively lower during galactopoiesis and early involution but was markedly increased during mammogenesis and late involution, 2 stages in which tissue remodeling is especially pronounced. The localization of proteins for MMP-1, MMP-14, and TIMP-2 showed a similar trend with strong staining intensity in cytoplasm of mammary duct and alveolar epithelial cells during pubertal mammogenesis and late involution. Interestingly, MMP-2 protein was localized only in the cytoplasm of endothelial cells during late involution. Our study demonstrated clearly that expression of extracellular matrix-degrading proteinases coincides with a concomitant expression of their inhibitors. High expression levels of MMP, TIMP, and PA family members seem to be a typical feature of the nonlactating mammary gland.

The up‐regulation of type I plasminogen activator inhibitor in human gingival fibroblasts stimulated with cyclosporin A

Cyclosporin A is used as an immunosuppressive agent and its prominent side-effect is the induction of fibrous gingival overgrowth. The progression of fibrous gingival overgrowth results from the accumulation of extracellular matrix. Type I plasminogen activator inhibitor (PAI-1) acts as the inhibitor of extracellular matrix degradation and is involved in some fibrotic diseases. However, little is known about the correlation between PAI-1 and cyclosporin A-induced gingival overgrowth. The aim of this study was to investigate the effects of cyclosporin A on the expression of PAI-1 mRNA and protein in human gingival fibroblasts human gingival fibroblasts in vitro and to compare PAI-1 expression in normal healthy gingival tissues and cyclosporin A-induced gingival overgrowth specimens in vivo. Quantitative reverse transcription-polymerase chain reaction and western blot assay were used to investigate the effects on human gingival fibroblasts exposed to cyclosporin A. In addition, 10 cyclosporin A-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Investigations of the time dependence of PAI-1 mRNA expression in human gingival fibroblasts treated with 200 ng/ml of cyclosporin A revealed a rapid accumulation of the transcript: a significant signal was first detectable after 1 h of exposure and the signal remained elevated throughout the 24-h incubation period (p < 0.05). Cyclosporin A was also found to up-regulate PAI-1 protein in a time-dependent manner (p < 0.05). The PAI-1 staining in gingival tissue was stronger in the cyclosporin A-induced gingival overgrowth group than in the normal gingival group (p < 0.05). In the cyclosporin A-induced gingival overgrowth group, intensive staining for PAI-1 expression was observed mainly in the cytoplasm of fibroblasts, endothelial cells and inflammatory cells. These findings suggest that the up-regulation of PAI-1 may play an important part in the molecular pathogenesis of cyclosporin A-induced gingival overgrowth.

Modulation of apelin and APJ receptor in normal and preeclampsia-complicated placentas.

Apelin is an endogenous ligand of the human orphan receptor APJ. This peptide is produced through processing from the C-terminal portion in the pre-pro-protein consisting of 77 amino acid residues and exists in multiple molecular forms. Although the main physiological functions of apelin have not yet been clarified, it is known that apelin is involved in the regulation of blood pressure, blood flow and central control of body fluid homeostasis in different organs. Since human placenta is a tissue where vasculogenesis, blood pressure and flow are dramatically important to allow a normal embryonic and fetal growth and development, the aim of the present study was to investigate the immunohistochemical distribution of apelin and APJ in normal placentas throughout pregnancy and in preeclampsia-complicated placentas. Specifically, we observed that in normal placentas the expression levels of apelin decreased from the first to the third trimester of gestation in both cytotrophoblast and syncytiotrophoblast cells and in the stroma of placental villi, in contrast with increased expression levels of APJ in the cytoplasm of cytotrophoblast cells and in the cytoplasm of endothelial cells of normal placenta samples. In contrast, in preeclampsia-complicated pregnancies, we observed a very strong increase of expression levels of both apelin and APJ receptor in all the placental compartments, cytotrophoblast, syncytiotrophoblast and stroma with a particular increase in endothelial cells inside preeclamptic placental villi. Our data seem to indicate an important role of apelin and APJ in the regulation of fetal development through a correct regulation of human placenta formation during pregnancy. Moreover, the strong expression levels of apelin and APJ in preeclamptic placentas, suggest their possible involvement in the onset of this pathology.

Strong suppression of high-sensitivity C-reactive protein level and its mediated pro-atherosclerotic effects with simvastatin: In vivo and in vitro studies

Background We hypothesize that high-sensitivity C-reactive protein (hs-CRP) levels and cell adhesion molecules (CAMs) significantly reflect serial changes in patients with atherosclerotic-risk factors undergoing simvastatin therapy. We further hypothesize that the site specificity of CRP on the expression of CAMs, which can be inhibited by simvastatin, is in the cytoplasm of endothelial cells (EC). Methods and results Serum hs-CRP levels and vascular (V) CAM-1 were measured (on days 0, 30, 90, 180 and 270) in 59 study subjects with at least one atherosclerotic-risk factor and low density lipoprotein (LDL) cholesterol > 100 gm/dL. Simvastatin (40 mg/day) was given to each study subjects for 6 months. The inflammatory mediators were evaluated in 30 healthy subjects. Human umbilical vein (HUV) ECs were incubated with 1) culture medium alone, 2) added CRP for stimulation, and 3) simvastatin for 4 h before CRP was added. The hs-CRP and VCAM-1 levels were significantly higher on day 0 in study subjects than in healthy subjects (all p < 0.0001). These inflammatory markers declined to a significantly lower level on day 90 (all p < 0.001) and to an even lower level by day 180 (all p < 0.0001). After simvastatin therapy was withdrawn, the hs-CRP level was once again significantly higher on day 270 than on day 180 ( p < 0.05), but VCAM-1 did not differ between day 180 and day 270. 25 μmol/L simvastatin markedly suppressed the CRP effect on VCAM-1 and intercellular CAM-1 expressions of EC. Immunocytochemical staining identified CRP in HUVEC cytoplasm. However, pretreatment with simvastatin reduced the intensity of CRP in cytoplasm. Conclusions CRP-mediated inflammation is inhibited by simvastatin.

Expression of vascular endothelial growth factor (VEGF) and its receptors KDR, Flt1 in lung cancer and their relationship to prognosis

It is well known that vascular endothelial growth factor (VEGF) and its receptors are closely related to tumor angiogenesis, but the exact relationship with patients' prognosis is unclear till now. The aim of this study is to explore the expression of VEGF and its receptors KDR, Flt1 in pulmonary carcinoma and their relationship with patients' prognosis. The expression of VEGF, KDR and Flt1 was examined immunohistochemically by PV-9000 method in 75 cases of pulmonary carcinoma with complete follow-up records. There was an extensive expression of VEGF, KDR and Flt1, mainly in the cytoplasm of tumor cells (TCs), fibroblasts (FBs), and endothelial cells. The distribution of VEGF, KDR and Flt1 was heterogeneous, mainly located at periphery of the tumor mass or necrosis. The positive rate of VEGF, KDR and Flt1 in the TCs was all significantly higher than that in the FBs (P < 0.01, P < 0.02, P < 0.02). Both in TCs and FBs, the positive expression of VEGF, KDR and Flt1 was related to the postoperative survival of patients (P < 0.01, P < 0.01, P < 0.01; P < 0.01, P < 0.01, P < 0.05). The survival time in patients with positive VEGF, KDR or Flt-1 in TCs was significantly lower than that in those with corresponding negative one respectively (P < 0.0001, P < 0.0005, P < 0.0005). There was a positive correlation between VEGF and Flt1 in TCs (P < 0.01), between VEGF in FBs and Flt1 in TCs (P < 0.01), and also between VEGF and KDR or Flt1 in FBs (P < 0.01, P < 0.01). VEGF may act as a considerable promoting growth factor on tumor cells via Flt1, mainly in autocrine and less in paracrine manner. VEGF, KDR and Flt1 may exert important roles in prognosis of patients with pulmonary carcinoma.

Finite-Element Stress Analysis of a Multicomponent Model of Sheared and Focally-Adhered Endothelial Cells

Hemodynamic forces applied at the apical surface of vascular endothelial cells may be redistributed to and amplified at remote intracellular organelles and protein complexes where they are transduced to biochemical signals. In this study we sought to quantify the effects of cellular material inhomogeneities and discrete attachment points on intracellular stresses resulting from physiological fluid flow. Steady-state shear- and magnetic bead-induced stress, strain, and displacement distributions were determined from finite-element stress analysis of a cell-specific, multicomponent elastic continuum model developed from multimodal fluorescence images of confluent endothelial cell (EC) monolayers and their nuclei. Focal adhesion locations and areas were determined from quantitative total internal reflection fluorescence microscopy and verified using green fluorescence protein-focal adhesion kinase (GFP-FAK). The model predicts that shear stress induces small heterogeneous deformations of the endothelial cell cytoplasm on the order of <100 nm. However, strain and stress were amplified 10-100-fold over apical values in and around the high-modulus nucleus and near focal adhesions (FAs) and stress distributions depended on flow direction. The presence of a 0.4 microm glycocalyx was predicted to increase intracellular stresses by approximately 2-fold. The model of magnetic bead twisting rheometry also predicted heterogeneous stress, strain, and displacement fields resulting from material heterogeneities and FAs. Thus, large differences in moduli between the nucleus and cytoplasm and the juxtaposition of constrained regions (e.g. FAs) and unattached regions provide two mechanisms of stress amplification in sheared endothelial cells. Such phenomena may play a role in subcellular localization of early mechanotransduction events.

Microrheology and ROCK Signaling of Human Endothelial Cells Embedded in a 3D Matrix

Cell function is profoundly affected by the geometry of the extracellular environment confining the cell. Whether and how cells plated on a two-dimensional matrix or embedded in a three-dimensional (3D) matrix mechanically sense the dimensionality of their environment is mostly unknown, partly because individual cells in an extended matrix are inaccessible to conventional cell-mechanics probes. Here we develop a functional assay based on multiple particle tracking microrheology coupled with ballistic injection of nanoparticles to measure the local intracellular micromechanical properties of individual cells embedded inside a matrix. With our novel assay, we probe the mechanical properties of the cytoplasm of individual human umbilical vein endothelial cells (HUVECs) embedded in a 3D peptide hydrogel in the presence or absence of vascular endothelial growth factor (VEGF). We found that VEGF treatment, which enhances endothelial migration, increases the compliance and reduces the elasticity of the cytoplasm of HUVECs in a matrix. This VEGF-induced softening response of the cytoplasm is abrogated by specific Rho-kinase (ROCK) inhibition. These results establish combined particle-tracking microrheology and ballistic injection as the first method able to probe the micromechanical properties and mechanical response to agonists and/or drug treatments of individual cells inside a matrix. These results suggest that ROCK plays an essential role in the regulation of the intracellular mechanical response to VEGF of endothelial cells in a 3D matrix.

Quantitative immunogold study of increased expression of metallothionein-I/II in the brain perivascular areas of diabetic scrapie-infected mice

Quantitative immunogold procedure was used to study the distribution of metallothionein I/II (MT-I/II) at the ultrastructural level in the perivascular areas, including microvascular endothelial cells (ECs) and astrocytes with their perivascular end-feet, in brains of scrapie-infected hyperglycemic (diabetic) and normoglycemic (non-diabetic) mice. Samples of the fronto-parietal cortex obtained from diabetic and non-diabetic scrapie-infected, as well as from non-infected (control) SJL/J mice, were processed for immunocytochemical examination. In control mice, the labelling of the ECs was of low intensity, restricted to few immunogold particles in the cytoplasm. More intense labelling was present in the cytoplasm of astrocytic perivascular processes and perikarya, where it was associated with endoplasmic reticulum and fibrils. A few immunosignals were also present inside the nuclei of astrocytes. In diabetic mice the labelling of the EC cytoplasm was slightly increased, whereas in the cytoplasm of perivascular processes and pericarya of astrocytes, including their nuclei, there was significant enhancement of labelling. In these cells the density of immunosignals was highest in the areas of cytoplasm containing bundles of fibrils. In non-diabetic, scrapie-infected mice the intensity of immunolabelling was higher than in control mice but slightly lower than in diabetic mice. These results are similar to those in Alzheimer's disease reported by other authors, and suggest that neurodegenerative diseases as well as metabolic stress enhance the metallothionein expression in perivascular regions of brain cerebral cortex, predominantly in astrocytes.

Pharmacological vascular reactivity in isolated diabetic rabbit ciliary artery

Impairment of the ocular circulation induced by diabetes mellitus has not been fully defined, but is thought to be related to hemodynamic changes in the ocular circulation. The purpose of the present study is to investigate the functional and morphological changes occurring in the ciliary artery wall of rabbits with alloxan-induced diabetes mellitus. A single intravenous bolus injection of alloxan (100 mg/kg) was given to each of 26 10-week-old rabbits and 16 sham-injected control rabbits. Twenty weeks later, control rabbits and diabetic rabbits were sacrificed, and their ciliary arteries were mounted in a myograph system. The responses of these arteries to high K + solution (K-Krebs solution), phenylephrine and carbachol were investigated using isometric tension recording. L-NAME ( NG-nitro- l-arginine methyl ester; 100 μM) and indomethacin (1 μM) were also used to test the mechanism causing the carbachol induced relaxation. The arteries were also examined morphologically. The maximum tensions induced by K-Krebs solution in this tissue were not significantly different: 17.2 ± 0.8 mN ( n = 16) in the control rabbits and 17.6 ± 0.8 mN ( n = 23) in the diabetic rabbits ( P = 0.36). Phenylephrine caused dose-dependent contraction with EC 50 values of 1.3 ± 0.4 μM ( n = 6) in the control and 5.1 ± 2.3 μM ( n = 6) in the diabetic rabbits, but there was no significant difference between the two ( P = 0.36). Carbachol induced dose-dependent relaxations in segments precontracted with K-Krebs solution. These relaxations were significantly reduced in the diabetic rabbits. The maximum relaxation induced by carbachol was 77.0 ± 2.4% (10 μM) and 66.4 ± 2.5% (100 μM) in the control and diabetic rabbits, respectively. These values were significantly different ( P = 0.0076). The IC 50 value for carbachol was 396.3 ± 58.4 nM ( n = 16) in the control, and 443.6 ± 141.1 nM ( n = 23) in the diabetic rabbit ( P = 0.87). Application of a 100 μM nitric oxide synthase inhibitor, L-NAME, significantly inhibited the amplitude of relaxations evoked by carbachol ( P = 0.0066). However, these relaxations were not inhibited by pretreatment with 1 μM indomethacin ( P = 0.60). Histologically, the frequency of invaginations was less in the diabetic arterioles with a flattening of the lamina in the diabetic rabbits than in the controls. The cytoplasm of endothelial cells contained large vacuoles, indicating weak adhesion to the lamina. Some endothelial cells even showed vacuolar degeneration due to breakdown of the cell membranes. However, the smooth muscle cells were well preserved in the diabetic rabbit. These results suggest that the mechanism of impairment of ocular circulation induced by diabetes mellitus is mainly the reduction of NO synthase due to endothelial cell dysfunction. Furthermore, the characteristics of rabbits with alloxan-induced diabetes mellitus probably make them a useful model for investigating ocular complications induced by diabetic mellitus.

Phosphorylation-dependent Regulation of Unique Nuclear and Nucleolar Localization Signals of LIM Kinase 2 in Endothelial Cells

LIM kinases (LIMKs) regulate actin dynamics through cofilin phosphorylation and also have a function in the nucleus. Recently we have shown that LIMK2 shuttles between cytoplasm and nucleus in endothelial cells and that nuclear import is inhibited by protein kinase C-mediated phosphorylation of Ser-283. Here we aimed to identify the structural features of LIMK2 responsible for nuclear import. We found that the kinase domain of LIMK2 is localized exclusively in the nucleus and, in contrast to the kinase domain of LIMK1, it accumulated in the nucleolus. Through site-directed mutagenesis, we identified the basic amino acid-rich motif KKRTLRKNDRKKR (amino acids 491-503) as the functional nuclear and nucleolar localization signal of LIMK2. After fusing this motif to enhanced green fluorescent protein, the fusion protein localized exclusively in the nucleus and nucleolus. Mutagenesis studies showed that phosphorylation of Thr-494, a putative protein kinase C phosphorylation site identified within the nuclear localization signal, inhibits nuclear import of the enhanced green fluorescent protein-PDZ kinase domain of LIMK2. After inhibiting nuclear export with leptomycin B, phosphorylation of either Ser-283 or Thr-494 reduced the nuclear import of LIMK2. Phosphorylation of both Ser-283 and Thr-494 sites inhibited nuclear import completely. Our findings identify a unique basic amino acid-rich motif (amino acids 491-503) in LIMK2 which is not present in LIMK1 that serves to target the protein not only to the nucleus but also to the nucleolus. Phosphorylation of Thr-494 within this motif negatively regulates nuclear import of LIMK2.

Thymidine phosphorylase expression as a prognostic marker for predicting recurrence in primary superficial bladder cancer

No prognostic marker for predicting recurrence in primary superficial bladder cancer has been established. The aim of this study was to investigate the expression levels of thymidine phosphorylase (TP) by enzyme-linked immunosorbent assay (ELISA) and the immunohistological localization of TP in primary superficial bladder cancer tissue to assess its association with tumor recurrence and stage progression as well as the pathological parameters of the tumor. TP expression in cancer tissues from 77 patients was measured by sandwich-type ELISA. Clinicopathological factors and the clinical prognosis were examined in relation to the expression levels of TP. To clarify the clinicopathological implication of TP localization in primary superficial bladder cancer tissue, the immunohistochemically determined TP expression was assessed for histological grades 1-3. The TP expression in primary superficial bladder cancer significantly increased with the histological grade and stage. The TP expression in patients with a shift to invasive cancer was significantly higher than in those without invasive cancer. Patients with low TP expression had a significant longer postoperative tumor-free period than those with high TP expression (P=0.011). High TP expression was an independent prognostic factor for tumor recurrence (P=0.0441). Strong immunoreactivity for TP was observed in the cytoplasms of tumor cells and vascular endothelial cells. This study suggests that elevated TP expression may be a prognostic marker for predicting recurrence in primary superficial bladder cancer, and that high TP expression may be associated with the marked proliferation of tumor cells and increased vascular endothelial cells, showing strong immunoreactivity for TP.

Cortical expression of nuclear factor κB after human brain contusion

The aim of current study was to analyze the binding activity and the temporal and cellular expression of nuclear factor kappa B (NF-κB) in human contused brain. Eighteen contused brain samples were obtained from 17 patients undergoing surgery for brain contusions 5–80 h after trauma. NF-κB binding activity was detected by electrophoretic mobility shift assay (EMSA), and temporal and cellular expression of NF-κB subunits p65 and p50 was analyzed by immunohistochemistry. The results showed that a progressive upregulation of NF-κB activity occurred in the area surrounding the injured brain with the time from brain trauma to operation. The maximal expression of NF-κB was detected after 48 h postinjury. The expression of NF-κB p65 was mainly located at glial and vascular endothelial cells without expression at neurons. The expression of NF-κB p50 was mainly located at glial cells, a little at neurons and no expression at vascular endothelial cells. Within 24 h postinjury, both NF-κB p65 and p50 immunoreactivity was mainly observed in the nucleus of cells. After 24 h postinjury, NF-κB p65 labeling was found in the both nucleus and cytoplasm of glial and endothelial cells; otherwise, p50 labeling was primarily found in the nucleus of glial cells and in the nucleus, cytoplasm and process of neurons. It is concluded that NF-κB could be highly upregulated at human contused brain and the cellular pattern of p65 and p50 expression might be closely associated with the cell functions.

Descending vasa recta endothelium is an electrical syncytium

We examined gap junction coupling of descending vasa recta (DVR). DVR endothelial cells or pericytes were depolarized to record the associated capacitance transients. Virtually all endothelia and some pericytes exhibited prolonged transients lasting 10-30 ms. Carbenoxolone (100 microM) and 18beta-glycyrrhetinic acid (18betaGRA; 100 microM) markedly shortened the endothelial transients. Carbenoxolone and heptanol (2 mM) reduced the pericyte capacitance transients when they were prolonged. Lucifer yellow (LY; 2 mM) was dialyzed into the cytoplasm of endothelial cells and pericytes. LY spread diffusely along the endothelial monolayer, whereas in most pericytes, it was confined to a single cell. In some pericytes, complex patterns of LY spreading were observed. DVR cells were depolarized by voltage clamp as fluorescence of bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC(4)(3)] was monitored approximately 200 microm away. A 40-mV endothelial depolarization was accompanied by a 26.1 +/- 5.5-mV change in DiBAC(4)(3) fluorescence. DiBAC(4)(3) fluorescence did not change after 18betaGRA or when pericytes were depolarized. Similarly, propagated cytoplasmic Ca(2+) responses arising from mechanical perturbation of the DVR wall were attenuated by 18betaGRA or heptanol. Connexin (Cx) immunostaining showed predominant linear Cx40 and Cx43 in endothelia, whereas Cx37 stained smooth muscle actin-positive pericytes. We conclude that the DVR endothelium is an electrical syncytium and that gap junction coupling in DVR pericytes exists but is less pronounced.

Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): Proposed mechanism for modulation of endothelial cell response to VEGF

We have recently demonstrated that thrombin-activated FXIII (FXIIIA-subunit), a plasma transglutaminase, activates VEGFR-2 by crosslinking it with the α vβ 3 integrin on the surface of endothelial cells (EC), thereby stimulating angiogenesis. Tissue transglutaminase (tTG), which is functionally and structurally related to FXIIIA, is expressed by numerous cell types, among them EC. However, its role in EC function has not been fully characterized. In the present study, we investigated the potential involvement of tTG in angiogenesis. Using co-immunoprecipitation and immunofluorescent staining experiments, we observed that tTG forms a complex with VEGFR-2 on the cell surface and within the cytoplasm of EC. Stimulation of EC with VEGF resulted in translocation of the tTG–VEGFR-2 complex from the cytoplasm to the nucleus. In VEGF-treated cells, tTG–VEGFR-2 interaction resulted in incorporation of VEGFR-2 into high molecular weight crosslinked complex (es), as revealed by an antibody against γ-glutamyl-ε-lysine isopeptide bond. tTG –VEGFR-2 association was inhibited by a specific VEGFR-2 protein tyrosine kinase inhibitor (PTKI ), as well as by cystamine, inhibitor of the transglutaminase activity of tTG, but not by bacitracin which inhibits the protein-disulfide isomerase (PDI) activity of tTG. Furthermore, cystamine completely abolished the VEGF-induced nuclear translocation of the tTG–VEGFR-2 complex. Blockade of the crosslinking activity of tTG by cystamine enhanced VEGF-induced migration of EC in Boyden chamber by 31% ( P < 0.02), and prolonged VEGF-induced signaling response, as demonstrated by sustained activation of the MAP kinase ERK. Taken together, our findings suggest that endothelial cell tTG might be involved in modulation of the cellular response to VEGF by forming an intracellular complex with VEGFR-2, and mediating its translocation into the nucleus upon VEGF stimulation.

In Vivo Low-Density Lipoprotein Exposure Induces Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1 Correlated With Activator Protein-1 Expression

We tested the hypothesis that direct native low-density lipoprotein (LDL) injection into LDL receptor-deficient (LDLR(-/-)) mice would induce the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in their aortic endothelial cells, and that transcriptional regulation of this pathway involved activator protein-1 (AP-1) but not nuclear factor kappaB (NF-kappaB). Using tail vein injection of LDL into LDLR(-/-) mice, we were able to maintain atherogenic LDL blood levels, which induced ICAM-1 and VCAM-1 expression in their aortic endothelial cells after 24 hours. We were able to visualize and quantify this expression using immunohistochemistry and confocal microscopy. Under conditions in which ICAM-1 and VCAM-1 were expressed, the regulatory AP-1 proteins c-Fos and c-Jun were also highly expressed in the endothelial cell cytoplasm and observed within the cell nucleus. The NF-kappaB protein P65, although expressed in the endothelial cell cytoplasm after LDL injection, was not observed within the cell nucleus. Elevated LDL blood levels, maintained in vivo, increased the expression of the adhesion molecules ICAM-1 and VCAM-1 in aortic endothelial cells. This effect appeared to correlate with AP-1 but not NF-kappaB.