Endothelial cell

A large proportion of the beneficial effects that oestrogens demonstrate on the vasculature are believed to be mediated via direct effects on the vascular wall. In this study we compared a number of oestrogenic compounds isolated from pregnant mare's urine including 17b-oestradiol and oestrone, in terms of their abilities to inhibit stimulated endothelin-1 release from normal human coronary artery endothelial cells (CAEC). We also examined their ability to stimulate expression of constitutive endothelial nitric oxide synthase (eNOS) and explored their effects on cellular angiotensin converting enzyme (ACE). All the oestrogens tested were able to inhibit serum-stimulated ET-1 release. Oestrone and 17a-dihydroequilenin failed to significantly affect cellular eNOS levels. 17b-oestradiol and oestrone significantly increased cellular ACE levels while 17b,D 8,9-dehydroestradiol decreased cellular ACE. We discuss these observations in terms of their potential clinical relevance and use as a means of screening novel oestrogen-like compounds.

Endothelin (ET) 1 is a potent vasoconstrictor peptide produced by vascular endothelial cells and implicated in various pathophysiologic states involving abnormal vascular tone. Homozygous ET-1 null mice have craniofacial and cardiac malformations that lead to neonatal death. To study the role of ET-1 in adult vascular physiology, we generated a mouse strain (ET-1 flox/flox ;Tie2-Cre mice) in which ET-1 is disrupted specifically in endothelial cells. ET-1 peptide levels in plasma, heart, lung, kidney, and brain homogenates were reduced by 65% to 80% in these mice. mRNA levels for ET receptors were unaltered except that the ET A receptor mRNA was upregulated in the heart. ET-1 flox/flox ;Tie2-Cre mice had mean blood pressures 10 to 12 mm Hg lower than genetic controls. In contrast, the blood pressure of mice systemically heterozygous for the ET-1 null allele (ET-1 dlox/ϩ mice) was unchanged compared with wild-type littermates. Despite the lower basal blood pressure, acute pharmacological responses to angiotensin II, captopril, phenylephrine, bradykinin, N G -nitro-L-arginine methyl ester, and exogenous ET-1 were normal in ET-1 flox/flox ;Tie2-Cre mice. These results support an essential role of endothelial-derived ET-1 in the maintenance of basal vascular tone and blood pressure. Normal pharmacological responses of ET-1 flox/flox ;Tie2-Cre mice suggest that the renin-angiotensin system, the adrenergic system, and NO are not significantly altered by endothelial ET-1. Taken in conjunction with other lines of genetically altered mice, our results provide evidence for a paracrine vasoregulatory pathway mediated by endothelial cell-derived ET-1 acting on the vascular smooth muscle ET A receptor. (Hypertension. 2010;56:121-128.) Key Words: hypertension Ⅲ endothelium Ⅲ ET A Ⅲ ET B Ⅲ cre/loxP Ⅲ blood pressure Ⅲ gene knockout E ndothelin (ET) 1, originally identified as a potent vaso-

Multi-organic failure in the context of autoimmune diseases is a multi-factorial condition where different pathways concur to produce a global system breakdown. Some of these pathways include the coagulation, fibrinolysis, kinin and complement cascades which in normal conditions work together to provide a comprehensive response to injury. In pathologic conditions these regulatory mechanisms are replaced by positive feed-back loops. The common response pattern is the activation of the immune system via endothelium activation. Furthermore, these different plasma-driven mechanisms may induce standardised endothelial cell responses of which the most relevant are the activation of p38, JNK, NF-kβ and IRF-3 pathways. In this paper we review the common points between these major pathways and how they become activated, contributing to a global clinical picture. We present two examples of apparently different clinical settings, caused by the same global dysfunction: the Macrophage Activation Syndrome and the iatrogenic "cytokine storm" triggered by the administration of anti-CD28 monoclonal antibody TGN1412 in a phase 1 trial.

The chronic inflammation of arterial walls is associated with the development of atherosclerosis. Earlier we reported that avenanthramide (Avn) s-enriched extract of oats (AvnsO) significantly suppressed interleukin (IL)-1β-stimulated secretion of proinflammatory cytokines, such as IL-6, IL-8, and MCP-1, by human aortic endothelial cells (HAEC). The main objective of the current study was to determine if the mechanism of inhibitory effect of these polyphenols from oats on the expression of proinflammatory cytokines is mediated through modulation of nuclear factor κB (NF-κB)-dependent transcription. Confluent HAEC monolayers were treated for 24 h with AvnsO, and synthetically prepared Avn-c suppressed IL-β-stimulated activation of NF-κB in a concentration-dependent manner. CH3-Avn-c, a synthetically prepared methyl ester derivative of Avn-c with a high biological potency, significantly and dose dependently decreased mRNA expression and secretion of IL-6, IL-8, and MCP-1 by HAEC as determined by real-time RT-PCR and ELISA, and it inhibited IL-1β-and TNFα-stimulated NF-κB activation as determined by a NF-κB DNA binding assay and a NF-κB luciferase reporter assay. AvnsO and Avn-c as well as CH3-Avn-c also inhibited the NF-κB-dependent reporter gene expression activated by TNFR-associated factor 2 and 6 (TRAF2, TRAF6) and NFκB-inducing kinase (NIK). CH3-Avn-c also significantly and dose dependently decreased the phosphorylation level of IκB kinase (IKK) and IκB, and prevented IκB degradation as measured by Western blotting. In addition, CH3-Avn-c markedly increased the overall levels of high mass ubiquitin-conjugated protein levels while it mildly inhibited proteasome activity. These observations suggest that Avns, unique polyphenols from oats, decrease the expression of endothelial proinflammatory cytokines at least in part through inhibition of NF-κB activation by inhibiting the phosphorylation of IKK and IκB, and by suppressing proteasome activity.

In a prospective study we used the change of central and peripheral (12 o'clockposition) corneal thickness (CT) after no-stitch small incision cataract surgery as a parameter of tissue traumatisation (33 eyes) and compared the values to a series of cases (32 eyes) with conventional 3.5 mm scleral step incision. In both groups the peripheral measurements showed a higher increase in corneal thickness than the central. After 1 month all eyes regained their central preoperative thickness. Increase in corneal thickness (ACTc, ACTp) after the different postoperative periods were correlated. The values of the central cornea showed no significant difference between the two groups. 1, 7 and 30 days after surgery the increase of peripheral CT was significantly higher in the no-stitch group. This fact was underlined by the clinical aspect at the slit lamp and is due to the anatomical and surgical characteristic of this procedure. One month postoperatively there was no increased endothelial cell loss in the no-stitch group (3%). No-stitch cataract surgery surgery provides a lot of intra-and postoperative advantages. The problem of increased swelling of the peripheral corneal entry seems to be a secondary one as corneal thickness decreases with time. Concerning the prospective endothelial cell loss it is mandatory to study the long term results.

Vascular responses to hypoxia are heterogeneous and involve the release of vasodilators substances such as nitric oxide (NO) and prostacyclin (PGI 2). In vitro studies have shown that Vitamin K 1 modulates the release of arachidonic acid (AA) in vascular cells, and thus inhibits the capacity of blood vessels to synthesise vasodilator AA metabolites. The aim of our work was to investigate the effects of Vitamin K 1 on the hypoxia-induced vasorelaxation. Hypoxia was induced by changing the gas from 95% O 2 /5% CO 2 to a mixture containing 95% N 2 /5% CO 2. Rat carotid arteries were pre-contracted with phenylephrine (Phe, 10 −8 mol/l) and when the contraction reached a plateau, the bath was bubbled with 95% N 2 /5% CO 2 for 15 min. In intact rings, there was a total relaxation after 15 min of exposure to hypoxia. Removal of the endothelium strongly reduced hypoxia-induced relaxation. In intact rings, indomethacin and l-NAME reduced the hypoxic relaxation after 5 min of exposure but not after 10 or 15 min. Exposure of endothelium-intact rings to Vitamin K 1 (5×10 −6 and 5×10 −5 mol/l), l-NAME +indomethacin as well as the combination of l-NAME +indomethacin + Vitamin K 1 reduced the hypoxic relaxation after 5 and 10 min of exposure but not after 15 min. At 5 × 10 −7 mol/l Vitamin K 1 did not attenuate hypoxia-induced relaxation. It was also found that Vitamin K 1 (5×10 −6 and 5×10 −5 mol/l) inhibited ACh-induced relaxation in normoxic conditions. These results show that the effect of Vitamin K 1 on attenuating hypoxia-induced vasorelaxation is concentration-dependent and probably related to its action on endothelial cells.

The earliest signs of the lymphatic vascular system are the lymph sacs, which develop adjacent to specific embryonic veins. It has been suggested that sprouts from the lymph sacs form the complete lymphatic vascular system. We have studied the origin of the jugular lymph sacs (JLS), the dermal lymphatics and the lymph hearts of avian embryos. In day 6.5 embryos, the JLS is an endothelial-lined sinusoidal structure. The lymphatic endothelial cells (LECs) stain (in the quail) positive for QH1 antibody and soybean agglutinin. As early as day 4, the anlagen of the JLS can be recognized by their Prox1 expression. Prox1 is found in the jugular section of the cardinal veins, and in scattered cells located in the dermatomes along the cranio-caudal axis and in the splanchnopleura. In the quail, such cells are positive for Prox1 and QH1. In the jugular region, the veins co-express the angiopoietin receptor Tie2. Quail-chick-chimera studies show that the peripheral parts of the JLS form by integration of cells from the paraxial mesoderm. Intravenous application of DiI-conjugated acetylated low-density lipoprotein into day 4 embryos suggests a venous origin of the deep parts of the JLS. Superficial lymphatics are directly derived from the dermatomes, as shown by dermatome grafting. The lymph hearts in the lumbo-sacral region develop from a plexus of Prox1-positive lymphatic capillaries. Both LECs and muscle cells of the lymph hearts are of somitic origin. In sum, avian lymphatics are of dual origin. The deep parts of the lymph sacs are derived from adjacent veins, the superficial parts of the JLS and the dermal lymphatics from local lymphangioblasts.

1. Laube HR, Duwe J, Rutsch W, Konertz W. Clinical experience with autologous endothelial cell-seeded polytetrafluorethylene coronary artery bypass grafts. J Thorac Cardiovasc Surg 2000;120:134 -41. 2. Tiwari A, Salacinski HJ, Hamilton G, Seifalian AM. Tissue engineering of vascular bypass grafts: role of endothelial cell extraction. Eur J Vasc Endovasc Surg 2001;21:193-201. 3. Kleisli T, Cheng W, Jacobs MJ, et al. In the current era, complete revascularization improves survival after coronary artery bypass surgery. J Thorac Cardiovasc Surg 2005;129: 1283-91. 4. Kouchoukos NT, Wareing TH, Murphy SF, Pelate C, Marshall WG Jr. Risks of bilateral internal mammary artery bypass grafting. Ann Thorac Surg 1990;49:210 -9. 5. Sajja LR, Mannam G, Pantula NR, Sompalli S. Role of radial artery graft in coronary artery bypass grafting. Ann Thorac Surg 2005;79:2180 -8. 6. Hirose H, Amano A, Takanashi S, Takahashi A. Coronary artery bypass grafting using the gastroepiploic artery: 1,000 cases. Ann Thorac Surg 2002;73:1371-9. 7. Herring M, Gardner A, Glover J. A single-staged technique for seeding vascular grafts with autogenous endothelium. Surgery 1978;84:498-504. 8. Deutsch M, Meinhart J, Fischlein T, Preiss P, Zilla P. Clinical autologous in vitro endothelialization of infrainguinal ePTFE grafts in 100 patients: a 9-year experience. Surgery 1999;126:847-55. 9. Lembcke A, Hein PA, Dohmen PM, et al. Pictorial review: electron beam computed tomography and multislice spiral computed tomography for cardiac imaging. Eur J Radiol 2006;57:356 -67.

Nitric oxide (NO) and reactive oxygen species (ROS) are emerging as important regulators of angiogenesis. NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in endothelial cells. Similarly, hydrogen peroxide induces VEGF synthesis and recent studies indicate the involvement of ROS in signaling downstream of VEGF stimulation. VEGF synthesis can not only be enhanced by gene transfer of VEGF but also by overexpression of NO synthase genes. Here, we examined the possibility of augmentation of VEGF production by gene transfer of copper/zinc superoxide dismutase (CuZnSOD, SOD1). Overexpression of human SOD1 in mouse NIH 3T3 fibroblasts increased SOD activity, enhanced intracellular generation of H 2 O 2 and significantly stimulated VEGF production as determined by increase in VEGF promoter activity, VEGF mRNA expression and VEGF protein synthesis. The stimulatory effect on VEGF synthesis induced by SOD1 gene transfer was reverted by overexpression of human catalase. The effect of H 2 O 2 produced by engineered cells is mediated by activation of hypoxia-inducible factor response element (HRE) as well as Sp1 recognition site of VEGF promoter. This data suggest the feasibility of stimulation of angiogenesis by overexpression of SOD1. (Mol Cell Biochem 264: [169][170][171][172][173][174][175][176][177][178][179][180][181] 2004)

Disabled-2 (DAB2) is an adaptor protein implicated in signal transduction pathways and in protein traffic regulation. Here, we show that DAB2 is highly expressed in human endothelial cells. DAB2 silencing in endothelial cells by lentiviral-mediated small hairpin RNA expression affects cell migration and differentiation into capillary-like structures while increasing cell proliferation and viability. DAB2 knockdown causes activation of the Src-FAK signal pathway, extracellular-signal regulated kinase and c-Jun NH 2 -terminal kinase activation, and inhibition of p38 phosphorylation. In DAB2 silenced endothelial cells, pharmacological inhibition of Src with its specific inhibitor PP2 abolishes focal adhesion kinase activation and restores differentiation of endothelial cells. These results suggest that DAB2, via Src and focal adhesion signaling, plays a role in human endothelial cell function.

Objective: Mechanisms that drive innate immune cell recruitment into atherosclerotic lesions are still not well defined. We tested the role of haemoglobin (Hb) to promote chemotaxis, adhesion to endothelial cells and transendothelial migration of human monocytes and monocyte-derived immature dendritic cells (iDCs) and its possible role in atherogenic cell recruitment.

Background: Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction.

Numerous studies have suggested relationships between myeloperoxidase, inflammation, and atherosclerosis. MPO-derived reactive chlorinating species (RCS) attack membrane plasmalogens releasing ␣-chloro-fatty aldehydes (␣-Cl-FALDs) including 2-chlorohexadecanal (2-ClHDA). The molecular targets of ␣-Cl-FALDs are not known. The current study demonstrates 2-ClHDA adducts with ethanolamine glycerophospholipids and Fmoc-lysine. Utilizing electrospray ionization mass spectrometry, chlorinated adducts were observed that are apparent Schiff base adducts. Reduction of these Schiff base adducts with sodium cyanoborohydride resulted in a novel, stable adduct produced by the elimination of HCl. NMR further confirmed this structure. 2-ClHDA adducts with ethanolamine glycerophospholipids were also substrates for phospholipase D (PLD). The hydrolysis products were derivatized to pentafluorobenzoyl esters, and further structurally confirmed by GC-MS. Multiple molecular species of 2-ClHDA-N-modified ethanolamine glycerophospholipids were observed in endothelial cells treated with 2-ClHDA. These results show novel Schiff base adducts of ␣-Cl-FALDs with primary amines, which may represent an important fate of ␣-Cl-FALDs.

Most in vitro studies of capillary permeability focus on endothelial cell (MVEC) monolayers and ignore the second cell that forms the capillary wall: the microvascular pericyte (PC). We describe a model to study the permeability characteristics of MVEC, PC, and MVEC:PC cocultures.

In order to create a hemodynamic environment that can simulate the physiological condition of arteries, an in vitro experiment apparatus was designed whose key modules were compliance chamber and after-load. These two modules were developed based on the theories of hemodynamics. Both the normal and shear stress to which endothelial cells are exposed can be controlled with these modules, thus facilitating the research of endothelial cells subjected to stresses. Ó

Recent evidences suggest a significant role of Plasmacytoid dendritic cells (PDC) role in the pathogenesis of lupus erythematosus (LE) via production of type I IFN. Taking advantage on the availability of multiple reagents (CD123, BDCA2, and CD2ap) specifically recognizing PDC on fixed tissues, we investigated the occurrence of PDC in a cohort of 74 LE patients. The large majority of LE biopsies (67/74; 90.5%) showed cutaneous infiltration of PDC. PDC were more frequently observed (96.4 vs 72.2) and numerous in cutaneous LE compared to systemic LE (SLE) and correlated with the density of the inflammatory infiltrate (r ¼ 0.40; po0.001). PDC reduction in SLE might be related to a broader tissue distribution of this cellular population, as indicated by their occurrence in kidneys in 11 out of 24 (45.8%) cases studied. The distribution of cutaneous PDC showed two distinct patterns. More commonly, PDC were observed within perivascular inflammatory nodules in the dermis, associated with CD208+ mature DC and T-bet+ cells [D-PDC]. A second component was observed along the dermal-epithelial junction [J-PDC], in association with cytotoxic T-cells in areas of severe epithelial damage. Notably, chemerin reactivity was observed in 64% of LE biopsies on endothelial cells and in the granular layer keratinocytes. Cutaneous PDC in LE strongly produced type I IFN, as indicated by the diffuse MxA expression, and the cytotoxic molecule granzyme B.

Pseudomonas aeruginosa, a common agent of septicemia, enters into human endothelial cellsin vitro but the effects of bacterial infection have not been addressed properly. In this study, human umbilical vein endothelial cells (HUVEC) were infected by the noninvasive PA103 and ...

Genetic studies have recently revealed a role for transforming growth factor-beta-I (TGF-~I) and its receptors (TGF-~Rs I and II as well as endoglin) in embryonic vascular assembly and in the establishment and maintenance of vessel wall integrity. The purpose of this review is threefold: first, to reassess previous studies on TGF-/~ and endothelium in the light of these recent findings; second, to describe some of the well-established as well as controversial issues concerning TGF-/~ and its regulatory role in angiogenesis; and third, to explore the notion of 'context' with respect to TGF-/~ and endothelial cell function. Although the focus of this review will be on the endothelium, other vascular wall cells are also likely to be important in the pathogenesis of the vascular lesions revealed by genetic studies. ~

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. Hypoxia has been shown to be a major inducer of VEGF gene transcription. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are highaffinity VEGF receptors. The role of VEGF in developmental angiogenesis is emphasized by the finding that loss of a single VEGF allele results in defective vascularization and early embryonic lethality. VEGF is critical also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with various VEGF inhibitors in a variety of malignancies are ongoing. Very recently, an anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the Food and Drug Administration as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration. (Endocrine Reviews 25: 581-611, 2004) I. Introduction II. Historical Note on Angiogenic Factors III. Identification of VEGF IV. Activities of VEGF A. Mitogenesis, angiogenesis, and endothelial survival B. Effects of VEGF on bone marrow cells and hematopoiesis C. Enhancement of vascular permeability and hemodynamic effects V. VEGF Isoforms VI. Regulation of VEGF Gene Expression A. Oxygen tension B. Growth factors, hormones, and oncogenes VII. VEGFRs A. VEGFR-1 (Flt-1) B. VEGFR-2 (KDR, human; Flk-1, mouse) C. Neuropilin (NP)1 and NP2 VIII. Role of VEGF in Physiological Angiogenesis A. Embryonic and postnatal development B. Skeletal growth and endochondral bone formation C. Angiogenesis in endocrine glands IX. Role of VEGF in Pathological Conditions A. Solid tumors B. Hematological malignancies C. Intraocular neovascular syndromes D. Inflammatory disorders and brain edema E. Pathology of the female reproductive tract X. VEGF and Therapeutic Angiogenesis XI. Perspectives

Palladium-based nanoparticles immobilized in polymeric matrices were applied to the reductive dechlorination of 3,3′,4,4′-tetrachlorobiphenyl (PCB77) at room temperature. Two different dechlorination platforms were evaluated using (1) Pd nanoparticles within conductive polypyrrole films; or (2) immobilized Fe/Pd nanoparticles within polyvinylidene fluoride microfiltration membranes. For the first approach, the polypyrrole film was electrochemically formed in the presence of perchlorate ions that were incorporated into the film to counterbalance the positive charges of the polypyrrole chain. The film was then incubated in a solution containing tetrachloropalladate ions, which were exchanged with the perchlorate ions within the film. During this exchange, reduction of tetrachloropalladate by polypyrrole occurred, which led to the formation of palladium nanoparticles within the film. For the second approach, the membranesupported Fe/Pd nanoparticles were prepared in three steps: polymerization of acrylic acid in polyvinylidene fluoride microfiltration membrane pores was followed by ion exchange of Fe 2+ , and then chemical reduction of the ferrous ions bound to the carboxylate groups. The membranesupported iron nanoparticles were then soaked in a solution of tetrachloropalladate resulting in the deposition of Pd on the Fe surface. The nanoparticles prepared by both approaches were employed in the dechlorination of PCB77. The presence of hydrogen was required when the monometallic Pd nanoparticles were employed. The results indicate the removal of chlorine atoms from PCB77, which led to the formation of lower chlorinated intermediates and ultimately biphenyl. Toxicity associated with vascular dysfunction by PCB77 and biphenyl was compared using cultured endothelial cells. The data strongly suggest that the dechlorination system used in this study markedly reduced the proinflammatory activity of PCB77, a persistent organic pollutant.

We have investigated the expression of leucocyte markers, phenotypic characteristics and cellular relationships of the normal human fetal hyaloid vasculature using immunohistochemistry, light and electron microscopy. Antibodies against von Willebrand Factor, alpha-smooth muscle actin, glial fibrillary acidic protein, vimentin, major histocompatibility complex classes-I and-II, CD45 (leucocytecommon antigen) and calcitonin gene-related peptide were used to identify the cellular constituents of the hyaloid vasculature in whole mounts. Additional morphological features were described at the ultrastructural level. Endothelial cells throughout the hyaloid system were immunoreactive to von Willebrand Factor and major histocompatibility complex class-I antibodies. Pericytes were immunoreactive to alpha-smooth muscle actin antibody ; labeled cells were distributed along large branches of the hyaloid artery, vasa hyaloidea propria, tunica vasculosa lentis and pupillary membrane but no immunoreactivity was detected on small connecting capillaries. Vessel and non-vessel-associated hyalocytes on the hyaloid artery, vasa hyaloidea propria, tunica vasculosa lentis, pupillary membrane and vitreous were immunoreactive to major histocompatibility complex classes-I and-II, CD45 and calcitonin gene-related peptide antibodies. Antiglial fibrillary acidic protein reactivity was detected on Bergmeister's papilla but not on the hyaloid artery. Cells immunoreactive for vimentin were present throughout the hyaloid vasculature including small connecting capillaries. Ultrastructural observations of the hyaloid vasculature revealed junctional complexes, including zonulae adherens, macula adherens and possible zonulae occludens, between adjacent endothelial cells. Fenestrae were not observed in the gestational ages included in the present study. The use of whole mounts in conjunction with specific antisera has provided novel immunohistochemical definitions of the structure and cellular constituents of the human hyaloid. The results indicate that hyalocytes are a heterogeneous population of leucocyte-lineage cells.

4-Hydroxynonenal (HNE) is a major aldehydic product formed by peroxidation of omega 6-unsaturated fatty acids and is regarded as a specific marker of lipid peroxidation. In this paper we demonstrate that there is a physiological steady-state concentration of HNE in human venous blood plasma. For the quantitative determination of HNE a modified version of an existing, but tedious and time-consuming HPLC method was developed. The extraction of aldehydic hydrazones from plasma was performed using an Extrelut column and the separation step by thin-layer chromatography was replaced by column chromatography on silica gel. The concentration of HNE in human blood plasma was in the same range as the concentration that was found to inhibit the proliferation of cells of the peripheral tissues, i.e., endothelial cells and fibroblasts in vitro. In an experiment with reduced peripheral blood flow a temporary significant increase of HNE was observed during reperfusion. It was concluded that lipid ...

5-Hydroxy-2-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione (5HPP-33: 10), which was obtained during our previous structural development studies on thalidomide, was revealed to possess potent anti-angiogenic activity in a human umbilical vein endothelial cell (HUVEC) assay. Thalidomide (1) and its metabolite, 5-hydroxythalidomide (5-HT: 2), which possesses a hydroxyl group at the position corresponding to that of 5HPP-33, as well as IMiDs (immunomodulatory derivatives of thalidomide: 3 and 5), also showed weak or moderate activity in the same assay.

Bone marrow derived hematopoietic stem cells can function as endothelial progenitor cells. They are recruited to malignant tumors and differentiate into endothelial cells. This mechanism of neovascularization termed vasculogenesis is distinct from proliferation of preexisting vessels. To better understand vasculogenesis we developed a cell culture model with expansion and subsequent endothelial differentiation of human CD133 + progenitor cells in vitro. a v b 3-integrins are expressed by endothelial cells and play a role in the attachment of endothelial cells to the extracellular matrix. We investigated the effect of Cilengitide, a peptide-like, high affinity inhibitor of a v b 3-and a v b 5-integrins in our in vitro system. We could show expression of a v b 3-integrin on 60 ± 9% of non-adherent endothelial progenitors and on 91 ± 7% of differentiated endothelial cells. a v b 3-integrin was absent on CD133 + hematopoietic stem cells. Cilengitide inhibited proliferation of CD133 + cells in a dose-dependent manner. The development of adherent endothelial cells from expanded CD133 + cells was reduced even stronger by Cilengitide underlining its effect on integrin mediated cell adhesion. Expression of endothelial antigens CD144 and von Willebrand factor on differentiating endothelial precursors was decreased by Cilengitide. In summary, Cilengitide inhibits proliferation and differentiation of human endothelial precursor cells underlining its anti-angiogenic effects.

Computational blood flow analysis through glycocalyx located over the endothelial cells in human capillary vascular system is performed. Stable numerical schemes are developed for obtaining approximate solutions to the mixed problem to the partial differential equation with variable space operator to model the blood flow. Numerical techniques are developed by applying a procedure of the solution to the linear difference equation with matrix coefficients. The flow equations inside the core flow region and porous region are established. Discretization is done and the flow velocities in both regions are calculated. The effect of the flow over the glycocalyx is investigated. The wall shear stresses and the drag force over the glycocalyx are calculated.

Using a novel cellular impedance biosensor and Using a novel cellular impedance biosensor and confocal confocal microscopy, this study has examined the dynamic and microscopy, this study has examines the dynamic and steady steady state cellular impedance response of porcine pulmonary state response of porcine pulmonary endothelial cells to endothelial cells to varying doses of cytochalasin D. varying doses of cytochalasin D. Cytochalasin D was Endothelial cell monolayer impedances were obtained using an chosen since it specifically inhibits actin polymerization. array of gold microelectrodes coated with fibronectin to facilitate endothelial cell adhesion. Impedance measurements The combnatlion of confocal imaging and cellular were acquired at 5.6 kHz by phase sensitive detection from a impedance analysis giveS us the opportunity to quantify lock-in amplifier. The electrically measured cytochalasin D pharmacological changes in actin filaments structure with dose dependant actin disruption was successfully correlated changes in endothelial cell barrier function.

Rapid translocation of P-selectin from WPB to the surface of endothelial cells is crucial for early neutrophil recruitment to acute inflammatory lesions. Likewise, the chemokine CXCL8/IL-8 is sorted to WPB in human endothelial cells, but little is known about its functional importance in lack of a suitable animal model. Here, we explored the distribution of the functional IL-8 homologues CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX in resting and inflamed murine vessels by confocal microscopy and paired immunostaining with markers of WPB, discovering that these chemokines did not localize to WPB but displayed a granular pattern in a subset in healthy skin compatible with sorting to the type 2 endothelial compartment for regulated secretion. Moreover, all chemokines colocalized with VWF and P-selectin in platelets, suggesting that their storage in platelet ␣-granules might represent an alternative source of rapidly available, neutrophil-recruiting chemokines. In conclusion, WPB appear not to be involved in regulated secretion of chemokines in the mouse, and instead, the possible existence of type 2 granules and the role of platelets in rapid leukocyte adhesion deserve further attention. J. Leukoc. Biol. 87: 000 -000; 2010.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5Gl. 1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5Gl. 1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5Gl. 1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5Gl. 1 scFv also effectively blocked C5a generation. Finally. an intact humanized h5Gl.l antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5Gl. 1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G 1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases. (0 1997 Elsevier Science Ltd. All rights reserved.

The reversibility of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is strongly associated with the degree of intimal proliferation, vessel narrowing, and number of circulating endothelial cells (CECs). Circulating endothelial cells may arise from either endothelial damage or accelerated turnover during vessel remodeling, but nothing is known about endothelial microparticles (EMPs) and other biomarkers reflecting endothelial alterations. This study aimed to document endothelial markers further according to the irreversibility of PAH secondary to CHD. The study investigated soluble markers of endothelial damage or activation (thrombomodulin, soluble endothelial protein C receptor, and soluble E-selectin), inflammation (interleukin-6), and angiogenic cytokine levels [vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)] in 26 patients with CHD, 16 with reversible PAH (median age, 2 years) and 10 with irreversible PAH (median age, 9 years). Endothelial activation/apoptosis was evaluated by measuring EMP levels. Plasma procoagulant activity also was measured. The results show that the levels of soluble markers indicating endothelial activation were not predictors of PAH irreversibility. Lower levels of PlGF were observed in reversible compared with irreversible PAH but were not associated with the CEC level, the mean pulmonary artery pressure (mPAP), or age. No significant difference in procoagulant activity or EMP level was found between irreversible and reversible PAH. Among a large panel of biomarkers reflecting endothelial activation, regeneration, and injury, the high CEC levels previously described proved to be the only marker allowing discrimination between reversible and irreversible PAH secondary to CHD.

Previously, we found a positive correlation between the expression of platelet-type 12-lipoxygenase (12-LOX) and the progression of human prostate adenocarcinoma (PCa; Gao et al., Urology, 46: 227-237, 1995). To determine the role of 12-LOX in PCa progression, we generated stable 12-LOX-transfected PC3 cells, which synthesize high levels of 12-LOX protein and 12(S)-hydroxyeicosatetraenoic acid metabolite. In vitro, 12-LOX-transfected PC3 cells demonstrated a proliferation rate similar to neo controls. However, following s.c. injection into athymic nude mice, 12-LOX-transfected PC3 cells formed larger tumors than did the controls. Decreased necrosis and increased vascularization were observed in the tumors from 12-LOX-transfected PC3 cells. Both endothelial cell migration and Matrigel implantation assays indicate that 12-LOX-transfected PC3 cells were more angiogenic than their neo controls. These data indicate that 12-LOX stimulates human PCa tumor growth by a novel angiogenic mecha...

inflammatory factor 1-3 . It reduces organ damage in animal models of sepsis, ischemic injury and stroke 1,4,5 and substantially reduces mortality in patients with severe sepsis 6 . It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect 5,7 is secondary to its anticoagulant and anti-inflammatory effects 1-3 . We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells 8,9 . Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR 10 . This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells.

METHODS: In a prospective randomized clinical trial, phacoemulsification was performed in 2 groups (high vacuum and low vacuum) with 3C nuclear sclerosis. The stop-and-chop technique was used with the Sovereign machine. Machine parameters during the chop stage were vacuum 400 mm Hg in the high-vacuum group and 200 mm Hg in the low-vacuum group and flow rate, 40 cc/min and 20 cc/min, respectively. Endothelial cell density preoperatively before and 1, 6, and 12 weeks postoperatively were compared.

Poly(N-isopropylacrylamide) (PIPAAm) brush surfaces with different layer thickness on polystyrene substrates were prepared by surfaceinitiated atom transfer radical polymerization (ATRP). Surface characteristics of PIPAAm brushes and their influence on adhesion and detachment of bovine carotid artery endothelial cells (ECs) were controlled by PIPAAm layer thickness. Surface hydrophilicity increased with PIPAAm layer thickness at 37 C because PIPAAm brush surfaces with higher thickness provide more extended chain conformations with relatively high chain mobility, and accompanying polymer chain hydration. These surface property alterations lead to negligible cell adhesion through minimal matrix protein adsorption and also modified surface modulus. By adjusting polymerization reaction conditions and time, polymer layers supporting confluent cultures of ECs were possible. Confluent EC monolayers spontaneously detached as contiguous cell sheets from PIPAAm brush surfaces at reduced temperatures. Thermoresponsive cell adhesion and detachment behavior were analyzed from the standpoint of surface physicochemical characteristics. Thermoresponsive surfaces prepared by surface-initiated ATRP techniques allow surface selection in preparing cell sheets from attachment-dependent cells having relatively strong adhesive property for tissue engineering applications.

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a “shear stress receptor”?Are “mechanical receptors” and the final signaling pathways they evoke similar to other stimulus–response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that activates and directs the migration of leukocytes that have CXCR4, which is the unique receptor for SDF-1. Although SDF-1/CXCR4 interaction has been implicated in various inflammatory conditions, its role in modulating ...

sis and septic shock associated with vascular damage and multiple organ failure. Apoptosis appears to play a key role during sepsis, and the ability of S. aureus to induce apoptosis in endothelial cells might contribute to metastatic infection. In contrast to leukocytes, in human umbilical vein endothelial cells and two endothelial cell lines neither purified a a a a -toxin nor staphylococcal supernatants were sufficient to induce apoptosis. Apoptosis induction instead required staphylococcal invasion as well as signals from metabolically active intracellular staphylococci. Only strongly haemolytic and invasive staphylococci, but not non-invasive strains induced apoptosis that was caspase-dependent but Fas-independent. However, only a subgroup of clinical isolates with an invasive and haemolytic phenotype induced apoptosis. Expression of a a a a -toxin in a non-haemolytic strain partially restored apoptosis induction, suggesting a role of a a a a -toxin as a trigger of apoptosis. Furthermore, infection of endothelial cells with isogenic mutants of various regulator genes revealed that apoptosis induction was dependent on the global regulator agr and the alternative sigma factor sigB , but not influenced by sarA . Together, our results indicate that the ability of S. aureus to induce apoptosis in endothelial cells is determined by multiple virulence factors.

The aim of this study is to elucidate the role of integrins in transducing fluid shear stress into intracellular signals in vascular endothelial cells, a fundamental process in vascular biology. We demonstrated that shear stress activates specific integrins in endothelial cells plated on substrates containing the cognate extracellular matrix (ECM) ligands. The shear stress-induced mechanotransduction, as manifested by integrin-Shc association,

To document the 1-year safety, efficacy, and stability results of 355 eyes treated in the multicenter study of conductive keratoplasty (CK) used to correct low to moderate hyperopia. Design: Nonrandomized comparative (self-controlled) trial. Participants: Twenty surgeons at 13 centers performed CK on the eyes of all patients enrolled in a multicenter, 2-year, U.S. phase III clinical trial. Treated eyes had ϩ0.75 to ϩ3.00 diopters (D) of hyperopia and Յ0.75 D of cylinder. Patients were 40 years of age or older. Intervention: Low-energy, high-frequency current was applied directly into the peripheral corneal stroma through a delivery tip inserted at 8 to 32 treatment spots. The number of treatment spots was increased for increasing levels of hyperopia, but the amount of radiofrequency energy remained constant. Emmetropia was intended. All eyes were treated once (there were no retreatments). Main Outcome Measures: Data from 355 eyes with 1 year of follow-up were analyzed for safety and stability, and data from 318 eyes were analyzed for efficacy and predictability, as well as stability and safety. All patients reported on satisfaction and quality of vision after surgery. Results: At 1 year, uncorrected visual acuity was Յ20/20 in 56%, Յ20/25 in 75%, and Յ20/40 in 92% of eyes. The manifest refractive spherical equivalent refraction was within 0.50 D in 63%, within Ϯ1.00 D in 89%, and within Ϯ2.00 D in 99%. Seven of 355 eyes lost 2 lines of best spectacle-corrected visual acuity at 1 year, but no eye lost Ͼ2 lines. One eye of 355 had induced cylinder of Ͼ2.00 D. The cycloplegic refractive spherical equivalent changed a mean of 0.25 Ϯ 0.50 D between months 3 and 6, 0.11 Ϯ 0.41 D between months 6 and 9, and 0.11 Ϯ 0.35 D between months 9 and 12. Refractive stability seemed to be attained by 6 months and remained stable through 12 months. Histology and confocal microscopy showed deep penetration of the treatment into the stroma. Endothelial cell counts were not changed by the treatment. Conclusions: CK seems to be safe, effective, and stable for correcting low to moderate spherical hyperopia in patients 40 years old or older. Treatment penetration is deep and cylindrical in shape, and it does not damage the corneal endothelium. Uncorrected visual acuity, predictability, and stability are as good as or better than those obtained with other techniques used to correct hyperopia.