Human Saphenous Vein Endothelial Cells Research Articles

Effect of Shear Stress on the Expression of Coagulation and Fibrinolytic Factors in Both Smooth Muscle and Endothelial Cells in a Co-Culture Model

Blood vessels are subjected to forces due to the flow. Endothelial cells (EC) are recipients, cross-talk with smooth muscle cells (SMC), and regulate physiology. It was hypothesized that both EC and SMC respond to shear stress, which alters the expression of factors in coagulation and fibrinolysis. Methods: A co-culture of human saphenous vein EC (HSVEC) and human saphenous vein SMC (HSVSMC) was exposed to shear, following which the cells were separated. Gene expression of tissue factor, thrombomodulin (TM), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) were analyzed with real-time RT-PCR. Protein expression was studied with ELISA. In HSVEC, the expression of PAI-1 (×2.1), tPA (×1.8), uPA (×1.6), tissue factor (×2.5) and TM (×1.9) was upregulated after 4 h of shear compared to controls. After 24 h of shear, expression was still upregulated in tPA (×2.3) and TM (×1.6). In HSVSMC, change in expression of PAI-1 (×2.1) was present after 4 h and in uPA (×2.1), and TM (×0.4) after 24 h. Both HSVEC and HSVSMC responded to shear, which led to altered expression of coagulation and fibrinolytic factors. This indicates that SMC, and interactions between EC and SMC, are more important in the regulation of vascular wall hemostasis than earlier studies have reported.

Peptide-presenting two-dimensional protein matrix on supported lipid bilayers: An efficient platform for cell adhesion

Understanding and controlling cell adhesion to biomaterials and synthetic materials are important issues in basic research and applied sciences. Supported lipid bilayers (SLBs) functionalized with cell adhesion peptides linked to lipid molecules are popular platforms of cell adhesion. In this paper, an alternative approach of peptide presentation is presented in which peptides are stereo-selectively linked to proteins self-assembling in a rigid two-dimensional (2D) matrix on SLBs. Annexin-A5 (Anx5) was used as prototype protein for its known properties of forming stable and rigid 2D matrices on lipid surfaces. Two types of Anx5-peptide complexes, containing either a RGD or an IKVAV sequence, were synthesized. The authors show that both Anx5-peptide complexes present the same properties of binding and 2D organization on lipid surfaces as Anx5, when investigated by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and transmission electron microscopy techniques. Anx5-RGD and Anx5-IKVAV 2D matrices were found to promote specific adhesion of human saphenous vein endothelial cells and mouse embryonic stem cells, respectively. The influence of the surface density of exposed peptides on cell adhesion was investigated, showing that cells attach to Anx5-peptide matrices when the average distance between peptides is smaller than about 60 nm. This cell adhesion platform provides control of the orientation and density of cell ligands, opening interesting possibilities for future applications.

Abstract 2278: Isoform Specific PKC Responses Modulate ET-1 and Hypoxia Reperfusion Induced Injury

Background: Hypoxia and reperfusion (H/R) injury is a predominant cause of allograft failure following heart transplantation. Several investigators have shown that endothelin-1 (ET) reduces myocardial function and survival via modulation of protein kinase C (PKC). We examined the role of ET and PKC modulation on human saphenous vein endothelial cells (HSVEC). Methods: HSVEC were submitted to 24 hours of hypoxia (PO 2 <0.1%) and 24 hour of reperfusion (PO 2 =21%). Normoxic controls were incubated at PO 2 =21%. Cells were incubated with either ET:100nM, BOS (ET antagonist):10μM, ET+BOS or PBS during H/R. Cells were also treated with PKCδ, PKCλ and PKCϵ inhibitors. Caspase activity was measured. Free radical (ROS) and NO production were measured. Protein expression and translocation of PKC isoforms and eNOS were assessed using Western Blot analysis. Results: Apoptosis (35% increase in caspase 3,8,9 activity) was observed following H/R compared to normoxia. BOS therapy prevented apoptosis while ET exposure increased caspase activity by 75%, p<0.01. H/R resulted in PKCδ and ϵ translocation while reducing PKCλ activity. BOS exposure prevented H/R induced PKCδ and ϵ activation and PKCλ inhibition while incubation with ET enhanced H/R induced PKC effects. PKCδ and ϵ inhibition reduced caspase activity after H/R and abrogated ET induced apoptosis, p<0.01. PKCλ inhibition enhanced apoptosis. NO production was impaired following H/R, p<0.05. BOS therapy restored NO homeostasis while ET exposure worsened NO production compared to H/R alone, p<0.05. PKCδ and ϵ inhibition improved NO production while PKCλ inhibition worsened NO release. H/R driven ROS production was inhibited by BOS and enhanced by ET exposure, p<0.01. PKCλ inhibition raised ROS production while PKCδ and ϵ inhibition reduced ROS release, p<0.01. eNOS protein expression was reduced by H/R while BOS treatment prevented downregulation. Conclusions: Our findings revealed that ET plays a crucial role in cellular injury following H/R via specific PKC isoforms. ET via PKC modulation leads to endothelial dysfunction and cell death. Therefore, targeted isoform specific PKC modulation as well as BOS may prove to be optimal strategy to prevent endothelial injury during allograft storage and transplantation.

Human vascular smooth muscle cells and endothelial cells cocultured on polyglycolic acid (70/30) scaffold in tissue engineered vascular graft

Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid (70/30) has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha). Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grow vascular patch (Groups A and B respectively) and cocultured in vitro to grow into vascular patch (Group C). Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1alpha in the culturing solutions were examined by radioimmunology to measure endothelial function. Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were (234 +/- 29) pg/ml and (428 +/- 98) pg/ml respectively in Group C and (196 +/- 30) pg/ml and (346 +/- 120) pg/ml in Group B; both significantly higher than in Groups A and D (blank control group, all P < 0.05). Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro.

Insulin does not regulate glucose transport and metabolism in human endothelium

Although endothelial cells express insulin receptors, it is controversially discussed whether the endothelium represents an insulin-responsive tissue. Since available data are primarily restricted to animal endothelial cells, this study tested (i) whether insulin affects glucose metabolism in human endothelium; (ii) whether insulin sensitivity is different in micro- versus macrovascular endothelial cells; and (iii) whether glucose concentration in the incubation medium affects the cells' response to insulin. Human umbilical vein endothelial cells (HUVECs), human adult saphenous vein endothelial cells (HAVECs), human aortic endothelial cells (HAEC), and human retinal endothelial cells (HRECs) as well as human smooth muscle cells were incubated with/without insulin (0.3 nmol L(-1) or 1 micromol L(-1)). Glucose transport, glycogen synthesis, glycogen content, lactate release, and expression of phospho-Akt, Akt, and endothelial nitric oxide synthase (eNOS) were determined. In HUVECs and HRECs, insulin (1 micromol L(-1)) increased (P < 0.05) eNOS expression by ~70% and doubled Akt phosphorylation, but the latter was by far more pronounced in human smooth muscle cells (+1093 +/- 500%, P < 0.05). In human smooth muscle cells, insulin (1 micromol L(-1)) stimulated glycogen synthesis by 67 +/- 11% (P < 0.01). In human micro- (HRECs) and macrovascular endothelial cells (HUVECs, HAVECs and HAECs), insulin, however, failed to stimulate glucose transport, glycogen synthesis, glycogen content, or lactate release under various conditions, i.e. after glucose deprivation or in medium with normal (5.5 mmol L(-1)) or high glucose (30 mmol L(-1)). Insulin stimulated glycogen synthesis and Akt phosphorylation in human smooth muscle cells. In human micro- and macrovascular endothelial cells, insulin, however, failed to affect glucose uptake and metabolism under all experimental conditions applied, whereas it increased Akt phosphorylation and eNOS expression.

Gene Response in Endothelial Cells Cultured on Engineered Surfaces Is Regulated by Shear Stress

In vitro endothelialization of small-diameter synthetic vascular prostheses confluently lined with cultured autologous endothelial cells (ECs) before implantation has been shown to increase their patency. Many authors have studied the effects of shear stress on EC gene response seeded on various substrates showing different gene expression profiles according to cell type, flow times, or shear type with different molecular biology techniques, but few studies have reported any EC gene response to shear stress when cells are seeded on vascular grafts. The purpose of this in vitro study was to investigate whether ECs were able to transduce shear stress at the level of the nucleus. Human saphenous vein ECs were seeded on glass slides coated with gelatin or fibrin glue or on 6-mm fibrin-glue-coated grafts. Then cells were exposed to 12 dyn/cm(2) for 4 h and ribonucleic acid (RNA) were extracted. The relative messenger RNA (mRNA) expression was studied using real-time quantitative polymerase chain reaction for the following mRNAs: von Willebrand Factor, tissue-plasminogen activator, CD31, vascular endothelial (VE)-cadherin, beta(1) integrin, and vascular endothelial growth factor receptor type 2. From parallel flow chambers, results have shown similar EC gene response on gelatin and fibrin glue under laminar shear stress with downregulation of prothrombotic genes, as well as upregulation of nonthrombotic genes and upregulation of adhesion molecules such as VE-cadherin, but some discrepancies are noted, with a downregulation of CD31 and kinase insert domain receptor (KDR) for the former, without significant variation for the latter. In comparison, results show upregulation of tissue type plasminogen activator gene and downregulation of KDR, VE-cadherin, and beta(1) integrin genes in ECs lining grafts. To conclude, the major finding of our study is to show that human saphenous vein ECs seeded on fibrin glue (in planar flow chambers or in tubular grafts) can be regulated using shear stress via gene expression changes in a nonthrombotic way.

Impact of diabetic serum on endothelial cells: An in-vitro-analysis of endothelial dysfunction in diabetes mellitus type 2

Diabetic endothelial dysfunction was characterized by altered levels of adhesion molecules and cytokines. Aim of our study was to evaluate the effects of diabetic serum on cell-growth and proinflammatory markers in human saphenous vein endothelial cells (HSVEC) from diabetic and non-diabetic patients. Diabetic serum showed (1) complementary proliferative activity for non-diabetic and diabetic HSVEC, (2) unchanged surface expression of adhesion molecules, and (3) elevated levels of sICAM-1 in HSVEC of all donors. The concentration of sVCAM-1 was increased only in diabetic cells. The proinflammatory state of diabetic HSVEC characterized by increased levels of cytokines was compensated. We concluded that even under normoglycemic conditions the serum itself contains critical factors leading to abnormal regulation of inflammation in diabetics. We introduced an in vitro model of diabetes representing the endothelial situation at the beginning of diabetes (non-diabetic cells/diabetic serum) as well as the diabetic chronic state (diabetic cells/diabetic serum).

Development of a disease-specific model to evaluate endothelial dysfunction in patients with diabetes mellitus

Diabetic patients have an increased cardiovascular risk. We propose to characterize the endothelial dysfunction in a disease-specific in vitro model. Human saphenous vein endothelial cells (HSVEC) were isolated from coronary artery bypass patients without and with non-insulin-dependent diabetes mellitus. Growth kinetics and proinflammatory responses (expression of adhesion molecules, cytokines) were documented under non-stimulating conditions. Diabetic HSVEC showed delayed growth kinetics with reduced cell densities of about 40%. During exponential growth of diabetic EC, the surface expression of adhesion molecules was increased 10-fold (p⩽0.05). However, in a monolayer the expression adapted to low levels of non-diabetic EC. In addition, diabetic EC produced significantly more soluble E-selectin, VCAM-1, IL-6 and MCP-1. Our results suggest a link between the pathologically proinflammatory basic state of diabetic EC and the endothelial dysfunction in diabetic disease. Therefore, this in vitro model could be used for investigating early dysfunction and environmental effects on pathological endothelium.

MTOR-inhibitors simultaneously inhibit proliferation and basal IL-6 synthesis of human coronary artery endothelial cells

Divergent results regarding the immunosuppressive effects of mammalian-target-of-rapamycin-(mTOR)-inhibitors on venous endothelial cells (ECs) have highlighted the importance of an accurate EC-model. The purpose of this study was to determine mTOR-inhibitor effects at a specific site of action – the human coronary-artery-ECs (HCAECs) – and to compare these data with results gained from cultures of human saphenous vein ECs (HSVECs). This EC-model could enable us to gain insight into site-specific pharmacodynamics and the immunosuppressive management of transplant vasculopathy. ECs were cultivated with rising concentrations of mTOR-inhibitors in the presence/absence of tumor necrosis factor (TNF). Cell counts, DNA-synthesis, cytotoxicity and concentrations of the cytokine IL-6 as well as the chemokines IL-8 and MCP-1 were measured. Half-maximal inhibitory effects on cell growth were reached after about 30 h incubation and both cell types showed equal responses regarding cell growth and DNA-synthesis after 48 h incubation time. mTOR-inhibitors failed to suppress basal/TNF-induced secretion of IL-8 and MCP-1, but IL-6 synthesis after TNF-induction was reduced to 35%. In contrast to human saphenous vein ECs (HSVECs), mTOR-inhibitors also reduced basal IL-6-secretion of HCAECs (to 55%) and cell proliferation was simultaneously inhibited within the same concentration range. Taking everything into account, we conclude that EC-proliferation is inhibited at concentrations needed to suppress TNF-stimulated IL-6 synthesis. Furthermore, the specific suppression of basal arterial IL-6-secretion and the delayed onset of the mTOR-inhibitor effect on HCAEC-proliferation (maximum reached after about 36 h) might be of relevance for the prevention of transplant vasculopathy at its initial stage, e.g. as a component of cardioplegic solutions.

Human endothelial progenitor cell attachment to polysaccharide-based hydrogels: A pre-requisite for vascular tissue engineering

A hydrogel was prepared from polysaccharides (pullulan/dextran/fucoidan) and evaluated as a novel biomaterial for Endothelial Progenitor Cell (EPC) culture. Using a cross-linking process with sodium trimetaphosphate in aqueous solution, homogeneous, transparent and easy to handle gels were obtained with a water content higher than 90%. Circular scaffolds (6 mm diameter and 2 mm thickness discs) were used for cell culture. Different types of EPCs were used: CD34+ derived ECs from cord blood and two sorts of CD133+ derived ECs from human bone marrow, old (30 days) and young (4 days) cells. EPCs were characterised as endothelial cells by immunofluorescent stainings for CD31 and Dil-Ac-LDL. CD133+ derived ECs from bone marrow were characterized by RT-PCR for CD31, VE-cadherin and KDR. HSVECs (Human Saphenous Vein Endothelial Cells) were used as control cells. We evaluated whether different kinds of EPCs could adhere on this novel hydrogel 4 h and 24 h after seeding, by a colorimetric quantitative test. EPCs adhered to hydrogels in serum- free conditions with values being over than 80% for young CD133+ cells at 4 h and 24 h. This pullulan-based hydrogel could constitute a suitable support for vascular cell adhesion as a pre-requisite for vascular tissue engineering.

Vascular bed origin dictates flow pattern regulation of endothelial adhesion molecule expression

Endothelial cell phenotypes markedly differ, depending upon function and vascular bed of origin. Differences might account for specific susceptibility to pathological conditions. As leukocyte adhesion to activated endothelium is the initiating event in a range of diseases, we compared the influence of vascular bed-specific flow patterns on adhesion molecule expression in human saphenous vein (HSVEC) and coronary artery endothelial cells (HCAEC). In vitro, immune cell attachment was increased 1.6-fold when tumor necrosis factor (TNF)-alpha-stimulated HSVEC were exposed to coronary artery flow in place of physiological venous flow and 1.9-fold higher compared with attachment to cytokine-stimulated HCAEC exposed to coronary artery flow. This was associated with increased concentrations of soluble E-selectin, VCAM-1, and ICAM-1 in supernatants of HSVEC exposed to coronary artery flow compared with HCAEC exposed to the same flow pattern. Venous and coronary artery flow both increased TNF-alpha-induced E-selectin and ICAM-1 expression on HSVEC, but only coronary artery flow increased VCAM-1 expression. In marked contrast to HSVEC, venous and coronary artery flow attenuated TNF-alpha-induced E-selectin and VCAM-1 expression on HCAEC, whereas coronary artery flow further induced ICAM-1 on cytokine-stimulated HCAEC. With the exception of cytokine-induced ICAM-1, adhesion molecule expression on HSVEC exposed to coronary artery flow exceeded expression on HCAEC. Thus ICAM-1 expression involves complex flow-dependent and -independent pathways with marked dissimilarities between the two endothelial cell types studied. Interestingly, Kruppel-like factor (KLF) 4 overexpression in HCAEC and HSVEC significantly reduced TNF-alpha-induced E-selectin and VCAM-1 expression in static conditions, while ICAM-1 expression remained constant. Furthermore, both flow patterns induced KLF2 and KLF4 expression in HCAEC and HSVEC. Venous and coronary artery flow differentially influence endothelial adhesion molecule and transcription factor expression, depending on the vascular bed of origin. Differences in adhesion molecule expression and subsequent immune cell adhesion between HSVEC and HCAEC may contribute to different susceptibility to pathological conditions.

Signal Transduction and Procoagulant State of Human Cord Blood—Progenitor-Derived Endothelial Cells after Interleukin-1α Stimulation

Isolation of endothelial progenitors from human umbilical cord blood generated great hope in vascular tissue engineering. However, before clinical use, progenitor derived endothelial cells (PDECs) have to be compared with mature endothelial cells (ECs). The aim of this study was to explore the behavior of PDECs exposed to a proinflammatory cytokine (interleukin-1alpha; IL-1alpha) according to the mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB signal transduction pathways as well as procoagulant activity (PCA). CD34(+) mononuclear cells were isolated using magnetic beads, cultured, and compared with human saphenous vein ECs (HSVECs). PDECs express endothelial markers: CD31, VE-cadherin, von Willebrand factor, KDR, and incorporate acetylated low-density lipoprotein (Dil-Ac-LDL). IL-1alpha similarly activates c-Jun N-terminal protein kinase (JNK) and p38 pathways in HSVECs and PDECs, whereas extracellular signal-related kinase (ERK)1/2 phosphorylation is lower in PDECs than in HSVECs. Low ERK1/2 phosphorylation in PDECs was specific to IL-1alpha as vascular endothelial growth factor (VEGF) similarly stimulated ERK1/2 pathway. With respect to inhibitor of NF-kappa B (Ikappa B) degradation, NF-kappa B translocation and phosphorylation, the NF-kappa B pathway is comparable in HSVECs and PDECs after stimulation. PCA and tissue factor level induced by IL-1alpha are lower in PDECs than in HSVECs. Thus, our data show that PDECs display the characteristics of functional mature ECs under IL-1alpha stimulation. However, we observed significant differences between PDECs and HSVECs related to both ERK1/2 pathway activation and tissue factor production.

Phosphatidylinositol 3-kinase/protein kinase Akt negatively regulates plasminogen activator inhibitor type 1 expression in vascular endothelial cells

Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolytic activity and mediates vascular atherothrombotic disease. Endothelial cells (ECs) synthesize and secrete PAI-1, but the intracellular signaling pathways that regulate PAI-1 expression are not entirely known. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt pathway, which regulates endothelial function, could modulate PAI-1 expression in ECs. Cultured bovine aortic and human saphenous vein ECs were stimulated with TNF-alpha, ANG II, insulin, or serum, and PAI-1 expression was determined by Northern and Western analyses. Inhibition of PI3K with wortmannin or LY-294002 enhanced PAI-1 expression induced by these extracellular stimuli. Similarly, overexpression of a dominant-negative mutant of PI3K or Akt increased TNF-alpha- and insulin-induced PAI-1 expression. The increase in PAI-1 was due to transcriptional and posttranscriptional mechanisms as PI3K inhibitors increased PAI-1 promoter activity and mRNA stability. The induction of PAI-1 by TNF-alpha and insulin is mediated, in part, by ERK and p38 MAPK. PI3K inhibitors augmented TNF-alpha- and insulin-induced phosphorylation of these MAPKs. Simvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, which is known to activate PI3K/Akt, blocks TNF-alpha- and insulin-induced PAI-1 expression. Treatment with PI3K inhibitors reversed the inhibitor effects of simvastatin on TNF-alpha- and insulin-induced PAI-1 expression. These findings indicate that the PI3K/Akt pathway acts as a negative regulator of PAI-1 expression in ECs, in part, through the downregulation of MAPK pathways. These results suggest that factors that activate the PI3K/Akt pathway in ECs may have therapeutic benefits for atherothrombotic vascular disease.

The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKCε inhibition

A high intake of the omega-3 fatty acid docosahexaenoate [docosahexaenoic acid (DHA)] has been associated with systemic antiinflammatory effects and cardiovascular protection. Cyclooxygenase (COX)-2 is responsible for the overproduction of prostaglandins (PG) at inflammatory sites, and its expression is increased in atheroma. We studied the effects of DHA on COX-2 expression and activity in human saphenous vein endothelial cells challenged with proinflammatory stimuli. A>or=24-h exposure to DHA reduced COX-2 expression and activity induced by IL-1, without affecting COX-1 expression. DHA effect depended on the NF-kappaB-binding site in the COX-2 promoter. EMSAs confirmed that DHA attenuated NF-kappaB activation. Because MAPK, PKC, and NAD(P)H oxidase all participate in IL-1-mediated COX-2 expression, we also tested whether these enzymes were involved in DHA effects. Western blots showed that DHA blocked nuclear p65 NF-kappaB subunit translocation by decreasing cytokine-stimulated reactive oxygen species and ERK1/2 activation by effects on both NAD(P)H oxidase and PKCepsilon activities. Finally, to address the question whether DHA itself or DHA-derived products were responsible for these effects, we inhibited the most important enzymes involved in polyunsaturated fatty acid metabolism, showing that 15-lipoxygenase-1 products mediate part of DHA effects. These studies provide a mechanistic basis for antiinflammatory and possibly plaque-stabilizing effects of DHA.

Why a Diet Rich in Seafood Is Healthy

Omega-3 fatty acids, which are found in marine organisms, are known for their beneficial health effects. One mechanism for their antiinflammatory effects is competitive inhibition of the enzymatic activity of cyclooxygenase (COX), which is the rate-limiting enzyme in the biosynthesis of prostaglandins. Massaro et al . report that exposure of endothelial cells to the omega-3 fatty acid docosahexaenoate (DHA) for periods long enough to allow incorporation of this lipid into cellular membranes inhibits the activation of nuclear factor κB (NF-κB) and subsequently COX-2 expression and prostaglandin production in response to proinflammatory signals. Treatment of human saphenous vein endothelial cells (HSVECs) with DHA for up to 48 hours maximally inhibited COX activation in response to subsequent exposure of the cells to interleukin 1α (IL-1α) or a phorbol ester. Western blot and Northern analysis showed that DHA treatment inhibited the IL-1α- or phorbol ester-stimulated expression and abundance of COX-2 (the inducible isoform) without affecting that of COX-1 (the constitutively expressed isoform). Reporter gene analysis demonstrated that DHA-mediated inhibition of COX-2 required the NF-κB binding site in the COX-2 promoter, and DHA pretreatment decreased IL-1α stimulation of NF-κB DNA binding (measured by electrophoretic mobility shift assay) and nuclear translocation (measured by cell fractionation followed by Western blotting). DHA treatment inhibited several steps in the pathway activated by IL-1α that leads to NF-κB activation. In cells treated with DHA, subsequent responses to IL-1α showed (i) decreased extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation but no change in activation of p38 mitogen-activated protein kinase (p38 MAPK), (ii) decreased reactive oxygen species production through inhibition of membrane association of the p47 phox subunit of NADPH oxidase, and (iii) decreased membrane association of protein kinase Cε (PKCε) but not PKCα or PKCζ. Thus, it appears that the long-term beneficial effects of omega-3 fatty acids may be due to alteration of membrane lipid composition leading to reduced signaling in response to inflammatory stimuli. M. Massaro, A. Habib, L. Lubrano, S. Del Turco, G. Lazzerini, T. Bourcier, B. B. Weksler, R. De Caterina, The omega-3 fatty acid docosahexaenoate attenuates endothelial cyclooxygenase-2 induction through both NADP(H) oxidase and PKCε inhibition. Proc. Natl. Acad. Sci. U.S.A. 103 , 15184-15189 (2006). [Abstract] [Full Text]

Effects of fibulin-5 on attachment, adhesion, and proliferation of primary human endothelial cells

Background Fibulin-5 is a novel extracellular protein that is thought to act as a bridging peptide between elastin fibers and cell surface integrins in blood vessel wall. Fibulin-5 binding to endothelial cell (EC) surface integrins may effect cell proliferation and cell attachment to extracellular matrix (ECM) or to artificial surfaces. In this paper, we describe the effects of fibulin-5 on attachment, adhesion, and proliferation of primary human EC. After demonstrating that fibulin- 5 over-expression inhibited EC proliferation, we tested the hypothesis that co-expression of fibulin-5 and VEGF 165 will lead to unique EC phenotype that will exhibit increased adherence properties and retain its proliferation capacity. Methods and results Fibulin-5 and VEGF 165 gene transfer to primary human saphenous vein endothelial cells was accomplished using retroviral vectors encoding the two genes. Transgene expression was verified using immunohistochemistry, Western blotting, and ELISA. Fibulin 5 over-expression tended to improve immediate EC attachment (30 min after seeding) and improved significantly adhesion (>40%) under shear stress tested 24 h after EC seeding. The effects of fibulin-5 and VEGF 165 on EC proliferation in the presence or absence of basic FGF were also tested. EC expressing fibulin-5 had reduced proliferation while VEGF 165 co-expression ameliorated this effect. Conclusion Fibulin-5 improved EC attachment to artificial surfaces. Dual transfer of fibulin-5 and VEGF 165 resulted in EC phenotype with increased adhesion and improved proliferation. This unique EC phenotype can be useful for tissue engineering on endovascular prostheses.

C-Reactive Protein Upregulates Receptor for Advanced Glycation End Products Expression in Human Endothelial Cells

The receptor for advanced glycation end products (RAGE) may play an important role in inflammatory processes and endothelial activation, likely to accelerate the processes of coronary atherosclerotic development, especially in diabetic patients. The factors that regulate arterial expression of RAGE are not completely clear. C-reactive protein (CRP) is identified as a key proinflammatory cytokine in patients with atherosclerosis. Therefore, we tested the hypothesis that RAGE expression in endothelial cells can be upregulated by CRP. Human saphenous vein endothelial cells were incubated with human recombinant CRP, free of sodium azide and endotoxin. RAGE protein expression was measured by flow-cytometric analysis and Western blotting. CRP caused a significant increase in RAGE protein expression at a dose as low as 5 mug/mL, with expression peaking at 24 to 48 hours after CRP incubation. The effects of modified monomeric CRP on RAGE protein expression were comparable with that of native pentameric CRP. At the mRNA level, CRP not only increased RAGE gene expression but also attenuated the degradation of RAGE mRNA. Furthermore, RNA interference of RAGE gene expression significantly decreased the level of macrophage chemoattractant protein 1, a key downstream mediator of CRP activity. Therefore, CRP at concentrations known to predict future vascular events upregulates RAGE expression in human endothelial cells at both the protein and mRNA level. Silencing of the RAGE gene prevents CRP-induced macrophage chemoattractant protein 1 activation. These data reinforce the mechanistic links among inflammation, endothelial dysfunction, and atherothrombosis.

Engineering of Blood Vessels from Acellular Collagen Matrices Coated with Human Endothelial Cells

Small-caliber synthetic grafts used for coronary bypass artery grafting are compromised by thrombogenicity and accelerated intimal thickening, resulting in early graft occlusion. Herein we describe the fabrication and physical properties of small-caliber blood vessels using decellularized porcine aortic segments. These vessels were further coated with human saphenous vein endothelial cells (HSVECs) for future clinical applications. Chemical staining of decellularized vessels showed that they preserved their native matrix architecture, including several collagen layers in between internal and external elastin layers. The burst pressure for the decellularized vessels was higher than 1,000 mmHg. HSVECs, seeded on the luminal side, adhered to the matrix and formed a uniform monolayer. HSVEC-seeded vessels produced prostaglandin I2 and released vasoactive agents in response to the calcium ionophore A23187. These results show that engineered blood vessels coated with the host endothelial cells possess morphologic and functional characteristics of human small-caliber vessels. Tissue-engineered vessels may potentially be useful clinically as vascular grafts.

Elevated Endothelin-1 Levels Impair Nitric Oxide Homeostasis Through a PKC-Dependent Pathway

Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms. Human saphenous vein endothelial cells (HSVECs) (n=8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKCdelta and PKCalpha translocation compared with control, whereas translocation of PKClambda was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control. Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKClambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

Expression of Indoleamine 2,3-Dioxygenase (IDO) by Endothelial Cells: Implications for the Control of Alloresponses

Indoleamine 2,3-dioxygenase (IDO) is an important enzyme in the regulation of immune responses; cells that express IDO can suppress T-cell responses and promote tolerance. Because of the critical role of endothelial cells in graft rejection, we have investigated the role of IDO expression by vascular endothelial cells and its consequence on immunoregulation. We compared the expression of IDO by primary human umbilical vein endothelial cells (HUVECs), human saphenous vein endothelial cells (HSVECs) and arterially derived endothelial cells using reverse transcriptase PCR, Western blotting and assays for enzymatic activity. In HUVECs IDO is upregulated by incubation with cytokines or in mycoplasma-infected cells. On the other hand HSVECs and arterially derived endothelial cells express little IDO, which is poorly upregulated upon activation (except by mycoplasma). Inhibition of IDO activity improved the ability of HUVECs to stimulate allogeneic T-cell responses. If either HUVECs or HSVECs are transfected with the gene encoding IDO, then they are incapable of stimulating allogeneic T-cell responses and induce anergy in allospecific T cells (which can also act as regulatory cells). The variable expression of IDO in different endothelial cells is important not only in understanding the role of endothelial cells in the regulation of graft rejection, but also as a potential therapeutic strategy.