Relaxin inhibits early steps in vascular inflammation

Increased expression of endothelial adhesion molecules, high levels of the monocyte chemoattractant protein-1 (MCP-1) and enhanced VLA4 integrin/VCAM-1 and CCR-2/MCP-1 interactions are initial steps in vascular inflammation. We sought to determine whether relaxin, a potent vasodilatory and anti-fibrotic agent, mitigates these early events compromising endothelial integrity. The effect of relaxin coincubation on the TNF-α-stimulated expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin; the MCP-1 expression by human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HAoSMC); as well as on direct monocyte–endothelium cell adhesion was quantified by ELISA or adhesion assay. CCR-2 and PECAM expression on HUVEC and THP-1 monocytes was investigated by FACS analysis. Relaxin treatment suppressed significantly TNF-α-induced upregulation of VCAM-1 and PECAM, CCR-2, and MCP-1 levels and direct monocyte adhesion to HUVEC. Our findings identify relaxin as a promising inhibitory factor in early vascular inflammation. By attenuating the upregulation of VCAM-1, key adhesion molecule in early vascular inflammation, and of MCP-1, a chemokine pivotal to monocyte recruitment, relaxin decreased initial monocyte–endothelium contact. This may be of relevance for the prevention and treatment of atherosclerosis and of other pro-inflammatory states.     

Endogenous prostaglandin D(2) synthesis decreases vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells

We examined the role of prostaglandin D(2) (PGD(2)) in the expression of vascular cell adhesion molecule-1 (VCAM)-1 following interleukin-1beta (IL-1) stimulation in human umbilical vein endothelial cells (HUVEC) transfected with lipocaline-type PGD(2) synthase (L-PGDS) genes. HUVEC were isolated from human umbilical vein and incubated with 20 U/ml IL-1 and various concentrations of authentic PGD(2). The isolated HUVEC were also transfected with L-PGDS genes by electroporation. The L-PGDS-transfected HUVEC were used to investigate the role of endogenous PGD(2) in IL-1-stimulated VCAM-1 biosynthesis. We also used an anti-PGD(2) antibody to examine whether an intracrine mechanism was involved in VCAM-1 production. PGD(2) and VCAM-1 levels were determined by radio- and cell surface enzyme-immunoassay, respectively. VCAM-1 mRNA was assessed by RT-PCR. IL-1-stimulated VCAM-1 expression by HUVEC was dose-dependently inhibited by authentic PGD(2). L-PGDS gene-transfected HUVEC produced more PGD(2) than HUVEC transfected with the reporter gene alone. IL-1 induced increases in VCAM-1 expression in HUVEC transfected with reporter genes alone. However, this effect was significantly attenuated in the case of IL-1 stimulation of HUVEC transfected with L-PGDS genes, and accompanied by an apparent suppression of VCAM-1 mRNA expression. Neutralization of extracellular PGD(2) by anti-PGD(2)-specific antibody influenced neither VCAM-1 mRNA expression nor VCAM-1 biosynthesis. In conclusion, HUVEC transfected with L-PGDS genes showed increased PGD(2) synthesis. This increase was associated with attenuation of both VCAM-1 expression and VCAM-1 mRNA expression. The results suggest that endogenous PGD(2) decreases VCAM-1 expression and VCAM-1 mRNA expression, probably through an intracrine mechanism.     

Butyrate inhibits cytokine-induced VCAM-1 and ICAM-1 expression in cultured endothelial cells: the role of NF-kappaB and PPARalpha

Adhesion and migration of leukocytes into the surrounding tissues is a crucial step in inflammation, immunity, and atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. Butyrate, a natural short-chain fatty acid produced by bacterial fermentation of dietary fiber, has been attributed with anti-inflammatory activity in inflammatory bowel disease. Butyrate in vitro is active in colonocytes and several other cell types. We have studied the effect of butyrate on expression of endothelial leukocyte adhesion molecules by cytokine-stimulated human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with butyrate-inhibited tumor necrosis factor-alpha (TNFalpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) in a time and concentration-dependent manner. Butyrate at 10 mM/L inhibited interleukin-1 (IL-1)-stimulated VCAM-1 and ICAM-1 expression. The effect of butyrate on cytokine-stimulated VCAM-1 expression was more pronounced than in the case of ICAM-1. Butyrate decreased TNFalpha-induced expression of mRNA for VCAM-1 and ICAM-1. Suppressed expression of VCAM-1 and ICAM-1 was associated with reduced adherence of monocytes and lymphocytes to cytokine-stimulated HUVEC. Butyrate inhibited TNFalpha-induced activation of nuclear factor-kappaB (NF-kappaB) in HUVEC. Finally, butyrate enhanced peroxisome proliferator-activated receptor-alpha (PPARalpha) expression in HUVEC. These results demonstrate that butyrate may have anti-inflammatory properties not only in colonocytes but also in endothelial cells. The anti-inflammatory and (perhaps) antiatherogenic properties of butyrate may partly be attributed to an effect on activation of NF-kappaB and PPARalpha and to the associated expression of VCAM-1 and ICAM-1. The present findings support further investigations on the therapeutic benefits of butyrate in several pathological events involving leukocyte recruitment.     

Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells.

Vascular endothelial cell adhesion molecule 1 (VCAM-1) is an adherence molecule that is induced on endothelial cells by cytokine stimulation and can mediate binding of lymphocytes or tumor cells to endothelium. Because these interactions often occur at the level of the microvasculature, we have examined the regulation of expression of VCAM-1 in human dermal microvascular endothelial cells (HDMEC) and compared it to the regulation of VCAM-1 in large vessel human umbilical vein endothelial cells (HUVEC). Both cell populations were judged pure as assessed by expression of von Willebrand factor and uptake of acetylated low density lipoprotein. Expression of VCAM-1 was not detectable on either unstimulated HDMEC or HUVEC when assessed by ELISA or flow cytometry. Stimulation of either HDMEC or HUVEC with TNF-alpha resulted in a time- and dose-dependent induction of VCAM-1. However, although TNF-alpha-induced cell surface and mRNA expression of VCAM-1 in HDMEC was transient, peaking after 16 h of stimulation, TNF stimulation led to persistently elevated cell surface expression of VCAM-1 on HUVEC. IL-1 alpha also induced cell surface expression of VCAM-1 on HUVEC in a time- and dose-dependent manner, but stimulation of HDMEC with IL-1 alpha at doses up to 1000 U/ml failed to induce significant cell surface expression. However, IL-1 alpha induced time- and dose-dependent increases in ICAM-1 on HDMEC. Similarly, IL-4 induced VCAM-1 expression and augmented TNF-alpha-induced expression on HUVEC but did not affect VCAM-1 expression on HDMEC. Binding of Ramos cells to cytokine-stimulated endothelial cell monolayers correlated with VCAM-1 induction. Increased binding was seen after stimulation of HDMEC with TNF-alpha, which was blocked by anti-VCAM-1 mAb, but no increases in binding were noted after stimulation of HDMEC monolayers with IL-1 alpha. These data provide additional evidence for the existence of endothelial cell heterogeneity and differences in cell adhesion molecule regulation on endothelial cells derived from different vascular beds.     

天然和氧化低密度脂蛋白对人脐静脉内皮细胞VCAM-1表达的影响

目的探讨天然和氧化低密度脂蛋白(n-LDL,ox-LDL)对人脐静脉内皮细胞(HUVEC)VCAM-1表达的影响。方法将n-LDL,ox-LDL作用于培养的HUVEC,用细胞酶联免疫吸附试验(cellELISA)检测VCAM-1蛋白的表达,用原位分子杂交技术检测VCAM-1 mRNA的表达。结果正常培养的HUVEC可表达VCAM-1,n-LDL和ox-LDL均可增强培养的HUVEC表达VCAM-1,尤以ox-LDL作用更明显。结论n-LDL、ox-LDL可能通过促进血管内皮细胞表达VCAM-1而在动脉粥样硬化的早期事件中起作用。     

三氧化二砷对人脐静脉内皮细胞凋亡及VCAM-1/ICAM-1 表达的影响

目的:观察三氧化二砷(As2O3)对血管内皮细胞增殖、凋亡及VCAM-1/ICAM-1表达的影响,探讨As2O3对血管内皮细胞增殖生长以及炎症反应的影响。方法:人脐静脉内皮细胞(HUVEC)体外培养,以不同As2O3浓度及时间对其进行干预。采用CCK-8测定细胞增殖活性,流式细胞仪AnnexinⅤ/PI双染法检测细胞的凋亡率,实时荧光定量PCR检测VCAM-1mRNA表达,酶联免疫吸附试验(ELISA)检测细胞间黏附分子(VCAM-1)及血管细胞黏附分子(ICAM-1)的表达情况。结果:当As2O3浓度在3μmol.L-1时HUVEC培养24 h的的凋亡率为(0.134±0.03)%,48 h为(3.305±0.53)%,72 h为(3.748±0.84)%(P<0.05),凋亡率均在一较低水平。当As2O3浓度>3μmol.L-1时HUVEC凋亡率明显增加(P<0.01)。不同浓度As2O3作用HUVEC48 h后检测上清液中ICAM-1与VCAM-1浓度时发现1μmol.L-1时VCAM-1表达即开始增加(123.32±3.78 mmol.L-1,P<0.01),而HUVEC表达ICAM-1含量与对照组相比差异并不明显(38.94±2.59 mmol.L-1,P>0.05),随着As2O3浓度的增加,HUVEC表达ICAM-1/VCAM-1的量均增加但敏感性不同。对照组及(1.0、2.0、3.0、4.0、5.0)μmol.L-1As2O3作用于HUVEC 48 h实时荧光定量PCR法检测VCAM-1mRNA表达量明显增加,与对照组相比实验组的表达量分别为(1.657±0.287,1.858±0.241,2.321±0.280,3.012±0.235,3.508±0.342)(P<0.01)。结论:As2O3可直接降低细胞活性,诱导细胞凋亡,并且呈一定的时间-浓度依赖性。在较低浓度时VCAM-1/ICAM-1的表达在一个相对较低的水平,随着As2O3浓度的逐渐升高,内皮细胞凋亡率增高,VCAM-1/ICAM-1表达增加,并且VCAM-1/ICAM-1对As2O3的敏感性呈现一定的差异性。     

Effect of high glucose concentrations on expression of ELAM-1, VCAM-1 and ICAM-1 in HUVEC with and without cytokine activation

Diabetes mellitus is associated with an increased prevalence of endothelial dysfunction and development of atherosclerotic vascular diseases. We demonstrate here that hyperglycemia results in the expression of adhesion molecules on endothelial cells in vitro. Incubation of human umbilical vein endothelial cells (HUVEC) in a culture medium with 11.0 mM, 16.5 mM and 22.0 mM glucose concentrations induced the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). This effect was detectable after 24 h incubation of HUVEC with a high glucose concentration. The effect of high glucose concentration on TNF-alpha induced expression of ELAM-1, VCAM-1 and ICAM-1 was negligible, if at all. These results show that even a short-term exposure of endothelial cells (ECs) to high glucose concentration leads to their activation associated with increased expression of adhesion molecules such as ELAM-1, VCAM-1 and ICAM-1.     

Effects of 17beta-estradiol on growth and apoptosis in human vascular endothelial cells: influence of mechanical strain and tumor necrosis factor-alpha

Vascular endothelial cell (EC) integrity is key to arterial health; endothelial dysfunction is linked to atherogenesis. Atherosclerosis shows a male preponderance, possibly related to the protective effect of estrogens in women. This study examined the effect of estrogens on growth, apoptosis and adhesion molecule expression in cultured human EC. The effects of 17beta-estradiol (E2) were studied in human umbilical vein endothelial cells (HUVEC) under normal culture conditions, and following exposure to cyclic mechanical strain or tumor necrosis factor alpha (TNFalpha). E2 enhanced HUVEC growth in serum-enriched media, in a concentration-dependent manner. This up-regulation of EC growth by E2 was associated with an increase in telomerase activity, assessed by PCR-based TRAP analysis. Cyclic strain enhanced [(3)H]-thymidine incorporation into DNA, and increased activation of mitogen-activated protein (MAP) kinase ERK1/2 and expression of early growth genes (Egr-1 and Sp-1); E2 attenuated the strain-induced ERK1/2 activation but not the early growth gene expression or DNA synthesis. TNFalpha (20 ng/mL) induced apoptosis in HUVEC, causing a decrease in DNA synthesis, increase in floating and Annexin-V-stained cell numbers, and morphological changes. TNFalpha also upregulated ERK1/2 activity and expression of adhesion molecules (ICAM-1, VCAM-1 and E-selectin). E2 significantly attenuated the effects of TNFalpha on ERK1/2 activity, apoptosis, and E-selectin expression in the cells. Thus, estradiol enhances growth and reduces TNFalpha-induced apoptosis in EC; enhanced EC growth may be mediated via upregulation of telomerase activity. These effects are possible cellular mechanisms underlying female gender-associated cardiovascular protection.     

Lack of modulatory effect of simvastatin on indoxyl sulfate-induced activation of cultured endothelial cells

AimsEndothelial dysfunction is a common manifestation of chronic kidney disease (CKD). The protein-bound uremic toxins have emerged as important factors associated with cardiovascular disease and the outcome of CKD. The effect of indoxyl sulfate (IS) on endothelial cells remains unclear.Main methodsHuman umbilical endothelial cells (HUVEC) were incubated using IS at two concentrations: 100 μM and 1000 μM over two periods of time: 16 and 48 h. HUVEC were also pre-treated with simvastatin to examine its effect. RT-PCR was used to assess changes in the gene expression of intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), Monocyte chemotactic protein-1 (MCP-1), E-selectin, and angiotensin receptor type 1 (AT1R). Protein abundance of the investigated molecules was assessed by immunoblotting.Key findingsTreatment with 100 μM IS for 16 h induced a 2-fold increase in the expression of ICAM-1, VCAM-1, and MCP-1. At a concentration of 1000 μM, there was a 2–3-fold increase. An extended treatment period at low concentrations was associated with a 2–3 fold increase and the increase of ICAM-1 and VCAM-1 was more prominent under high concentration. Results of immunoblotting confirmed an increase in the abundance of ICAM-1, VCAM-1 and MCP-1. No significant change was noted in E-selectin and AT1R according to concentration or treatment duration. Pre-treatment with simvastatin did not alter IS-induced changes.SignificanceIS increased the expression of adhesion molecules of endothelial cells exhibiting a concentration and duration dependent pattern. Simvastatin did not demonstrate any effect on IS-associated endothelial activation.     

Endothelial cell preservation at hypothermic to normothermic conditions using clinical and experimental organ preservation solutions

Introduction

Endothelial barrier function is pivotal for the outcome of organ transplantation. Since hypothermic preservation (gold standard) is associated with cold-induced endothelial damage, endothelial barrier function may benefit from organ preservation at warmer temperatures. We therefore assessed endothelial barrier integrity and viability as function of preservation temperature and perfusion solution, and hypothesized that endothelial cell preservation at subnormothermic conditions using metabolism-supporting solutions constitute optimal preservation conditions.Methods: Human umbilical vein endothelial cells (HUVEC) were preserved at 4–37 °C for up to 20 h using Ringer's lactate, histidine–tryptophan–ketoglutarate solution, University of Wisconsin (UW) solution, Polysol, or endothelial cell growth medium (ECGM). Following preservation, the monolayer integrity, metabolic capacity, and ATP content were determined as positive parameters of endothelial cell viability. As negative parameters, apoptosis, necrosis, and cell activation were assayed. A viability index was devised on the basis of these parameters.Results: HUVEC viability and barrier integrity was compromised at 4 °C regardless of the preservation solution. At temperatures above 20 °C, the cells' metabolic demands outweighed the preservation solutions' supporting capacity. Only UW maintained HUVEC viability up to 20 °C. Despite high intracellular ATP content, none of the solutions were capable of sufficiently preserving HUVEC above 20 °C except for ECGM.Conclusion: Optimal HUVEC preservation is achieved with UW up to 20 °C. Only ECGM maintains HUVEC viability at temperatures above 20 °C.     

Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium.

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.     

Dynamic interplay between breast cancer cells and normal endothelium mediates the expression of matrix macromolecules,proteasome activity and functional properties of endothelial cells

Background

Breast cancer–endothelium interactions provide regulatory signals facilitating tumor progression. The endothelial cells have so far been mainly viewed in the context of tumor perfusion and relatively little is known regarding the effects of such paracrine interactions on the expression of extracellular matrix (ECM), proteasome activity and properties of endothelial cells.

Methods

To address the effects of breast cancer cell (BCC) lines MDA-MB-231 and MCF-7 on the endothelial cells, two cell culture models were utilized; one involves endothelial cell culture in the presence of BCCs-derived conditioned media (CM) and the other co-culture of both cell populations in a Transwell system. Real-time PCR was utilized to evaluate gene expression, an immunofluorescence assay for proteasome activity, and functional assays (migration, adhesion and invasion) and immunofluorescence microscopy for cell integrity and properties.

Results

BCC-CM decreases the cell migration of HUVEC. Adhesion and invasion of BCCs are favored by HUVEC and HUVEC-CM. HA levels and the expression of CD44 and HA synthase-2 by HUVEC are substantially upregulated in both cell culture approaches. Adhesion molecules, ICAM-1 and VCAM-1, are also highly upregulated, whereas MT1-MMP and MMP-2 expressions are significantly downregulated in both culture systems. Notably, the expression and activity of the proteasome β5 subunit are increased, especially by the action of MDA-MB-231-CM on HUVEC.

Conclusions and general significance

BCCs significantly alter the expression of matrix macromolecules, proteasome activity and functional properties of endothelial cells. Deep understanding of such paracrine interactions will help to design novel drugs targeting breast cancer at the ECM level. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

A fibronectin-fibrinogen-tropoelastin coating reduces smooth muscle cell growth but improves endothelial cell function

Reendothelialization of the stent surface after percutaneous coronary intervention (PCI) is known to be an important determinant of clinical outcome. We compared the effects of biological stent coatings, fibronectin, fibrinogen and tropoelastin, on human umbilical vein endothelial cell (HUVEC) and vascular smooth muscle cell (VSMC) characteristics. Umbilical cord arterial segments were cultured on coated surfaces and VSMC outgrowth (indicating proliferation and migration) was measured after 12 days. mRNA was isolated from HUVEC and VSMC cultured on these coatings and gene expression was profiled by QPCR. Procoagulant properties of HUVEC were determined by an indirect chromogenic assay which detects tissue factor activity. The varying stent coatings influence VSMC outgrowth: 31.2 ± 4.0 mm(2) on fibronectin, 1.6 ± 0.3 mm(2) on tropoelastin and 8.1 ± 1.5 mm(2) on a mixture of fibronectin/fibrinogen/tropoelastin, although HUVEC migration remains unaffected. Culturing HUVEC on tropoelastin induces increased expression of VCAM-1 (13.1 ± 4.4 pg/ml), ICAM-1 (5.1 ± 1.3 pg/ml) and IL-8 (11.6 ± 3.1 pg/ml) compared to fibronectin (0.7 ± 0.2, 0.8 ± 0.2, 2.3 ± 0.5 pg/ml, respectively), although expression levels on fibronectin/fibrinogen/tropoelastin remain unaltered. No significant differences in VCAM-1, ICAM-1 and IL-8 mRNA expression are found in VSMC. Finally, HUVEC cultured on tropoelastin display a fivefold increased tissue factor activity (511.6 ± 26.7%), compared to cells cultured on fibronectin (100 ± 3.9%) or fibronectin/fibrinogen/tropoelastin (76.3 ± 25.0%). These results indicate that tropoelastin inhibits VSMC migration but leads to increased inflammatory and procoagulant markers on endothelial cells. Fibronectin/fibrinogen/tropoelastin inhibits VSMCs while compensating the inflammatory and procoagulant effects. These data suggest that coating a mixture of fibronectin/fibrinogen/tropoelastin on a stent may promote reendothelialization, while keeping unfavourable processes such as restenosis and procoagulant activity limited.     

Mast cells are potent regulators of endothelial cell adhesion molecule ICAM-1 and VCAM-1 expression

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM-1 and VCAM-1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose-dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM-1 was observed. The ICAM-1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF-α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM-1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF-α; thus other substances released by MC may contribute to VCAM-1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM-1 and VCAM-1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM-enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T-cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM-1 or VCAM-1. Intradermal injection of allergen in human atopic volunteers known to develop late-phase allergic reactions led to marked expression of both ICAM-1 and VCAM-1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM-1 and VCAM-1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley-Liss Inc.