Induction of tissue factor production but not the upregulation of adhesion molecule expression by ceramide in human vascular endothelial cells

Binding of tumor necrosis factor-alpha (TNF-alpha) to p60 TNF-alpha receptor induces the activation of sphingomyelinase to generate ceramide, which in turn activates certain protein kinases and phosphatases, resulting in various TNF-alpha-mediated biological effects. We have investigated the role for the sphingomyelin/ceramide pathway in the TNF-alpha-induced upregulation of adhesion molecule expression and tissue factor production of human endothelial cells. TNF-alpha stimulated human umbilical vascular endothelial cells (HUVECs) to upregulate the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and HLA class I molecules in addition to the induction of procoagulant tissue factor production. C2-ceramide, a highly cell-permeable ceramide analog, was able to stimulate HUVECs to produce tissue factor activity as well as TNF-alpha. However, C2-ceramide did not stimulate HUVECs to upregulate the expression of VCAM-1, ICAM-1 and HLA class I molecules. These results suggest that there exist both the ceramide-dependent and -independent pathways in TNF-alpha signal transduction system in human vascular endothelial cells.     

过氧化物酶增殖物激活受体α激动剂降低高糖诱导的黏附因子的表达

目的探讨过氧化物酶增殖物激活受体(PPAR)α激动剂对高糖条件下人血管内皮细胞细胞间黏附因子-1(ICAM-1)和血管黏附因子-1(VCAM-1)表达的影响及相关机制。方法体外培养人脐静脉内皮细胞和HL-60细胞,酶联免疫吸附试验(ELISA)、HL-60细胞黏附试验及逆转录-聚合酶链反应(RT-PCR)方法检测ICAM-1和VCAM-1的表达,Western blotting和共聚焦显微镜方法探讨NF-κB通路IκB、磷酸化-IκB蛋白水平及p65亚基的移位,流式细胞仪和共聚焦显微镜方法检测细胞内活性氧离子(ROS)水平,化学发光法测定烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的活性。结果 (1)高糖(33 mmol/L)干预24 h能够显著增加内皮细胞ICAM-1和VCAM-1的表达;(2)PPARα激动剂非诺贝特、NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)和NADPH氧化酶抑制剂二联苯碘(DPI)显著抑制高糖诱导的ICAM-1和VCAM-1的表达;(3)高糖能够诱导内皮细胞IκBα的降解、磷酸化-IκBα蛋白水平的增加;另外,高糖可以显著增加内皮细胞NADPH氧化酶活性和细胞内ROS水平;非诺贝特则呈浓度依赖性的逆转高糖引起的上述效应。结论 PPARα激动剂非诺贝特通过抑制NF-κB通路和NADPH氧化酶的激活降低高糖诱导的人血管内皮细胞炎症因子ICAM-1和VCAM-1的表达。     

内皮脂酶对人脐静脉内皮细胞黏附分子表达的影响

目的研究人脐静脉内皮细胞（HUVECs）表达黏附分子与内皮脂酶（EL）的关系，以及EL对内皮细胞黏附分子表达的影响。方法以肿瘤坏死因子-α（TNF-α）10μg／L刺激HUVECs，采用逆转录-聚合酶链反应（RT—PCR）检测细胞间黏附分子-1（ICAM-1）、血管内皮黏附分子-1（VCAM-1）和E-选择素的mRNA表达变化，以50μg／L的抗EL抗体进行预处理，再检测黏附分子mRNA表达的变化。利用凝胶成像系统拍照、分析、计算黏附分子产物相对含量。结果TNF-α作用于HUVECs后，黏附分子ICAM-1、VCAM-1和E-选择素的mRNA表达均增高，与正常对照组比较差异有统计学意义（P均〈0．01）；该作用可被50μg／L的抗EL抗体所拮抗（P〈0．05或P〈0．01）。结论EL参与了内皮细胞黏附分子表达的调控，推测它可能通过影响内皮细胞黏附分子表达而参与动脉粥样硬化的病理生理过程。     

Rac1 and superoxide are required for the expression of cell adhesion molecules induced by tumor necrosis factor-alpha in endothelial cells

Oxidative signals play an important role in the regulation of endothelial cell adhesion molecule expression. Small GTP-binding protein Rac1 is activated by various proinflammatory substances and regulates superoxide generation in endothelial cells. In the present study, we demonstrate that adenoviral-mediated expression of dominant negative N17Rac1 (Ad.N17Rac1) suppresses tumor necrosis factor-alpha (TNF-alpha)-induced vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin gene expression in a dose-dependent manner. Ad.N17Rac1 did not inhibit TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) binding activity or inhibitor of NF-kappaB-alpha degradation. In contrast, Ad.N17Rac1 inhibited TNF-alpha-induced NF-kappaB-driven HIV(kappaB)(4)-CAT and p288VCAM-Luc promoter activity, suggesting that N17Rac1 inhibits TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 through suppressing NF-kappaB-mediated transactivation. In addition, expression of superoxide dismutase by adenovirus suppressed TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 mRNA accumulation. However, adenoviral-mediated expression of catalase only partially inhibited TNF-alpha-induced E-selectin gene expression and had no effect on VCAM-1 and ICAM-1 gene expression. These data suggest that Rac1 and superoxide play crucial roles in the regulation of expression of cell adhesion molecules in endothelial cells.     

In vitro effects of cyclosporin A on the expression of adhesion molecules on human umbilical vein endothelial cells.

BACKGROUND: Among the different factors playing crucial roles in endothelial cell activation, cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been reported to demonstrate profound effects on this cell type. It has been shown that the increased release of IFN-alpha/gamma and TNF-alpha causes structural and functional modulations of the endothelial cell. These cytokines participate in the recruitment and activation of the immune system. CsA is an immunosuppressive drug that is necessary at high levels in human recipients of vascularised xenografts. This drug could contribute to a prolonged graft survival by modulation of endothelial cell activation. METHODS: The present study deals with the effects of cyclosporin A on adhesion molecule expression (i.e. ICAM-1, VCAM-1, E-selectin, P-selectin, PECAM-1 and the L-selectin ligand CD 34) on the surface of cytokine stimulated HUVECs. The in vitro model described herein mimics the stimulation of endothelial cells by cytokines as seen during inflammatory processes after transplantation. Therefore, HUVECs were activated either with TNF-alpha, IL-1beta or with a cytokine mixture consisting of those stimulants present at an elevated level in sera of patients during allograft rejection (i.e. IL-1beta, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma). RESULTS: The results obtained show that the immunosuppression of CsA is not only achieved by inhibiting lymphocyte proliferation, but also by decreasing the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process. CONCLUSION: Co-incubation of stimulated endothelial cells with a final CsA concentration of 5 microg/ml revealed a significant down-regulating influence on the surface expression of E-selectin and VCAM-1.     

Increased expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured endothelial cells exposed to serum from type 1 diabetic patients: no effects of high glucose concentrations

Activation of the arterial endothelium may play an important role in the development of an atherosclerosis-prone vascular wall in diabetes. The induction of the adhesion molecules VCAM-1 and E-selectin on activated endothelial cells is crucial in monocyte recruitment during the atherogenic process. In the present study, we investigated whether sera from type 1 diabetic patients and non-diabetic persons are capable of inducing expression of VCAM-1 and E-selectin in human endothelial cells cultured in vitro. First, it was found that the addition of serum from non-diabetics to the cultures resulted in expression of adhesion molecules above basal level and also increased the cellular response to the cytokine tumor necrosis factor-alpha (TNF-alpha), a strong inducer of both adhesion molecules. Moreover, it was found that, on average, sera from 17 diabetic males induced a higher expression of VCAM-1 in the endothelial cells after 6 h of incubation than samples from 20 non-diabetic age-matched males (p < 0.05). No difference between the diabetic and non-diabetic group was seen in the expression of E-selectin. Likewise, no differences were observed between the effects of the sera to induce TNF-alpha responsivity. A series of experiments showed that alterations in the glucose concentrations of the growth medium (5.5-13.5 mmol/L) did not change the cellular content of either VCAM-1 or E-selectin before and after TNF-alpha treatment. In conclusion, it has been shown that sera from diabetic patients contain component(s), capable of inducing VCAM-1 expression in endothelial cells independent of hyperglycemia. Augmented induction of endothelial VCAM-1 expression by circulating factor(s) may play a role in the development of atherosclerosis in diabetes.     

Cilostazol suppresses superoxide production and expression of adhesion molecules in human endothelial cells via mediation of cAMP-dependent protein kinase-mediated maxi-K channel activation

This study shows whether increased intracellular cAMP level by cilostazol is directly coupled to its maxi-K channel activation in human endothelial cells. Cilostazol (1 microM) increased the K+ currents in the human endothelial cells by activating maxi-K channels, which was abolished by iberiotoxin (100 nM), a maxi-K channel blocker. On incubation of human coronary artery endothelial cells with tumor necrosis factor-alpha (TNF-alpha) (50 ng/ml), monocyte adhesion significantly increased with increased superoxide generation and expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) accompanied by increased degradation of inhibitory kappaBalpha in cytoplasm and activation of nuclear factor-kappaB p65 in nucleus. All these variables were significantly suppressed by cilostazol (10 microM), which was antagonized by iberiotoxin (1 microM) and (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-l] [1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) (300 nM, cAMP-dependent protein kinase inhibitor), but not by (9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindo-lo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823) (300 nM, cGMP-dependent protein kinase inhibitor). In the human endothelial cells transfected with siRNA-targeting maxi-K channels, cilostazol did not suppress the superoxide generation, VCAM-1 and MCP-1 expressions, and monocyte adhesion as contrasted with the wild-type cells. These findings were similarly evident with (3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indole-2-one (BMS-204352), a maxi-K channel opener, and forskolin and dibutyryl cAMP. In conclusion, increased cAMP level by cilostazol is directly coupled to its maxi-K channel opening action via protein kinase activation in human endothelial cells, thereby suppressing TNF-alpha-stimulated superoxide production and expression of adhesion molecules.     

Native low density lipoprotein-induced calcium transients trigger VCAM-1 and E-selectin expression in cultured human vascular endothelial cells.

Low density lipoprotein (LDL) interactions with the endothelium are thought to play a major role in the development of atherosclerosis. The mechanism(s) involved are not fully understood, although several lines of evidence support the idea that oxidation of LDL increases its atherogenicity. In this study we report for the first time that native LDL (n-LDL) binding to the LDL receptor (100-700 mug/ml) triggers a rise in intracellular calcium which acts as a second messenger to induce vascular cell adhesion molecule-1 (VCAM-1) expression in human coronary artery (HCAEC) and pig aortic endothelial cells (PAEC) and VCAM-1 and E-selectin expression in human aortic (HAEC) endothelial cells. Preincubation of HCAEC with a monoclonal antibody (IgGC7) to the classical LDL receptor or pretreatment with pertussis toxin blocked the n-LDL-induced calcium transients. Preincubation of each of the endothelial cell lines with the calcium chelator 1,-2-bis(o-aminophenoxy)ethane-N,N,N'', N''-tetraacetic acetomethyl ester (BAPTA/AM) prevented the expression of VCAM-1 and E-selectin. The increase in VCAM-1 by n-LDL results in increased monocyte binding to HCAEC which can be attenuated by inhibiting the intracellular calcium rise or by blocking the VCAM-1 binding sites. These studies in human and pig endothelial cells link calcium signaling conferred by n-LDL to mechanisms controlling the expression of endothelial cell adhesion molecules involved in atherogenesis.     

西洛他唑对人脐静脉内皮细胞血管细胞黏附分子1和细胞间黏附分子1mRNA表达的影响

目的观察西洛他唑对人脐静脉内皮细胞（HUVECs）血管细胞黏附分子1（VCAM-1）和细胞间黏附分子1（ICAM-1）mRNA表达的影响,探讨西洛他唑可能的抗动脉粥样硬化作用机制。方法将HUVECs用不同浓度的西洛他唑（0μg/L、0.05μg/L、0.1μg/L、1.0μg/L、10μg/L）溶液处理1小时后,用肿瘤坏死因子α（TNF-α）10μg/L诱导24小时。半定量复合逆转录聚合酶链反应（RT-PCR）测定黏附分子VCAM-1和ICAM-1mRNA的表达。结果 TNF-α能上调VCAM-1和ICAM-1的表达,西洛他唑在一定程度上可抑制上述作用,随着西洛他唑浓度的增加,ICAM-1mRNA表达水平逐步下降,分别为0.239±0.012、0.205±0.012、0.166±0.010、0.136±0.008,VCAM-1mRNA表达水平也逐步下降,分别为0.114±0.048、0.093±0.051、0.083±0.045、0.068±0.039。结论西洛他唑可抑制TNF-α诱导的HUVECs的黏附分子VCAM-1和ICAM-1mRNA表达,提示西洛他唑的抗动脉粥样硬化作用可能是通过阻止血单核细胞向血管内皮细胞聚集和黏附实现的。     

Nifedipine and diltiazem but not verapamil up-regulate endothelial nitric-oxide synthase expression

We have recently shown that felodipine, a long-acting dihydropyridine L-type calcium channel blocker (CCB), up-regulates nitric oxide (NO) production and endothelial NO synthase (eNOS) expression and activity in cultured endothelial cells as well as in animals with chronic renal failure. This study was intended to compare the effects of prototypes of the three classes of L-type CCBs on the NO system in cultured human coronary artery endothelial cells. Thus, cultured endothelial cells were incubated either with nifedipine, diltiazem, or verapamil for 24 h at 10(-5) to 10(-7) M concentrations. Cells incubated with inactive vehicle served as controls. NO production, as discerned from total nitrate plus nitrite recovered in the medium, was significantly increased by nifedipine (P <.03) and by diltiazem (P <.05). However, NO production remained unchanged with verapamil (P = NS). Similarly, eNOS protein abundance was increased significantly by nifedipine (P <.05) and diltiazem (P <.05). In contrast, eNOS expression was not changed by verapamil (P = NS). Likewise, NOS activity, as measured from [(3)H]L-arginine to [(3)H]L-citrulline conversion, significantly increased with nifedipine (P <.01) and diltiazem (P <.01). However, incubation with verapamil failed to alter NOS activity of the cultured endothelial cells (P = NS). We concluded that prototypes of dihydropyridine and benzothiazepine classes, but not phenylalkylamine class of CCBs, up-regulate the NO system. This may, in part, account for the different biological properties of these agents.     

核因子κB的反义寡核苷酸对肿瘤坏死因子α诱导的血管细胞黏附分子-1表达的影响

目的：探讨人脐静脉内皮细胞（HUVECs）ECV304细胞株中，核因子κB（NF—κB）的反义寡核苷酸（AODNs）对肿瘤坏死因子α（TNF—α）诱导血管细胞黏附分子（VCAM-1）表达的影响。方法：将培养的人脐静脉内皮细胞株ECV304分为3组，其中2组用脂质体介导法转染寡核苷酸（ODNs）分别转染AODNs、正义寡核苷酸（SODNs）：另1组（阳性对照组）不转染ODNs。流式细胞仪荧光活细胞计数检测转染效率及NF-κB的表达情况，逆转录-聚合酶链反应、流式细胞仪检测及免疫组织化学染色测定AODNs对TNF—α诱导的VCAM—1表达的影响。结果：脂质体介导的转染方法能将ODNs有效地转染至HUVECs中，且AODNs可有效抑制NF-κB的复制合成。NF—κBP65的AODNs可使TNF—α刺激的人脐静脉内皮VCAM-1mRNA表达下调33．08％，蛋白质水平的表达下调48．27％，并与免疫组织化学染色显示结果一致。结论：NF—κB的AODNs可显著下调NF—κB调控的、与动脉粥样硬化相关的黏附分子表达。     

Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF- R75, and by receptor type-specific agonists, binding exclusively to TNF- R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha- dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF- R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences.     

Tumor necrosis factor-alpha stimulates attachment of small cell lung carcinoma to endothelial cells

Tumor cell attachment to endothelial cells (ECs) is an important step in the metastasis of small cell lung carcinoma (SCLC). Tumor necrosis factor-alpha (TNF-alpha) stimulation of ECs increases the attachment of some malignant cell types to ECs by affecting the expression of cell adhesion molecules (CAMs). Similarly, the inhibition of EC protein kinase C (PKC) and tyrosine kinase (TK) pathways modulates TNF-alpha-mediated effects on CAM expression. We hypothesized that TNF-alpha would increase SCLC attachment to ECs by affecting CAM expression through activation of PKC and TK pathways. To test this hypothesis, human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-alpha (0 to 500 U/mL) for variable time periods (1 to 24 hours), and the attachment of H82 cells (an SCLC cell line) to the HUVECs was quantified. TNF-alpha stimulation of the HUVECs increased H82 attachment from 28.1% +/- 1.6% to 48.8% +/- 1.7% (P < .05). Preincubation of HUVECs with the PKC inhibitors bis-indolylmaleimide (BIN) or calphostin C or the TK inhibitors genistein or herbimycin A (HMA) blocked the TNF-alpha-induced increase in H82 cell attachment. The addition of antibodies to vitronectin (Vn) or beta1-integrin to TNF-alpha-activated HUVECs before the addition of the H82 cells also significantly decreased H82 attachment, whereas the addition of antibodies to E-selectin, P-selectin, vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), neural cell adhesion molecule (NCAM), sialyl-Lewis(x), fibronectin (Fn), alpha(v)-integrin, alpha3-integrin, alpha4-integrin, or alpha5-integrin had no effect on SCLC attachment. In summary, the TNF-alpha-mediated increase in SCLC attachment to ECs appears to be mediated by the activation of EC PKC and TK pathways as well as through effects on the function or expression of EC Vn and beta1 integrin.     

Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells.

Accumulation of monocyte-derived foam cells in focal areas of the arterial intima is one of the key events in early atherogenesis. We have examined the effect of lysophosphatidylcholine (lyso-PC; lysolecithin), a major phospholipid component of atherogenic lipoproteins, on the expression of adhesion molecules for monocytes, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), in cultured human and rabbit arterial endothelial cells. Cultured rabbit aortic endothelial cells treated with lyso-PC showed increased mRNA and cell surface expression of VCAM-1 and ICAM-1, which was associated with increased adhesion of monocytes and monocyte-like cells (THP-1, U937). In cultured human iliac artery endothelial cells, lyso-PC similarly induced both VCAM-1 and ICAM-1, whereas in umbilical vein endothelial cells only ICAM-1 was up-regulated. In all endothelial cells examined, the effect of lyso-PC on E-selectin (endothelial-leukocyte adhesion molecule-1) expression was negligible, thus differentiating this stimulus from other endothelial activators, such as interleukin 1, tumor necrosis factor, or lipopolysaccharide. We conclude that lyso-PC can selectively induce VCAM-1 and ICAM-1 in arterial endothelial cells and that this action, in addition to its monocyte chemoattractant activity, may play an important role in monocyte recruitment into atherosclerotic lesions.     

In vitro effects of mycophenolic acid on the nucleotide pool and on the expression of adhesion molecules of human umbilical vein endothelial cells

The immunosuppressive drug mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid (MPA) selectively inhibit inosine 5'-monophosphate dehydrogenase (IMPDH), and therefore interfere with cellular guanine nucleotide biosynthesis. IMPDH is additionally involved in the synthesis of membrane glycoproteins, some of which are adhesion receptors known to play an active part in the regulation of cell-cell contacts, which are crucial in the process of recruitment and transendothelial infiltration of activated leucocytes in the transplanted organ. As a consequence, MPA leads to a reduction of cellular infiltrates in the course of transplant rejection. In the present study, the effects of MPA on human umbilical vein endothelial cells (HUVEC) are investigated at both molecular and cellular levels. In our experiments, HUVECs are treated with tumor necrosis factor-alpha (TNF-alpha; 10 ng/ml) in order to mimic activation occurring at a rejection crisis. The dose-dependent influence of concomitant incubation with MPA (5-20 micromol/l; 48 h, 37 degrees C, 5% CO2) on their intracellular nucleotide profile is observed by determining the concentrations of purine and pyrimidine nucleotides, using a HPLC method based on solvent generated ion-exchange. The possibility of synergistic effects is investigated by incubating endothelial cells with mixtures of three different immunosuppressants (mycophenolic acid; cyclosporin A, 100 ng/ml; prednisolone, 1 micromol/l)--a combination commonly used after transplantation--varying the amount of MPA (5-20 micromol/l). Stimulation with TNFalpha does not significantly modulate the intracellular levels of nucleotides quantitated. In the presence of MPA concentrations of at least 5 micromol/l, GTP levels (68+/-12%) are significantly decreased compared to controls (100%). At a concentration of 20 micromol/l MPA, the GTP amount is reduced to 58+/-7%. In contrast to these observations, the levels of UDP and UTP are increasing significantly under coincubation with MPA concentrations greater than 5 micromol/l. At 20 micromol/l MPA, UDP and UTP are increased to 147+/-19% and 114+/-11%, respectively. All other nucleotides (CTP, ADP, ATP) reveal no significant alterations in their intracellular concentrations under the conditions applied. Incubation of TNFalpha-treated HUVEC monolayers, with a mixture of three immunosuppressive drugs varying the amount of MPA, show no significant differences compared with the data observed after incubation with MPA alone. In addition, the influence of MPA (10 micromol/l) on a cellular level is observed by measuring the cell surface expression of adhesion molecules on cytokine-stimulated HUVECs, using TNFalpha (10 ng/ml), interferon-gamma (100 ng/ml), interleukin-1beta (10 ng/ml) and interleukin-8 (20 ng/ml). Expression of the intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1) was assessed by flow cytometry. Activation of endothelial cell monolayers with TNFalpha significantly increases the mean fluorescence intensity of VCAM-1 (361+/-14%) and ICAM-1 (429+/-47%) surface expression, compared to controls, and additionally induces E-selectin expression (2919+/-134%). The same tendencies, but in a lesser degree, are observed under stimulation of cells with either IFNgamma or IL-1beta. Incubation with a combination of TNFalpha and MPA leads to a significant reduction in VCAM-1 (329+/-13%) and E-selectin (2613+/-167%) expression, compared to the values obtained for HUVEC incubated with the cytokine alone. Treatment of the cells with IL-1beta/MPA also reduces the expression of VCAM-1 to a level significantly lower than the level observed after stimulation with IL-1beta. Incubation with MPA alone reveals no significant modulation in the expression of all surface molecules tested compared to the values of unstimulated HUVECs. The experiments show that the immunosuppressive action of MPA not only inhibits lymphocyte proliferation but also decreases the expression of adhesion molecules on endothelial cells, which are the first target of the cellular rejection process.     

Plasma concentration-response relationships for some cardiovascular effects of dihydropyridines in healthy subjects

The pharmacokinetics and some hemodynamic effects of three dihydropyridine (DHP) calcium channel blockers were studied in healthy subjects. In a randomized order, each subject was given 24 micrograms/kg nifedipine, 40 micrograms/kg nitrendipine, or 10 micrograms/kg nisoldipine intravenously. The three DHPs differed as to their total clearance and volume of distribution (nifedipine less than nisoldipine less than nitrendipine) but showed similar values for elimination half-lives. All three drugs evoked increases in heart rate and forearm blood flow (FBF) and small decreases in blood pressure. Whereas the observed peak changes in heart rate were virtually identical for the three drugs (about 45% above baseline), the peak changes in FBF were more pronounced in response to nitrendipine and nisoldipine (greater than 200% above baseline) than in response to nifedipine (79% above baseline). The heart rate and FBF responses to the DHPs were related directly to the drug concentrations in plasma. The plasma level-response curves obeyed Hill's equation. They showed that the DHPs differ mainly in their potencies at dilating the forearm resistance vessels (nifedipine less than nitrendipine less than nisoldipine).