Monocyte chemoattractant protein-1 enhances and interleukin-10 suppresses the production of inflammatory cytokines in adult rat cardiomyocytes

Objective Chemokines control the migration of leukocytes to inflamed tissue, and in particular monocyte chemoattractant protein (MCP)-1 has been implicated in the pathogenesis of several cardiovascular disorders such as chronic heart failure (CHF) and myocarditis. We hypothesised that MCP-1 may directly contribute to an inflammatory response in the cardiomyocytes, and in the present study we examined in adult rat cardiomyocytes: (i) the effect of tumour necrosis factor (TNF)a on MCP-1 production, (ii) the effect of MCP-1 on production of other inflammatory cytokines, and (iii) if the anti-inflammatory cytokine interleukin (IL)-10 could suppress any TNFα-induced MCP-1 production. Methods We used enzyme immunoassays, RNase protection assays and slot blot analysis to measure protein and mRNA levels of various cytokines in adult rat cardiomyocyte cultures. Results (i) We found a ∼6.4-fold increase of the MCP-1 level accompanied by an increase in MCP-1 mRNA accumulation in cardiomyocyte cultures after TNFα stimulation. (ii) In contrast, TNFα had no effect on IL-10 and only a modest effect on IL-1β and IL-6 levels in these cells. (iii) Importantly, MCP-1 stimulated inflammatory response in cardiomyocytes by enhancing IL-1β and IL-6 levels in these cells as found at both the protein and mRNA level. (iv) Co-stimulation with IL-10 resulted in a ∼55 % reduction in TNFα-stimulated MCP-1 levels in cardiomyocyte culture supernatants. Conclusion The present study demonstrates for the first time that MCP-1 can directly affect cardiomyocytes, and we introduce MCP-1 as a potential enhancer and IL-10 as a potential suppresser of inflammatory responses within the myocardium. Received: 10 October 2000 / Returned for 1. revision: 2 November 2000 / 1. Revision received: 5 December 2000 / Returned for 2. revision: 2 January 2001 / 2. Revision received: 5 January 2001 / Accepted: 8 January 2001     

Identification and characterization of endothelial glycoprotein Ib using viper venom proteins modulating cell adhesion

The expression and function of a glycoprotein Ib (GPIb) complex on human umbilical vein endothelial cells (HUVECs) is still a matter of controversy. We characterized HUVEC GPIb using viper venom proteins: alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin is an antagonist, and alboaggregins act as agonists of the platelet GPIb complex. Botrocetin is a venom protein that alters von Willebrand factor (vWF) conformation and increases its binding affinity for the GPIb complex. Echistatin is a disintegrin that blocks alphavbeta3. Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese hamster ovary (CHO) cells transfected with alphavbeta3. We found the following: (1) Binding of monoclonal antibodies against GPIbalpha to HUVECs was moderately increased after stimulation with cytokines and phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both echicetin and echistatin, an alphavbeta3 antagonist, inhibited the adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The inhibitory effect was additive when both proteins were used together. (3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this effect was completely abolished by echicetin, but not by echistatin. (4) CHO cells expressing GPIbalphabeta/IX adhered to vWF (in the presence of botrocetin) and to alboaggregins; GPIbalpha was required for this reaction. Echicetin, but not echistatin, inhibited the adhesion of cells transfected with GPIbalphabeta/IX to immobilized vWF. (5) HUVECs adhered strongly to immobilized vWF and alboaggregins with extensive spreading, which was inhibited by LJ1b1, a monoclonal antibody against GPIb. The purified alphavbeta3 receptor did not interact with the alboaggregins, thereby excluding the contribution of alphavbeta3 in inducing HUVEC spreading on alboaggregins. In conclusion, our data confirm the presence of a functional GPIb complex expressed on HUVECs in low density. This complex may mediate HUVEC adhesion and spreading on immobilized vWF and alboaggregins.     

Effects of C-reactive protein on the release of von Willebrand factor, E-selectin, thrombomodulin and intercellular adhesion molecule-1 from human umbilical vein endothelial cells.

Increased levels of various plasma molecules, including C-reactive protein (CRP), and endothelial markers von Willebrand factor (vWF), E-selectin, intercellular adhesion molecule-1 (ICAM-1) and thrombomodulin all predict the development and/or progression of cardiovascular disease. As CRP has been shown to upregulate the expression of adhesion molecules on the surface of human umbilical vein endothelial cells (HUVECs) in vitro, we hypothesized that CRP would induce the release of increased levels of the endothelial markers from HUVECs. Accordingly, recombinant human CRP was added to the culture medium of confluent monolayers of HUVECs at physiological to pathological doses of 1-50 microg/ml for 3-48 h. Markers were measured in supernatants by commercial enzyme-linked immunosorbent assays. We found that increased release of thrombomodulin was induced by 20 and 50 microg/ml CRP after 48 h. Increased ICAM-1 was induced by 50 microg/ml CRP after 24 and 48 h. There was no clear influence of CRP on E-selectin, but 20 and 50 microg/ml CRP inhibited the release of vWF. Our data provide further evidence of a link between increased levels of ICAM-1, thrombomodulin and CRP in atherosclerosis, but they counter reports of a direct, possibly causal, relationship between CRP and increases in E-selectin or vWF.     

Regulated von Willebrand factor secretion is associated with agonist-specific patterns of cytoskeletal remodeling in cultured endothelial cells

von Willebrand factor (vWF), an adhesive glycoprotein involved in primary hemostasis, is stored and released from endothelial secretory granules called Weibel-Palade bodies. Regulated secretion occurs in reaction either to [Ca(2+)](i)-raising agents (histamine or thrombin) or to cAMP-raising agents (epinephrine, adenosine, or forskolin). We investigated the pattern of release and the cytoskeletal requirements for secretion in response to these 2 classes of agonists. Secretion induced by [Ca(2+)](i)-raising agents involves peripheral and central granules and is inhibited by colchicine-induced microtubule disruption. It is accompanied by Rho-dependent stress fiber formation and cell retraction. Secretion and remodeling occur in the same individual cells. However, secretion is potentiated by cytochalasin E and C3 toxin, indicating that stress fiber formation antagonizes vWF secretion. In contrast, vWF secretion induced by cAMP-raising agents involves the release of only peripheral granules (implying less vWF release on a per cell basis) and is not inhibited by microtubule disruption. cAMP-mediated secretion is accompanied by disruption of stress fibers, strengthening of the cortical actin rim, and preservation of cell-cell contacts. It is unaffected by cytochalasins or C3 toxin. In contrast to [Ca(2+)](i)-raising agents, cAMP-raising agents induce secretion without cell retraction/intercellular gap formation. Thus, they are likely to play a physiological role in the regulation of endothelial vWF secretion and, therefore, of plasma vWF levels.     

Antiendothelial antibodies in sera of patients with infective endocarditis

Infective endocarditis is characterized by the colonization of endocardium by microorganisms. Except for Staphylococcus aureus, microorganisms are not able to adhere to and grow on endocardial cells; prior damage, e. g., by shear stress or other mechanical factors, is necessary. But other causes may well have a share. This study was, therefore, designed to identify immunological factors, especially antibodies against endothelial cells, which could contribute to the initiation of endocardial injury. Sera of patients with infective endocarditis and healthy controls were investigated for the presence of antibodies against endothelial antigen. As the antigen source human umbilical vein endothelial cells were used. Antibodies against endothelial cells were detected by indirect immunofluorescence, ELISA, immunoblotting, antibody dependent cellular cytotoxicity, and antibody mediated cytotoxicity. Antibodies against endothelial cells were found in seven out of fifteen patients. These antibodies were directed against cytoplasmic structures and only appeared in the course of the disease. A correlation between the presence of these antibodies and disease activity or the outcome of disease was not observed. These antibodies may develop as a consequence of damage to endocardial cells (thereby exposing intracellular antigen to the immune system) and do not seem to play a role in the pathogenesis of infective endocarditis. Received: 23 March 2000, Returned for revision: 2 May 2000, Revision received: 9 June 2000, Accepted: 27 June 2000     

Genetic induction of a releasable pool of factor VIII in human endothelial cells

Although it is known that factor VIII (FVIII) plasma levels increase rapidly in response to a number of stimuli, the biological stimuli behind this release is less clear. Previously, we showed that FVIII can traffic together with von Willebrand factor (vWF) into storage granules in a pituitary tumor cell line, AtT-20; however, AtT-20 cells could not be used to address the release or functional activity of released FVIII. To investigate the regulated secretion of stored FVIII, endothelial cells with intact agonist-stimulated release pathways were used. Human umbilical vein endothelial cells (HUVECs) were transduced with retroviral FVIII construct [hFVIII(V)] to create a FVIII/vWF storage pool. Immunofluorescent staining of transduced cells demonstrated FVIII in Weibel-Palade bodies. In contrast, the transduction of hFVIII(V) into HT-1080 and HepG2 cells displayed FVIII only in the cytoplasm. We studied the regulated release of both FVIII and vWF from endothelial cells after agonist-induced stimulation and demonstrated a parallel release of FVIII and vWF proteins. This released FVIII was functionally active. Hence, endothelial cells transduced with hFVIII(V) store FVIII together with vWF in Weibel-Palade bodies, creating a releasable storage pool of both proteins. Because FVIII secretion can be physiologically regulated by agonists in culture, this may explain the pharmacological agonist-induced release of FVIII by drugs such as desmopressin in vivo and suggests vascular endothelium as a reasonable target of gene therapy of hemophilia A.     

Tumor necrosis factor-alpha is expressed by monocytes/macrophages following cardiac microembolization and is antagonized by cyclosporine

The time course of expression of TNF-α in myocardial wound healing following ischemic injury was investigated in the porcine heart. Microembolization was used to induce focal ischemia and necrosis in hearts of 39 adult pigs. The animals were sacrified after 3, 6, 12, 24 h, 3 and 7 days, and after 4 weeks, and the myocardial tissue was studied by immunofluorescence using specific antibodies. TNF-α containing cells were identified as monocytes/macrophages by double staining with a muramidase antibody. Monocytes/macrophages were the only source of TNF-α. Microembolization caused multiple necrotic foci with loss of myocytes in the left ventricular myocardium. These foci contained numerous monocytes/macrophages and showed an inflammatory reaction typical of wound healing followed by replacement with scar tissue. The number of TNF-α positive cells increased after 24 h, peaked between 3–7 days and slowly decreased thereafter. Expression of TNF-α in monocytes/macrophages was significantly reduced after pretreatment of pigs with cyclosporine or dexamethasone. It is concluded that 1.) in myocardial tissue monocytes/macrophages are the only cell type expressing TNF-α, 2.) TNF-α is involved in wound healing after ischemia, and 3.) synthesis of TNF-α and inflammatory angiogenesis can be inhibited be treatment with either cyclosporine or dexamethasone. Received: 23 June 1997, Returned for revision: 20 August 1997, Revision received: 10 December 1997, Accepted: 17 January 1998     

Serum IL-6, TNFα, p55 srTNFα, p75 srTNFα, srIL-2α Levels and Disease Acitivity in Systemic Lupus Erythematosus

The aim of this study was to determine whether the levels of serum cytokines IL-6 and TNFα and of the soluble receptors p55 srTNFα, p75 srTNFα and srIL-2α are valuable markers of disease activity in patients with systemic lupus erythematosus (SLE) compared with the established parameters of anti-dsDNA, C3, C4 and CH₅₀. Forty patients with SLE, 19 ambulatory and 21 hospitalised, were included in this study. On the day of blood sampling a clinical examination was performed and SLEDAI and ECLAM disease activity scores were used to assess disease activity. Nineteen patients had inactive disease and 21 patients had active disease. Thirteen patients from the second group developed nephritis. In these patients the blood sampling and disease activity assessment were performed twice (at presentation and 6 months after treatment). Serum levels of cytokines and soluble receptors were measured by ELISA. Serum levels of cytokines and soluble receptors of patients with active disease were significantly higher than in patients with inactive disease (IL-6 p = 0.0004, TNFαp = 0.0015, srIL-2αp<0.0001, p55 srTNFαp<0.0001, p75 srTNFαp<0.0001). Serum soluble receptor levels of patients with inactive disease were higher than those of healthy controls (p55 srTNFαp<0.0001, p75 srTNFαp = 0.0002, srIL-2αp = 0.0012). No significant difference was found for TNFα and IL-6 (TNFαp = 0.015, IL-6 p = 0.019). Serum TNFα levels and especially srIL-2α, p55 srTNFα and p75 srTNFα levels correlated strongly with SLEDAI and ECLAM disease activity scores, anti-dsDNA, C3, C4 and CH₅₀ (p<0.0001). Serum soluble receptor (srIL-2α, p55 srTNFα, p75 srTNFα) levels were higher in patients with nephritis before treatment and decreased significantly 6 months after treatment (p = 0.005). The same trend was noticed with SLEDAI and ECLAM disease activity scores (p = 0.005) and anti-dsDNA (p = 0.008). In contrast, no significant differences were observed for C3 and C4 levels before and after treatment, which suggests that soluble receptors of cytokines are more sensitive markers of disease activity than C3 or C4 in predicting improvement. Serum levels of srIL-2α, p55 srTNFα and p75 srTNFα could provide useful information about disease activity in SLE patients, especially in cases where the other markers do not. Received: 3 March 1998 / Accepted: 13 July 1998     

Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1)

Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras^61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21^Cip1/Waf1. Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-α for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2 % vs. 4.3 %, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58 % of Ras-infected cells stained positive for senescence-associated β-galactosidase activity as opposed to 2 % in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21^Cip1/Waf1 in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-α induced-apoptosis. Surprisingly, senescence-associated β-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21^Cip1/Waf1. Future studies have to investigate a potential role of Ras in human vascular biology. Received: 5 June 2001, Returned for revision: 28 June 2001, Revision received: 6 July 2001, Accepted: 31 July 2001     

Expression of adhesion molecules in endothelial cells during allogeneic bone marrow transplantation

Abstract: Endothelial cell activation during allogeneic bone marrow transplantation, mainly in acute graft-versus-host disease (aGvHD) was studied in 23 recipients and 5 controls using anti-von Willebrand factor (vWF) antibody, antibodies to endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and anti-HLA-DQ antibody, by immunohistological staining of skin. vWF extravasation, ELAM-1 and VCAM-1 expression were present in most recipients with a cutaneous rash which was confirmed as an aGvHD by histological examination (documented aGvHD) (p = 0.005 for vWF extravasation and ELAM-1 expression and p = 0.03 for VCAM-1 expression in comparison with the controls). In recipients with a rash, the cases displaying vWF extravasation and ELAM-1 expression were significantly more numerous in those with a documented aGvHD than in those without histological features of aGvHD (p = 0.01). vWF extravasation and ELAM-1 occurred concomitantly (p<0.01). This study demonstrates that, during the course of skin aGvHD following bone marrow transplantation, there is transient expression of ELAM-1 and VCAM-1 by endothelial cells and simultaneous vWF extravasation, indicating an intense inflammation with endothelial cell participation.     

Regulation of von willebrand factor of human endothelial cells exposed to laminar flows: an in vitro study

The effect of laminar flow on the regulation of von Willebrand Factor (vWF) of cultured human umbilical vein endothelial cells (HUVECs) was studied. Confluent endothelial monolayers were exposed to shear stresses (0.2 and 1.0 Pa) from 2 to 24 h. vWF was labelled with indirect immunofluorescence method and observed with 3D fluorescence microscopy. The distribution of vWF and the cytoskeleton organization were observed simultaneously by double fluorescence labelling. More actin stress fibers and an increased release of vWF appeared in the cells exposed to flow at the same time. The qualitative and quantitative results showed that there was not only a shear-dependent regulation but also a time-dependent modification. For a short-time shear stimulation, both 0.2 Pa and 1.0 Pa shear stresses induced a release of vWF from the endothelial cells. In contrast, after 24 h exposure to 1.0 Pa shear flow, vWFs were much more in the cells than that in the cells exposed to 0.2 Pa for 24 h (p < 0.01) or that in the control cells (p < 0.05). TNF-alpha caused a decrease of vWF and Weibel-Palade bodies in the cells.     

Integrins αvβ3 and αvβ5 mediate VSMC migration and are elevated during neointima formation in the rat aorta

Neointima formation involves tissue expression of matrix proteins and growth factors. The role of α_vβ₃, but not α_vβ₅ integrin in vascular cells has been sufficiently investigated. The aim of the present study was to determine and compare the function of α_vβ₃ and α_vβ₅ integrins in rat aortic (RASMC) and human coronary vascular smooth muscle cells (HCSMC) and to characterize their expression accompanying neointima formation in vivo. RASMC and HCSMC express α_vβ₃ and α_vβ₅ integrin subunits. The α_vβ₅ integrin predominantly mediated adhesion of RASMCs to vitronectin and spreading on vitronectin via RGD-binding sequences. In contrast, the α_vβ₃ integrin did not contribute to the adhesion and spreading on fibronectin, vitronectin, gelatin or collagen I coated layers. PDGF-directed migration through gelatin coated membranes involved both α_vβ₃ and α_vβ₅ integrins. Selective blocking antibodies for α_vβ₃ and α_vβ₅ inhibited migration of RASMC and HCSMC by more than 60% (p < 0.01). Integrin expression was studied in vivo in thoracic aorta of Sprague Dawley rats before and after balloon injury. In situ hybridization demonstrated low signals for α_vβ₃ and β₅ mRNA in uninjuried aorta, which increased significantly at 14 days, localized predominantly in the neointima. Northern analysis of aorta after 14 days of injury also demonstrated an upregulation of α_vβ₃ and β₅ mRNA compared to uninjured aorta. Consistent with the increase in message levels, increased inegrin protein expression was seen in the neointima after 7 and 14 days. This study provides evidence that α_vβ₃ and α_vβ₅ are elevated during neointima formation in the rat and indicates a novel role for α_vβ₅ participating in mechanisms regulating smooth muscle cell migration. Received: 30 May 2000, Returned for revision: 10 July 2000, Revision received: 27 July 2000, Accepted: 16 August 2000     

Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum

Intracellular protein transport in endothelial cells is selectively inhibited by homocysteine, a thiol amino acid associated with both thrombosis and atherosclerosis. In a previous study, homocysteine decreased cell surface expression of the surface transmembrane glycoprotein thrombomodulin without decreasing secretion of another endothelial cell protein, plasminogen activator inhibitor-1. To define further the effects of homocysteine on protein transport, we examined the processing and secretion of the multimeric glycoprotein von Willebrand factor (vWF) in human umbilical vein endothelial cells. Incubation with 2 mmol/L homocysteine resulted in complete loss of vWF multimers and prevented asparagine-linked oligosaccharide maturation, propeptide cleavage, and secretion; these effects are consistent with impaired exit from the endoplasmic reticulum (ER). Dimerization was only partially inhibited, suggesting that homocysteine causes retention of provWF in the ER without preventing dimer formation. In pulse-chase incubations, intracellular provWF was degraded before exiting the ER in homocysteine-treated cells. Homocysteine also inhibited the processing and secretion of a carboxyl-terminal truncation mutant of human provWF expressed in rat insulinoma cells, indicating that retention in the endoplasmic reticulum can be mediated by regions of provWF apart from the carboxyl-terminal 20-Kd segment. These results suggest that retention of secretory proteins in the ER is regulated by redox mechanisms and imply that the intracellular transport of multiple endothelial cell proteins may be altered in patients with homocystinuria.     

Potential role of endothelin-1 and endothelin antagonists in cardiovascular diseases

The endothelins comprise a family of three isopeptides ET-1, ET-2 and ET-3, whereby ET-1 appears to be the most relevant in humans. They act in a paracrine manner on ET_A and ET_B receptors. ET-1 plays an important role in the cardiovascular system. In addition, it modulates vasomotion and growth processes, and it participates in thrombogenesis and neutrophil adhesion. This review summarizes some of the current literature pertaining to the physiological and pathophysiological significance of ET-1, focusing the assets and drawbacks of elevated ET-1 levels. In this regard, modulation of the endothelin system by either receptor blockade or by inhibition of endothelin converting enzyme is expected to provide novel therapeutic drug strategies. Received: 13 September 1999, Returned for revision: 2 November 1999, Revision received: 2 March 2000, Accepted: 23 March 2000     

Muscular levels of proinflammatory cytokines correlate with a reduced expression of insulinlike growth factor-I in chronic heart failure

Objective: Chronic heart failure (CHF) is associated with metabolic abnormalities leading to a catabolic syndrome in advanced stages of the disease. To assess the role of proinflammatory cytokines for the local expression of insulin-like growth factor-I (IGF-I) in this process, muscular and systemic levels of interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα) and IGF-I were analyzed in an animal model of CHF. Methods: Ligation of the left coronary artery or sham operation was performed in adult Wistar Kyoto rats. After 12 weeks, all animals were assessed by echocardiography and cardiac catheterization. Serum levels of IGF-I, IL-1β, TNFα and IL-6 were determined by ELISA. In the quadriceps muscle, the expression of IGF-I, IGF-I receptor, IL-1β and TNFα were assessed by RT-PCR and quantitative immunohistochemistry. Alterations in muscle fiber morphology were analyzed microscopically. Results: The local expression of IGF-I decreased significantly in animals with CHF (0.47 ± 0.07 versus 0.77 ± 0.09; p < 0.01). This reduction correlated with a decreased muscle fiber cross-sectional area (r = 0.62; p < 0.01) and inversely with the local expression of IL-1β (r = −0.49; p < 0.05). IGF-I serum levels showed no significant differences between the two groups. Conclusions: In CHF, the local IGF-I expression is reduced in the presence of normal serum levels of IGF-I. Both elevated proinflammatory cytokines and reduced local IGF-I expression contribute to a catabolic metabolism that may finally result in skeletal muscle atrophy and cardiac cachexia. Received: 17 December 2002, Returned for 1. revision: 13 January 2003, 1. Revision received: 28 January 2003, Returned for 2. revision: 10 February 2003, 2. Revision received: 10 March 2003, Accepted: 11 March 2003, Published online: 16 April 2003 Drs. Schulze and Gielen, Both contributed equally to this work Correspondence to: P. C. Schulze, MD     

Cancer cell-derived IL-1α induces IL-8 release in endothelial cells

Purpose Cancer cells release a multitude of cytokines and growth factors that influence neighboring cells and help establish a favorable environment for tumor development. As part of our studies designed to elucidate the complex cellular interactions within the tumor microenvironment that facilitate tumor development, we investigated cancer cell-induced changes in gene expression in endothelial cells. Methods After treatment of human umbilical vein endothelial cells (HUVEC) with conditioned medium (CM) of SNUC5 colon cancer cells, gene expression profile in HUVEC was analyzed using cDNA microarray. Neutralizing antibodies against pro-inflammatory cytokines were used to identify the major effecter in SNUC5 CM. Results IL-8 was one of the four genes up-regulated over fourfold, and IL-1α in SNUC5 CM was revealed as a major effecter of IL-8 over-expression and release, which was nearly completely neutralized by anti-IL-1α antibody. Constitutive secretion of IL-1α was confirmed in many other human cancer cells. Conclusions IL-1α is constitutively expressed in many human cancer cells and directly induces IL-8 secretion in neighboring endothelial cells.     

c-Jun N-terminal kinases inhibitor suppresses the TNF-α induced MCP-1 expression in human umbilical vein endothelial cells

Monocyte chemoattractant protein-1 (MCP-1) is a 76-amino-acid chemokine that is considered to be an important chemotactic factor for monocytes. MCP-1 is expressed in the macrophage-rich areas of atherosclerotic lesions. A recent report indicated that MCP-1 expression in human umbilical vein endothelial cells (HUVECs) is induced by the stimulation of tumor necrosis factor (TNF)-α via the c-Jun N-terminal kinases (JNK) pathway. In this study, we examined the effects of JNK inhibitor (JNKI-1), on MCP-1 expression. The results of this study indicated that the expression of MCP-1 mRNA and protein were stimulated in the presence of TNF-α. TNF-α stimulated the phosphrylation of JNK, however, JNKI-1 inhibited the TNF-α stimulated MCP-1 secretion and gene expression. As expected, JNKI-1 blocked the stimulatory effect of TNF-α on the MCP-1 promoter activity. In conclusion, JNKI-1 partially inhibits the TNF-α-induced MCP-1 expression in HUVECs, and therefore JNKI-1 may be of therapeutic value in the treatment of diseases such as atherosclerosis.     

Effect of stimulation by interferonγ and tumor necrosis factorα on CD54 expression and homotypic aggregation of blasts of acute lymphoblastic leukemias

 CD54 (intercellular adhesion molecule-1, ICAM-1) surface expression on the blasts in ten cases of acute lymphoblastic leukemia (ALL) and its up-regulation by human recombinant interferon-γ (IFNγ) and/or tumor necrosis factor-α (TNFα) were studied in a serum-free culture system. In addition, the function of CD54 was assessed by a cellular aggregation assay. Prior to in vitro culture, only 3/10 ALL cases had more than 20% CD54-positive blasts. A 24-h incubation in serum-free medium alone induced CD54 positivity in another two cases. In these two ALLs, stimulation with IFNγ and/or TNFα further enhanced CD54 positivity. In addition, TNFα induced CD54 expression in one further case. In the remaining four cases no CD54 expression was induced by either cytokine. Of the six cases with constitutive or inducible CD54 expression, only five displayed CD54-dependent cellular aggregation. Taken together, the ALLs studied were heterogeneous with respect to their constitutive and cytokine-driven CD54 expression, while TNFα seemed to be more effective than IFNγ. In most cases, the CD54 molecule was functionally active, in that CD54 expression was paralleled by CD54-dependent homotypic aggregation of the blasts. Received: 25 August 1997 / Accepted: 10 October 1997     

The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells

The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.