Vascular Cell Adhesion Molecule-1 Transcription Research Articles

Abstract 654: Isoleukotoxin Promotes Autophagy and Reduces Vascular Cell Adhesion Molecule-1 Expression in Endothelial Cells

Rapamycin is a widely used autophagy inducer and has been shown to suppress development of atherosclerosis in animal models. However, the side effect is severe including hyperlipidemia. We identified isoleukotoxin, a metabolite of linoleic acid, as an autophagy inducer and hypothesized that it can be used to reduce inflammation of endothelial cells. To test this hypothesis we treated human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor α (TNFα) along with isoleukotoxin. Rapamycin was used as a positive control to induce autophagy. Isoleukotoxin promotes autophagic flux in HUVECs as well as rapamycin. Isoleukotoxin does not inhibit activity of mammalian target of rapamycin complex, while it inhibits activity of the downstream kinase, S6K. Isoleukotoxin modulates TNFα-induced cell adhesion molecules expression in HUVECs with a similar pattern as rapamycin. In this pattern, only vascular cell adhesion molecule-1 (VCAM-1) expression is reduced but not intercellular cell adhesion molecule-1 (ICAM-1). More interestingly, time-course analysis shows that VCAM-1 expression is only reduced after 20 hours treatment, which is different from that of inhibitors of VCAM-1 transcription such as SB202190. By qPCR we confirmed that isoleukotoxin does not inhibit VCAM-1 transcription. To determine whether isoleukotoxin reduces VCAM-1 expression by inhibiting S6K-mediated protein translation, we compared the effect of isoleukotoxin with S6K siRNA. S6K siRNA reduces expression of VCAM-1 slightly and also ICAM-1, which is different from that of isoleukotoxin. After excluding the regulation of transcription and translation, we hypothesize that isoleukotoxin reduces VCAM-1 expression by promoting VCAM-1 degradation via autophagy. This hypothesis is supported by blocking of autophagy and endocytosis. Blocking of autophagy by Atg5 siRNA enhances VCAM-1 expression, suggesting that VCAM-1 may be degraded by autophagy. Blocking of endocytosis by dynasore blocks the effect of isoleukotoxin on VCAM-1 expression, suggesting that VCAM-1 is degraded after endocytosis. Here we propose that isoleukotoxin is an anti-inflammation and a potential therapeutic molecule for inflammatory vascular diseases such as atherosclerosis.

IRF-5 accelerates leukocyte adhesion to endothelial cells in ischemia-reperfusion injury through regulating the transcription of VCAM-1

Ischemia-reperfusion injury (IRI) has been implicated in many pathological conditions, including cardiovascular diseases. Adhesion of leukocytes to the surface of endothelial cells has been considered as one of the principle steps in the pathological cascade of inflammatory tissue damage during IRI. The role of the transcriptional factor interferon regulatory factor-5 (IRF-5) in endothelial physiology remains unknown. Here, we report that IRF-5 is expressed in human umbilical vein endothelial cells (HUVECs) and is rapidly upregulated in response to IRI, mediated by the JAK2/STAT3 pathway. Importantly, IRF-5 is involved in IRI-induced attachment of THP-1 leukocytes to HUVECs. Mechanistically, it was found that IRF-5 targeted the expression of vascular cell adhesion molecule 1 (VCAM-1) at the transcriptional level by binding to its promoter. In conclusion, we identify IRF-5 as a new regulator and thus a therapeutic target in IRI-driven cardiovascular pathologies.

Abstract 211: Response Gene to Complement 32 Deficiency Protects Endothelial Cell From Inflammation and Attenuates Atheroslcerosis

Response gene to complement 32 (RGC-32) is involved in diet-induced obesity and hepatic steatosis. Therefore, we hypothesized that RGC-32 plays a role in atherosclerosis. Our current study showed that RGC-32 expression was dramatically induced in endothelial cells (ECs) of the atherosclerotic lesions from both apolipoprotein E-deficient (ApoE -/- ) mice and diseased human arteries. RGC-32 deficiency (Rgc32 -/- ) significantly attenuated the high-fat diet-induced and spontaneously developed atherogenesis in ApoE -/- mice without affecting the serum lipid profiles. In addition, Rgc32 -/- decreased the macrophage content without affecting fibrous cap size. Rgc32 -/- in bone marrow cells had no effect on atherosclerosis development, and Rgc32 -/- mice transplanted with wild-type (WT) bone marrow cells exhibited decreased atherosclerotic lesion area and macrophage infiltration, suggesting that RGC-32 in resident vascular cells plays a critical role in the atherogenesis. Rgc32 -/- decreased the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in ECs both in vivo and in vitro , resulting in a decreased monocyte-EC interaction. Mechanistically, RGC-32 regulated ICAM-1 and VCAM-1 transcription via activation of nuclear factor (NF)-κB. It appeared that RGC-32 interacted with NF-κB, which facilitated NF-κB binding to ICAM-1 and VCAM-1 promoters. Indeed, blockade of NF-κB activity by its selective inhibitor ammonium pyrrolidinedithiocarbamate blocked RGC-32-induced ICAM-1 and VCAM-1 expression. In conclusion, RGC-32 promoted atherogenesis by inducing monocyte-EC interaction through NF-κB-dependent induction of endothelial ICAM-1 and VCAM-1 expression.

Abstract 2805: LPA-dependent regulation of VCAM-1 in the ovarian cancer metastatic microenvironment and its impact on mesothelial invasion.

Abstract Ovarian cancer is the leading cause of gynecological cancer deaths in the United States due in large part to the advanced stage of disease at diagnosis. The poor prognosis is attributed to ovarian cancer metastasis to the peritoneal cavity and the accumulation of ascites, which contains elevated levels of several growth factors and cytokines including lysophosphatidic acid (LPA), a bioactive lipid that promotes ovarian cancer proliferation and survival as well as activities relevant to metastasis including migration and invasion. Ovarian cancer metastasis is a complex process that involves attachment to and invasion through the mesothelium and has been associated with vascular cell adhesion molecule 1 (VCAM-1). Previously we have shown that VCAM-1 is preferentially expressed on the mesothelium of patients with ovarian cancer where it functions to promote mesothelial invasion. Inhibition of VCAM-1 function reduces tumor burden and increases survival in a mouse model, implicating VCAM-1 as an important target for the treatment of ovarian cancer. Since LPA promotes ovarian cancer migration and invasion, we questioned whether it also influenced the mesothelium to regulate invasion. Using a co-culture system to measure ovarian cancer cell invasion of the mesothelium, treatment with a VPC51299, a LPA1/3 receptor antagonist, which targets both the cancer cells and the mesothelium, resulted a dose dependent decrease in mesothelial invasion. Importantly, specific targeting of the mesothelium using siRNA directed at the LPA1 receptor also diminished ovarian cancer cell invasion of the mesothelium. Additionally, we observed that siRNA knockdown of the LPA receptor diminished VCAM-1 expression; re-expression of VCAM-1 to endogenous levels in cells lacking LPA receptor expression restored mesothelial invasion. To determine how LPA regulated VCAM-1 expression, human mesothelial cells derived from ovarian cancer patients (LP9 or LP3) were subjected to siRNA inhibition of LPA1 receptor expression and protein and mRNA expression were evaluated. The results revealed a significant decrease in VCAM-1 protein expression but no impact on VCAM-1 mRNA levels, indicating that LPA does not have a major role in the regulation of VCAM-1 transcription as has been reported in other systems. Rather, it implicates LPA in the regulation of VCAM-1 protein expression. To investigate whether LPA regulates VCAM-1 protein stability, LP9 cells were treated with a eukaryotic translation inhibitor, cycloheximide, for increasing periods of time. VCAM-1 protein half-life decreased after treatment with cycloheximide and LPA1 knockdown compared to control. Taken together, these observations indicate that in addition to targeting the cancer cells, LPA influences the tumor microenvironment to promote mesothelial invasion in part by regulating VCAM-1 protein stability. Citation Format: Timothy A. Raines, Kevin R. Lynch, Jill K. Slack-Davis. LPA-dependent regulation of VCAM-1 in the ovarian cancer metastatic microenvironment and its impact on mesothelial invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2805. doi:10.1158/1538-7445.AM2013-2805

Rhein Inhibits the Expression of Vascular Cell Adhesion Molecule 1 in Human Umbilical Vein Endothelial Cells with or without Lipopolysaccharide Stimulation

Reducing the expression of endothelial cell adhesion molecules (ECAMs) is known to decrease inflammation-induced vascular complications. In this study, we explored whether rhein can reduce the inflammation-induced expression of ECAMs in human umbilical vein endothelial cells (HUVECs) with or without lipopolysaccharide (LPS) stimulation. HUVECs were treated with different concentrations of rhein with or without 2.5 μg/ml LPS stimulation. Cell viability was assayed using the MTT method. Real-time PCR and Western blot analysis were used to measure the transcription and expression levels of ECAMs, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-SELECTIN and related signaling proteins. The results indicated that rhein (0-20 μmol/L) and LPS (0-10 μg/ml) had no effect on the viability of HUVECs. LPS could promote the expression of VCAM-1, ICAM-1 and E-SELECTIN. Rhein appeared to target VCAM-1, ICAM-1 and E-SELECTIN, with the transcription and expression of all three factors being reduced by the rhein treatment (10 and 20 μmol/L). The transcription and expression of VCAM-1 were also reduced by treatment with rhein (10 and 20 μmol/L) in the presence of LPS stimulation. In conclusion, rhein treatment reduced the expression of VCAM-1 in HUVECs via a p38-dependent pathway.

In Vivo Quantification of Vcam-1 Expression in Renal Ischemia Reperfusion Injury Using Non-Invasive Magnetic Resonance Molecular Imaging

Rationale and ObjectiveVascular cell adhesion molecule-1 (VCAM-1) is upregulated in ischemia reperfusion injury (IRI), persisting after restoration of blood flow. We hypothesized that microparticles of iron oxide targeting VCAM-1 (VCAM-MPIO) would depict “ischemic memory” and enable in vivo assessment of VCAM-1 expression.Methodology and FindingsMice subject to unilateral, transient (30 minutes) renal ischemia and subsequent reperfusion received intravenous VCAM-MPIO (4.5 mg iron/kg body weight). Contrast agent bound rapidly (<30 minutes) in IRI-kidneys and appeared as intensely low signal areas by MRI in vivo. Automated segmentation and quantification yielded MPIO contrast volumes of 5991±354×106 µm3 in IRI vs. 87±7×106 µm3 in kidneys with no surgical intervention (P<0.001); 90±8×106 µm3 in IRI kidneys exposed to control (IgG-MPIO) and 625±80×106 µm3, in IRI kidneys pre-treated with a blocking dose of VCAM-1 antibody (P<0.001). In keeping with quantitative MRI data, VCAM-1 mRNA expression in IRI was 65-fold higher than in kidneys without surgical intervention (3.06±0.63 vs. 0.05±0.02, P<0.001). Indeed VCAM-1 mRNA expression and VCAM-MPIO contrast volume were highly correlated (R2 = 0.901, P<0.01), indicating that quantification of contrast volume reflected renal VCAM-1 transcription. Serial imaging showed VCAM-MPIO accumulation at target within 30 minutes, persisting for ≥90 minutes, while unbound VCAM-MPIO was cleared rapidly from blood, with sequestration by mac-3 positive Kupffer cells in the liver and monocyte/macrophages in the spleen.Conclusions(1) VCAM-MPIO detected VCAM-1 expression and defined its 3-dimensional distribution, revealing “ischemic memory” in renal IRI; (2) automated volumetric quantification of VCAM-MPIO accurately reflected tissue levels of VCAM-1 mRNA; and (3) VCAM-MPIO bound rapidly to target with active sequestration of unbound MPIO in the liver and spleen.

Inhibition of TNF-α induced ICAM-1, VCAM-1 and E-selectin expression by Momordica charantia L.

The initiation of an atherosclerotic lesion involves establishment of an endothelial cell pro-inflammatory state that recruits leukocytes and promotes their movement across the endothelium. These processes require endothelial expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin). Tumor necrosis factor-α (TNF-α) is a powerful inducer of these adhesion molecules [1]. The fruit of Momordica charantia L. (MC) is a common vegetable in tropical areas of Asia and Africa. MC also has been traditionally used as a bitter stomachic and an antidiabetic [2]. Experiments were performed to test whether MC alters TNF-α-induced expression of these adhesion molecules. Human umbilical vein endothelial cells (HUVEC) were treated for 18h with or without an extract and fractions of MC and TNF-α. ICAM-1, VCAM-1 and E-selectin were detected by cell-based ELISA, Western blots and RT-PCR. MC significantly inhibited TNF-α-induced expression of each adhesionα molecule in a dose-dependent manner. The chloroform fraction of MC significantly inhibited TNF-α-induced expression of each adhesion molecule in a dose-dependent manner. Nuclear factor-κB (NF-κB) is required for transcription of ICAM-1, VCAM-1 and E-selectin adhesion molecule genes [3]. Western blot analysis revealed that the chloroform fraction of MC inhibits translocation of the p65 subunit of NF-κB to the nucleus. Thus, the chloroform fraction of MC inhibited TNF-α-mediated induction of ICAM-1, VCAM-1 and E-selectin in HUVEC by inhibiting NF-κB, and lipophilic components of MC may suppress inflammation and modulate the immune response.

Pulmonary CXCR2 regulates VCAM-1 and antigen-induced recruitment of mast cell progenitors

Chemokine receptors regulate the trafficking of leukocytes by mediating chemotaxis and by their influence on the expression and/or affinity of leukocyte integrins. Using blocking mAb, we showed that antigen-induced recruitment of mast cell progenitors (MCp) to the lung requires interaction of a4 integrins on the MCp with endothelial vascular cell adhesion molecule 1 (VCAM-1). In seeking a chemokine component, we found that CXCR2-deficient but not CCR3- or CCR5-deficient sensitized and antigen-challenged mice have significantly fewer lung MCp 1 day after challenge and fewer tracheal intraepithelial MC 1 week after challenge, implying that recruited MCp provide the source for these mature MC. Unexpectedly, reconstitution of sensitized, sublethally irradiated +/+ and -/- mice with bone marrow cells of either genotype indicated that expression of CXCR2 by the migrating MCp was not required. Instead, receptor function by resident lung cells was required because normal BM did not reconstitute MCp recruitment in irradiated CXCR2(-/-) mice. The reduced MCp influx into the lung of CXCR2(-/-) mice was accompanied by reduced induction of VCAM-1 transcripts and reduced endothelial surface expression. Thus, these studies demonstrate a role for a chemokine receptor in regulating endothelial VCAM-1 expression, MCp migration, and the level of intraepithelial MC in the lung of aerosolized, antigen-challenged mice.

Propionyl-L-Carnitine Prevents Age-Related Myocardial Remodeling in the Rabbit

Age-related cardiac remodeling is characterized by changes in myocardial structure, which include fibrosis (ie, increased collagen concentration). The pathogenetic mechanisms of age-related cardiac changes and possible pharmacologic interventions are still a matter of investigation. A morphometric analysis of collagen accumulation was performed in Sirius Red-stained left ventricular sections of 3-month-old and 5-6-year-old animals after a 9-month period of propionyl-L-carnitine treatment (PLC; 120 mg Kg(-1) day(-1) per os); aged rabbits showed decreased interstitial collagen accumulation and no changes in cellularity and apoptotic rate compared to controls. Age-related expression of vascular cell adhesion molecule-1 (VCAM-1)-positive microvessels was also reduced in PLC-treated rabbits. In vitro, the 16-hour, 10-microM PLC treatment reduced collagen type 1 and VCAM-1 transcripts, which were investigated by reverse transcription-polymerase chain reaction, more markedly in cardiac fibroblasts from aged donors. In the latter, the anti-VCAM-1 antibody treatment was found to be associated with a reduction in collagen type I transcripts. Our results demonstrated that long-term PLC treatment partially prevents age-related interstitial remodeling and suggests that a more complex interstitial cell-to-cell signaling regulates senescent myocardium properties.

Dual Pathways for Nuclear Factor κB Activation by Angiotensin II in Vascular Smooth Muscle

Activation of nuclear factor (NF)-kappaB by angiotensin II (Ang II) plays an essential role in stimulating expression of vascular adhesion molecules, which are essential for vascular inflammation. We report that Ang II activates NF-kappaB by phosphorylating its p65 subunit via a pathway mediated partially by ribosomal S6 kinase (RSK). In investigating other pathway(s) that may be involved, we found that the ability of Ang II to activate NF-kappaB in mouse embryonic fibroblast is suppressed (approximately 70%) either by deletion of IkappaB Kinase (IKK) or by inhibiting or knocking down IKK in vascular smooth muscle cells using a dominant-negative IKK adenovirus or small interference RNA to IKKbeta. Thus, Ang II also stimulates NF-kappaB via IKK. In vitro, we found that Ang II stimulates IKK to phosphorylate myelin basic protein and the p65 subunit of NF-kappaB. The mechanism by which Ang II activates IKK is to increase phosphorylation of IKKbeta in its activation loop (Ser181) rather than IkappaB phosphorylation. Inhibiting both the RSK and IKK pathways completely blocks the Ang II-induced p65 phosphorylation and NF-kappaB activation. These 2 pathways are independent: inhibiting IKK does not block Ang II-induced phosphorylation of RSK, whereas inhibiting mitogen-activated protein kinase 1 does not affect phosphorylation of IKK. Finally, we found that Ang II can induce expression of vascular adhesion molecules by 2 pathways; both IKK and RSK lead to phosphorylation of the p65 subunit of NF-kappaB to increase vascular cell adhesion molecule-1 transcription. The 2 pathways are functionally important because inhibiting IKK and RSK in vascular smooth muscle cells blocks Ang II-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 to limit vascular inflammation.

Diverse effects of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase on the expression of VCAM-1 and E-selectin in endothelial cells

The expression of monocyte adhesion molecules, such as VCAM-1 (vascular cell adhesion molecule-1) and E-selectin, on the surface of the endothelium is an important step in the initiation and progression of atherosclerotic lesions. We hypothesized that the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in endothelial cells could influence the expression of VCAM-1 and E-selectin. Using cultured human umbilical vein endothelial cells, we found that mevastatin (0.1–1μM) significantly reduced the expression of VCAM-1 protein in cells activated by tumour necrosis factor-α (TNF-α) for 7h. In contrast, TNF-α-induced E-selectin protein expression was augmented after mevastatin treatment. Mevastatin inhibited the mRNA expression of both VCAM-1 and E-selectin in TNF-α-stimulated endothelial cells. The activity of the transcription factor nuclear factor-κB, which is known to regulate the transcription of VCAM-1 and E-selectin, was significantly reduced after incubation with mevastatin. Analysis of the time-dependent variation in the TNF-α-induced expression of E-selectin, and estimation of the rate of surface disappearance of E-selectin together with measurement of the amounts of E-selectin molecules secreted, indicated that mevastatin inhibited the surface removal of E-selectin. This is compatible with the observed increase in E-selectin expression after statin treatment. All observed effects of mevastatin were reversed by mevalonate, the product of the HMG-CoA reductase reaction. In conclusion, inhibition of HMG-CoA reductase in endothelial cells attenuates VCAM-1 expression, but increases E-selectin expression, after cytokine induction. These diverse effects are associated with changes in the transcriptional regulation of the two adhesion molecule genes and modulation of the surface removal of E-selectin.

Diverse effects of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase on the expression of VCAM-1 and E-selectin in endothelial cells.

The expression of monocyte adhesion molecules, such as VCAM-1 (vascular cell adhesion molecule-1) and E-selectin, on the surface of the endothelium is an important step in the initiation and progression of atherosclerotic lesions. We hypothesized that the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in endothelial cells could influence the expression of VCAM-1 and E-selectin. Using cultured human umbilical vein endothelial cells, we found that mevastatin (0.1-1 microM) significantly reduced the expression of VCAM-1 protein in cells activated by tumour necrosis factor-alpha (TNF-alpha) for 7 h. In contrast, TNF-alpha-induced E-selectin protein expression was augmented after mevastatin treatment. Mevastatin inhibited the mRNA expression of both VCAM-1 and E-selectin in TNF-alpha-stimulated endothelial cells. The activity of the transcription factor nuclear factor-kappa B, which is known to regulate the transcription of VCAM-1 and E-selectin, was significantly reduced after incubation with mevastatin. Analysis of the time-dependent variation in the TNF-alpha-induced expression of E-selectin, and estimation of the rate of surface disappearance of E-selectin together with measurement of the amounts of E-selectin molecules secreted, indicated that mevastatin inhibited the surface removal of E-selectin. This is compatible with the observed increase in E-selectin expression after statin treatment. All observed effects of mevastatin were reversed by mevalonate, the product of the HMG-CoA reductase reaction. In conclusion, inhibition of HMG-CoA reductase in endothelial cells attenuates VCAM-1 expression, but increases E-selectin expression, after cytokine induction. These diverse effects are associated with changes in the transcriptional regulation of the two adhesion molecule genes and modulation of the surface removal of E-selectin.

MRNA expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in acute renal allograft rejection.

The intercellular adhesion molecule-1 (ICAM-1) and the vascular cell adhesion molecule-1 (VCAM-1) show a form of complementary distribution in normal and grafted kidneys. The molecular mechanism by which ICAM-1 and VCAM-1 are increased or induced on vascular cells during acute renal allograft rejection has not been clearly defined. We examined ICAM-1 and VCAM-1 mRNA expression in 17 renal allograft biopsies with (n=12) and without (n=5) features of acute rejection, and four control renal biopsies with no detectable abnormalities by RNA in situ hybridization. The expression of ICAM-1 and VCAM-1 protein was also assessed by immunohistochemical staining of frozen sections. In controls and nonrejecting graft biopsies, the signals of the ICAM-1 and VCAM-1 transcripts in vascular cells were almost negligible. Specific signals of ICAM-1 and VCAM-1 mRNAs were detected on the endothelial cells of small muscular arteries in most cases with acute renal allograft rejection. The messages for ICAM-1 and VCAM-1 were also detected on arterial smooth muscle cells in all the five cases with severe type III rejection. These results suggest that the induced appearance of ICAM-1 and VCAM-1 on the vascular cells of acutely rejecting renal transplants was related to actual cellular synthesis and that both adhesion molecules could act together in the rejection process. They also suggest that the expression of ICAM-1 and VCAM-1 genes by arterial smooth muscle cells may be an important cause of transmural arteritis in severe acute renal allograft rejection.

PPARalpha activators inhibit cytokine-induced vascular cell adhesion molecule-1 expression in human endothelial cells.

Adhesion molecule expression on the endothelial cell (EC) surface is critical for leukocyte recruitment to atherosclerotic lesions. Better understanding of transcriptional regulation of adhesion molecules in ECs may provide important insight into plaque formation. Peroxisome proliferator-activated receptor-alpha (PPARalpha), a member of the nuclear receptor family, regulates gene expression in response to certain fatty acids and fibric acid derivatives. The present study investigated PPARalpha expression in human ECs and their regulation of vascular cell adhesion molecule-1 (VCAM-1). Immunohistochemistry revealed that human carotid artery ECs express PPARalpha. Pretreatment of cultured human ECs with the PPARalpha activators fenofibrate or WY14643 inhibited TNF-alpha-induced VCAM-1 in a time- and concentration-dependent manner, an effect not seen with PPARgamma activators. Both PPARalpha activators decreased cytokine-induced VCAM-1 mRNA expression without altering its mRNA half-life. Transient transfection of deletional VCAM-1 promoter constructs and electrophoretic mobility shift assays suggest that fenofibrate inhibits VCAM-1 transcription in part by inhibiting NF-kappaB. Finally, PPARalpha activators significantly reduced adhesion of U937 cells to cultured human ECs. Human ECs express PPARalpha, a potentially important regulator of atherogenesis through its transcriptional control of VCAM-1 gene expression. Such findings also have implications regarding the clinical use of lipid-lowering agents, like fibric acids, which can activate PPARalpha.

α-Lipoic acid reduces expression of vascular cell adhesion molecule-1 and endothelial adhesion of human monocytes after stimulation with advanced glycation end products

Advanced glycation end products (AGEs) have been identified as relevant mediators of late diabetic complications such as atherosclerotic disease. The endothelial migration of monocytes is one of the first steps in atherogenesis and monocyte-endothelial interaction itself is linked to the expression of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1). Recently, stimulation of VCAM-1 by AGEs has been demonstrated. Since endothelial stimulation by AGEs is followed by generation of oxygen free radicals with subsequent activation of nuclear transcription factor kappaB, we investigated the influence of alpha-lipoic acid on the expression of VCAM-1 and monocyte adherence to endothelial cells in vitro by means of cell-associated chemiluminescence assays and quantitative reverse transcriptase polymerase chain reaction using a constructed recombinant RNA standard. We found that alpha-lipoic acid was able to decrease the number of VCAM-1 transcripts from 41. 0+/-11.2 to 9.5+/-4.7 RNA copies per cell in AGE-stimulated cell cultures. Furthermore, expression of VCAM-1 was suppressed in a time- and dose-dependent manner by alpha-lipoic acid as shown by chemiluminescence endothelial cell assay. Pretreatment of endothelial cells with 0.5 mM or 5 mM alpha-lipoic acid reduced AGE-induced endothelial binding of monocytes from 22.5+/-2.9% to 18. 3+/-1.9% and 13.8+/-1.8% respectively. Thus, we suggest that extracellularly administered alpha-lipoic acid reduces AGE-albumin-induced endothelial expression of VCAM-1 and monocyte binding to endothelium in vitro. These in vitro results may contribute to the understanding of a potential antioxidative treatment of atherosclerosis.

Establishment of a quantitative RT-pCR for detection of vascular cell adhesion molecule-1 transcripts in endothelial cells after stimulation with advanced glycation endproducts.

Advanced glycation endproducts (AGE) are supposed to increase endothelial expression of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1) by inducing an intracellular stress with subsequent activation of nuclear transcription factor NF-kappa-B. Quantitative analysis of VCAM-1-transcription has not been demonstrated concerning this topic. Thus, the aim of this study was to establish quantitative reverse transcription polymerase chain reaction (RT-PCR) assays using a spacer gene in order to measure the amounts of specific mRNA for VCAM-1 in human umbilical vein endothelial cells (HUVEC) which were stimulated with AGE-albumin (AGE-BSA). A recombinant RNA-standard was synthesized and used as internal RT-PCR standard. The amount of VCAM-1-mRNA in unstimulated HUVEC was found to be 2.2 +/- 2.7 copies per cell. After stimulation with AGE-BSA, mRNA-levels were elevated to 38.9 +/- 10.9 copies per cell. Positive controls (stimulated with lipopolysaccharide) revealed mRNA-levels of 78.7 +/- 27.5 copies per cell. We conclude that quantitative RT-PCR using the spacer gene technique is a valid and reliable method for the measurement of small amounts of specific

Postinduction transcriptional repression of E-selectin and vascular cell adhesion molecule-1.

TNF-alpha induction of the E-selectin and vascular cell adhesion molecule-1 (VCAM-1) genes leads to transient accumulation of high levels of mRNA in endothelial cells. The increase in these mRNAs after induction is due to an increase in the rate of gene transcription, which is maintained for several hours in the continuous presence of cytokine. Cytokine-induced transcriptional activation of these genes requires the transcription factor, nuclear factor-kappaB. Following removal of TNF-alpha, there is rapid postinduction transcriptional repression common to both of these genes. The repression is protein synthesis dependent and correlates with protein synthesis-dependent loss of both the p50 and p65 subunits of nuclear factor-kappaB from the nucleus. IkappaBalpha is capable of specifically displacing endothelial-derived heterodimeric p50/p65 from the E-selectin and VCAM-1 kappaB elements, while having no effect on binding of p50 homodimer. In the presence of agents that block proteasomal degradation of IkappaBalpha, endogenous IkappaBalpha can be visualized in the nucleus of both resting and TNF-alpha-activated endothelial cells. Endogenous IkappaBalpha is readily detected in the nucleus of HeLa cells, and its nuclear localization is increased following removal of TNF-alpha. Repression of E-selectin and VCAM-1 transcription following cytokine removal requires the loss of nuclear p50 and p65, and involves IkappaBalpha. This postinduction transcription repression mechanism may be one component of a program that prevents inappropriate and prolonged expression of adhesion molecules.

Very late antigen-4/vascular cell adhesion molecule-1 (VLA-4/VCAM-1) pathway is involved in the transendothelial migration of lymphocytes in bancroftian filariasis.

The role of endothelial vascular cell adhesion molecule-1 (VCAM-1) in the trafficking of lymphocytes from the vascular circulation through endothelium and into sites of filarial inflammation was investigated in asymptomatic microfilaremic (MF; n = 16) and in patients with filaria-associated lymphatic pathology (LP; n = 23). When compared, by human umbilical vein endothelial cell ELISA, with PBMC supernatants generated from MF patients, filarial Ag (Brugia malayi adult Ag (BmA))-stimulated supernatants from LP patients stimulated over 50% more VCAM-1 (lymphatic pathology (LP) = 0.377 relative ELISA units vs MF = 0.246; p = 0.02). No difference was seen between patient groups with nonfilarial Ag (streptolysin O)-stimulated supernatant (LP = 0.160 relative ELISA units vs MF = 0.166). BmA-stimulated supernatants from LP patients stimulated significantly more T cell migration (percentage of relative migration LP = 79.9% vs MF = 25.8%; p = 0.01) through tightly confluent human umbilical vein endothelial cell monolayers cultured on collagen gels. Anti-VCAM-1 inhibited the increased T cell migration induced by BmA-stimulated supernatants by 97.7%, anti-VLA-4 by 74.7%, and blockade of NF-kappa B-dependent VCAM-1 transcription with 50 microM pyrrolidine dithiocarbamate resulted in 87.7% inhibition. Biopsies from 87.5% of the LP patients, but only 38.5% of the MF patients, demonstrated VCAM-1 on vascular endothelium. BmA-stimulated supernatants pretreated with anti-IL-1 alone resulted in VCAM-1 expression that was 23.7% of that with untreated supernatants while anti-IL-4, anti-IFN-gamma, or anti-TNF alone had essentially no effect on VCAM-1 expression.

Differential regulation of vascular cell adhesion molecule-1 gene transcription by tumor necrosis factor alpha and interleukin-1 alpha in dermal microvascular endothelial cells

As part of the inflammatory response, the localization of leukocytes depends to an important degree on cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells (EC). We have previously shown that VCAM-1 expression is induced on human umbilical vein EC (HUVEC) by both tumor necrosis factor alpha (TNF- alpha) and interleukin-1 alpha (IL-1 alpha), whereas on human dermal microvascular EC (HDMEC) only TNF alpha results in VCAM-1 expression. To explore molecular mechanisms responsible for these contrasting patterns of VCAM-1 induction in HUVEC versus HDMEC, we performed transcriptional activation studies with VCAM-1-based reporter constructs and in vitro binding assays using two adjacent NF-kappa B binding sequences of the VCAM-1 promoter as a DNA probe. Previous studies have established that these NF-kappa B motifs are required for cytokine-induced VCAM-1 transcription, and may further mediate cell- specific VCAM-1 gene activation by cytokines. The findings reported here demonstrate a significant HDMEC-specific attenuation of VCAM-1 gene transcription in response to IL-1 alpha, but not TNF alpha. An upstream VCAM-1 gene regulatory region distinct from the NF-kappa B sites appears to function as an IL-1 alpha-mediated transcriptional repressor within HDMEC. This repressor region conveys IL-1 alpha- dependent, but not TNF alpha-dependent, inhibition of transcription driven by a heterologous cytokine response element and promoter.

Differential Regulation of Vascular Cell Adhesion Molecule-1 Gene Transcription by Tumor Necrosis Factor α and Interleukin-1α in Dermal Microvascular Endothelial Cells

As part of the inflammatory response, the localization of leukocytes depends to an important degree on cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells (EC). We have previously shown that VCAM-1 expression is induced on human umbilical vein EC (HUVEC) by both tumor necrosis factor alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha), whereas on human dermal microvascular EC (HDMEC) only TNF alpha results in VCAM-1 expression. To explore molecular mechanisms responsible for these contrasting patterns of VCAM-1 induction in HUVEC versus HDMEC, we performed transcriptional activation studies with VCAM-1-based reporter constructs and in vitro binding assays using two adjacent NF-kappa B binding sequences of the VCAM-1 promoter as a DNA probe. Previous studies have established that these NF-kappa B motifs are required for cytokine-induced VCAM-1 transcription, and may further mediate cell-specific VCAM-1 gene activation by cytokines. The findings reported here demonstrate a significant HDMEC-specific attenuation of VCAM-1 gene transcription in response to IL-1 alpha, but not TNF alpha. An upstream VCAM-1 gene regulatory region distinct from the NF-kappa B sites appears to function as an IL-1 alpha-mediated transcriptional repressor within HDMEC. This repressor region conveys IL-1 alpha-dependent, but not TNF alpha-dependent, inhibition of transcription driven by a heterologous cytokine response element and promoter.