ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of cholesterol efflux from cells to apolipoproteins, whereas sterol-responsive element-binding protein 2 (SREBP2) is the key protein regulating cholesterol synthesis and uptake. We investigated the regulation of ABCA1 by SREBP2 in vascular endothelial cells (ECs). Our results showed that sterol depletion activated SREBP2 and increased its target, low density lipoprotein receptor mRNA, with a concurrent decrease in the ABCA1 mRNA. Transient transfection analysis revealed that sterol depletion decreased the ABCA1 promoter activity by 50%, but low density lipoprotein receptor promoter- and the sterol-responsive element-driven luciferase activities were increased. Overexpression of the N terminus of SREBP2 (SREBP2(N)), an active form of SREBP2, also inhibited the ABCA1 promoter activity. Functionally adenovirus-mediated SREBP2(N) expression increased cholesterol accumulation and decreased apoA-I-mediated cholesterol efflux. The conserved E-box motif was responsible for the SREBP2(N)-mediated inhibition since mutation of the E-box increased the basal activity of the ABCA1 promoter and abolished the inhibitory effect of SREBP2(N). Furthermore sterol depletion and SREBP2(N) overexpression induced the binding of SREBP2(N) to both consensus and ABCA1-specific E-box. Chromatin immunoprecipitation assay demonstrated that serum starvation enhanced the association of SREBP2 and the ABCA1 promoter in ECs. To correlate this mechanism pathophysiologically, we found that oscillatory flow caused the activation of SREBP2 and therefore attenuated ABCA1 promoter activity in ECs. Thus, this SREBP-regulated mechanism may control the efflux of cholesterol, which is a newly defined function of SREBP2 in ECs in addition to its role in cholesterol uptake and biosynthesis.

A decrease in the bioavailability of endothelium-derived nitric oxide (NO) is linked to hypercholesterolemia. However, the mechanism by which low density lipoprotein (LDL) mediates endothelial NO synthase (eNOS) dysfunction remains controversial. We investigate the effect of LDL on eNOS regulation in human endothelial cells (ECs). In cultured ECs, a high level of LDL increased the abundance of eNOS and caveolin-1 (Cav-1) in the membrane caveolae and the association of eNOS with Cav-1. Furthermore, it decreased the basal level of NO and blocked NO production stimulated by the calcium ionophore A23187. LDL exposure also increased the formation of stress fibers and the membrane translocation of eNOS. These effects can be blocked by cytochalasin D, an actin cytoskeleton disruptor. In revealing the mechanism underlying the translocation of eNOS, we found that a high level of LDL increased the level of membrane-associated and GTP-formed RhoA and activated the RhoA downstream kinase ROCK-1 activity. Y-27632, a specific inhibitor of ROCK-1, blocked LDL-induced stress fiber formation, eNOS translocation and NO production. In conclusion, a high level of LDL increases the movement of eNOS to membrane caveolae via the increased stress fibers. The RhoA-mediated pathway may play a crucial role in this process in vascular ECs.

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (*NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O(2)(*-)), which inactivates *NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for *NO, O(2)(*-), phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O(2)(*-) by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by approximately 34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (approximately 85%) and cytosol (approximately 15%). Only 3-6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O(2)(*-), which scavenges *NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O(2)(*-).

Low density lipoprotein (LDL) induces intercellular adhesion molecule-1 (ICAM-1) gene expression and leads to endothelial cell (EC) leukocyte adhesion. However, the transcriptional mechanism for LDL-induced EC perturbation remains to be fully explained. Activator protein-1 (AP-1) is induced after the exposure of ECs to LDL. In the present study, a regulated adenovirus expressing a dominant-negative mutant of c-Jun (TAM-67) was used to examine the role of AP-1 in the LDL-induced ICAM-1 activation. Overexpression of TAM-67 specifically inhibited AP-1 activation and prevented the LDL-activated surface expression of ICAM-1 protein in human umbilical vein ECs and human coronary artery ECs. Northern analyses and promoter transactivation assays indicated that this effect of TAM-67 was likely mediated through a suppression of the transcriptional regulation of the ICAM-1 gene. Functionally, TAM-67 attenuated leukocyte adherence to ECs in response to LDL. Furthermore, electrophoresis mobility shift assays and site-directed mutagenesis suggested that an AP-1-like motif in the promoter region of the human ICAM-1 gene was a critical cis element for LDL induction. These results, for the first time, provide evidence suggesting that AP-1 is a major regulatory mechanism leading to endothelial activation.

Endothelial dysfunction is a major atherogenic proinflammatory event. LDL causes the activation and phenotypic changes of cultured vascular endothelial cells (ECs). We previously reported that LDL activates c-Jun and AP-1 in ECs. In this study, we demonstrated that p38-ATF-2 is activated by LDL in human ECs and that this activation is mediated by Ras. When ECs are incubated with LDL in pathophysiological concentrations, the p38-mediated ATF-2 phosphorylation and ATF-2 transactivation are increased in a time- and dose-dependent manner. To elucidate the upstream mechanism in LDL-activated p38 in ECs, we demonstrate that LDL increases Ras translocation from the cytoplasm to the cellular membrane, with concurrent increases in Ras binding activity to GST-Raf-1. Overexpression of RasN17, a dominant negative mutant of Ras, attenuates the LDL-induced increases in (1) phosphorylation of ATF-2, (2) phosphorylation of c-Jun, (3) AP-1 binding, and (4) AP-1-driven luciferase activity. To study the effect of p38 in the regulation of an LDL targeting gene, we show that a specific p38 inhibitor attenuates LDL-induced E-selectin at the mRNA level. Thus, LDL activates both p38 and JNK signaling pathways through Ras activation, and furthermore, these events may play an important role in LDL-induced endothelial activation.

Hypercholesterolemia is a major risk factor for atherosclerosis, but the mechanism by which cholesterol activates the endothelium remains undocumented. The present investigation was undertaken to investigate the role of cholesterol, one of the bioactive moieties of the low-density lipoprotein (LDL) particle, in initiating of intracellular signaling in endothelial cells (ECs) and culminating in increased abundance of the intercellular adhesion molecule-1 (ICAM-1). Cholesterol was delivered to human umbilical vein ECs (HUVECs) via cholesterol-enriched liposomes. In HUVECs, the cellular cholesterol:phospholipid ratio increased after 1 h of exposure to cholesterol. The level of ICAM-1 increased in both mRNA and protein after 24 h of cholesterol exposure. ICAM-1 mRNA half-life was not affected by cholesterol exposure. Promoter studies showed greater than two-fold activation of the ICAM-1 gene expression after cholesterol exposure. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) activity substantially increased after 2 h of exposure to cholesterol. In contrast, cholesterol did not affect nuclear factor-κB (NF-κB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-κB-dependent expression was not affected. The AP-1/Ets (−891 to −908) site, one of the three AP-1-like sites in the ICAM-1 promoter, was most responsive to cholesterol. These data demonstrate for the first time that cholesterol enrichment phenotypically modulates ECs by transcriptionally upregulating ICAM-1 expression.

HomeCirculation ResearchVol. 88, No. 12Ref-1 and Transcriptional Control of Endothelial Apoptosis Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBRef-1 and Transcriptional Control of Endothelial Apoptosis Nanping Wang and Michael B. Stemerman Nanping WangNanping Wang From the Division of Biomedical Sciences, University of California, Riverside, Calif. and Michael B. StemermanMichael B. Stemerman From the Division of Biomedical Sciences, University of California, Riverside, Calif. Originally published22 Jun 2001https://doi.org/10.1161/hh1201.093162Circulation Research. 2001;88:1223–1225Vascular endothelium, when unperturbed, provides a surface to the blood vessel, which is passive to the development of thrombosis, and potentially adherent blood cells. This characteristic is the quintessence of vascular homeostasis.1 However, endothelial cells (ECs) can undergo apoptosis in vitro in response to a variety of pathophysiological conditions including hypoxia, proinflammatory cytokines, bacterial endotoxins, and atherogenic risk factors such as homocysteine and lipoproteins (reviewed in Stefanec2 and Dimmeler and Zeiher3 ). These cellular perturbations have in common the generation of intracellular reactive oxygen intermediates, referred to as oxidative stress. ECs respond to these adverse conditions by altering their intracellular reduction/oxidization (redox) state and making their ultimate decision between adaptation (survival) and apoptosis (see Figure). Understanding the precise mechanisms controlling such a process is an important component to our knowledge of cardiovascular diseases. In this issue of Circulation Research, Hall et al4 provide novel evidence for a critical role of Ref-1, a redox-sensitive regulator, in affecting EC apoptosis.Ref-1 was cloned as Redox factor, also known as apurinic (apyrimidinic) endonuclease (APE).5 As a ubiquitously expressed multifunctional 36-kDa protein, Ref-1 is involved in the repair of DNA damage as well as in the transcriptional regulation of genes. Its 5′AP-endonuclease functions in base excision repair, and its 3′-diesterase activity removes phosphoglycolate residues from DNA damaged by genotoxic stresses. In addition, Ref-1 is also important for the activation of transcription factors, such as activator protein-1 (AP-1),67 nuclear factor-κB (NF-κB),8 p53,910 and hypoxia-inducible factor-1α (HIF-1α).11 Activation of transcription factors, which occurs via a redox-based mechanism, pertains to its 6-kDa N-terminal domain. Following its discovery, Xanthoudakis and Curran12 identified Ref-1 as a reductive activator of c-Fos and c-Jun (two major components of AP-1) via a reduction of the conserved cysteine residues in their DNA binding domains. Interestingly, Ref-1 also acts as a transcriptional repressor of its own gene and other genes such as that coding for the parathyroid hormone. Although it has been observed that a decrease in Ref-1 protein level precedes apoptotic changes in rodent models for ischemic or traumatic brain injury,13 a role of Ref-1 in EC apoptosis has not been investigated previously. In the present study, hypoxia resulted in decrease in Ref-1 protein expression in both human umbilical vein ECs and bovine pulmonary artery ECs. Moreover, overexpression of Ref-1 rescued both hypoxia-and tumor necrosis factor (TNF-α)–induced apoptosis. This demonstrates that the decline in Ref-1 is a cause of, but not a response to, hypoxia-induced apoptosis.4 Further, Ref-1 appears to be an antiapoptotic factor in ECs. This agrees with a recent report showing a protective effect for Ref-1 in dopamine-induced neuron apoptosis.14Numerous agents are categorized as having pro- or anti-EC apoptotic properties. What remains as inconclusive and perhaps controversial are the roles of specific transcription factors controlling EC response to these various perturbations. Transcription factor NF-κB has, for years, been recognized as a central mediator of gene expression induced by proinflammatory cytokines and pathogens. It is thought to play a pivotal role in cardiovascular diseases including atherosclerosis (see review by Collins and Cybulsky15 ). Activation of NF-κB has been linked to apoptosis, with the factor playing either an antiapoptotic or proapoptotic role, depending on the cell type. Activation of NF-κB is essential to protect TNF-α–induced apoptosis,16 which appears to be a common mechanism in many cell types. Although how NF-κB protects against apoptosis is far from established, it is believed that a major mechanism by which the transcription factor inhibits cell death is to induce the expression of antiapoptotic genes whose products, in turn, provide protection to the cells under adverse conditions. A number of such protective genes that are induced by NF-κB have been identified, including inhibitors of apoptosis (IAPs), TNF-receptor–associated factor-1 and –2 (TRAF-1 and TRAF-2), Bcl-2–like factors and A20, a zinc-finger protein that was originally identified as a TNF-inducible gene in ECs.17 Although NF-κB protects ECs from TNF-α–induced apoptosis, this survival pathway seems to provide little protection against some other apoptotic stimuli such as lipopolysaccharide (LPS), interleukin-1β,18 and hypoxia, despite the fact that NF-κB is also activated in these scenarios.1819 In addition, certain endothelial survival factors such as Bcl-2, Bcl-XL,20 and A20, which although suppressing NF-κB, can override cellular apoptotic signaling and make NF-κB dispensable in EC protection.21 Because NF-κB is a key transcription factor governing a variety of proinflammatory genes including chemokines and adhesion molecules, an NF-κB–independent antiapoptotic pathway can protect endothelial integrity without converting the endothelium to a proinflammatory state. Such a mechanism is desirable for therapeutic intervention for many clinical conditions such as reperfusion injury and xenotransplantation.17 In the present study, Ref-1 rescues ECs from apoptosis via both an NF-κB–dependent and –independent mechanism, depending on whether it is triggered by TNF-α or by hypoxia.4 This result reinforces the concept that transcriptional mechanisms regulating EC responses are context-specific.AP-1 is also a transcription factor regulated by Ref-1 during redox change. AP-1 complexes are composed of various dimers between Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, Fra-1, and Fra-2).22 Mounting evidence has implied that activation of AP-1 is also associated with an apoptotic response in ECs. Many EC-perturbing agents, such as inflammatory cytokines, LPS, reactive oxygen, and oxidized LDL (oxLDL) induce EC apoptosis as well as c-jun expression.23 Sustained activation of c-Jun N-terminal kinase (JNK), an immediate c-Jun–activating molecule, induces EC apoptosis.24 More direct evidence pointing to a proapoptotic role for c-Jun originates from the observation that overexpression of c-Jun in ECs triggers marked apoptosis. However, an N-terminal truncate of c-Jun, missing the transactivation domain, not only loses its proapoptotic property but also provides protection against hydrogen peroxide–induced EC death.23 A recent report demonstrated the involvement of JNK–c-Jun pathway in oxLDL-induced apoptosis in human coronary artery ECs.25What appears to be more paradoxical is that Ref-1 is also known as a potent activator for the tumor suppressor p53, which, when activated in cells, can induce either cell cycle arrest or apoptosis. The p53 is activated in response to genotoxic stresses and is associated with hypoxia-induced EC death.26 Gaiddon et al9 recently showed that Ref-1 enhances the proapoptotic functions of p53 in a transformed cell line. It thus seems to be in apparent contradiction that in the present study ECs were prevented from undergoing apoptosis by overexpression of Ref-1, which, on the other hand, may activate p53. However, it should be pointed out that, in the study of Gaiddon et al, Ref-1 increased the ability of p53 to induce apoptosis only when exogenous p53 and Ref-1 were both overexpressed by cotransfection. Thus, it is unclear whether Ref-1 activation of endogenous p53 is to induce apoptosis or, alternatively, to arrest cell cycle. In fact, laminar shear stress, known to promote EC survival, can cause sustained activation of p53 and endothelial growth arrest.27 Given that Ref-1 possesses dual functions as transcriptional regulator and DNA repair enzyme, it is rational to speculate that these two domains of Ref-1, although they can function independently, may act in concert to protect cells from oxidative damage: one activates p53 to ensure efficient cell-cycle arrest for the other to fix the DNA damage. Nevertheless, precise interactions between Ref-1 and certain transcription factors as well as their functional readouts under specific endothelial conditions would be of considerable importance in understanding the transcriptional regulation of EC apoptosis. The need to understand this effect of Ref-1 is underscored by the increasing number of transcription factors that have been found to interact with Ref-1. On this expanding list are HIF-1α, HIF-like factor (HLF), activating transcription factor (ATF), cAMP response element–binding protein (CREB), the oncogene Myb, nuclear factor-Y (NF-Y), and early growth response-1 gene (Egr-1), Pax-5, and Pax-8.28 Although the consequences of activating these transcription factors remain poorly understood, it can be hypothesized that Ref-1 may play a pivotal role in integrating the transcriptional response and, thus, control EC fate under specific oxidative conditions. However, an important caveat must be considered regarding the pathogenetic importance of EC apoptosis. Most reports studying EC programmed cell death have examined the process in cell culture. Although a few studies have shown in situ detection of EC apoptosis in microvessels29 and transplant coronary artery disease,30 it is yet uncertain as to its importance in major circulatory disorders such as atherosclerosis. Until such in vivo studies are carried out, the role for EC apoptosis in vascular diseases remains speculative.Finally, the finding that Ref-1 increases EC survival under conditions of hypoxia and TNF-α stimulation has potential clinical relevance to vascular diseases. For example, upregulation of Ref-1 by either gene transfer or pharmacological agonists can be expected to promote angiogenesis that is therapeutically desirable for ischemic diseases and wound healing. On the other hand, antagonizing Ref-1 may exert an angiostatic effect and, in turn, inhibit tumor growth.The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.Download figureDownload PowerPoint Figure 1. Role of Ref-1 in endothelial apoptosis. Various pathophysiological conditions cause oxidative stress and intracellular redox change in ECs. Diverse signaling pathways activate transcription factors in both a cell type– and context-specific manner. Successive regulation of pro- or antiapoptotic gene expression controls EC apoptosis or survival. As a DNA repair protein (exhibiting 5′AP-endonuclease activity and 3′-phosphodiesterase) and a regulator of transcription (via redox-based activation of transcription factors (eg, AP-1, p53, and NF-κB), Ref-1 may play a pivotal role in modulating EC fate under oxidative stress.FootnotesCorrespondence to Michael B. Stemerman, MD, Division of Biomedical Sciences, University of California Riverside, Riverside, CA 92521. E-mail [email protected] References 1 Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, Pober JS, Wick TM, Konkle BA, Schwartz BS, Barnathan ES, McCrae KR, Hug BA, Schmidt AM, Stern DM. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood.1998; 91:3527–3561.MedlineGoogle Scholar2 Stefanec T. Endothelial apoptosis: could it have a role in the pathogenesis and treatment of disease? Chest.2000; 117:841–854.CrossrefMedlineGoogle Scholar3 Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res.2000; 87:434–439.CrossrefMedlineGoogle Scholar4 Hall JL, Wang X, Adamson V, Zhao Y, Gibbons GH. Overexpression of Ref-1 inhibits hypoxia and tumor necrosis factor–induced endothelial cell apoptosis through nuclear factor-κB–independent and –dependent pathways. Circ Res.2001; 88:1247–1253.CrossrefMedlineGoogle Scholar5 Demple B, Herman T, Chen DS. Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes. Proc Natl Acad Sci U S A.1991; 88:11450–11454.CrossrefMedlineGoogle Scholar6 Xanthoudakis S, Curran T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO J.1992; 11:653–665.CrossrefMedlineGoogle Scholar7 Xanthoudakis S, Miao G, Wang F, Pan YC, Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J.1992; 11:3323–3335.CrossrefMedlineGoogle Scholar8 Mitomo K, Nakayama K, Fujimoto K, Sun X, Seki S, Yamamoto K. Two different cellular redox systems regulate the DNA-binding activity of the p50 subunit of NF-κB in vitro. Gene.1994; 145:197–203.CrossrefMedlineGoogle Scholar9 Gaiddon C, Moorthy NC, Prives C. Ref-1 regulates the transactivation and pro-apoptotic functions of p53 in vivo. EMBO J.1999; 18:5609–5621.CrossrefMedlineGoogle Scholar10 Jayaraman L, Murthy KG, Zhu C, Curran T, Xanthoudakis S, Prives C. Identification of redox/repair protein Ref-1 as a potent activator of p53. Genes Dev.1997; 11:558–570.CrossrefMedlineGoogle Scholar11 Ema M, Hirota K, Mimura J, Abe H, Yodoi J, Sogawa K, Poellinger L, Fujii-Kuriyama Y. Molecular mechanisms of transcription activation by HLF and HIF1α in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. 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Science.1996; 274:782–784.CrossrefMedlineGoogle Scholar17 Bach FH, Hancock WW, Ferran C. Protective genes expressed in endothelial cells: a regulatory response to injury. Immunol Today.1997; 18:483–486.CrossrefMedlineGoogle Scholar18 Zen K, Karsan A, Stempien-Otero A, Yee E, Tupper J, Li X, Eunson T, Kay MA, Wilson CB, Winn RK, Harlan JM. NF-κB activation is required for human endothelial survival during exposure to tumor necrosis factor-α but not to interleukin-1β or lipopolysaccharide. J Biol Chem.1999; 274:28808–28815.CrossrefMedlineGoogle Scholar19 Stempien-Otero A, Karsan A, Cornejo CJ, Xiang H, Eunson T, Morrison RS, Kay M, Winn R, Harlan J. Mechanisms of hypoxia-induced endothelial cell death: role of p53 in apoptosis. J Biol Chem.1999; 274:8039–8045.CrossrefMedlineGoogle Scholar20 Badrichani AZ, Stroka DM, Bilbao G, Curiel DT, Bach FH, Ferran C. Bcl-2 and Bcl-XL serve an anti-inflammatory function in endothelial cells through inhibition of NF-κB. J Clin Invest.1999; 103:543–553.CrossrefMedlineGoogle Scholar21 Lee EG, Boone DL, Chai S, Libby SL, Chien M, Lodolce JP, Ma A. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science.2000; 289:2350–2354.CrossrefMedlineGoogle Scholar22 Karin M, Liu Z, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol.1997; 9:240–246.CrossrefMedlineGoogle Scholar23 Wang N, Verna L, Hardy S, Zhu Y, Ma KS, Birrer MJ, Stemerman MB. c-Jun triggers apoptosis in human vascular endothelial cells. Circ Res.1999; 85:387–393.CrossrefMedlineGoogle Scholar24 Hu YL, Li S, Shyy JY, Chien S. Sustained JNK activation induces endothelial apoptosis: studies with colchicine and shear stress. Am J Physiol.1999; 277:H1593–H1599.MedlineGoogle Scholar25 Napoli C, Quehenberger O, De Nigris F, Abete P, Glass CK, Palinski W. Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells. 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Ali I, Nanchal R, Husnain F, Audi S, Konduri G, Densmore J, Medhora M and Jacobs E (2013) Hypoxia Preconditioning Increases Survival and Decreases Expression of Toll‐Like Receptor 4 in Pulmonary Artery Endothelial Cells Exposed to Lipopolysaccharide, Pulmonary Circulation, 10.1086/674337, 3:3, (578-588), Online publication date: 1-Sep-2013. Hang X, Li P, Li Z, Qu W, Yu Y, Li H, Shen Z, Zheng H, Gao Y, Wu Y, Deng M, Sun Z and Zhang C (2009) Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array, BMC Genomics, 10.1186/1471-2164-10-126, 10:1, Online publication date: 1-Dec-2009. 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Kin H, Wang N, Halkos M, Kerendi F, Guyton R and Zhao Z (2006) Neutrophil Depletion Reduces Myocardial Apoptosis and Attenuates NFκB Activation/TNFα Release After Ischemia and Reperfusion, Journal of Surgical Research, 10.1016/j.jss.2006.02.019, 135:1, (170-178), Online publication date: 1-Sep-2006. Tell G, Damante G, Caldwell D and Kelley M (2005) The Intracellular Localization of APE1/Ref-1: More than a Passive Phenomenon?, Antioxidants & Redox Signaling, 10.1089/ars.2005.7.367, 7:3-4, (367-384), Online publication date: 1-Mar-2005. Wei Y and Au J Role of Tumour Microenvironment in Chemoresistance Integration/Interaction of Oncologic Growth, 10.1007/1-4020-3414-8_17, (285-321) Yu E, Li Y, Liu X, Kagan E and McCarron R (2004) Antiapoptotic action of hypoxia-inducible factor-1α in human endothelial cells, Laboratory Investigation, 10.1038/labinvest.3700071, 84:5, (553-561), Online publication date: 1-May-2004. Johnson N, Sengupta S, Saidi S, Lessan K, Charnock‐Jones S, Scott L, Stephens R, Freeman T, Tom B, Harris M, Denyer G, Sundaram M, Sasisekharan R, Smith S and Print C (2003) Endothelial cells preparing to die by apoptosis initiate a program of transcriptome and glycome regulation, The FASEB Journal, 10.1096/fj.03-0097fje, 18:1, (188-190), Online publication date: 1-Jan-2004. Zhao H, Miller M, Pfeiffer K, Buras J and Stahl G (2003) Anoxia and reoxygenation of human endothelial cells decreases ceramide glucosyltransferase expression and activates caspases, The FASEB Journal, 10.1096/fj.02-0806fje, 17:6, (723-724), Online publication date: 1-Apr-2003. June 22, 2001Vol 88, Issue 12 Advertisement Article InformationMetrics © 2001 American Heart Association, Inc.https://doi.org/10.1161/hh1201.093162 Originally publishedJune 22, 2001 Keywordsendotheliumapoptosisoxidative stresstranscription factorPDF download Advertisement

—To explore the role of LDL in caveolin-Ras regulation in human endothelial cells (ECs), we incubated confluent human umbilical vein endothelial cells (HUVECs) with LDL. This resulted in a high steady-state caveolin-1 (Cav-1) expression at both the mRNA and protein levels. LDL exposure appeared not to regulate the abundance of Cav-1. Immunofluorescence staining showed that Cav-1 protein migrated from the cytoplasm to the cell membrane after LDL exposure. Cav-1 protein and cholesterol partitioned mainly into the caveola fractions, and LDL increased both Cav-1 and cholesterol in these fractions. Ras protein in caveola fractions was also increased by LDL. Increased Ras was detected in Cav-1 immunoprecipitated samples, and conversely, increased Cav-1 was found in Ras-immunoprecipitated samples. We also demonstrated LDL-increased Ras activity in HUVECs by measuring the GTP/GTP+GDP ratio of Ras with [ 32 P]orthophosphate labeling in the cells. Finally, we determined the binding of [ 3 H]-labeled free cholesterol and recombinant H-Ras to Cav-1 fusion proteins in vitro. Both cholesterol and Ras bound to full-length GST–Cav-1, scaffolding domain (61–101), and C-terminal (135–178) Cav-1 fusion peptides. Addition of cholesterol enhanced Ras binding to the full-length and scaffolding domain of Cav-1 but not to the C-terminal Cav-1. These findings strongly suggest a role for Cav-1 in cholesterol trafficking and cholesterol-mediated intracellular signaling, which may mediate EC activation by LDL.

HomeCirculation ResearchVol. 86, No. 7Lipoprotein Effects on the Vessel Wall Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBLipoprotein Effects on the Vessel Wall Michael B. Stemerman Michael B. StemermanMichael B. Stemerman From the Department of Biomedical Sciences, University of California, Riverside, Calif. Originally published14 Apr 2000https://doi.org/10.1161/01.RES.86.7.715Circulation Research. 2000;86:715–716Accumulation of lipids within the arterial wall is a distinguishing characteristic of the atherosclerotic lesion and is seen in virtually all stages of plaque development. This process is directly correlated with the serum level of some lipids, especially cholesterol, LDL, and other lipoprotein particles. In contrast, an increased serum level of HDL is protective against plaque formation. Evidence has steadily accumulated showing that HDL acts as a "sink" for cholesterol, presumably removing it from tissue. In any discussion of trafficking of materials between blood and the arterial wall, the endothelium must be considered pivotal because of its critical location at the junction between blood and blood vessel. Penetration of lipoproteins into the arterial wall has been shown both quantitatively and qualitatively in experimental animal models. Areas of lipid accumulation appear at localized sites along the arterial lumen in these animal models, consistent with the focal nature of plaque development in humans. The mechanism for the predilection of some localized areas to accumulate plaque has been under debate for a considerable time and is likely multifactorial. In some instances, this process seems related to a focal transient loosing of the tight association between adjoining endothelial cells (ECs). In these areas, there may be easy access to underlying vascular tissue. However, because such gaps appear only occasionally, it is likely that some ECs are actively involved in the accumulation of lipids.1 These topics are at the center of the study by Rutledge et al2 described in this issue of Circulation Research.In the present study, VLDL was fluorescently labeled and perfused into rat arteries. Attachment of VLDL at the arterial surface was modest, but with the addition of lipoprotein lipase and subsequent VLDL lipolysis, there was accumulation of lipids in the arterial wall in focal areas, or "lakes." After addition of HDL, the lipid-lake accumulation was ameliorated. It seems that HDL acts by both interfering with endothelial permeability and increasing lipid removal rate from the arterial wall. The implication of the former observation is that the permeability of endothelium can be modified by its milieu, and of the latter is that HDL is actively involved in the removal of lipids from tissue. The study provides ex vivo evidence of the ability of HDL to remove lipids from a cellular deposition site and confirms in vitro reports gathered from cell culture studies. The study is particularly important because the site modified by HDL is the arterial wall.The hypothesis that atherosclerotic progression can be reduced by either improving the barrier function of the arterial wall or modifying blood lipoprotein concentration is the basis of a great deal of current preventative research. Risk factors for atherosclerosis, such as hypertension, smoking, mechanical injury, dense LDL, and genetic predisposition, may affect the barrier function of the arterial wall, predisposing to atherosclerosis. The study by Rutledge et al2 focuses on the modifications of VLDL by lipoprotein lipase and HDL and their influence on the arterial wall and endothelial function.The concept that alteration in EC function may play a pivotal role in the pathobiology of atherogenesis has been the subject of numerous investigations. Consequently, many of the responses of these cells to stimuli, both physiological and nonphysiological, have begun to provide a mechanistic motif for understanding intracellular EC reaction pathways. With the current hypothesis of atherosclerotic plaque formation centering on the roles of circulating lipoproteins, an understanding of how these lipoproteins affect EC function has begun to emerge. Such studies may not only convey insight into the pathogenesis of plaque formation but also provide important therapeutic implications.Triglyceride-rich lipoproteins, including VLDL, chylomicrons, and their remnants, are acknowledged as cardiovascular disease risk factors, and studies have implicated VLDL pathogenically in atherosclerosis. For example, triglyceride-rich remnants have been shown to impair vasorelaxation,3 and plasma triglyceride levels show a direct correlation with plasminogen activator inhibitor (PAI-1) levels. Incubation of ECs with VLDL induces the synthesis of PAI-1. Because elevated PAI-1 levels may interfere with fibrinolysis and therefore predispose to excessive thrombosis, high-serum VLDL levels may play a role in the development of thrombotic disorders and cardiovascular disease. Hence, investigations have been carried out to understand the mechanism of this effect. Studies using HepG2 cells have investigated the intracellular signaling pathway induced by VLDL exposure.4 VLDL seems to induce protein kinase C activity, resulting in activation of mitogen-activated protein kinase. Studies carried out in ECs indicate that VLDL can also induce nuclear factor-κB (NF-κB),5 a transcription factor that has been shown to play an important role in the phenotypic modulation of ECs to a proinflammatory condition. When rats were infused with VLDL, there ensued an increase in expression of RelA, a component of NF-κB, and an induction of cell adhesion molecules in the arterial endothelium. Thus, evidence is accumulating that VLDL directly affects the endothelium, which may help explain why VLDL is proatherogenic.HDL seems to protect against plaque formation.6 This observation has been documented in both experimental animal studies and human epidemiological investigations. Animal studies describe protection against the development of lipid deposits when HDL is abundant in the plasma. Rutledge et al2 provide an additional demonstration of the effectiveness of HDL in protecting against plaque formation by showing that HDL can remove lipid buildup from the vessel. Cell culture studies have examined the salutary effect of this lipoprotein on the endothelium. To understand the potential cellular effect of HDL as a vascular protective agent, ECs were incubated with HDL during exposure to a potent inducer of EC dysfunction, tumor necrosis factor-α (TNF-α).7 HDL inhibited TNF-α–induced expression of adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular cell adhesion molecule-1 (ICAM-1). TNF-α is a well-recognized activator of ECs and seems to have its primary effect in upregulating adhesion molecule expression by induction of NF-κB. However, the ability of HDL to moderate the TNF-α effect on ECs may be caused by enhancement of Cox-2 expression. By inducing this enzyme, ECs exposed to HDL produce an abundance of prostacyclin, which is recognized for its ability to inhibit leukocyte function. Thus, HDL can modulate a potent activator of ECs and TNF-α; and interestingly, the mechanism seems to be independent of the transcriptional regulation of NF-κB.LDL has been shown to have a strong correlation with atherosclerotic vascular disease. How LDL is handled by the blood vessel is a major concern for understanding arterial wall plaque development. Elevated LDL levels are associated with a lack of vasorelaxation.8 With a rapid decrease in the serum level of LDL using apheresis, vascular reactivity in humans can be restored rapidly.9 The endothelial response to LDL has been examined using cell culture. The EC phenotype can be modulated by LDL, especially when the concentrations of LDL in the culture medium are similar to those identified with the development of atherosclerosis.LDL has numerous effects on the endothelium, including effects on PAI-1,10 arachidonate metabolism,11 and induction of adhesion molecule expression. Of particular interest to the inflammatory component of plaque development is the upregulation of both ICAM-112 and VCAM-1.13 In examining the mechanism underlying this modulation, it has been determined that exposure of ECs to LDL causes the rapid activation of Ras.14 Ras activation leads to induction of the signaling cascade that activates JNK and the expression of AP-1, which in turn can upregulate ICAM-1. In contrast to VLDL-induced signaling, NF-κB seems to have little, if any, role in the induction of adhesion molecule formation by LDL. Indeed, LDL is the only physiological substance known to behave in this fashion. Recent studies have pursued this finding and indicate that most of this activation resides in the free-cholesterol component of LDL.15 If this is the case, it can be hypothesized that cholesterol is a biologically active molecule carried by LDL, which initiates cellular activation. One of the implications of this notion is the likelihood not only that LDL and serum cholesterol are risk factors for developing atherosclerosis, but also that lipids can act directly as atherogenic factors. Hence, our ability to comprehensively understand the varying roles of lipoproteins as they affect the arterial vasculature has major ramifications for understanding and controlling atherosclerosis.The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.FootnotesCorrespondence to Michael B. Stemerman, Division of Biomedical Sciences, B605 Statistics Road, University of California, Riverside, CA 92521-0121. E-mail [email protected] References 1 Nielsen LB. Transfer of low density lipoprotein into the arterial wall and risk of atherosclerosis. Atherosclerosis.1996; 123:1–15.CrossrefMedlineGoogle Scholar2 Rutledge JC, Mullick AE, Gardner G, Goldberg IJ. Direct visualization of lipid deposition and reverse lipid transport in a perfused artery: roles of VLDL and HDL. Circ Res.2000; 86:768–773.CrossrefMedlineGoogle Scholar3 Doi H, Kugiyama K, Ohgushi M, Sugiyama S, Matsumura T, Ohta Y, Nakano T, Nakajima K, Yasue H. Remnants of chylomicron and very low density lipoprotein impair endothelium-dependent vasorelaxation. Atherosclerosis.1998; 137:341–349.CrossrefMedlineGoogle Scholar4 Banfi C, Mussoni L, Ris P, Cattaneo MG, Vicentini L, Battaini F, Galli C, Tremoli E. Very low density lipoprotein-mediated signal transduction and plasminogen activator inhibitor type 1 in cultured HepG2 cells. Circ Res.1999; 85:208–217.CrossrefMedlineGoogle Scholar5 Dichtl W, Nilsson L, Goncalves I, Ares MP, Banfi C, Calara F, Hamsten A, Eriksson P, Nilsson J. Very low-density lipoprotein activates nuclear factor-κB in endothelial cells. Circ Res.1999; 84:1085–1094.CrossrefMedlineGoogle Scholar6 Tall AR. An overview of reverse cholesterol transport. Eur Heart J. 1998;19(suppl A):A31–A35.Google Scholar7 Cockerill GW, Saklatvala J, Ridley SH, Yarwood H, Miller NE, Oral B, Nithyanathan S, Taylor G, Haskard DO. High-density lipoproteins differentially modulate cytokine-induced expression of E-selectin and cyclooxygenase-2. Arterioscler Thromb Vasc Biol.1999; 19:910–917.CrossrefMedlineGoogle Scholar8 Howes LG, Abbott D, Straznicky NE. Lipoproteins and cardiovascular reactivity. Br J Clin Pharmacol.1997; 44:319–324.CrossrefMedlineGoogle Scholar9 Mellwig KP, Baller D, Gleichmann U, Moll D, Betker S, Weise R, Notohamiprodjo G. Improvement of coronary vasodilatation capacity through single LDL apheresis. Atherosclerosis.1998; 139:173–178.CrossrefMedlineGoogle Scholar10 Levin EG, Miles LA, Fless GM, Scanu AM, Baynham P, Curtiss LK, Plow EF. Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial cells. Arterioscler Thromb.1994; 14:438–442.CrossrefMedlineGoogle Scholar11 Pritchard KA Jr, Wong PY, Stemerman MB. Atherogenic concentrations of low-density lipoprotein enhance endothelial cell generation of epoxyeicosatrienoic acid products. Am J Pathol.1990; 136:1383–1391.MedlineGoogle Scholar12 Smalley DM, Lin JH, Curtis ML, Kobari Y, Stemerman MB, Pritchard KAJ. Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol.1996; 16:585–590.CrossrefMedlineGoogle Scholar13 Lin JH, Zhu Y, Liao HL, Kobari Y, Groszek L, Stemerman MB. Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. Atherosclerosis.1996; 127:185–194.CrossrefMedlineGoogle Scholar14 Zhu Y, Liao H, Wang N, Verna L, Ma K-S, Zhang S-X, Liao JK, Stemerman MB. Low-density lipoprotein activates Ras-dependent signaling pathway in human endothelial cells. Circulation. 1999;100(suppl I):I-693. Abstract.Google Scholar15 Yuan Y, Wang N, Zhu Y, Verna L, Stemerman MB. Cholesterol causes human vascular endothelial cell (EC) activation. FASEB J..2000; 14:A414. Abstract 306.28.Google Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetailsCited By Shen W, Lu F, Yang Y, Wu J, Chang Y, Huang Y and Chang C (2017) The relationship between high-density lipoprotein cholesterol levels and arterial stiffness in a Taiwanese population, Nutrition, Metabolism and Cardiovascular Diseases, 10.1016/j.numecd.2017.10.003, 27:12, (1136-1142), Online publication date: 1-Dec-2017. Lee H, Paudel K, Jeong J, Wi A, Park W, Kim D and Oak M (2016) Antiatherogenic Effect of Camellia japonica Fruit Extract in High Fat Diet-Fed Rats , Evidence-Based Complementary and Alternative Medicine, 10.1155/2016/9679867, 2016, (1-8), . Gutsche I, Coulibaly F, Voss J, Salmon J, d'Alayer J, Ermonval M, Larquet E, Charneau P, Krey T, Mégret F, Guittet E, Rey F and Flamand M (2011) Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein, Proceedings of the National Academy of Sciences, 10.1073/pnas.1017338108, 108:19, (8003-8008), Online publication date: 10-May-2011. Goldberg R (2006) Type 2 Diabetes Comprehensive Management of High Risk Cardiovascular Patients, 10.3109/9781420019667.006, (187-254), Online publication date: 1-Oct-2006. Beklen A, Laine M, Ventä I, Hyrkäs T and Konttinen Y (2016) Role of TNF-α and Its Receptors in Pericoronitis, Journal of Dental Research, 10.1177/154405910508401216, 84:12, (1178-1182), Online publication date: 1-Dec-2005. Giacconi R, Caruso C, Lio D, Muti E, Cipriano C, Saba V, Boccoli G, Gasparini N, Malavolta M and Mocchegiani E (2005) 1267 HSP70-2 polymorphism as a risk factor for carotid plaque rupture and cerebral ischaemia in old type 2 diabetes-atherosclerotic patients, Mechanisms of Ageing and Development, 10.1016/j.mad.2005.03.007, 126:8, (866-873), Online publication date: 1-Aug-2005. Ziouzenkova O and Plutzky J (2004) Lipolytic PPAR activation: new insights into the intersection of triglycerides and inflammation?, Current Opinion in Clinical Nutrition & Metabolic Care, 10.1097/01.mco.0000134358.46159.61, 7:4, (369-375), Online publication date: 1-Jul-2004. Hannuksela M, Liisanantti M and Savolainen M (2008) Effect of Alcohol on Lipids and Lipoproteins in Relation to Atherosclerosis, Critical Reviews in Clinical Laboratory Sciences, 10.1080/10408360290795529, 39:3, (225-283), Online publication date: 1-Jan-2002. Napoli C and Lerman L (2001) Involvement of Oxidation-Sensitive Mechanisms in the Cardiovascular Effects of Hypercholesterolemia, Mayo Clinic Proceedings, 10.4065/76.6.619, 76:6, (619-631), Online publication date: 1-Jun-2001. Burnett J and Watts G (2001) Therapeutic considerations for postprandial dyslipidaemia, Diabetes, Obesity and Metabolism, 10.1046/j.1463-1326.2001.00155.x, 3:3, (143-156), Online publication date: 1-Jun-2001. Napoli C and Lerman L (2001) Involvement of Oxidation-Sensitive Mechanisms in the Cardiovascular Effects of Hypercholesterolemia, Mayo Clinic Proceedings, 10.1016/S0025-6196(11)62413-0, 76:6, (619-631), Online publication date: 1-Jun-2001. April 14, 2000Vol 86, Issue 7 Advertisement Article InformationMetrics © 2000 American Heart Association, Inc.https://doi.org/10.1161/01.RES.86.7.715 Originally publishedApril 14, 2000 KeywordslipoproteinsVLDLLDLHDLendotheliumPDF download Advertisement

P75 Inhibitors of PDE 3 and/or PDE4 block smooth muscle cell (SMC) proliferation/migration in vitro and reduce restenosis. To evaluate chronic regulation of SMC PDEs, rats were subjected to BAL. At several times after BAL (30 min to 10days/10D), medial SMC were isolated from the thoracic aortae. Tissues were fractionated for total RNA (analyzed by Northern blots/ RNAase protection) or cytosolic protein (Western blots or low Km [100 nM] cAMP PDE activity). BAL-dependent increases in PDE4B mRNA were biphasic: 2.7-fold at 1hr, which declined by 24hr below control values, followed by 2-3-fold increases by 4-7D (p<0.05 vs controls, n=3-6). PDE4B and c-myc mRNAs were selectively superinduced in cycloheximide-treated rats. Despite modest changes in PDE3 mRNAs, both 80 and 120 kDa PDE3A proteins were detected and only the 80kDa increased 7D post-BAL (10-fold increase, p<0.05, n=6). PDE4B2 (80kDa) persistently increased 2.5-3-fold (p<0.05, n=4-6) from 24 hr to 10D post-BAL, while 2-fold increases in PDE4D3 (90kDa) and PDE4B1 (104kDa) were evident at 24hr or 7D, respectively. PDE activity increased 50-60% (p<0.05; n=4) at 24 hr and 7D after BAL. Thus PDE4B resembles an immediate-early gene. Selective induction of the PDE4 family is associated with 2 waves of SMC proliferation in vivo, the latter of which is also accompanied by induction of PDE3A/80kDa. Upregulation of specific SMC PDEs following angioplasty represents an important response to injury that may be a useful therapeutic target in vascular disease.

The antioxidant agent pyrrolidine dithiocarbamate (PDTC) has been shown to protect endothelial cells (EC) from pro-inflammatory-induced and pro-oxidant-induced NF-kappaB activation. It also perturbs EC by altering activator protein-1 (AP-1) status and inducing ICAM-1. Experiments were performed to investigate the upstream mechanism by which PDTC produces these effects. We have demonstrated that PDTC not only induced AP-1 binding and ICAM-1 expression by itself, but it also augmented AP-1 activation and ICAM-1 induction in low-dose IL-1alpha treated cells. To dissect the mechanism of these effects, we measured c-Jun and c-Fos expression, and the activity of c-Jun NH2-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) in human umbilical vein endothelial cells (HUVEC). We detected an increase in JNK activity in PDTC-treated HUVEC. Following cotransfection with JNK[K-M], a kinase-deficient JNK1, the PDTC-increased AP-1-driven-luciferase activity was attenuated. Utilizing a specific trans-reporting system we confirmed c-Jun activation by upstream signaling mechanisms. The results show that c-Jun activity was increased 9-fold after PDTC treatment. In addition, PDTC promoted more transient activation in ERK-c-fos. In contrast, PDTC produced sustained JNK-c-Jun activation, which translated into long-lasting ICAM-1 production. These results suggest that an antioxidant may contribute to chronic vascular endothelial activation.

We have reported previously that native low-density lipoprotein (LDL) activates c-Jun and transcription factor AP-1 in human umbilical vein endothelial cells (HUVEC). The aim of this study was to elucidate the upstream signaling mechanisms mediating LDL activation of c-Jun/AP-1. Using a c-Jun NH2-terminal kinase (JNK) activity assay, we have detected an increase in JNK activity in LDL-exposed HUVEC, which started at 15 min and reached maximum activity after 1–2 h. This JNK activity, increased by LDL, occurred in a dose-dependent fashion starting at a concentration of 80 mg/dl of LDL and reaching maximum activation at a concentration of 160–240 mg/dl. Following cotransfection, the increase of AP-1-driven luciferase activity by LDL was attenuated 54% by a kinase-deficient JNK1. Furthermore, a specific trans-reporting system was utilized to confirm c-Jun activation by upstream signal mechanisms. The results show c-Jun activity increased by 3-fold after LDL exposure when compared with respective controls. In contrast, LDL exposure did not affect the activation of extracellular signal regulated kinase 1 and 2 (ERK1/2), even though phorbol 12-myristate 13-acetate treatment remarkably increased the activity of these kinases. Thus, this study demonstrates, for the first time, that JNK mediates LDL-induced endothelial cell activation.

In this study, the effect of low density lipoproteins (LDL) on the ability of the vascular endothelium to respond to vascular cell adhesion molecule 1 (VCAM-1) activation by a cytokine was investigated. After a 4-day pre-exposure to 240 mg/dl of LDL, human umbilical vein endothelial cells (HUVECs) were hyperresponsive to minute amounts of interleukin 1 alpha (IL-1 alpha) as demonstrated by an augmentation of VCAM-1 gene expression. Furthermore, in response to LDL exposure, endothelial recruitment of monocytes induced by minute amounts of IL-1 alpha was increased. This enhancing effect was blocked by an anti-VCAM antibody. The increased response appears not to be due to changes in IL-1 binding affinity or induction of endogenous IL-1 alpha. Transient transfection of HUVECs with a reporter driven by the VCAM promoter showed that LDL increased cellular response to IL-1 alpha by 46%. LDL itself does not increase NF-kappa B binding in endothelial cells (ECs). However, after a 2-day LDL incubation, NF-kappa B binding could be induced by over 63% with a very low dose of IL-1 alpha. IL-1 alpha at this dose (which activates NF-kappa B, but not AP-1) also enhanced LDL-activated AP-1 binding. This cross-enhanced effect may be an important intracellular signaling mechanism for EC activation. The results from this study provide new clues to understanding the mechanisms governing combined risk factors for atherosclerosis.

As distal targets and mediators of signal transduction pathways, activator protein-1 (AP-1), c-Jun, and c-Fos are among the primary regulators of genes involved in cell function, proliferation, and differentiation. By using adenovirus-mediated gene transfer, we show that overexpression of AP-1 proteins directly causes coinduction of gene expression of an adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and a chemokine, monocyte chemoattractant protein-1 (MCP-1), in human vascular endothelial cells (ECs). The AP-1-induced gene expression occurs through a mechanism independent of nuclear factor-kappaB. Because the induced expression of ICAM-1 and MCP-1 in ECs has been implicated in endothelial activation and a number of important vascular disorders, it is suggested that AP-1 activation may play an important role in the pathogeneses of inflammation, angiogenesis, and atherogenesis.