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CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-κB (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16–CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis.

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.

We investigated the effect of shear stress on the sterol regulatory element–binding protein 1 (SREBP1) in vascular endothelial cells (ECs) and the mechanotransduction mechanism involved. Application of a shear stress (12 dyn/cm2) caused the proteolytic cleavage of SREBP1 and the ensuing translocation of its transcription factor domain into the nucleus. As a result, shear stress increased the mRNAs encoding the low density lipoprotein receptor (LDLR), as well as the binding of125I-LDL. Using a step flow channel, we showed that SREBP1 activation in ECs under laminar flow is transient, but disturbed flow causes sustained activation. In studying the shear stress–elicited molecular signaling that activates SREBP1, we found that blocking the β1-integrin with the AIIB2 blocking-type monoclonal antibody inhibited SREBP1 activation induced by shear stress. EC attachment to fibronectin or the activation of β1-integrin in the suspended ECs by the TS2/16 monoclonal antibody was sufficient for SREBP1 activation. Furthermore, transient transfection assays showed that dominant-negative mutants of focal adhesion kinase and c-Src attenuated the shear stress–increased LDLR promoter activity. These results demonstrate that integrin signaling plays a critical role in the modulation of SREBP in ECs in response to shear stress.

Abstract —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.

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.

Abstract —In endothelial cells (ECs), the transcription factor c-Jun is induced by a variety of stimuli that perturb EC function. To extend our understanding of the role of c-Jun in EC physiology, we have directed overexpression of c-Jun in human umbilical vein ECs by using a tetracycline-regulated adenoviral expression system. In this study, we report a novel observation using this system. Specific expression of c-Jun is a sufficient trigger for ECs to undergo apoptosis, as demonstrated by a set of combined assays including an ELISA specific for histone-associated DNA fragmentation, DNA laddering, and TdT-mediated dUTP nick end labeling (TUNEL). Tetracycline can effectively shut off c-Jun overexpression and prevent EC apoptosis. Cleavage of poly(ADP-ribose) polymerase was also detected in ECs overexpressing c-Jun. Moreover, inhibitors of cysteine proteases blocked the apoptosis, suggesting a caspase-associated mechanism involved in proapoptotic effects of c-Jun. To gain further insight into the role of c-Jun as a pathophysiological regulator of EC death, TAM67, a dominant-negative mutant of c-Jun, was overexpressed in human umbilical vein ECs to abrogate endogenous c-Jun/activator protein-1 activation. H 2 O 2 -triggered apoptosis was largely attenuated in ECs overexpressing TAM67. Together, these results suggest that c-Jun, as a proapoptotic molecule, may play a role in mediating the cell death program in vascular endothelium.

Abstract— 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-κB. 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.

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.

Lysophosphatidylcholine (lyso-PC) selectively upregulates the mRNA level of intercellular adhesion molecule-1 (ICAM-1) but not that of vascular cell adhesion molecule-1 (VCAM-1) in cultured human umbilical vein endothelial cells. Transfection studies show that lyso-PC activates the ICAM-1 promoter but not the VCAM-1 promoter. Gel mobility shift assays document an increase in NF-kappa B binding in cells treated with lyso-PC. The increases of ICAM-1 mRNA and NF-kappa B binding were inhibited by the protein tyrosine kinase inhibitors, genistein and lavendustin A, but not by inhibitors for cyclic AMP-dependent protein kinases or protein kinase C. Our results suggest that lyso-PC induces ICAM-1 expression most likely by activating NF-kappa B, and that the effect appears to be protein tyrosine kinase-dependent.

Oxytocin (OT) is a potent uterine agonist. Its receptor (OTR) is a G protein-coupled receptor that is downregulated by prolonged exposure to OT. We hypothesized that activation of PKC mediated this OT-induced decrease in OTR expression. Diminished PKC activity in late pregnancy could underlie the increased expression of uterine OTR preceding labor onset. Using cell cultures of transformed human uterine myocytes, we determined the effects of PKC agonists and antagonists on the expression of OTR. We also explored the effects of overexpression of activator protein-1 (AP-1, a mediator of many PKC- and phorbol ester-induced effects) using adenoviral expression vectors for the AP-1 subunits c-Jun and c-Fos. Stimulation of PKC using the phorbol ester 12- O-tetradecanoylphorbol 13-acetate caused a rapid, significant (P ≤ 0.05) increase in c-Jun and c-Fos concentrations but a significant decrease in mRNA for OTR within 6 h followed by a significant decrease in OT binding by 24 h. Adenoviral infection of the cells with expression vectors for c-Jun and c-Fos increased the AP-1 subunits but had no effect on OTR expression. Furthermore, there were no changes in c-Fos or c-Jun levels in human intrauterine tissues around the time of labor onset, as measured by Western analyses. We conclude that phorbol ester treatment decreases OTR expression, likely through a mechanism that does not involve AP-1.

Objective— We tested the hypothesis that direct native low-density lipoprotein (LDL) injection into LDL receptor–deficient (LDLR −/− ) mice would induce the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in their aortic endothelial cells, and that transcriptional regulation of this pathway involved activator protein-1 (AP-1) but not nuclear factor κB (NF-κB). Methods and Results— Using tail vein injection of LDL into LDLR −/− mice, we were able to maintain atherogenic LDL blood levels, which induced ICAM-1 and VCAM-1 expression in their aortic endothelial cells after 24 hours. We were able to visualize and quantify this expression using immunohistochemistry and confocal microscopy. Under conditions in which ICAM-1 and VCAM-1 were expressed, the regulatory AP-1 proteins c-Fos and c-Jun were also highly expressed in the endothelial cell cytoplasm and observed within the cell nucleus. The NF-κB protein P65, although expressed in the endothelial cell cytoplasm after LDL injection, was not observed within the cell nucleus. Conclusions— Elevated LDL blood levels, maintained in vivo, increased the expression of the adhesion molecules ICAM-1 and VCAM-1 in aortic endothelial cells. This effect appeared to correlate with AP-1 but not NF-κB.

Objective: ATP-binding cassette transporter-1 (ABCA1) mediates the active efflux of cholesterol and phospholipids, playing an important role in cholesterol homeostasis and atherogenesis. Oxidized low density lipoprotein (oxLDL) is an atherogenic molecule associated with the vascular endothelial dysfunction and development of atherosclerotic plaque. This report describes the effect of copper-catalyzed oxLDL on the regulation of ABCA1 in human endothelial cells (ECs). Methods and results: oxLDL downregulated ABCA1 at both mRNA and protein levels in a dose-dependent manner. This inhibitory effect of oxLDL was observed with both minimally and extensively oxLDL. Transfection of the ABCA1 promoter luciferase revealed oxLDL to substantially decrease ABCA1 promoter activity at basal conditions and after stimulation by overexpressing the liver X receptor LXRa and retinoid X receptor RXRa. oxLDL also attenuated LXR activation by blocking LXR ligand binding and interfering with the generation of 27hydroxycholesterol, an LXR endogenous ligand. Furthermore, oxLDL inhibited exogenous cholesterol- and oxysterol-induced endothelial ABCA1 induction. Conclusion: oxLDL downregulated ABCA1 by inhibiting LXR activation in endothelial cells. Such an effect may contribute to endothelial dysfunction and plaque formation.

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.

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-kappaB (NF-kappaB) activity. Results of trans-reporting assay revealed 2.5-fold increased expression of the AP-1-dependent reporter gene after cholesterol exposure whereas NF-kappaB-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.

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 criticalciselement for LDL induction. These results, for the first time, provide evidence suggesting that AP-1 is a major regulatory mechanism leading to endothelial activation.

Abstract —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.

Low-density lipoprotein (LDL) is a well-established risk factor for atherosclerosis. When endothelial cells are incubated with this lipoprotein in pathophysiologic amounts, the cells are activated. Among the documented cellular responses to LDL is increased recruitment of monocytes, which are believed to play a major role in promoting intimal plaque formation. The findings presented here link an atheogenic lipoprotein, LDL, with the induction of an adhesion molecule important in atherogenesis Human LDL induces the vascular cell adhesion molecule-1 (VCAM-1) transcriptionally with an increase in mRNA levels through activation of the VCAM promoter. This effect is blocked by anti-VCAM antibodies. After a 2-day incubation in LDL, the binding of NF-kappa B, which is believed to be a key oxidative-stress sensor for VCAM regulation, remains at basal level. In contrast, the binding activities of AP-1 and GATA, on the other hand, are increased by LDL. Thus, a component of LDL-enhanced endothelial recruitment of monocytes is attributed to VCAM-1 expression, which appears to be mediated through AP-1 and GATA. These data identify LDL as a VCAM-inducer possibly distinct from cytokines and endotoxin.

Abstract To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O 2 − ) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O 2 − by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [ 3 H]citrulline assay. N ω -Nitro- l -arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O 2 − by >300% but decreases LDL-treated EC (LDL-EC) release by >95%. l -Arginine inhibits the release of O 2 − from LDL-ECs by >95% but did not effect C-EC release of O 2 − . Indomethacin and SKF 525A partially attenuate LDL-induced increases in O 2 − production by ≈50% and 30%, respectively. Thus, n-LDL increases O 2 − and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO − ), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO − , and tyrosine by ≈50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs. These data indicate that n-LDL directly perturbs endothelial oxidative metabolism and uncouples l -arginine metabolism from NO release to increase eNOS generation of O 2 − . Such changes may represent one of the earliest EC perturbations in atherogenesis.

Accumulation 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 …

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

Transmural concentration profiles of 125I-albumin in vivo were measured across the normal and balloon catheter-deendothelialized rabbit descending thoracic aorta as a function of time following intravenous injection. A tracer was injected 5 or 60 minutes after deendothelialization, and the animals were sacrificed after circulation times of 10, 30 or 60 minutes. The aorta was immediately excised and frozen flat between glass slides. Samples were serially sectioned parallel to the intimal surface in a refrigerated microtome, washed with trichloroacetic acid (TCA), and counted. Relative tissue concentration profiles of TCA-precipitable radioactivity from the media of control animals showed entry from both luminal and adventitial sides, as previously found with conscious normal rabbits, but spatial gradients at both luminal and medial-adventitial borders were less steep. Relative concentration levels in ballooned animals were 10- to 40-fold higher than in controls, and the profiles were flatter. Uptake rates at equivalent circulation times were greater in experiments initiated 60 minutes, as compared with 5 minutes, after deendothelialization, suggesting that progressive medial edema may have occurred following balloon injury. These results show that the intact endothelium is the dominant mass transfer resistance for 125I-albumin transport across the aortic wall. The data also suggest that the incomplete monolayer of platelets adherent to the subendothelium after balloon deendothelialization is not a substantial resistance to transport, as compared to that of the media, and that convection plays a more important role than diffusion for 125I-albumin transport across the deendothelialized aortic wall.

The basement membrane of the human umbilical vein was studied by electron microscopy with respect to its ultrastructure, susceptibility to digestion by collagenase or trypsin, and reactivity with human platelets. Electron microscopic examination of this vessel showed a continuous reticulated basement membrane which morphologically resembled those of mammalian capillaries and rabbit heart valves. The vascular endothelium was removed by freezing and thawing, thus uncovering the underlying connective tissue. The vessels were sliced into rings which were incubated with collagenase or trypsin. The basement lamella appeared to be susceptible to digestion by either enzyme. Platelet interaction with exposed vascular basement membrane was studied by rotating frozen-thawed everted and non-everted rings in anticoagulated whole human blood. In heparinized or citrated blood, large aggregates of degranulated platelets adhered to collagenous controls; in contrast, the test rings with exposed basement membrane were partially covered with a monolayer of platelets which appeared to retain discoid or spherical shape and granules. In EDTA-anticoagulated blood, the collagen control rings accumulated a platelet monolayer, whereas little or no adhesion occurred on the basement membrane surface. In this system the basement membrane of the human umbilical vein appears to be a poor platelet reactive surface as compared to collagen.

Biochemical and morphological evidence is presented to support the characterization of a rat bone-marrow-derived cell line (RPM) as an analog of the promegakaryoblast. The conditions for in vitro growth and maturation of the RPM line are described. Rapid proliferation of the RPM line is readily achieved when cultures are supplemented with moderate levels of fetal bovine serum (FBS). The proliferative compartment is a small blast-like cell. Immunofluorescent staining demonstrates that the RPM cells contain factor VIII:antigen and fibrinogen in their cytoplasm. The cells secrete, into their conditioned medium, a potent mitogenic activity for rat aortic smooth muscle cells. When incubated under conditions of relative serum deprivation, the cells stop proliferating and undergo a process of maturation. The sequence of maturation is described as stage I (promegakaryoblast--the proliferative compartment); stage II (immature megakaryocyte or promegakaryocyte); stage III (mature megakaryocyte). The stage III cells release, from their cytoplasm, small membrane-bound vesicular bodies containing lavender granules and cytoplasmic organelles. These have been designated stage IV. The RPM line may provide a useful model for the in vitro study of megakaryocyte maturation and the synthesis of megakaryocyte-specific proteins.

Age is a risk factor in the development of atherosclerosis. In this study we investigated the hypothesis that proliferation of vascular smooth muscle cells (SMCs), an integral part of atherosclerotic plaque formation, changes with age. SMC growth kinetics of old rats (21-24 months) were compared to those of young adult rats (3-4 months). Rat aortas were denuded of their endothelium and the animals were killed after [3H]thymidine and Evans blue injections at 0-28 days after denudation. Incorporation of [3H]thymidine into SMC peaked in the young animals by day 2, whereas the older animals responded to endothelial removal with greater incorporation at day 2 and a more sustained rate of incorporation peaking at day 4. The [3H]thymidine incorporation curves decreased sharply from their peaks at 2 and 4 days, respectively, and paralleled each other after day 7. [3H]Thymidine uptake reflected the subsequent SMC intimal growth as measured morphometrically, with old animals showing greater numbers of intimal SMC than did the younger animals. The difference in response of SMC to injury with age suggests that aging produces a change in the vascular SMC that enhances proliferation. This change in response implies that the more pronounced atherosclerotic plaque growth seen with aging may be a result of an age-related increase in response to injury rather than merely the accumulation of time-related intimal change.

Electron microscopic studies were performed on rabbit heart valves, and all four valves were found to be lined with a continuous basement membrane (BM). The valvular BM was liable to digestion by either trypsin or collagenase, but brief exposure to trypsin was suitable for desquamating endothelial cells, while leaving BM exposed and intact. Such preparations were used to determine their platelet reactivity with heparinized and citrated whole blood or with platelet-rich plasma. Although various conditions of exposure were used, including shaking and centrifugation, little or no platelet adhesion to the BM was observed. With similar conditions of exposure, preparations of collagen showed massive platelet adhesion accompanied by aggregation. This vascular BM was similarly nonreactive to platelets when endothelial cell removal was accomplished by mechanical methods. Rabbit valvular BM appears to be a poorly thrombogenic surface.

Arteriosclerotic lesions have been produced in monkeys (Macaca nemestrina) by selective removal of the vascular endothelium with an intra-arterial balloon catheter. Immediately after de-endothelialization a platelet layer covers the denuded area. This thrombus is gradually removed and by 7 days the vessel appears to be largely reendothelialized. Beginning at day 4, smooth muscle cells undergo modification and migrate through fenestrae in the internal elastic lamina into the intima where they proliferate. By 28 days, the intimal lesion consists of multiple layers of smooth muscle cells surrounded by collagen and elastic fibers and basement-like material. After 3 months the lesions are markedly hyperplastic and contain new extracellular connective tissue elements. In contrast, with no further injury after 6 months the lesion has decreased markedly in size suggesting that it may be reversible in the absence of continued endothelial injury. The importance of endothelial "injury" exposing medial smooth muscle to plasma constituents may be the principal factors associated with the migration and proliferation of the smooth muscle cells into the intima resulting in the lesion. The smooth muscle cells do not contain lipid. The similarities of this lesion to the fibromusculo-elastic lesion or preatherosclerotic intimal hyperplasia in man makes it a useful model for the further study of atherosclerosis.

The continual remodeling of the artery wall significantly narrows the lumen. It contributes to the development of atherosclerotic plaque through the incorporation of lipid into the arterial intima. The steps in the repair of arterial intima include platelet accumulation followed by leukocyte attachment to denuded areas, smooth muscle migration from the media, deposition of extracellular connective tissue components, and endothelial proliferation from undamaged artery branches. Intimal growth and regression seem related to re-endothelialization, platelets, the pituitary, and unidentified factors.

The growth of a thombus is determined by the combined influences of blood flow, blood constituents, and blood vessel (Virchow, 1860). Major attention has been given to the circulating elements of the blood which participate in thrombus formation. Studies of these elements have led to a detailed in vitro description of blood coagulation in terms of the physiologic reactivity of procoagulants and platelets. Recently, the biologic application of numerous synthetic materials has brought those concerned with thrombus formation to inquire in a detailed manner into the influence of blood flow. The use of these materials has attracted the expertise of bioengineers interested in the field of fluid mechanics. The effect has been an emerging, comprehensive description of the behavior of physiologic fluids relating to the pathophysiology of thrombus formation. Results from these disciplines have underscored the need to consider the role of the remaining affector of thrombogenesis — the vascular surface.