The present study was undertaken to examine the effect of vitamin B12 on serum cholesterol levels and development of aortic atherosclerosis in New Zealand White male rabbits fed a high cholesterol diet. Twenty rabbits were divided into 4 groups matched for their baseline serum cholesterol values. Groups 1 and 2 received a normal diet, whereas groups 3 and 4 had cholesterol mixed with their chow. After 5 weeks, the serum cholesterol of groups 3 and 4 increased by approximately 12 fold compared to groups 1 and 2. At that point, vitamin B12 was administered in the drinking water (1000 micrograms/l) to rabbits in groups 2 and 4. At week 8, the Vitamin B12 dose was increased to 2400 micrograms/l for group 4. At 12 weeks into the study, rabbits in group 4 (high cholesterol diet plus vitamin B12) had a serum cholesterol level of 919 +/- 270 mg/dl while those of group 3 (high cholesterol diet) had a serum cholesterol level of 1417 +/- 250 mg/dl. At the end of the study, rabbit aortas were excised, fixed, and stained with fat-red-B, a dye for lipid. Comparison of the aortas of rabbits that received the lipid rich diet revealed that animals given additional vitamin B12 had significantly decreased surface area of fat deposition. These data suggest the potential role for vitamin B12 as a safe and inexpensive pharmacological intervention for the modification of the progression of human atherosclerosis.

Increased expression and activity of 12/15-lipoxygenase (12/15-LO) in vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetes and vascular complications. However, the consequences of 12/15-LO overexpression for VSMC migration and inflammatory gene expression are not known. In this study, 12/15-LO was overexpressed using adeno- and baculoviral vectors in human VSMC (HVSMCs) and proatherogenic responses compared with control enhanced green fluorescent protein (EGFP)-expressing cells. HVSMCs transduced with 12/15-LO viruses expressed high levels of enzymatically active protein and produced increased levels of the LO product, 12(S)-hydroxyeicosatetraenoic acid. 12/15-LO-overexpressing HVSMCs exhibited increased oxidant stress, activation of p38 mitogen-activated protein kinase, migration and inflammatory gene expression relative to HVSMCs expressing EGFP. Furthermore, inflammatory gene expression induced by 12/15-LO overexpression was abolished by anti-oxidants, siRNAs targeting p65 (nuclear factor-ĸB), or new-generation baculoviruses expressing inhibitory IĸBα or IĸBα superrepressor mutant. Thus, we have used novel viral vector delivery systems, including baculoviruses, for the first time to deliver foreign genes into VSMCs and thereby demonstrated that 12/15-LO overexpression increases oxidant stress, mitogen-activated protein kinase activation, migration and inflammatory genes in VSMCs and that NF-ĸB is a key downstream effector. Enhanced proatherogenic responses in VSMCs triggered by increased 12/15-LO levels under pathological conditions may contribute to vascular dysfunction.

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 kappaB (NF-kappaB). 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-kappaB 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-kappaB.

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-kappaB (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.

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 < or = 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: 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 LXR alpha and retinoid X receptor RXR alpha. oxLDL also attenuated LXR activation by blocking LXR ligand binding and interfering with the generation of 27-hydroxycholesterol, 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. (c) 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-activated nuclear receptor that has an essential role in adipogenesis and glucose homeostasis. PPAR-gamma is expressed in vascular tissues including endothelial cells (ECs). PPAR-gamma activity can be regulated by many pathophysiological and pharmacological agonists. However, the role of PPAR-gamma activation in ECs remains unclear. In this study, we examined the effect of the constitutive activation of PPAR-gamma on the phenotypic modulation of ECs. Adenovirus-mediated expression of a constitutively active mutant of PPAR-gamma resulted in significant ligand-independent activation of PPAR-gamma and specific induction of the PPAR-gamma target genes. However, PPAR-gamma activation significantly suppressed the expression of vascular adhesion molecules in ECs and the ensuing leukocyte recruitment. Furthermore, constitutive activation of PPAR-gamma resulted in simultaneous repression of AP-1 and NF-kappaB activity, which suggests that PPAR-gamma may reduce pro-inflammatory phenotypes via, at least in part, suppression of the AP-1 and NF-kappaB pathways. Therefore, using a gain-of-function approach, our study provides novel evidence showing that constitutive activation of PPAR-gamma is sufficient to prevent ECs from converting into a pro-inflammatory phenotype. These results also suggest that, in addition to pharmacological agonists, the genetic modification of the PPAR-gamma activity in ECs may be a potential approach for therapeutic intervention in various inflammatory disorders.

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.

Low density lipoprotein (LDL) has been shown to perturb endothelial cells, with manifestations ranging from alterations in free radicals and arachidonate metabolism to stress fiber formation and monocyte recruitment. Some of these changes are regulated by LDL at the transcriptional level. Using mobility shift assays with consensus sequences for various transcription factors, we have detected an increase in activator protein 1 (AP-1), but not nuclear factor-kB (NF-kB), binding in human umbilical vein endothelial cells exposed to LDL. Following transfection, AP-1–driven chloramphenicol acetyltransferase and AP-1–driven-luciferase are upregulated by LDL. In contrast, there is no effect on NF-kB–driven chloramphenicol acetyltransferase. AP-1 increases in a biphasic fashion, with the first peak occurring 6 hours after and the second 48 hours after exposure to LDL. This AP-1 binding increase involves c-Jun, but not c-Fos, as shown by gel supershift, Northern hybridization, and Western blotting analyses. c-Jun mRNA levels are elevated by 9 hours after and remain so until at least 24 hours after exposure to LDL. c-Jun protein levels increase at 12 hours and continue to rise for 24 hours after exposure to LDL. Moreover, this LDL-increased AP-1 binding is suppressed by several protein kinase (PK) inhibitors: the PKC inhibitor calphostin C, the cAMP-dependent PK inhibitor H89, and the tyrosine PK inhibitors genistein and lavendustin A. This study demonstrates that (1) LDL is an endothelial agonist distinct from other cell stimulators, such as cytokines, endotoxin, and phorbol 12-myristate 13-acetate, because LDL appears to activate human umbilical vein endothelial cells predominantly through the transcription factor AP-1 and not NF-kB; and (2) LDL increases AP-1 via mechanisms involving multiple kinase activities and c-Jun transcription. (Arterioscler Thromb Vasc Biol. 1998;18:473-480.)

Human umbilical vein endothelial cells (EC) metabolize arachidonic acid (AA) through three major pathways—cyclooxygenase, lipoxygenase and cytochrome P450 (P450) isozymes. Previously, we have shown that pathophysiological concentrations of native low density lipoprotein (n-LDL) increase EC P450-dependent epoxyeicosatrienoic acid (EET) production. The present study was designed to identify putative P450 isozymes involved in EC epoxidation of AA. Reverse transcription—polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were employed to detect P450 2 family cDNA from EC mRNA. Degenerate primers complimenting 2 homologous regions from 6 different P450 2 families were designed to capture a 440 bp cDNA fragment corresponding to the heme binding region of P450 2 isozymes. RT-PCR of EC total RNA with these primers amplified a 440 bp fragment. After gel purification, the fragment was cloned, sequenced and found to share a high degree of identity with human liver P450 2C8 and 2C9. New primers were designed based on the nucleotide sequences of the 440 bp fragment and a third homologous upstream region from the 440 bp fragment. RT-PCR was used to capture 5′ sequences upstream from the 440 bp fragment and lock-docking 3′-RACE was used to capture sequences downstream from the 440 bp fragment. These steps successfully expanded the number of nucleotides captured and sequenced to 1.4 kb. The partial clone was a homologous to human liver P450 2C8, sharing a >99.8% identity, and >86.1% with P450 2C9. When sequence analysis of independently cloned 440 bp fragments was performed, no other P450 2 isozymes were detected. To determine if P450 2C isozyme family members were capable of AA epoxidation, ECV-304 cells, which usually generate low levels of EETs were transfected with pcDNA3 plasmids containing DNA encoding functional P450 2C9. Transient P450 2C9 transfection of this eternal endothelial cell line increased EET production levels up to 4-fold, which was similar in magnitude to EET production by EC exposed to native low density lipoprotein (n-LDL). These data indicate that EC epoxidize AA by a P450 2C8 isozyme.

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O2- release by EC was not detectable. EC exposure to Bk (10(-6) to 10(-5) M) resulted in a rapid release of O2-. The release of O2- occurred within 5 minutes of exposure. O2- release was partially inhibited by indomethacin (63 +/- 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O2- production. EC O2- release may be an important component in the pathophysiologic actions of Bk on vascular function.

The influence of intimal de-endothelialization on stress fiber expression in regenerating rat and rabbit aortic endothelium was studied using immunofluorescence microscopy. Rat thoracic and abdominal aortae were balloon de-endothelialized, and endothelial cell shape and stress fiber expression was studied in both uninjured and de-endothelialized animals. In control animals, the majority of thoracic endothelial cells did not contain stress fibers while the majority of abdominal endothelial cells did. One week after injury, all the endothelial cells distal to the regenerating edge contained very prominent stress fibers. In areas directly adjacent to the still de-endothelialized surface, the endothelial cells had an intense, diffuse cytoplasmic staining without stress fibers. Regenerating endothelium also had a substantially higher length-to-width ratio, but smaller cell areas. Six weeks after injury, the endothelium had completely regenerated, and stress fibers were lost from the majority of the thoracic endothelial cells. Changes in abdominal aorta stress fiber expression were not as marked. In the rabbit, all the control thoracic endothelial cells had stress fibers; however, cells at the leading edge of a narrow region of de-endothelialization had few stress fibers. The results suggest that stress fibers do not play a primary role in cellular migration in situ. The transient increase in stress fiber expression in the rat may result from a temporary demand for greater adhesive capabilities until the subendothelial extracellular matrix is remodeled.