COX-2 expression in atherosclerosis: the good, the bad or the ugly?

Cyclooxygenase (COX) is the rate limiting enzyme catalyzing the conversion of arachidonic acid into prostanoids, lipid mediators critically implicated in a variety of physiological and pathophysiological processes, including inflammation, vascular and renal homeostasis, and immune responses. Since the early 1990s it has been appreciated that two isoforms of COX exist, referred to as COX-1 and COX-2. Although structurally homologous, COX-1 and COX-2 are regulated by two independent and quite different systems and have different functional roles. In the setting of acute ischemic syndromes it has been recognized that COX pathway plays an important role; however, whereas the function of platelet COX-1 in acute ischemic diseases is firmly established, the role of COX-2 in atherothrombosis remains controversial. The complex role of COX-2 in this setting is also confirmed by the unexpected cardiovascular side effects of long-term treatment with COX-2 inhibitors. In this article, we review the pattern of expression of COX-2 in the cellular players of atherothrombosis, its role as a determinant of plaque vulnerability, the effects of the variable expression of upstream and downstream enzymes in the prostanoid biosynthesis on COX-2 expression and inhibition.

Cyclic AMP signalling pathway and cellular proliferation: induction of CREM during liver regeneration.

The CREM gene encodes both activators and repressors of cAMP-induced gene expression. An isoform of CREM encodes the powerful transcriptional repressor ICER (Inducible cAMP Early Repressor), which has been shown to be inducible by virtue of an alternative, intronic promoter. The CREM gene belongs to the early response class and displays a characteristic neuroendocrine cell- and tissue-specific expression. To date ICER inducibility has been described in non-replicating, terminally differentiated tissues. In this paper we document a robust induction of CREM expression in the regenerating rat liver after partial hepatectomy. This represents the first link of inducible CREM expression to the phenomenon of cellular proliferation. Furthermore, it represents the first example of transcriptional activation of a cAMP-responsive factor in the regenerating liver. This has significant physiological relevance since the adenylate cyclase signalling pathway is strongly implicated in liver regeneration. Finally, we show that the repressor ICER is inducible in the hepatoma cell line H35 upon activation of the adenylate cyclase and phosphorylation of the activator CREB.

Overexpression of functionally coupled cyclooxygenase-2 and prostaglandin E synthase in symptomatic atherosclerotic plaques as a basis of prostaglandin E(2)-dependent plaque instability.

BACKGROUND: Studies have implicated a role for prostaglandin (PG) E(2)-dependent matrix metalloproteinase (MMP) biosynthesis in the rupture of atherosclerotic plaque. Cyclooxygenase-2 (COX-2) and PGE synthase (PGES) are coregulated in nucleated cells by inflammatory stimuli. The aim of this study was to characterize the expression of COX-2 and PGES in carotid plaques and to correlate it with the extent of inflammatory infiltration and MMP activity and with clinical features of patients' presentation. METHODS AND RESULTS: Plaques were obtained from 50 patients undergoing carotid endarterectomy and divided into 2 groups (symptomatic and asymptomatic) according to clinical evidence of recent transient ischemic attack or stroke. Plaques were analyzed for COX-2, PGES, MMP-2, and MMP-9 by immunocytochemistry and Western blot, whereas zymography was used to detect MMP activity. Immunocytochemistry was used to identify CD68+ macrophages, CD3+ T lymphocytes, and HLA-DR+ cells. The percentage of macrophage-rich areas was larger (P<0.0001) in symptomatic plaques. COX-2, PGES, and MMPs were detected in all specimens; enzyme concentration, however, was significantly higher in symptomatic plaques. COX-2, PGES, and MMPs were especially noted in shoulders of symptomatic plaques, colocalizing with HLA-DR+ macrophages. All symptomatic plaques contained activated forms of MMPs. Finally, inhibition of COX-2 by NS-398 was accompanied by decreased production of MMPs that was reversed by PGE(2). CONCLUSIONS: This study demonstrates the colocalization of COX-2 and PGES in symptomatic lesions and provides evidence that synthesis of COX-2 and PGES by activated macrophages is associated with acute ischemic syndromes, possibly through metalloproteinase-induced plaque rupture.

Novel determinants of plaque instability.

Arachidonic acid metabolism plays an important role in acute ischemic syndromes affecting the coronary or cerebrovascular territory, as reflected by biochemical measurements of eicosanoid biosynthesis and the results of inhibitor trials in these settings. Two cyclooxygenase (COX)-isozymes have been characterized, COX-1 and COX-2, that differ in terms of regulatory mechanisms of expression, tissue distribution, substrate specificity, preferential coupling to upstream and downstream enzymes and susceptibility to inhibition by the extremely heterogeneous class of COX-inhibitors. While the role of platelet COX-1 in acute coronary syndromes and ischemic stroke is firmly established through approximately 20 years of thromboxane metabolite measurements and aspirin trials, the role of COX-2 expression and inhibition in atherothrombosis is substantially uncertain, because the enzyme was first characterized in 1991 and selective COX-2 inhibitors became commercially available only in 1998. In this review, we discuss the pattern of expression of COX-2 in the cellular players of atherothrombosis, its role as a determinant of plaque 'vulnerability,' and the clinical consequences of COX-2 inhibition. Recent studies from our group suggest that variable expression of upstream and downstream enzymes in the prostanoid biosynthetic cascade may represent important determinants of the functional consequences of COX-2 expression and inhibition in different clinical settings.

Elevated circulating levels of monocyte chemoattractant protein-1 in patients with restenosis after coronary angioplasty.

Inflammation plays a pathogenic role in the development of restenosis after percutaneous transluminal coronary angioplasty (PTCA). Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant of monocytes; however, its role in the pathophysiology of restenosis is still unclear. We set out to investigate the role of MCP-1 in restenosis after PTCA. In addition, we tested the hypothesis that MCP-1 exerts its effect, at least in part, by inducing O(2)(-) generation in circulating monocytes. Plasma levels of MCP-1 were measured before and 1, 5, 15, and 180 days after PTCA in 50 patients (30 males and 20 females, aged 62+/-5 years) who underwent PTCA and who had repeated angiograms at 6-month follow-up. Restenosis occurred in 14 (28%) patients. The MCP-1 level was no different at baseline between patients with or without restenosis. However, after the procedure, restenotic patients, compared with nonrestenotic patients, had statistically significant (P<0.0001) elevated levels of MCP-1. In contrast, plasma levels of other chemokines, such as RANTES and interleukin-8, did not differ between the 2 groups after PTCA. Higher MCP-1 throughout the study was correlated with restenosis. Moreover, increased MCP-1 was significantly correlated with increased monocyte activity, as reflected by enhanced O(2)(-) generation. Finally, multivariate regression analysis showed that the MCP-1 plasma level measured 15 days after PTCA was the only statistically significant independent predictor of restenosis (beta=0.688, P<0.0001). This study suggests that MCP-1 production and macrophage accumulation in the balloon-injured vessel may play a pivotal role in restenosis after PTCA. MCP-1 may induce luminal renarrowing, at least in part, by inducing O(2)(-) release in monocytes. Further understanding of the mechanism(s) by which MCP-1 is produced and acts after arterial injury may provide insight into therapies to limit the progression of atherosclerosis and restenosis after balloon angioplasty.

Relationship between reduced BCL-2 expression in circulating mononuclear cells and early nephropathy in type 1 diabetes.

Microalbuminuria is the earliest clinical evidence of diabetic nephropathy, but the mechanisms linking hyperglycemia and kidney complications are not clear. The aim of this study was to evaluate whether enhanced oxidative stress in patients with microalbuminuria can contribute to diabetic nephropathy development through downregulation of the antiapoptotic gene Bcl-2 that promotes in turn a pro-inflammatory status. We studied 30 patients with type 1 diabetes (15 with and 15 without microalbuminuria) compared to 15 matched healthy controls. Plasma oxidant status, and expression of Bcl-2, activated NF-kB, inducible Nitric Oxide synthase (iNOS), and monocyte chemoattractant protein (MCP)-1 in circulating monocytes were evaluated at baseline and after 8-week oral vitamin E treatment (600 mg b.i.d.). Bcl-2 expression was significantly reduced in microalbuminuric diabetic patients as a consequence of increased oxidant burden secondary to persistent hyperglycemia. Bcl-2 down-regulation was associated with enhanced expression of NF-kB, iNOS and MCP-1, and showed a strong correlation with the albumin excretion rate. Low Bcl-2 expression and high inflammatory status were normalized by vitamin E both in vivo and in vitro. Our study showed that Bcl-2 down-regulation in diabetic patients with poor glycemic control results in the activation of the NF-kB pathway leading to the development of nephropathy. Vitamin E might provide a novel form of therapy for prevention of nephropathy in diabetic patients in which an acceptable glycemic control is difficult to achieve despite insulin therapy.

Changes in oncogene expression in ascite tumour cells during ageing.

The expression of two oncogenes, c-myc and c-fos, was studied in an ascitic tumour (ATPC+) at different times after implantation. The specific mRNA synthesis was analysed by Northern blot analysis. The presence of the oncogene proteins was shown by immunofluorescence using flow cytometry and referred to the distribution of the cells in the different cell phases. The results show that both oncogenes are expressed by ATPC+ tumour cells. c-myc is expressed 5, 8 and 12 days after implantation, although with a different intensity, and the protein is mainly present in S or S+G2 phase cells. The c-fos oncogene is expressed only 12 days after tumour implantation and the cells labelled with the specific antibody are mainly in G1 phase. We conclude that c-myc is principally correlated with proliferative activity, whereas c-fos is expressed by non-cycling cells.

Cyclic AMP signalling and cellular proliferation: regulation of CREB and CREM.

In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREB) is modulated by phosphorylation by the cAMP-dependent protein kinase. The ICER (inducible cAMP early repressor) protein is the only inducible member of this family and is a product of the CREM gene. The induction of this powerful repressor is likely to be important for the transient nature of cAMP-induced gene expression. CREB proteins have been found to play an important role in the physiology of neuroendocrine functions. In addition, recent results indicate that CREB and CREM could be involved in the proliferation of hepatocytes which follows partial hepatectomy.

Stress-induced expression of transcriptional repressor ICER in the adrenal gland.

Second messenger cyclic AMP plays a central role in signalling within the hypothalamo-pituitary-adrenal (HPA) axis. Changes in gene expression are central to long-term adaptations made in response to stress in the adrenal gland. Here we demonstrate that expression of the cAMP inducible transcriptional repressor, ICER (Inducible cAMP Early Repressor), is rapidly and powerfully induced in response to surgical stress in the rat adrenal gland. Hypophysectomisation blocks stress-induced ICER expression. Finally we demonstrate that injection of the pituitary hormone ACTH (Adrenocorticotropin Hormone) induces robust ICER expression in the adrenal cortex. Thus, induction of the transcriptional repressor ICER is coupled to the HPA axis response to stress.

Promoter characterization and expression of the gene coding for the human GM2 activator protein.

Genomic clones of the human GM2 activator protein have been isolated and analyzed. The 5' region of the gene demonstrated promoter activity as ascertained by its ability to drive luciferase gene expression in transfected COS cells. This sequence contains GC rich region and several putative promoter elements were present, including Sp1, AP2, cAMP-responsive element, and B-cell-specific activating protein. Analysis of tissue distribution of the GM2 activator protein gene revealed tissue-specific variations in transcript levels. Placenta, bone marrow, mammary gland, bladder, lymph node, and spleen had the highest mRNA levels.

Tyrosine aminotransferase gene regulation during aging.

Tyrosine aminotransferase activity (TAT) and its expression were measured in rats of different ages (3, 12 and 24 months). The enzyme activity showed a circadian rhythm with a peak at midnight due to different levels of transcription during the day. The circadian rhythm was present at all ages studied but showed some differences: an age-related shift of the peak was more evident in the actual levels of transcript. Both specific mRNA and enzyme activity analyses provide better insight into the complex modifications in gene-expression of aging animals.

Hepatocyte odd protein shuttling (HOPS) is a bridging protein in the nucleophosmin-p19 Arf network.

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19(Arf), directs localization of p19(Arf) in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19(Arf), resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19(Arf) and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19(Arf), providing new mechanistic insight into how NPM and p19(Arf) will oppose tumor cell proliferation.

Enhanced soluble CD40 ligand contributes to endothelial cell dysfunction in vitro and monocyte activation in patients with diabetes mellitus: effect of improved metabolic control.

AIMS/HYPOTHESIS: Inflammation plays a pathogenic role in the development of accelerated atherosclerosis in diabetes. Soluble CD40 ligand (sCD40L) is enhanced in diabetes; however, the molecular mechanisms linking sCD40L to accelerated atherosclerosis in diabetes are still unclear. We tested the hypothesis that sCD40L may be involved in the vascular complications in diabetes and exerts its effect by triggering inflammatory reactions on mononuclear and endothelial cells (ECs). METHODS: We studied 70 patients, 40 with type 2 and 30 with type 1 diabetes, with a history or physical examination negative for cardiovascular disease, and 40 non-diabetic and 30 healthy subjects, matched with the type 2 and type 1 diabetic patients, respectively. Plasma and serum sCD40L, and plasma soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1, E-selectin and monocyte chemo-attractant protein-1 (MCP-1) were measured. Adhesion molecules and MCP-1 release, the ability to repair an injury in ECs, and O2- generation in monocytes were analysed in vitro after stimulation with serum from patients or controls. RESULTS: Type 2 and type 1 diabetic patients had significantly higher sCD40L levels than controls. Furthermore, high sCD40L was associated with in vitro adhesion molecules and MCP-1 release, impaired migration in ECs and enhanced O2- generation in monocytes. Improved metabolic control was associated with a reduction of plasma sCD40L by 37.5% in 12 type 1 diabetic patients. Furthermore, elevated sCD40L in diabetic patients was significantly correlated with HbA1c levels. CONCLUSIONS/INTERPRETATION: Upregulation of sCD40L as a consequence of persistent hyperglycaemia in diabetic patients results in EC activation and monocyte recruitment to the arterial wall, possibly contributing to accelerated atherosclerosis development in diabetes.

Variation of tyrosine aminotransferase expression during the day in rats of different ages.

The activity of the enzyme tyrosine aminotransferase and the synthesis of its specific mRNA were evaluated at different hours of the day in the liver of 3-, 12- and 24-month old BN rats. The enzyme activity has a circadian rhythm with a peak at midnight in 3- and 12-month old, which shifts to 03.00 hrs in 24-month old animals, in agreement with previous results. The expression of TATmRNA also changes during the day indicating circadian fluctuations which change with age. In 3-month old rats the TATmRNA peak is at 19.00 hrs, preceding that of the enzyme activity. In 12-month old rats the TATmRNA synthesis reaches a maximum at midnight and in 24-month old rats at 03.00 hrs. The results show that the circadian rhythm of tyrosine aminotransferase activity is due to a different gene expression throughout the day, which is influenced by age.

Different expression of tyrosine aminotransferase and serine deydratase in rat livers after partial hepatectomy.

Tyrosine aminotransferase activity in rat liver increases during the first 24 hrs after partial hepatectomy with two peaks, one at 10 hrs and another at 18 hrs. This behaviour is due to an increase in TATmRNA synthesis. Expression of serine deydratase is also enhanced during the first 5 hrs after hepatectomy. It is suggested that the enhanced expression of the two genes is due to an increase in hormone incretion particularly glucagon and glucocorticoids.

Transcription factor CREM coordinates the timing of hepatocyte proliferation in the regenerating liver.

The liver regenerates upon partial hepatectomy (PH) as terminally differentiated hepatocytes undergo a tremendous proliferative process. CREM gene expression is powerfully induced during liver regeneration. We show that cell proliferation is significantly reduced upon PH in CREM-/- mice. There is a reduction in DNA synthesis, in the number of mitosis and of phosphorylated histone H3-positive cells. The post-PH proliferation peak is delayed by 10 hr, indicating an altered hepatocyte cell cycle. Expression of cyclins A, B, D1, E, and cdc2, of c-fos and tyrosine aminotransferase is deregulated. CREM mutation results in delayed S-phase entry, impairing the synchronization of proliferation.

[Gonadotropin response to slow infusion of GN-RH in idiopathic ologospermia. Preliminary findings]. / Risposta gonadotropinica all'infusione lenta di GN-RH nella oligospermia idiopatica. Rilievi preliminari.

Gn-RH was administered i.v. to infertile patients presenting with idiopathic oligospermia at a constant rate (0,4 microgram) for 4 h. All the patients showed an increased FSH response, and LH levels higher than normal in both the first and the second peak of response. Our data indicate that an increased pituitary sensitivity and reserve of FSH and LH are present in idiopathic oligospermia, suggesting an involvement of both Leydig cells and seminiferous epithelium in this condition.

[Gonadotropin response to acute stimulation with LHFSHRH in idiopathic oligospermia. Preliminary results]. / Risposta gonadotropinica alla stimolazione acuta con GN-RH nell'oligosppermia idiopatica. Risultati preliminari.

Serum FSH and LH levels in basal conditions and after Gn-RH test were investigated in 15 idiopathic oligozoospermic patients. Basal FSH was significantly higher in oligospermic than in normal subjects and a negative relationship was found between basal FSH and sperm count. Basal LH was not different in oligozoospermic and in normal subjects. Both FSH and LH responses to GN-RH were significantly higher in oligozoospermic patients. In idiopathic oligozoospermia the presence of a testicular defect involving both the seminiferous epithelium and Leydig cells is suggested.

Different expression of beta-N-acetylhexosaminidase in mouse tissues.

The expression of genes encoding for the alpha and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase was investigated in different mouse tissues. It was found, using fluorogenic substrates, that the amounts of alpha and beta subunits were not the same in different tissues: alpha-subunit was more abundant in the brain, beta-subunit in epididymis and brain. The different isoenzyme patterns and specific activities in mouse tissues are due to the differences in the amount of hexosaminidase subunits. The mRNA, evaluated by Northern blotting analyses, revealed a greater expression of alpha-subunit in the testis and of beta-subunit in the brain and epididymis. The results indicate, therefore, that gene expression and the amount of subunits are in good relationship for beta-subunit, whereas there is no correlation for alpha-subunit.

[Abnormal origin of the left coronary artery from the pulmonary artery. Description of a rare case in adulthood]. / Origine anomala della coronaria sinistra dall'arteria polmonare. Descrizione di un raro caso in età adulta.

The anomalous origin of the left coronary artery from the pulmonary artery is a rare and usually fatal congenital malformation. The Authors present a case of anomalous left coronary artery arising from the pulmonary artery diagnosed in an adult patient.