Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.

Integrin-mediated adhesion and soluble ligand binding stabilize COX-2 protein levels in endothelial cells by inducing expression and preventing degradation.

Cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis, is highly expressed during inflammation and cellular transformation and promotes tumor progression and angiogenesis. We have previously demonstrated that endothelial cell COX-2 is required for integrin alphaVbeta3-dependent activation of Rac-1 and Cdc-42 and for endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047; Dormond, O., Bezzi, M., Mariotti, A., and Ruegg, C. (2002) J. Biol. Chem. 277, 45838-45846). In this study, we addressed the question of whether integrin-mediated cell adhesion may regulate COX-2 expression in endothelial cells. We report that cell detachment from the substrate caused rapid degradation of COX-2 protein in human umbilical vein endothelial cells (HUVEC) independent of serum stimulation. This effect was prevented by broad inhibition of cellular proteinases and by neutralizing lysosomal activity but not by inhibiting the proteasome. HUVEC adhesion to laminin, collagen I, fibronectin, or vitronectin induced rapid COX-2 protein expression with peak levels reached within 2 h and increased COX-2-dependent prostaglandin E2 production. In contrast, nonspecific adhesion to poly-L-lysine was ineffective in inducing COX-2 expression. Furthermore, the addition of matrix proteins in solution promoted COX-2 protein expression in suspended or poly-L-lysine-attached HUVEC. Adhesion-induced COX-2 expression was strongly suppressed by pharmacological inhibition of c-Src, phosphatidylinositol 3-kinase, p38, extracellular-regulated kinase 1/2, and, to a lesser extent, protein kinase C and by the inhibition of mRNA or protein synthesis. In conclusion, this work demonstrates that integrin-mediated cell adhesion and soluble integrin ligands contribute to maintaining COX-2 steady-state levels in endothelial cells by the combined prevention of lysosomal-dependent degradation and the stimulation of mRNA synthesis involving multiple signaling pathways.

Cyclooxygenase-2 controls energy homeostasis in mice by de novo recruitment of brown adipocytes.

Obesity results from chronic energy surplus and excess lipid storage in white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) efficiently burns lipids through adaptive thermogenesis. Studying mouse models, we show that cyclooxygenase (COX)-2, a rate-limiting enzyme in prostaglandin (PG) synthesis, is a downstream effector of beta-adrenergic signaling in WAT and is required for the induction of BAT in WAT depots. PG shifted the differentiation of defined mesenchymal progenitors toward a brown adipocyte phenotype. Overexpression of COX-2 in WAT induced de novo BAT recruitment in WAT, increased systemic energy expenditure, and protected mice against high-fat diet-induced obesity. Thus, COX-2 appears integral to de novo BAT recruitment, which suggests that the PG pathway regulates systemic energy homeostasis.

Cellular and molecular studies of the effects of a selective COX-2 inhibitor celecoxib in the cardiac cell line H9c2 and their correlation with death mechanisms

Cardiovascular disease is one of the leading causes of death worldwide, and evidence indicates a correlation between the inflammatory process and cardiac dysfunction. Selective inhibitors of cyclooxygenase-2 (COX-2) enzyme are not recommended for long-term use because of potentially severe side effects to the heart. Considering this and the frequent prescribing of commercial celecoxib, the present study analyzed cellular and molecular effects of 1 and 10 µM celecoxib in a cell culture model. After a 24-h incubation, celecoxib reduced cell viability in a dose-dependent manner as also demonstrated in MTT assays. Furthermore, reverse transcription-polymerase chain reaction analysis showed that the drug modulated the expression level of genes related to death pathways, and Western blot analyses demonstrated a modulatory effect of the drug on COX-2 protein levels in cardiac cells. In addition, the results demonstrated a downregulation of prostaglandin E2 production by the cardiac cells incubated with celecoxib, in a dose-specific manner. These results are consistent with the decrease in cell viability and the presence of necrotic processes shown by Fourier transform infrared analysis, suggesting a direct correlation of prostanoids in cellular homeostasis and survival.

Low-level Laser Therapy Improves Skeletal Muscle Performance, Decreases Skeletal Muscle Damage and Modulates mRNA Expression of COX-1 and COX-2 in a Dose-dependent Manner

We tested if modulation in mRNA expression of cyclooxygenase isoforms (COX-1 and COX-2) can be related to protective effects of phototherapy in skeletal muscle. Thirty male Wistar rats were divided into five groups receiving either one of four laser doses (0.1, 0.3, 1.0 and 3.0 J) or a no-treatment control group. Laser irradiation (904 nm, 15 mW average power) was performed immediately before the first contraction for treated groups. Electrical stimulation was used to induce six tetanic tibial anterior muscle contractions. Immediately after sixth contraction, blood samples were collected to evaluate creatine kinase activity and muscles were dissected and frozen in liquid nitrogen to evaluate mRNA expression of COX-1 and COX-2. The 1.0 and 3.0 J groups showed significant enhancement (P < 0.01) in total work performed in six tetanic contractions compared with control group. All laser groups, except the 3.0 J group, presented significantly lower post-exercise CK activity than control group. Additionally, 1.0 J group showed increased COX-1 and decreased COX-2 mRNA expression compared with control group and 0.1, 0.3 and 3.0 J laser groups (P < 0.01). We conclude that pre-exercise infrared laser irradiation with dose of 1.0 J enhances skeletal muscle performance and decreases post-exercise skeletal muscle damage and inflammation.

Celecoxib prevents tumor growth in an animal model by a COX-2 independent mechanism

Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to reduce cell growth in several tumors. Among these possible antineoplastic drugs are cyclooxygenase-2 (COX-2)-selective drugs, such as celecoxib, in which antitumoral mechanisms were evaluated in rats bearing Walker-256 (W256) tumor. W256 carcinosarcoma cells were inoculated subcutaneously (10(7) cells/rat) in rats submitted to treatment with celecoxib (25 mg kg(-1)) or vehicle for 14 days. Tumor growth, body-weight gain, and survival data were evaluated. The mechanisms, such as COX-2 expression and activity, oxidative stress, by means of enzymes and lipoperoxidation levels, and apoptosis mediators were also investigated. A reduction in tumor growth and an increased weight gain were observed. Celecoxib provided a higher incidence of survival compared with the control group. Cellular effects are probably COX-2 independent, because neither enzyme expression nor its activity, measured by tumoral PGE(2), showed significant difference between groups. It is probable that this antitumor action is dependent on an apoptotic way, which has been evaluated by the expression of the antiapoptotic protein Bcl-xL, in addition to the cellular changes observed by electronic microscopy. Celecoxib has also a possible involvement with redox homeostasis, because its administration caused significant changes in the activity of oxidative enzymes, such as catalase and superoxide dismutase. These results confirm the antitumor effects of celecoxib in W256 cancer model, contributing to elucidating its antitumoral mechanism and corroborating scientific literature about its effect on other types of cancer.

COX-2 Inhibitors for Cancer Treatment in Dogs

Cancer is one of the main causes of death in canines and felines, and this fact is probably related to the increase in the longevity of these species. The longer the animals live, the higher the exposure to carcinogenic agents will be. With the high incidence of cancer in companion animals, new studies are currently being performed with the aim of finding therapeutic options which make the complete inhibition of the development of neoplasms in animals possible in the future. The correlation of cyclooxygenase-2 (COX-2) whith the development of cancer opens the way for the use of new therapeutic approaches. This relationship has been suggested based on various studies which established an association between the chronic use of nonsteroidal anti-inflammatory drugs (NSAID) and a decrease in the incidence of colon carcinoma. As cancer progresses, COX-2 participates in the arachidonic acid metabolism by synthesizing prostaglandins which can mediate various mechanisms related to cancer development such as: increase in angiogenesis, inhibition of apoptosis, suppression of the immune response, acquisition of greater invasion capacity and metastasis. Accordingly, overexpression of this enzyme in tumors has been associated with the most aggressive, poor-prognosis cancer types, especially carcinomas. Therefore, treatments which use COX-2 inhibitors such as coxibs, whether administered as single agents or in combination with conventional antineoplastic chemotherapy, are an alternative for extending the survival of our cancer patients.

Late administration of a specific COX-2 inhibitor does not treat and/or prevent progression of gastric tumors in rats submitted to duodenogastric reflux procedure

PURPOSE:To assess whether late introduction of a specific COX-2 inhibitor (Meloxicam) can treat and/or prevent the progression of tumors in the stomach of rats submitted to duodenogastric reflux. METHODS: Seventy five male Wistar rats, weighing 150 grams, were submitted to the induction of duodenogastric reflux through the pylorus. At 36 weeks of follow-up were established three experimental groups: DGR36 sacrificed immediately, DGR54 and DGR54MLX both sacrificed at 54th week of follow-up . The animals of the latter group were fed with a rat chow premixed with Meloxicam (2.0 mg/ kg feed; 0.3 mg / kg bw / day) and the other two with standard rat chow. The lesions found in the pyloric mucosa and gastrojejunal anastomosis were analyzed macroscopically and histologically. For statistical analysis was adjusted a generalized linear model assuming a binomial distribution with LOGIT link function. RESULTS: No significant differences were found when comparing the incidences of benign tumor lesions (Adenomatous Hyperplasia), p=0.4915, or malignant (Mucinous Adenocarcinoma), p=0.2731, among groups. CONCLUSION: Late introduction of specific COX-2 inhibitor (Meloxicam) did not treat and was not able to prevent the progression of tumoral lesions induced by duodenogastric reflux in the rat stomachs.

Síntese e modelagem molecular do novo derivado indolinônico como candidato a antiinflamatório COX-2 seletivo

Anti-inflammatory drugs are known to be the most widely-marketed drugs in the world, despite their serious side effects, mainly on the gastrointestinal tract. Thus, there are constant efforts to discover new prototypes with improved therapeutic activity and safety for the patient. Since the advent of the computational chemistry, the theoretical study of the physiological behavior of a new compound and hence an understanding of its supposed mechanism of action have been made a lot more accessible. Thus, molecule-receptor mathematical modeling was applied to compound I (1-(2,6-dichlorophenyl)indolin-2-one), to predict theoretically its ability to inhibit, selectively, the COX-2 isoform of prostaglandin endoperoxide synthase (PGHS-2), and the best positions to introduce chemical groups and to make molecular modifications.

Healing Actions of Essential Oils from Citrus aurantium and d-Limonene in the Gastric Mucosa: The Roles of VEGF, PCNA, and COX-2 in Cell Proliferation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cellular and molecular studies of the effects of a selective COX-2 inhibitor celecoxib in the cardiac cell line H9c2 and their correlation with death mechanisms

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expressão da ciclooxigenase – 2 (COX-2) em carcinomas mamários espontâneos em cadelas

The immunohistochemistry regarding the expression of cyclooxygenase-2 (COX-2) for the woman’s breast cancer became a routine application due to the biological relevance. It is possible that COX-2 is closely related to tumor angiogenesis, inhibition of apoptosis mechanism, adhesion and metastasis. Herewith the COX-2 expression contributes to verify the malignant potential of the breast cancer. However it is slightly used in female dog’s spontaneous breast carcinomas. Keeping this in mind, the \cox-2 inhibitions appear as a promising perspective for the prevention and treatment of some sorts of cancer. The present dissertation had as main purpose to show the occurrence of the COX-2 expression in some of the female dog’s spontaneous breast carcinomas

Efeitos preventivos do treinamento físico na expressão das proteínas Cox-2 e Vegf de ratos tratados com dexametasona

Dexamethasone is a synthetic glucocorticoid widely used to treat allergic and inflammatory processes. This drug is used in three main situations, are used to contain acute or chronic inflammatory processes, or like immunosuppressive drug's. In these cases the patient will receive high doses for a chronic period and, therefore, has a much greater chance of adverse side effects, such as hypertension, diabetes and dyslipidemia. Dexamethasone promotes deleterious effects on the arachidonic acid pathway, when administered in high doses, because it is a potent anti-inflammatory drug. We recently demonstrated that dexamethasone significantly reduces the protein expression of vascular endothelial growth factor (VEGF) in both skeletal muscle and heart, but the mechanisms involved remain unclear. Meanwhile, exercise has been shown to be effective against high blood pressure, diabetes and dyslipidemia, promoting, among other factors, the increase in VEGF and angiogenesis. One possible explanation for these effects would be the creation of new vessels mediated by inflammation, or by the stimulation of the formation of products of the metabolism of arachidonic acid (AA), such as prostaglandin E2 (PGE2) and VEGF, by increasing the stimulation of the enzymes cyclooxygenase 1 and 2 (COX-1 and COX-2). Little is known about the preventive effects of training on the action of dexamethasone in the arachidonic acid pathway. Therefore, the aim of this study was to determine whether aerobic exercise training, performed before and concomitant treatment with dexamethasone, was able to prevent the effects of the dexamethasone in the protein expression of COX-2 and VEGF. For this, we used young Wistar rats (n = 40) which were randomly divided into 4 groups: sedentary control (SC), sedentary and treated with dexamethasone (SD), trained control (TC) and trained and treated with dexamethasone (TD). These rats performed aerobic exercise training, 60% of maximum capacity, 5

Assessment of MMP-9, TIMP-1, and COX-2 in normal tissue and in advanced symptomatic and asymptomatic carotid plaques

Abstract Background Mature carotid plaques are complex structures, and their histological classification is challenging. The carotid plaques of asymptomatic and symptomatic patients could exhibit identical histological components. Objectives To investigate whether matrix metalloproteinase 9 (MMP-9), tissue inhibitor of MMP (TIMP), and cyclooxygenase-2 (COX-2) have different expression levels in advanced symptomatic carotid plaques, asymptomatic carotid plaques, and normal tissue. Methods Thirty patients admitted for carotid endarterectomy were selected. Each patient was assigned preoperatively to one of two groups: group I consisted of symptomatic patients (n = 16, 12 males, mean age 66.7 ± 6.8 years), and group II consisted of asymptomatic patients (n = 14, 8 males, mean age 67.6 ± 6.81 years). Nine normal carotid arteries were used as control. Tissue specimens were analyzed for fibromuscular, lipid and calcium contents. The expressions of MMP-9, TIMP-1 and COX-2 in each plaque were quantified. Results Fifty-eight percent of all carotid plaques were classified as Type VI according to the American Heart Association Committee on Vascular Lesions. The control carotid arteries all were classified as Type III. The median percentage of fibromuscular tissue was significantly greater in group II compared to group I (p < 0.05). The median percentage of lipid tissue had a tendency to be greater in group I than in group II (p = 0.057). The percentages of calcification were similar among the two groups. MMP-9 protein expression levels were significantly higher in group II and in the control group when compared with group I (p < 0.001). TIMP-1 expression levels were significantly higher in the control group and in group II when compared to group I, with statistical difference between control group and group I (p = 0.010). COX-2 expression levels did not differ among groups. There was no statistical correlation between MMP-9, COX-2, and TIMP-1 levels and fibrous tissue. Conclusions MMP-9 and TIMP-1 are present in all stages of atherosclerotic plaque progression, from normal tissue to advanced lesions. When sections of a plaque are analyzed without preselection, MMP-9 concentration is higher in normal tissues and asymptomatic surgical specimens than in symptomatic specimens, and TIMP-1 concentration is higher in normal tissue than in symptomatic specimens.

Selektive und duale COX-1-, COX-2-Inhibitoren aus der Reihe der Methanone

Zusammenfassung: Prostaglandine (PG) sind wichtige biologische Entzündungsmediatoren, die aus der Arachidonsäure (AA) durch das Enzym Cyclooxygenase (COX) entstehen. Trotz einiger unerwünschter Wirkungen, sind Cyclooxygenase-Hemmer Mittel der Wahl zur Unterdrückung entzündlicher Prozesse. Von der Cyclooxygenase existieren zwei Isoenzyme: COX-1 und COX-2. Eine selektive Hemmung der COX-2 bzw. eine duale Hemmung der COX-1 und COX-2 wird als erfolgversprechendes Prinzip zur Behandlung von entzündlichen Erkrankungen diskutiert.Ziel der Arbeit war die Synthese und in vitro Testung sowie die Erstellung von Struktur-Wirkungs-Beziehungen selektiver bzw. dualer Hemmstoffe der COX-1/-2. Zusätzlich wurden die Substanzen auf inhibitorische Aktivität gegenüber der 5- und 12-Lipoxygenase untersucht.Ausgehend von der Struktur selektiver Hemmstoffe der COX-2 bzw. von dualen COX-1/ COX-2-Inhibitoren sowie von marktüblichen nichtsteroidalen Antirheumatika (NSAR), wurde das Diarylmethanon-Element als Basis gewählt. An diesem Strukturelement wurden Modifikationen vorgenommen, um selektive Hemmstoffe der COX-2 bzw. duale COX-1/ COX-2-Hemmstoffe zu erhalten.Die synthetisierten Verbindungen lassen sich in [4-(Methylsulfanyl)phenyl]- und [4-(Methylsulfonyl)phenyl](aryl)methanone, N-(Aroylphenyl)sulfonamide und -amide sowie (Hydroxyphenyl)(2-thienyl)methanone unterteilen.In der Reihe der [4-(Methylsulfanyl)phenyl](aryl)methanone sind potente Hemmstoffe sowohl der COX-1 als auch der COX-2 erhalten worden. Im Gegensatz dazu zeigen die [4-(Methylsulfonyl)phenyl](aryl)methanone gegenüber COX-1 und COX-2 keine inhibitorische Aktivität. Mit dem 2-Thienylderivat wurde ein potenter, dualer Hemmstoff beider Cyclooxygenase-Isoenzyme identifiziert, dessen Wirkstärke (bezüglich der COX-2) auf den Austausch von Phenyl gegen 2-Thienyl zurückzuführen ist.Die N-(Aroylphenyl)sulfonamide und -amide bilden die umfangreichste Gruppe bei den durchgeführten Untersuchungen, wobei besonders die regioisomeren N-(2-Aroylphenyl)sulfonamide und -amide eingehender studiert wurden. Auf der Basis der (2-Aroylphenyl)sulfonamide läßt sich für die Hemmung der COX-1 eine Struktur-Wirkungs-Beziehungen formulieren, die anhand Hilfe geeigneter Verbindungen überprüft wurde. Die Untersuchungen wurden zum Teil auch auf die 3- und 4-Regioisomeren ausgedehnt, wobei sich die erhaltenen Struktur-Wirkungs-Beziehungen bestätigten. Die Arylsulfonamide inhibieren bevorzugt die COX-1. Auch (4-Aroylphenyl)sulfonamide wurden auf mögliche inhibitorische Aktivität untersucht. Die Einbindung des Amidstickstoffs in ein Indolin- bzw. Tetrahydrochinolin-Ringsystem oder des Sulfonamids in ein 1,3-Propansultam führte in jedem Falle zu wenig aktiven Verbindungen gegenüber der COX-1. N-(2-Aroylphenyl)amide zeigten in Übereinstimmung mit der Hypothese an der COX-1 eine gute inhibitorische Aktivität.Aus der Reihe der (Hydroxyphenyl)(2-thienyl)methanone wurden die freien Alkohole, die Methylether und verschiedene Ester dargestellt und auf COX-1-Aktivität untersucht. Acetate, aber auch Phenole sind die potentesten Inhibitoren der COX-1. Als günstigte Positionen für die 2-Thienylcarbonyl-Einheit am Hydroxyphenylrest erweist sich die ortho- bzw. para-Position.