Cox 1 And Cox 2 Tissue Expression Implications And Predictions Pdf
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- Unresolved Issues in the Role of Cyclooxygenase-2 in Normal Physiologic Processes and Disease
- Analgésicos inibidores específicos da ciclooxigenase-2: avanços terapêuticos
- Analgésicos inibidores específicos da ciclooxigenase-2: avanços terapêuticos
Unresolved Issues in the Role of Cyclooxygenase-2 in Normal Physiologic Processes and Disease
Primary open-angle glaucoma POAG is the predominant form of chronic glaucoma, but the underlying pathologic mechanisms are largely unknown.
Because prostaglandins PGs have been introduced into POAG treatment with remarkable success, this study was undertaken to investigate whether a change in the expression of the PG-synthesizing enzymes cyclooxygenase COX -1 and -2 might be involved in the pathogenesis of POAG.
Expression of COX-1 and -2 was assessed by confocal laser microscopy, immunohistochemistry, Western blot analysis, and real-time RT-PCR in human eyes with different forms of glaucoma primary open-angle, angle-closure, congenital juvenile, and steroid-induced , as well as in age-matched control eyes. Additionally, PGE 2 was measured in aqueous humor by means of an enzyme-linked immunoassay as a product of COX activity. In normal eyes, ocular COX-1 and -2 expression were largely confined to the nonpigmented secretory epithelium of the ciliary body.
By immunohistochemistry, in the ciliary bodies of eyes in five patients with diagnosis of early POAG, eyes in two had complete loss of COX-2 expression and in three showed only a few remaining scattered COX-2—expressing cells. COX-2 expression in the ciliary body was also lost in patients with steroid-induced glaucoma and was reduced in patients receiving topical steroid treatment.
Eyes of patients with either congenital juvenile or angle-closure glaucoma showed COX-2 expression indistinguishable from control eyes. Both cyclooxygenase isoforms are constitutively expressed in the normal human eye. Specific loss of COX-2 expression in the nonpigmented secretory epithelium of the ciliary body appears to be linked to the occurrence of POAG and steroid-induced glaucoma. Purchase this article with an account.
Jump To Materials and Methods Results Discussion. Tamm ; Kay Brune. Author Affiliations. Alerts User Alerts. You will receive an email whenever this article is corrected, updated, or cited in the literature. You can manage this and all other alerts in My Account. This feature is available to authenticated users only. Get Citation Citation. Get Permissions. Primary open-angle glaucoma POAG is among the dominating ocular diseases in industrialized countries.
POAG shares several characteristics with corticosteroid hormone—induced secondary open-angle glaucoma. PG production involves two isoenzymes, COX-1 and In view of the potential importance of cyclooxygenases in glaucoma, the localization and expression of these enzymes in normal and glaucomatous human eyes was investigated.
Informed consent to eye and tissue donation was obtained and the study adhered to the tenets of the Declaration of Helsinki. Immunofluorescence and Confocal Laser Microscopy.
Anterior segments prepared from normal human donor eyes were embedded in optimal cutting temperature compound OCT; Tissue-Tek; Miles Laboratories, Elkhart, IN and snap frozen in isopentane-liquid nitrogen.
Sections were coverslipped with TBS-glycerol , pH 8. Ultrathin sections were incubated successively in drops of TBS, 0. After a rinse, sections were stained with uranyl acetate and examined with an electron microscope EM ; Leo, Oberkochen, Germany. Membranes were probed with polyclonal goat anti-human COX-2 antiserum diluted , followed by a horseradish peroxidase HRP —linked donkey anti-goat IgG secondary antibody diluted ; Santa Cruz Biotechnology.
Quantification was performed by determining the threshold cycle C T. Ten-micrometer-thick slices of respective eyes were cut. Afterward, ciliary processes were separated by means of microdissection and transferred into cups Eppendorf, Fremont, CA. All isolation steps were performed at room temperature in the same cup. The solution was then centrifuged at full speed for 5 minutes, and the supernatant was removed.
After the last wash, ethanol was totally removed and the pellet was air dried. Special primers were designed for the detection of potential fragmented RNA, according to reports in the respective literature. After sections were rinsed, they were incubated with a polyclonal goat anti-human COX-1 or -2 antibody diluted in TBS , with the biotinylated link antibody and the HRP-conjugated streptavidin, respectively, for 30 minutes each.
Negative controls included incubation of ocular tissues with nonimmunized goat serum, omitting the primary antibody, and incubation of negative control tissues muscle.
Positive tissue controls included sections of brain, kidney, and uterus data not shown. Aqueous humor obtained at eye surgery was immediately snap frozen in liquid nitrogen. The expression of COX-1 and -2 at different sites in the human eye was investigated with immunofluorescence and confocal laser microscopy Fig. By contrast, only minor expression of both cyclooxygenases was observed in other areas of the anterior eye segments.
The ultrastructural localization of COX-2 was revealed by immunoelectron microscopy. The strongest staining was found in the basolateral membranes of the nonpigmented secretory epithelial cells Fig.
Additionally, COXprotein was found in association with intercellular contacts maculae and zonulae adherentes, Figs. A schematic overview of the distribution of COX-2 in the ciliary epithelium based on immunoelectron microscopy is shown in Figure 2E. Constitutive expression of COXprotein was further confirmed by Western blot analysis using protein extracts from human ciliary body tissue.
The antibody raised against human COX-2 protein detected a double band of approximately 68 to 75 kDa, corresponding to the molecular weight of COX-2 Fig. The detected bands were also in the same range as a parallel-run positive control. Paraffin-embedded sections of surgically enucleated blind and painful glaucomatous eyes were obtained from the archives of the Department of Ophthalmology at the University of Erlangen, Germany.
This loss was very specific in two respects. First, loss of COX-2 expression was restricted to the nonpigmented ciliary epithelium and did not affect COX-2 expression in the connective tissue cells of the ciliary body and iris stroma 4D, inset. By contrast, all eyes with congenital juvenile and angle-closure glaucoma exhibited an expression of COX-2 very similar to that in normal control eyes Figs.
Equally processed and age-matched normal human eyes obtained from organ donors and eyes enucleated for small juxtapapillary malignant melanomas of the choroid not affecting anterior segment tissues served as control eyes Figs. The latter eyes are regarded as otherwise normal and have the advantage of a very short time until fixation of the tissue can begin.
Therefore, postmortem changes can be largely excluded. These eyes showed the same COX-2 expression as observed in normal human donor eyes. Because glucocorticoids are known to inhibit COX-2 expression in various cell types, 8 14 16 we investigated whether topical ocular steroid treatment affects the COX-2 expression in the ciliary epithelium. COX-2 expression was almost completely absent in glucocorticoid-treated eyes Fig.
Because the analysis was performed on paraffin-embedded sections from the archives of the Department of Ophthalmology at the University of Erlangen, the specimens were not suitable for quantitative investigations, such as Western blot analysis or RT-PCR.
Patients with clinically diagnosed glaucoma in various stages of the disease were registered in their lifetimes by the foundation. All donors agreed that at death their eyes could be immediately enucleated and committed to glaucoma research. After evaluation of the clinical data, five eyes All patients had typical glaucomatous visual field loss: one had already undergone glaucoma filtration surgery 2 years before his death.
The cup-to-disc ratio ranged between 0. In addition, we had access to two eyes with ocular hypertension without any clinical signs of glaucoma and one eye with steroid-induced glaucoma. COXprotein was completely absent in the eye with steroid-induced glaucoma Fig. To add further proof to our histologic and biochemical findings described later , we tried to assess the expression of COX-2 mRNA in paraffin-embedded tissue specimens.
We had access to two specimens obtained during eye surgery that were carefully prepared with the intention of using them for the analysis of mRNA expression. As far as possible, the specimens were processed in RNase-free conditions after enucleation of the eye. One eye with absolute end-stage POAG from an year-old woman was compared with an eye enucleated due to a small juxtapapillary malignant melanoma from a year-old man , which served as a control.
The respective ciliary processes were separated by microdissection, and afterward, RNA-extraction was performed. PGE 2 content was analyzed in the ocular aqueous humor in patients with different ocular diseases Fig. This study demonstrated a complete and selective loss of COX-2 expression in the ciliary nonpigmented epithelial layer of eyes with end-stage POAG.
This loss was anatomically highly selective. The changes in COX-2 expression were restricted to the secretory epithelium of the ciliary body, whereas COX-2 expression remained unchanged in other parts of the eye. COX-1 expression was unchanged throughout the eye. Although the results obtained from our specimens were very consistent, further investigations are needed to elucidate whether reduced COX-2 expression is a common characteristic of POAG or occurs only in a particular subtype of this disease.
In addition, this study showed that COX-2 expression in the ciliary epithelium was lost in steroid-induced glaucoma and was reduced in patients treated topically with glucocorticoids.
Moreover, aqueous humor of eyes treated topically with glucocorticoids contained significantly less PGE 2 than did control eyes. The proposed mechanism of corticosteroid-induced glaucoma includes morphologic and functional changes in the trabecular meshwork system. These changes are thought to be similar to those in POAG. Furthermore, the outflow resistance is markedly increased in eyes with POAG and steroid-induced glaucoma.
The PGE 2 concentration in the aqueous humor of glaucomatous eyes was found to be significantly lower than in control eyes.
However, the predominant prostanoid receptor in the human ciliary body is EP3. We suggest there would be a more dramatic reduction of PGE 2 if PG production in the ciliary epithelium alone could be measured. Nevertheless, our PG measurements agree with our immunohistochemical and molecular biological data and provide supportive evidence for a decreased COXexpression in POAG.
This is the first study that describes a constitutive expression of both COX isoforms in the anterior segments of normal human eyes. Neufeld et al. It was argued that COXderived PGs might play a role in the pathogenesis of glaucomatous optic neuropathy. In the anterior segments of the eye, the ability of the ciliary epithelium and the ciliary body to produce PGs has been previously described.
They suggested that this inhibition may be due to inhibition of phospholipase A 2. However, inhibition of phospholipase A 2 by glucocorticoids occurs only at very high dosages. Circadian oscillations of endogenous glucocorticoid plasma levels may well contribute to the circadian rhythm of IOP. In addition, ocular COX-2 expression appears to be regulated by glucocorticoids. Our findings further extend the concept 14 that COX-2 contributes constitutively to the physiological regulation in highly differentiated organ systems such as the central nervous system CNS , 15 16 the kidney, 17 and, now, the eye.
Analgésicos inibidores específicos da ciclooxigenase-2: avanços terapêuticos
Primary open-angle glaucoma POAG is the predominant form of chronic glaucoma, but the underlying pathologic mechanisms are largely unknown. Because prostaglandins PGs have been introduced into POAG treatment with remarkable success, this study was undertaken to investigate whether a change in the expression of the PG-synthesizing enzymes cyclooxygenase COX -1 and -2 might be involved in the pathogenesis of POAG. Expression of COX-1 and -2 was assessed by confocal laser microscopy, immunohistochemistry, Western blot analysis, and real-time RT-PCR in human eyes with different forms of glaucoma primary open-angle, angle-closure, congenital juvenile, and steroid-induced , as well as in age-matched control eyes. Additionally, PGE 2 was measured in aqueous humor by means of an enzyme-linked immunoassay as a product of COX activity. In normal eyes, ocular COX-1 and -2 expression were largely confined to the nonpigmented secretory epithelium of the ciliary body.
Abstract. It has been proposed that cyclooxygenase (COX)-1 and COX-2 subserve different physiologic functions largely because of the striking differences in their tissue expression and regulation. In animal models of inflammatory arthritis, COX-2 increases in parallel with PG production and clinical inflammation.
Analgésicos inibidores específicos da ciclooxigenase-2: avanços terapêuticos
Wilson Andrade Carvalho, M. This review aimed at discussing some current cycloxygenase biochemical aspects, which have provided the basis for the development of new analgesic and anti-inflammatory drugs. At least two COX isoforms have already been identified: COX-1, which is constitutively expressed in most tissues, and the inducible enzyme COX-2, which is primarily found in inflammatory cells and tissues. The discovery of COX-2 has enabled the development of more selective drugs to decrease inflammation without affecting COX-1 that protects stomach and kidneys and giving origin to a new generation of anti-inflammatory compounds called specific COX-2 inhibitors. Despite these potential side effects, these new drugs are being tested in different clinical conditions, especially in cancer prevention and Alzheimer's disease.
Metrics details. Cyclooxygenase COX -1 and COX-2 produce prostanoids from arachidonic acid and are thought to have important yet distinct roles in normal brain function. The major gene function affected in all genotype comparisons was 'transcriptional regulation'. Because certain anti-inflammatory and analgesic treatments are based on inhibition of COX activity, the specific alterations observed in this study further our understanding of the relationship of COX-1 and COX-2 with signaling pathways in brain and of the therapeutic and toxicologic consequences of COX inhibition. Prostaglandin H synthase, otherwise known as cyclooxygenase COX , catalyzes the first metabolic step in the transformation of arachidonic acid AA to the bioactive products prostaglandins and thromboxanes [ 1 ].
The role of cyclooxygenase-2 COX-2 in renin angiotensin physiologic processes.
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