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Differential metabolism of dihomo-gamma-linolenic acid and arachidonic acid by cyclo-oxygenase-1 and cyclo-oxygenase-2: implications for cellular synthesis of prostaglandin E1 and prostaglandin E2.

ABSTRACT: Prostaglandin (PG) E(1) has been shown to possess anti-inflammatory properties and to modulate vascular reactivity. These activities are sometimes distinct from those of PGE(2), suggesting that endogenously produced PGE(1) may have some beneficial therapeutic effects compared with PGE(2). Increasing the endogenous formation of PGE(1) requires optimization of two separate processes, namely, enrichment of cellular lipids with dihomo-gamma-linolenic acid (20:3 n-6; DGLA) and effective cyclo-oxygenase-dependent oxygenation of substrate DGLA relative to arachidonic acid (AA; 20:4 n-6). DGLA and AA had similar affinities (K(m) values) and maximal reaction rates (V(max)) for cyclo-oxygenase-2 (COX-2), whereas AA was metabolized preferentially by cyclo-oxygenase-1 (COX-1). To overcome the kinetic preference of COX-1 for AA, CP-24879, a mixed Delta(5)/Delta(6) desaturase inhibitor, was used to enhance preferential accumulation of DGLA over AA in cells cultured in the presence of precursor gamma-linolenic acid (18:3 n-6). This protocol was tested in two cell lines and both yielded a DGLA/AA ratio of approx. 2.8 in the total cellular lipids. From the enzyme kinetic data, it was calculated that this ratio should offset the preference of COX-1 for AA over DGLA. PGE(1) synthesis in the DGLA-enriched cells was increased concurrent with a decline in PGE(2) formation. Nevertheless, PGE(1) synthesis was still substantially lower than that of PGE(2). It appears that employing a dietary or a combined dietary/pharmacological paradigm to augment the cellular ratio of DGLA/AA is not an effective route to enhance endogenous synthesis of PGE(1) over PGE(2), at least in cells/tissues where COX-1 predominates over COX-2.

SUBMITTER: Levin G

PROVIDER: S-EPMC1222686 | biostudies-other | 2002 Jul

REPOSITORIES: biostudies-other

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Project description:BackgroundThe clinical features of keloids consist of aberrant proliferation, secretion, differentiation and apoptosis of keloid dermis-derived fibroblasts (KFBs). Notably, the apoptosis rate of KFBs is lower than the proliferation rate. Though the anti-fibrotic effect of adipose-derived stem cells (ADSCs) on keloids has become a hot topic of research, the exact anti-fibrotic mechanism of the paracrine effect remains unclear. This study aimed to find out how the conditioned medium of ADSCs (ADSC-CM) exerts an anti-fibrotic effect in KFBs.MethodsKFBs and ADSCs were extracted and cultured. Then, ADSC-CM was prepared. Whether ADSC-CM could inhibit KFB growth and induce apoptosis was verified by the use of a cell counting kit-8, an 5-Ethynyl-2-deoxyuridine (Edu) kit and flow cytometry. The expressions of cyclooxygenase-1 (COX-1), COX-2, caspase 3 and B-cell lymphoma-2 (Bcl-2) in ADSC-CM-cultured KFBs were tested by real-time PCR and western blotting. To clarify the role of COX-2 in ADSC-CM-induced KFB apoptosis, a specific COX-2 inhibitor, celecoxib, was applied to KFBs cultured in ADSC-CM. Moreover, we tested the production of arachidonic acid (AA) and prostaglandin E2 (PGE2) by ELISA. Then, we established a keloid transplantation model in a nude mouse to validate the therapeutic effect in vivo.ResultsThe proliferation ability of KFBs cultured in ADSC-CM was found to be weakened and apoptosis was significantly increased. Caspase 3 expression was significantly upregulated and Bcl-2 was downregulated in ADSC-CM-cultured KFBs. Furthermore, ADSC-CM strikingly elevated COX-2 mRNA and protein expressions, but COX-1 expression was unaltered. COX-2 inhibitors reduced ADSC-CM-induced apoptosis. Additionally, COX-2 inhibition blocked the elevation of caspase 3 and reversed the decrease in Bcl-2 expression. ADSC-CM increased PGE2 levels by 1.5-fold and this effect was restrained by COX-2 inhibition. In the nude mouse model, expressions of AA, COX-2 and PGE2 were higher in the translated keloid tissues after ADSC-CM injection than in the controls.ConclusionsWe showed activation of the COX-2/PGE2 cascade in KFBs in response to ADSC-CM. By employing a specific COX-2 inhibitor, COX-2/PGE2 cascade activation played a crucial role in mediating the ADSC-CM-induced KFB apoptosis and anti-proliferation effects.

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Differential metabolism of dihomo-gamma-linolenic acid and arachidonic acid by cyclo-oxygenase-1 and cyclo-oxygenase-2: implications for cellular synthesis of prostaglandin E1 and prostaglandin E2.

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