Limits...
Enhanced prostacyclin synthesis by adenoviral gene transfer reduced glial activation and ameliorated dopaminergic dysfunction in hemiparkinsonian rats.

Tsai MJ, Weng CF, Yu NC, Liou DY, Kuo FS, Huang MC, Huang WC, Tam K, Shyue SK, Cheng H - Oxid Med Cell Longev (2013)

Bottom Line: Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation.Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats.Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

View Article: PubMed Central - PubMed

Affiliation: Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei 11221, Taiwan.

ABSTRACT
Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinson's disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

Show MeSH

Related in: MedlinePlus

CAY10449 and AdPGIS transduction on MTT reduction in mixed glial cultures. CAY10449 is a high affinity ligand and functional antagonist for the human IP (prostacyclin) receptor. CAY10449 (500 nM) was added to cultured cells at 2 hr after AdPGIS transduction. Data were means ± SEM from 4 independent cultures done in duplicate. aP < 0.01 AdPGIS versus Control; bP < 0.05 AdPGIS + CAY10449 versus AdPGIS.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3649752&req=5

fig3: CAY10449 and AdPGIS transduction on MTT reduction in mixed glial cultures. CAY10449 is a high affinity ligand and functional antagonist for the human IP (prostacyclin) receptor. CAY10449 (500 nM) was added to cultured cells at 2 hr after AdPGIS transduction. Data were means ± SEM from 4 independent cultures done in duplicate. aP < 0.01 AdPGIS versus Control; bP < 0.05 AdPGIS + CAY10449 versus AdPGIS.

Mentions: To examine the effects of enhanced prostacyclin synthesis on cell proliferation, subconfluent glial cells were transduced with Ad-PGIS for 2 days. Tritiated thymidine was added to cultures 10 hr before cell harvest. Table 1 shows that overexpression of PGIS in cultured glial cells concurrently enhanced prostacyclin production (indicated by level of 6-keto-PGF1α, the product of PGI2 hydrolysis) and inhibited astroglial proliferation (indicated by thymidine incorporation). To examine whether the effect of overexpressing PGIS on cell proliferation was mediated via IP (prostacyclin) receptor, CAY10449 was added to mixed glial cells at 2 hr after AdPGIS transduction. As shown in Figure 2, AdPGIS transduction reduced degree of MTT reduction in mixed glial cultures. The high-affinity IP antagonist, CAY10449, at 500 nM partially but significantly abrogated prostacyclin effect on cell proliferation. LPS is a powerful immune challenge. LPS treatment has been shown to induce release of cytokines, NO, and proinflammatory factors from macrophages [34, 35]. Effect of overexpressing PGIS on LPS stimulation was examined in mixed glial cultures. Confluent glial cultures were transduced with AdGFP or AdPGIS. Three days later, cells were further treated with LPS at a dose of 600 ng/mL for 2 days. In our previous paper [20], we used 100 ng/mL LPS to stimulate neuron/glial cultures. However, LPS failed to affect mixed glial cells at 100 ng/mL and required higher concentration of LPS for effective cell stimulation. As shown in Figure 3, Ad-PGIS transduction enhanced the expression of PGIS whereas iNOS and COX-2 levels were barely detectable. By contrast, LPS treatment induced increase of iNOS and COX-2 expression in control and AdGFP-infected cells. AdPGIS transduction effectively reduced LPS-induced COX-2 and iNOS levels. Concurrently, LPS-stimulated nitrite releases were significantly inhibited in AdPGIS-transduced cells (Figure 3(b)) compared to control and Ad-PGK-infected cells. This indicates that enhanced prostacyclin synthesis in PGIS-overexpressing cells significantly inhibited iNOS expression and NO production.


Enhanced prostacyclin synthesis by adenoviral gene transfer reduced glial activation and ameliorated dopaminergic dysfunction in hemiparkinsonian rats.

Tsai MJ, Weng CF, Yu NC, Liou DY, Kuo FS, Huang MC, Huang WC, Tam K, Shyue SK, Cheng H - Oxid Med Cell Longev (2013)

CAY10449 and AdPGIS transduction on MTT reduction in mixed glial cultures. CAY10449 is a high affinity ligand and functional antagonist for the human IP (prostacyclin) receptor. CAY10449 (500 nM) was added to cultured cells at 2 hr after AdPGIS transduction. Data were means ± SEM from 4 independent cultures done in duplicate. aP < 0.01 AdPGIS versus Control; bP < 0.05 AdPGIS + CAY10449 versus AdPGIS.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649752&req=5

fig3: CAY10449 and AdPGIS transduction on MTT reduction in mixed glial cultures. CAY10449 is a high affinity ligand and functional antagonist for the human IP (prostacyclin) receptor. CAY10449 (500 nM) was added to cultured cells at 2 hr after AdPGIS transduction. Data were means ± SEM from 4 independent cultures done in duplicate. aP < 0.01 AdPGIS versus Control; bP < 0.05 AdPGIS + CAY10449 versus AdPGIS.
Mentions: To examine the effects of enhanced prostacyclin synthesis on cell proliferation, subconfluent glial cells were transduced with Ad-PGIS for 2 days. Tritiated thymidine was added to cultures 10 hr before cell harvest. Table 1 shows that overexpression of PGIS in cultured glial cells concurrently enhanced prostacyclin production (indicated by level of 6-keto-PGF1α, the product of PGI2 hydrolysis) and inhibited astroglial proliferation (indicated by thymidine incorporation). To examine whether the effect of overexpressing PGIS on cell proliferation was mediated via IP (prostacyclin) receptor, CAY10449 was added to mixed glial cells at 2 hr after AdPGIS transduction. As shown in Figure 2, AdPGIS transduction reduced degree of MTT reduction in mixed glial cultures. The high-affinity IP antagonist, CAY10449, at 500 nM partially but significantly abrogated prostacyclin effect on cell proliferation. LPS is a powerful immune challenge. LPS treatment has been shown to induce release of cytokines, NO, and proinflammatory factors from macrophages [34, 35]. Effect of overexpressing PGIS on LPS stimulation was examined in mixed glial cultures. Confluent glial cultures were transduced with AdGFP or AdPGIS. Three days later, cells were further treated with LPS at a dose of 600 ng/mL for 2 days. In our previous paper [20], we used 100 ng/mL LPS to stimulate neuron/glial cultures. However, LPS failed to affect mixed glial cells at 100 ng/mL and required higher concentration of LPS for effective cell stimulation. As shown in Figure 3, Ad-PGIS transduction enhanced the expression of PGIS whereas iNOS and COX-2 levels were barely detectable. By contrast, LPS treatment induced increase of iNOS and COX-2 expression in control and AdGFP-infected cells. AdPGIS transduction effectively reduced LPS-induced COX-2 and iNOS levels. Concurrently, LPS-stimulated nitrite releases were significantly inhibited in AdPGIS-transduced cells (Figure 3(b)) compared to control and Ad-PGK-infected cells. This indicates that enhanced prostacyclin synthesis in PGIS-overexpressing cells significantly inhibited iNOS expression and NO production.

Bottom Line: Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation.Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats.Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

View Article: PubMed Central - PubMed

Affiliation: Neural Regeneration Laboratory, Department of Neurosurgery, Neurological Institute, Taipei Veterans General Hospital, Taipei 11221, Taiwan.

ABSTRACT
Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinson's disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

Show MeSH
Related in: MedlinePlus