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Regulatory mechanism of endothelin receptor B in the cerebral arteries after focal cerebral ischemia.

Grell AS, Thigarajah R, Edvinsson L, Samraj AK - PLoS ONE (2014)

Bottom Line: Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels.It also significantly reduced the ETBR mediated cerebrovascular contractility.The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Experimental Research, Glostrup research institute, University of Copenhagen, Glostrup, Denmark.

ABSTRACT

Background and purpose: Increased expression of endothelin receptor type B (ETBR), a vasoactive receptor, has recently been implied in the reduced cerebral blood flow and exacerbated neuronal damage after ischemia-reperfusion (I/R). The study explores the regulatory mechanisms of ETBR to identify drug targets to restore normal cerebral artery contractile function as part of successful neuroprotective therapy.

Methods: We have employed in vitro methods on human and rat cerebral arteries to study the regulatory mechanisms and the efficacy of target selective inhibitor, Mithramycin A (MitA), to block the ETBR mediated contractile properties. Later, middle cerebral artery occluded (MCAO) rats were used to substantiate the observations. Quantative PCR, immunohistochemistry, western blot and wire myograph methods were employed to study the expression and contractile properties of cerebral arteries.

Results: Increased expression of specificity protein (Sp1) was observed in human and rat cerebral arteries after organ culture, strongly correlating with the ETBR upregulation. Similar observations were made in MCAO rats. Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels. It also significantly reduced the ETBR mediated cerebrovascular contractility. Detailed analysis indicated that ERK1/2 mediated phosphorylation of Sp1 might be essential for ETBR transcription.

Conclusion: Transcription factor Sp1 regulates the ETBR mediated vasoconstriction in focal cerebral ischemia via MEK-ERK signaling, which is also conserved in humans. The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

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Inhibition of transcription factor Sp1 blocks ETBR upregulation ex vivo in rat and human cerebral arteries.A. Representative western blot for Sp1 and ETBR protein levels in cultured cerebral arteries with and without 5 µM MitA at 24 hrs. B and C. Bar graphs show the statistical significance of protein expression and inhibition of Sp1 and ETBR in figure 2A (Fresh n = 7, OC n = 7, MitA n = 6, **P<0.01, *P<0.05). D and E. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in fresh and 24 hrs. cultured MCA segments with and without MitA treatment (n = 6 per group, **P<0.01, *P<0.05). F. Representative immunohistochemical stainings of MCAs cultured in the presence or absence of MitA at 24 hrs. (n = 4 per group). Scale bar is 50 µm. G. Immunohistochemical stainings of cultured human cerebral arteries show ETBR and Sp1 immunoreactivity with and without MitA treatment at 24 hrs. (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was done for figure B, C and D while Mann-Whitney test was done for figure E for statistical significance.
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pone-0113624-g002: Inhibition of transcription factor Sp1 blocks ETBR upregulation ex vivo in rat and human cerebral arteries.A. Representative western blot for Sp1 and ETBR protein levels in cultured cerebral arteries with and without 5 µM MitA at 24 hrs. B and C. Bar graphs show the statistical significance of protein expression and inhibition of Sp1 and ETBR in figure 2A (Fresh n = 7, OC n = 7, MitA n = 6, **P<0.01, *P<0.05). D and E. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in fresh and 24 hrs. cultured MCA segments with and without MitA treatment (n = 6 per group, **P<0.01, *P<0.05). F. Representative immunohistochemical stainings of MCAs cultured in the presence or absence of MitA at 24 hrs. (n = 4 per group). Scale bar is 50 µm. G. Immunohistochemical stainings of cultured human cerebral arteries show ETBR and Sp1 immunoreactivity with and without MitA treatment at 24 hrs. (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was done for figure B, C and D while Mann-Whitney test was done for figure E for statistical significance.

Mentions: DNA foot printing studies have shown that Sp1 competitively binds to GC rich motifs of various promoters [16]. MitA has been shown to selectively displace Sp1 from its binding site to inhibit the transcriptional activity [17],[18]. Therefore, the effect of Sp1 inhibition on ETBR upregulation was investigated by culturing cerebral arteries in the presence or absence of MitA. Western blot analysis showed a notable reduction in the ETBR expression of cerebral arteries cultured with MitA (5 µM). Among the two reactive bands for ETBR, expression of one band was notably reduced indicating a possible isoform-specific inhibition by MitA. Similar trend was observed in the expression pattern of Sp1 in the presence of MitA (Fig. 2A). Intensity measurements of the protein expression levels of Sp1 and ETBR are significantly altered in the presence or absence of MitA after organ culture (P<0.05) (Fig. 2B and C). Quantitative PCR analysis showed a significant reduction in the ETBR mRNA upon treatment (P<0.05) while the expression of Sp1 mRNA remained unaffected (Fig. 2D and E). In conjunction, immunohistochemical analysis showed a marked reduction in ETBR expression in MCAs incubated with MitA (Fig. 2F). No significant reduction in the expression of Sp1 was observed as MitA is expected to competitively displace Sp1 from the DNA not the protein expression. However, when compared to MCAs cultured in absence of the inhibitor, MitA-treated arteries showed a diffused Sp1 staining pattern, which probably is due to its displacement and subsequent degradation of the protein (Fig. 2F).


Regulatory mechanism of endothelin receptor B in the cerebral arteries after focal cerebral ischemia.

Grell AS, Thigarajah R, Edvinsson L, Samraj AK - PLoS ONE (2014)

Inhibition of transcription factor Sp1 blocks ETBR upregulation ex vivo in rat and human cerebral arteries.A. Representative western blot for Sp1 and ETBR protein levels in cultured cerebral arteries with and without 5 µM MitA at 24 hrs. B and C. Bar graphs show the statistical significance of protein expression and inhibition of Sp1 and ETBR in figure 2A (Fresh n = 7, OC n = 7, MitA n = 6, **P<0.01, *P<0.05). D and E. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in fresh and 24 hrs. cultured MCA segments with and without MitA treatment (n = 6 per group, **P<0.01, *P<0.05). F. Representative immunohistochemical stainings of MCAs cultured in the presence or absence of MitA at 24 hrs. (n = 4 per group). Scale bar is 50 µm. G. Immunohistochemical stainings of cultured human cerebral arteries show ETBR and Sp1 immunoreactivity with and without MitA treatment at 24 hrs. (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was done for figure B, C and D while Mann-Whitney test was done for figure E for statistical significance.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4257531&req=5

pone-0113624-g002: Inhibition of transcription factor Sp1 blocks ETBR upregulation ex vivo in rat and human cerebral arteries.A. Representative western blot for Sp1 and ETBR protein levels in cultured cerebral arteries with and without 5 µM MitA at 24 hrs. B and C. Bar graphs show the statistical significance of protein expression and inhibition of Sp1 and ETBR in figure 2A (Fresh n = 7, OC n = 7, MitA n = 6, **P<0.01, *P<0.05). D and E. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in fresh and 24 hrs. cultured MCA segments with and without MitA treatment (n = 6 per group, **P<0.01, *P<0.05). F. Representative immunohistochemical stainings of MCAs cultured in the presence or absence of MitA at 24 hrs. (n = 4 per group). Scale bar is 50 µm. G. Immunohistochemical stainings of cultured human cerebral arteries show ETBR and Sp1 immunoreactivity with and without MitA treatment at 24 hrs. (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was done for figure B, C and D while Mann-Whitney test was done for figure E for statistical significance.
Mentions: DNA foot printing studies have shown that Sp1 competitively binds to GC rich motifs of various promoters [16]. MitA has been shown to selectively displace Sp1 from its binding site to inhibit the transcriptional activity [17],[18]. Therefore, the effect of Sp1 inhibition on ETBR upregulation was investigated by culturing cerebral arteries in the presence or absence of MitA. Western blot analysis showed a notable reduction in the ETBR expression of cerebral arteries cultured with MitA (5 µM). Among the two reactive bands for ETBR, expression of one band was notably reduced indicating a possible isoform-specific inhibition by MitA. Similar trend was observed in the expression pattern of Sp1 in the presence of MitA (Fig. 2A). Intensity measurements of the protein expression levels of Sp1 and ETBR are significantly altered in the presence or absence of MitA after organ culture (P<0.05) (Fig. 2B and C). Quantitative PCR analysis showed a significant reduction in the ETBR mRNA upon treatment (P<0.05) while the expression of Sp1 mRNA remained unaffected (Fig. 2D and E). In conjunction, immunohistochemical analysis showed a marked reduction in ETBR expression in MCAs incubated with MitA (Fig. 2F). No significant reduction in the expression of Sp1 was observed as MitA is expected to competitively displace Sp1 from the DNA not the protein expression. However, when compared to MCAs cultured in absence of the inhibitor, MitA-treated arteries showed a diffused Sp1 staining pattern, which probably is due to its displacement and subsequent degradation of the protein (Fig. 2F).

Bottom Line: Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels.It also significantly reduced the ETBR mediated cerebrovascular contractility.The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Experimental Research, Glostrup research institute, University of Copenhagen, Glostrup, Denmark.

ABSTRACT

Background and purpose: Increased expression of endothelin receptor type B (ETBR), a vasoactive receptor, has recently been implied in the reduced cerebral blood flow and exacerbated neuronal damage after ischemia-reperfusion (I/R). The study explores the regulatory mechanisms of ETBR to identify drug targets to restore normal cerebral artery contractile function as part of successful neuroprotective therapy.

Methods: We have employed in vitro methods on human and rat cerebral arteries to study the regulatory mechanisms and the efficacy of target selective inhibitor, Mithramycin A (MitA), to block the ETBR mediated contractile properties. Later, middle cerebral artery occluded (MCAO) rats were used to substantiate the observations. Quantative PCR, immunohistochemistry, western blot and wire myograph methods were employed to study the expression and contractile properties of cerebral arteries.

Results: Increased expression of specificity protein (Sp1) was observed in human and rat cerebral arteries after organ culture, strongly correlating with the ETBR upregulation. Similar observations were made in MCAO rats. Treatment with MitA, a Sp1 specific inhibitor, significantly downregulated the ETBR mRNA and protein levels. It also significantly reduced the ETBR mediated cerebrovascular contractility. Detailed analysis indicated that ERK1/2 mediated phosphorylation of Sp1 might be essential for ETBR transcription.

Conclusion: Transcription factor Sp1 regulates the ETBR mediated vasoconstriction in focal cerebral ischemia via MEK-ERK signaling, which is also conserved in humans. The results show that MitA can effectively be used to block ETBR mediated vasoconstriction as a supplement to an existing ischemic stroke therapy.

Show MeSH
Related in: MedlinePlus