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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 Sp1 blocks ETBR mediated cerebrovascular contractility ex vivo.A. Graphs depict concentration-response curves of 24 hrs. organ cultured (cultured) and non-cultured (Fresh) MCA segments elicited by cumulative application of S6c (ETBR specific) in the presence or absence of MitA in a dose dependent manner (Fresh n = 6, OC n = 5, 2 µM MitA n = 3, 5 µM MitA n = 4). OC vs. 2 µM MitA; not significant. OC vs. 5 µM MitA; P = 0.0004. B. Graphs show concentration-response curves of cultured and fresh MCA segments elicited by cumulative application of ET-1 in the presence or absence of MitA (Fresh n = 5, OC n = 4, 5 µM MitA n = 5). No significant difference between the groups was observed. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was performed for statistical significance.
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pone-0113624-g004: Inhibition of Sp1 blocks ETBR mediated cerebrovascular contractility ex vivo.A. Graphs depict concentration-response curves of 24 hrs. organ cultured (cultured) and non-cultured (Fresh) MCA segments elicited by cumulative application of S6c (ETBR specific) in the presence or absence of MitA in a dose dependent manner (Fresh n = 6, OC n = 5, 2 µM MitA n = 3, 5 µM MitA n = 4). OC vs. 2 µM MitA; not significant. OC vs. 5 µM MitA; P = 0.0004. B. Graphs show concentration-response curves of cultured and fresh MCA segments elicited by cumulative application of ET-1 in the presence or absence of MitA (Fresh n = 5, OC n = 4, 5 µM MitA n = 5). No significant difference between the groups was observed. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was performed for statistical significance.

Mentions: Next we asked whether the inhibition of Sp1 with MitA could restore normal vascular contractile function by preventing the increase in ETBR-mediated vasoconstriction after in vivo; stroke and in vitro; organ culture. Changes in the contractile properties of MCA were investigated using an ETBR specific agonist, S6c, in a wire myograph system. The MCAs were cultured with and without varying concentrations of MitA for 24 hours in serum-free conditions. MCAs treated with MitA showed significant reduction in the contractile responses to S6c compared to vehicle-treated MCAs (Fig. 4A). A 2 µM concentration of the inhibitor was sufficient to bring noticeable reduction in ETBR-mediated contraction. A 5 µM concentration was sufficient to bring the S6c contraction to the basal level of contraction as evidenced in fresh MCAs (P<0.05, Fig 4A). Meanwhile, up to 10 µM of MitA, contractile responses mediated by ETAR remained unaltered suggesting that Sp1 specifically interferes with the ETBR regulation (Fig 4B).


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 Sp1 blocks ETBR mediated cerebrovascular contractility ex vivo.A. Graphs depict concentration-response curves of 24 hrs. organ cultured (cultured) and non-cultured (Fresh) MCA segments elicited by cumulative application of S6c (ETBR specific) in the presence or absence of MitA in a dose dependent manner (Fresh n = 6, OC n = 5, 2 µM MitA n = 3, 5 µM MitA n = 4). OC vs. 2 µM MitA; not significant. OC vs. 5 µM MitA; P = 0.0004. B. Graphs show concentration-response curves of cultured and fresh MCA segments elicited by cumulative application of ET-1 in the presence or absence of MitA (Fresh n = 5, OC n = 4, 5 µM MitA n = 5). No significant difference between the groups was observed. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was performed for statistical significance.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4257531&req=5

pone-0113624-g004: Inhibition of Sp1 blocks ETBR mediated cerebrovascular contractility ex vivo.A. Graphs depict concentration-response curves of 24 hrs. organ cultured (cultured) and non-cultured (Fresh) MCA segments elicited by cumulative application of S6c (ETBR specific) in the presence or absence of MitA in a dose dependent manner (Fresh n = 6, OC n = 5, 2 µM MitA n = 3, 5 µM MitA n = 4). OC vs. 2 µM MitA; not significant. OC vs. 5 µM MitA; P = 0.0004. B. Graphs show concentration-response curves of cultured and fresh MCA segments elicited by cumulative application of ET-1 in the presence or absence of MitA (Fresh n = 5, OC n = 4, 5 µM MitA n = 5). No significant difference between the groups was observed. Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnett's multiple comparison test was performed for statistical significance.
Mentions: Next we asked whether the inhibition of Sp1 with MitA could restore normal vascular contractile function by preventing the increase in ETBR-mediated vasoconstriction after in vivo; stroke and in vitro; organ culture. Changes in the contractile properties of MCA were investigated using an ETBR specific agonist, S6c, in a wire myograph system. The MCAs were cultured with and without varying concentrations of MitA for 24 hours in serum-free conditions. MCAs treated with MitA showed significant reduction in the contractile responses to S6c compared to vehicle-treated MCAs (Fig. 4A). A 2 µM concentration of the inhibitor was sufficient to bring noticeable reduction in ETBR-mediated contraction. A 5 µM concentration was sufficient to bring the S6c contraction to the basal level of contraction as evidenced in fresh MCAs (P<0.05, Fig 4A). Meanwhile, up to 10 µM of MitA, contractile responses mediated by ETAR remained unaltered suggesting that Sp1 specifically interferes with the ETBR regulation (Fig 4B).

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