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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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Sp1 inhibition downregulates ETBR expression and vascular contractility after MCAO.A. Representative ETBR and Sp1 immunostainings in the MCA on the occluded side (RMCA). Stainings show changes in the expression levels after treatment with MitA compared to vehicle (n = 4 per group). B. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of S6c (ETBR specific) of MitA and vehicle treated rats after MCAO. Vehicle vs. RMCA MitA, P<0.0001 and LMCA Vehicle vs. RMCA MitA, not significant. C. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of ET-1 of MitA and vehicle treated rats after MCAO. No significant difference between the groups (Vehicle, n = 4 and MitA, n = 6). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnet's multiple comparison test was performed to obtain statistical significance.
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pone-0113624-g005: Sp1 inhibition downregulates ETBR expression and vascular contractility after MCAO.A. Representative ETBR and Sp1 immunostainings in the MCA on the occluded side (RMCA). Stainings show changes in the expression levels after treatment with MitA compared to vehicle (n = 4 per group). B. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of S6c (ETBR specific) of MitA and vehicle treated rats after MCAO. Vehicle vs. RMCA MitA, P<0.0001 and LMCA Vehicle vs. RMCA MitA, not significant. C. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of ET-1 of MitA and vehicle treated rats after MCAO. No significant difference between the groups (Vehicle, n = 4 and MitA, n = 6). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnet's multiple comparison test was performed to obtain statistical significance.

Mentions: The findings from MitA inhibition and Sp1-regulated ETBR vasoconstriction of rat MCAs in vitro were then validated in experimental stroke. A profound decrease in the vascular smooth muscle ETBR receptor expression was observed after treatment with MitA (Fig. 5A). Furthermore, MitA treatment significantly prevented the ischemia-induced ETBR mediated vasoconstriction compared to vehicle treated rats (P<0.001, Fig 5B). In addition, confirming the in vitro data, MitA treatment did not significantly alter the contractile function mediated by the ETAR in MitA treated rats compared to vehicle treated rats indicating that MitA might be specifically interfering with the ETBR transcription mechanism (Fig. 5C). Taken together these results indicate that MitA could serve as a potential pharmacological agent to block ETBR mediated cerebrovascular contraction after I/R.


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)

Sp1 inhibition downregulates ETBR expression and vascular contractility after MCAO.A. Representative ETBR and Sp1 immunostainings in the MCA on the occluded side (RMCA). Stainings show changes in the expression levels after treatment with MitA compared to vehicle (n = 4 per group). B. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of S6c (ETBR specific) of MitA and vehicle treated rats after MCAO. Vehicle vs. RMCA MitA, P<0.0001 and LMCA Vehicle vs. RMCA MitA, not significant. C. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of ET-1 of MitA and vehicle treated rats after MCAO. No significant difference between the groups (Vehicle, n = 4 and MitA, n = 6). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnet's multiple comparison test was performed to obtain statistical significance.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4257531&req=5

pone-0113624-g005: Sp1 inhibition downregulates ETBR expression and vascular contractility after MCAO.A. Representative ETBR and Sp1 immunostainings in the MCA on the occluded side (RMCA). Stainings show changes in the expression levels after treatment with MitA compared to vehicle (n = 4 per group). B. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of S6c (ETBR specific) of MitA and vehicle treated rats after MCAO. Vehicle vs. RMCA MitA, P<0.0001 and LMCA Vehicle vs. RMCA MitA, not significant. C. Graphs depict concentration-response curves of the MCA segments elicited by cumulative application of ET-1 of MitA and vehicle treated rats after MCAO. No significant difference between the groups (Vehicle, n = 4 and MitA, n = 6). Statistics: Values are presented as mean ± S.E.M. One-way ANOVA and Dunnet's multiple comparison test was performed to obtain statistical significance.
Mentions: The findings from MitA inhibition and Sp1-regulated ETBR vasoconstriction of rat MCAs in vitro were then validated in experimental stroke. A profound decrease in the vascular smooth muscle ETBR receptor expression was observed after treatment with MitA (Fig. 5A). Furthermore, MitA treatment significantly prevented the ischemia-induced ETBR mediated vasoconstriction compared to vehicle treated rats (P<0.001, Fig 5B). In addition, confirming the in vitro data, MitA treatment did not significantly alter the contractile function mediated by the ETAR in MitA treated rats compared to vehicle treated rats indicating that MitA might be specifically interfering with the ETBR transcription mechanism (Fig. 5C). Taken together these results indicate that MitA could serve as a potential pharmacological agent to block ETBR mediated cerebrovascular contraction after I/R.

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