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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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Increased vascular smooth muscle expression of Sp1 and ETBR after MCAO and organ culture.A. Representative immunohistochemical stainings of MCA sections from MCAO rats after 48 hrs. of I/R (LMCA: non-occluded side and RMCA: occluded side) (n = 4 per group). Scale bar is 50 µm. B. Bar graphs show the statistical significance of intensity measurements of Sp1 and ETBR in figure 1A (***P<0.001, *P<0.05). C. Representative western blot shows protein levels of ETBR and Sp1 in cultured cerebral arteries at 24 hrs. (n = 4 per group). D. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in cultured cerebral arteries at 6 hrs. (n = 4 per group, **P<0.01). E. Immunohistochemical staining showing the expression and localization of Sp1 in fresh and 24 hrs. cultured MCAs (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. Mann-Whitney test was performed between two groups for statistical significance.
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pone-0113624-g001: Increased vascular smooth muscle expression of Sp1 and ETBR after MCAO and organ culture.A. Representative immunohistochemical stainings of MCA sections from MCAO rats after 48 hrs. of I/R (LMCA: non-occluded side and RMCA: occluded side) (n = 4 per group). Scale bar is 50 µm. B. Bar graphs show the statistical significance of intensity measurements of Sp1 and ETBR in figure 1A (***P<0.001, *P<0.05). C. Representative western blot shows protein levels of ETBR and Sp1 in cultured cerebral arteries at 24 hrs. (n = 4 per group). D. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in cultured cerebral arteries at 6 hrs. (n = 4 per group, **P<0.01). E. Immunohistochemical staining showing the expression and localization of Sp1 in fresh and 24 hrs. cultured MCAs (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. Mann-Whitney test was performed between two groups for statistical significance.

Mentions: MCAO was performed for 120 minutes followed by 48 hours of reperfusion. Immunohistochemical analysis was carried out to evaluate the expression of Sp1 and ETBR in the right MCA (RMCA, occluded side) and left MCA (LMCA, non-occluded side). We noted an increased Sp1 immunoreactivity, primarily in vascular smooth muscle cells of the MCA on the occluded side, correlating with the increased expression of ETBR (Fig. 1A). Fluorescence intensity measurements of the sections showed a significant increase in the expression of both proteins (Fig. 1B). An in vitro method, where the cerebral arteries were cultured in serum-free medium for a defined period of time, was employed to further study the regulatory mechanism of the receptor. The method is shown to mimic several of the vascular changes during focal cerebral ischemia [12]. This method is in general used for detailed analysis of molecular alterations in the vessel walls. Similar to the observations in cerebral arteries after MCAO, western blot analysis showed a notable increase in the expression of Sp1 and ETBR in cerebral arteries cultured for 24 hours (Fig. 1C). We noted two reactive bands in the blots for Sp1 and ETBR, indicating that the proteins might have undergone post-translational modification or are expressed in their respective isoforms [13], [14]. Quantitative PCR analysis after 6 hours culturing showed a significant increase in the ETBR mRNA and an inverse correlation between Sp1 mRNA and protein was observed (P<0.01, Fig. 1D). With immunohistochemistry we noted increased immunoreactivity for Sp1 in the smooth muscle cell layer of cultured MCAs supporting the western blot observation (Fig. 1E and fig. S1). Sp1 was primarily localized in the nucleus of the vascular smooth muscle cells. Such changes in the Sp1 protein expression without increased levels of mRNA indicate that Sp1 is probably regulated by cap-independent translation mechanisms [15].


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)

Increased vascular smooth muscle expression of Sp1 and ETBR after MCAO and organ culture.A. Representative immunohistochemical stainings of MCA sections from MCAO rats after 48 hrs. of I/R (LMCA: non-occluded side and RMCA: occluded side) (n = 4 per group). Scale bar is 50 µm. B. Bar graphs show the statistical significance of intensity measurements of Sp1 and ETBR in figure 1A (***P<0.001, *P<0.05). C. Representative western blot shows protein levels of ETBR and Sp1 in cultured cerebral arteries at 24 hrs. (n = 4 per group). D. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in cultured cerebral arteries at 6 hrs. (n = 4 per group, **P<0.01). E. Immunohistochemical staining showing the expression and localization of Sp1 in fresh and 24 hrs. cultured MCAs (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. Mann-Whitney test was performed between two groups for statistical significance.
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pone-0113624-g001: Increased vascular smooth muscle expression of Sp1 and ETBR after MCAO and organ culture.A. Representative immunohistochemical stainings of MCA sections from MCAO rats after 48 hrs. of I/R (LMCA: non-occluded side and RMCA: occluded side) (n = 4 per group). Scale bar is 50 µm. B. Bar graphs show the statistical significance of intensity measurements of Sp1 and ETBR in figure 1A (***P<0.001, *P<0.05). C. Representative western blot shows protein levels of ETBR and Sp1 in cultured cerebral arteries at 24 hrs. (n = 4 per group). D. Quantitative PCR analysis of ETBR and Sp1 mRNA levels in cultured cerebral arteries at 6 hrs. (n = 4 per group, **P<0.01). E. Immunohistochemical staining showing the expression and localization of Sp1 in fresh and 24 hrs. cultured MCAs (n = 4 per group). Scale bar is 50 µm. Statistics: Values are presented as mean ± S.E.M. Mann-Whitney test was performed between two groups for statistical significance.
Mentions: MCAO was performed for 120 minutes followed by 48 hours of reperfusion. Immunohistochemical analysis was carried out to evaluate the expression of Sp1 and ETBR in the right MCA (RMCA, occluded side) and left MCA (LMCA, non-occluded side). We noted an increased Sp1 immunoreactivity, primarily in vascular smooth muscle cells of the MCA on the occluded side, correlating with the increased expression of ETBR (Fig. 1A). Fluorescence intensity measurements of the sections showed a significant increase in the expression of both proteins (Fig. 1B). An in vitro method, where the cerebral arteries were cultured in serum-free medium for a defined period of time, was employed to further study the regulatory mechanism of the receptor. The method is shown to mimic several of the vascular changes during focal cerebral ischemia [12]. This method is in general used for detailed analysis of molecular alterations in the vessel walls. Similar to the observations in cerebral arteries after MCAO, western blot analysis showed a notable increase in the expression of Sp1 and ETBR in cerebral arteries cultured for 24 hours (Fig. 1C). We noted two reactive bands in the blots for Sp1 and ETBR, indicating that the proteins might have undergone post-translational modification or are expressed in their respective isoforms [13], [14]. Quantitative PCR analysis after 6 hours culturing showed a significant increase in the ETBR mRNA and an inverse correlation between Sp1 mRNA and protein was observed (P<0.01, Fig. 1D). With immunohistochemistry we noted increased immunoreactivity for Sp1 in the smooth muscle cell layer of cultured MCAs supporting the western blot observation (Fig. 1E and fig. S1). Sp1 was primarily localized in the nucleus of the vascular smooth muscle cells. Such changes in the Sp1 protein expression without increased levels of mRNA indicate that Sp1 is probably regulated by cap-independent translation mechanisms [15].

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