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tPA Deficiency in Mice Leads to Rearrangement in the Cerebrovascular Tree and Cerebroventricular Malformations.

Stefanitsch C, Lawrence AL, Olverling A, Nilsson I, Fredriksson L - Front Cell Neurosci (2015)

Bottom Line: Our analysis demonstrates that life-long deficiency of tPA is associated with rearrangements in the cerebrovascular tree, including a reduction in the number of vascular smooth-muscle cell covered, large diameter, vessels and a decrease in vessel-associated PDGFRα expression as compared to wild-type (WT) littermate controls.In addition, we found that ablation of tPA results in an increased number of ERG-positive endothelial cells and increased junctional localization of the tight junction protein ZO1.In addition, we found that tPA (-/-) mice displayed mild cerebral ventricular malformations, a feature previously associated with ablation of PDGF-C, thereby providing an in vivo link between tPA and PDGF signaling in central nervous system (CNS) development.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Division of Vascular Biology, Karolinska Institutet Stockholm, Sweden.

ABSTRACT
The serine protease tissue-type plasminogen activator (tPA) is used as a thrombolytic agent in the management of ischemic stroke, but concerns for hemorrhagic conversion greatly limits the number of patients that receive this treatment. It has been suggested that the bleeding complications associated with thrombolytic tPA may be due to unanticipated roles of tPA in the brain. Recent work has suggested tPA regulation of neurovascular barrier integrity, mediated via platelet derived growth factor (PDGF)-C/PDGF receptor-α (PDGFRα) signaling, as a possible molecular mechanism affecting the outcome of stroke. To better understand the role of tPA in neurovascular regulation we conducted a detailed analysis of the cerebrovasculature in brains from adult tPA deficient (tPA(-/-) ) mice. Our analysis demonstrates that life-long deficiency of tPA is associated with rearrangements in the cerebrovascular tree, including a reduction in the number of vascular smooth-muscle cell covered, large diameter, vessels and a decrease in vessel-associated PDGFRα expression as compared to wild-type (WT) littermate controls. In addition, we found that ablation of tPA results in an increased number of ERG-positive endothelial cells and increased junctional localization of the tight junction protein ZO1. This is intriguing since ERG is an endothelial transcription factor implicated in regulation of vascular integrity. Based on these results, we propose that the protection of barrier properties seen utilizing these tPA (-/-) mice might be due, at least in part, to these cerebrovascular rearrangements. In addition, we found that tPA (-/-) mice displayed mild cerebral ventricular malformations, a feature previously associated with ablation of PDGF-C, thereby providing an in vivo link between tPA and PDGF signaling in central nervous system (CNS) development. Taken together, the data presented here will advance our understanding of the role of tPA within the CNS and in regulation of cerebrovascular permeability.

No MeSH data available.


Related in: MedlinePlus

Increased number of ERG+ endothelial cells and enhanced tight junction staining in tPA−/− mice. (A) Immunofluorescent stainings of murine brain sections from tPA−/− mice (n = 5) and WT littermate controls (WT, n = 5) with antibodies for the endothelial transcription factor ERG showed what appeared to be increased number of endothelial cells in tPA−/− mice as compared to WT littermate controls. (B–C) This was confirmed through quantification of the number of ERG+ nuclei from four maximum intensity confocal Z-stacks per animal. This revealed that there was significantly more ERG-positive cells in tPA−/− brains compared to WT littermate controls (B), also when normalized to CD31 staining as quantified by pixel intensity (arbitrary units, A.U.) (C). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from two independent staining experiments. (D) Staining with antibodies against the tight junction protein ZO1 illustrated increased junctional expression in cerebral vessels in tPA−/− mice as compared to WT littermate controls. The images display maximum intensity projections generated from confocal Z-stacks (22 μm). Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test and **P < 0.01 relative to control. Scale bars (A,D) 20 μm.
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Figure 2: Increased number of ERG+ endothelial cells and enhanced tight junction staining in tPA−/− mice. (A) Immunofluorescent stainings of murine brain sections from tPA−/− mice (n = 5) and WT littermate controls (WT, n = 5) with antibodies for the endothelial transcription factor ERG showed what appeared to be increased number of endothelial cells in tPA−/− mice as compared to WT littermate controls. (B–C) This was confirmed through quantification of the number of ERG+ nuclei from four maximum intensity confocal Z-stacks per animal. This revealed that there was significantly more ERG-positive cells in tPA−/− brains compared to WT littermate controls (B), also when normalized to CD31 staining as quantified by pixel intensity (arbitrary units, A.U.) (C). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from two independent staining experiments. (D) Staining with antibodies against the tight junction protein ZO1 illustrated increased junctional expression in cerebral vessels in tPA−/− mice as compared to WT littermate controls. The images display maximum intensity projections generated from confocal Z-stacks (22 μm). Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test and **P < 0.01 relative to control. Scale bars (A,D) 20 μm.

Mentions: In order to characterize whether the cerebrovascular changes seen in tPA−/− mice were associated with altered number of endothelial cells we conducted immunofluorescent stainings using antibodies against the endothelial transcription factor ERG. Unexpectedly, we found an apparent increase in the number of ERG-positive cells in the brains of tPA−/− mice (n = 5) relative to WT littermate controls (n = 5; Figure 2A). This was confirmed by quantification of the number of ERG-positive cells, showing a significant (P < 0.01) overall increase in the tPA−/− mice (20.2 ± 1.2 ERG+ cells/field of view) compared to WT littermate controls (15.2 ± 0.6 ERG+ cells/field of view; Figure 2B). To ensure that the increase of ERG-positive cells was not due to an increase in the number of small vessels in tPA−/− brains, we normalized the number of ERG+ cells to the amount of CD31 staining. This verified that there were significantly more (P < 0.01) ERG+ cells in the cerebral vessels of tPA−/− mice (149 ± 11% of WT; Figure 2C). This was particularly interesting since ERG has been implicated in vascular development, control of vascular permeability and maintenance of the integrity of the endothelial junctions (Randi et al., 2009; Birdsey et al., 2015). We therefore investigated the appearance of endothelial tight junctions in tPA−/− mice by immunofluorescent stainings using antibodies against the tight junction protein ZO1. We found that ZO1 appeared to show an increased junctional localization in vessels in tPA−/− brains compared to WT littermate controls (Figure 2D), suggesting that tPA−/− mice might have tighter endothelial cell-cell junctions.


tPA Deficiency in Mice Leads to Rearrangement in the Cerebrovascular Tree and Cerebroventricular Malformations.

Stefanitsch C, Lawrence AL, Olverling A, Nilsson I, Fredriksson L - Front Cell Neurosci (2015)

Increased number of ERG+ endothelial cells and enhanced tight junction staining in tPA−/− mice. (A) Immunofluorescent stainings of murine brain sections from tPA−/− mice (n = 5) and WT littermate controls (WT, n = 5) with antibodies for the endothelial transcription factor ERG showed what appeared to be increased number of endothelial cells in tPA−/− mice as compared to WT littermate controls. (B–C) This was confirmed through quantification of the number of ERG+ nuclei from four maximum intensity confocal Z-stacks per animal. This revealed that there was significantly more ERG-positive cells in tPA−/− brains compared to WT littermate controls (B), also when normalized to CD31 staining as quantified by pixel intensity (arbitrary units, A.U.) (C). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from two independent staining experiments. (D) Staining with antibodies against the tight junction protein ZO1 illustrated increased junctional expression in cerebral vessels in tPA−/− mice as compared to WT littermate controls. The images display maximum intensity projections generated from confocal Z-stacks (22 μm). Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test and **P < 0.01 relative to control. Scale bars (A,D) 20 μm.
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Figure 2: Increased number of ERG+ endothelial cells and enhanced tight junction staining in tPA−/− mice. (A) Immunofluorescent stainings of murine brain sections from tPA−/− mice (n = 5) and WT littermate controls (WT, n = 5) with antibodies for the endothelial transcription factor ERG showed what appeared to be increased number of endothelial cells in tPA−/− mice as compared to WT littermate controls. (B–C) This was confirmed through quantification of the number of ERG+ nuclei from four maximum intensity confocal Z-stacks per animal. This revealed that there was significantly more ERG-positive cells in tPA−/− brains compared to WT littermate controls (B), also when normalized to CD31 staining as quantified by pixel intensity (arbitrary units, A.U.) (C). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from two independent staining experiments. (D) Staining with antibodies against the tight junction protein ZO1 illustrated increased junctional expression in cerebral vessels in tPA−/− mice as compared to WT littermate controls. The images display maximum intensity projections generated from confocal Z-stacks (22 μm). Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test and **P < 0.01 relative to control. Scale bars (A,D) 20 μm.
Mentions: In order to characterize whether the cerebrovascular changes seen in tPA−/− mice were associated with altered number of endothelial cells we conducted immunofluorescent stainings using antibodies against the endothelial transcription factor ERG. Unexpectedly, we found an apparent increase in the number of ERG-positive cells in the brains of tPA−/− mice (n = 5) relative to WT littermate controls (n = 5; Figure 2A). This was confirmed by quantification of the number of ERG-positive cells, showing a significant (P < 0.01) overall increase in the tPA−/− mice (20.2 ± 1.2 ERG+ cells/field of view) compared to WT littermate controls (15.2 ± 0.6 ERG+ cells/field of view; Figure 2B). To ensure that the increase of ERG-positive cells was not due to an increase in the number of small vessels in tPA−/− brains, we normalized the number of ERG+ cells to the amount of CD31 staining. This verified that there were significantly more (P < 0.01) ERG+ cells in the cerebral vessels of tPA−/− mice (149 ± 11% of WT; Figure 2C). This was particularly interesting since ERG has been implicated in vascular development, control of vascular permeability and maintenance of the integrity of the endothelial junctions (Randi et al., 2009; Birdsey et al., 2015). We therefore investigated the appearance of endothelial tight junctions in tPA−/− mice by immunofluorescent stainings using antibodies against the tight junction protein ZO1. We found that ZO1 appeared to show an increased junctional localization in vessels in tPA−/− brains compared to WT littermate controls (Figure 2D), suggesting that tPA−/− mice might have tighter endothelial cell-cell junctions.

Bottom Line: Our analysis demonstrates that life-long deficiency of tPA is associated with rearrangements in the cerebrovascular tree, including a reduction in the number of vascular smooth-muscle cell covered, large diameter, vessels and a decrease in vessel-associated PDGFRα expression as compared to wild-type (WT) littermate controls.In addition, we found that ablation of tPA results in an increased number of ERG-positive endothelial cells and increased junctional localization of the tight junction protein ZO1.In addition, we found that tPA (-/-) mice displayed mild cerebral ventricular malformations, a feature previously associated with ablation of PDGF-C, thereby providing an in vivo link between tPA and PDGF signaling in central nervous system (CNS) development.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Division of Vascular Biology, Karolinska Institutet Stockholm, Sweden.

ABSTRACT
The serine protease tissue-type plasminogen activator (tPA) is used as a thrombolytic agent in the management of ischemic stroke, but concerns for hemorrhagic conversion greatly limits the number of patients that receive this treatment. It has been suggested that the bleeding complications associated with thrombolytic tPA may be due to unanticipated roles of tPA in the brain. Recent work has suggested tPA regulation of neurovascular barrier integrity, mediated via platelet derived growth factor (PDGF)-C/PDGF receptor-α (PDGFRα) signaling, as a possible molecular mechanism affecting the outcome of stroke. To better understand the role of tPA in neurovascular regulation we conducted a detailed analysis of the cerebrovasculature in brains from adult tPA deficient (tPA(-/-) ) mice. Our analysis demonstrates that life-long deficiency of tPA is associated with rearrangements in the cerebrovascular tree, including a reduction in the number of vascular smooth-muscle cell covered, large diameter, vessels and a decrease in vessel-associated PDGFRα expression as compared to wild-type (WT) littermate controls. In addition, we found that ablation of tPA results in an increased number of ERG-positive endothelial cells and increased junctional localization of the tight junction protein ZO1. This is intriguing since ERG is an endothelial transcription factor implicated in regulation of vascular integrity. Based on these results, we propose that the protection of barrier properties seen utilizing these tPA (-/-) mice might be due, at least in part, to these cerebrovascular rearrangements. In addition, we found that tPA (-/-) mice displayed mild cerebral ventricular malformations, a feature previously associated with ablation of PDGF-C, thereby providing an in vivo link between tPA and PDGF signaling in central nervous system (CNS) development. Taken together, the data presented here will advance our understanding of the role of tPA within the CNS and in regulation of cerebrovascular permeability.

No MeSH data available.


Related in: MedlinePlus