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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

Lateral ventricular defects and abnormal ependymal lining in the brains of tPA−/− mice. Lateral ventricular abnormalities (arrowheads) and hypoplastic septum separating the lateral ventricles (LV; arrows) were seen in tPA−/− mice in (A)ex vivo sections visualized with DAPI and with (B)in vivo magnetic resonance imaging (MRI) of WT and tPA−/− adult mice. (B) Montage from MR scans of an adult WT female mouse (upper panel) and two tPA−/− mice (middle panels, images from a male adult tPA−/− mouse and lower panel, images from a female adult tPA−/− mouse). (C) Quantification of the ratio between the smallest to the largest ventricular size from the MRI scans confirmed that tPA−/− mice (n = 13) displayed a significant asymmetry in lateral ventricular size as compared to WT littermate controls (n = 10). The quantifications in (C) were made using Image J to trace the entire area of the right and left ventricle respectively. (D–H) Abnormal ependymal lining of the LV in tPA−/− mice were revealed through immunofluorescent stainings and confocal microscopy on brain sections from WT (n = 5) and tPA−/− (n = 5) mice. Analysis of the ependymal cell lining of the lateral wall of the LV was conducted using antibodies against S100B and Podocalyxin (Podo) (D), GLUT1 (E–F) and ZO1 (G–H). Note the thicker ependymal lining in tPA−/− mice (arrows, D) compared to WT littermate controls (arrowheads, D) and the punctate pattern of ZO1 staining in WT (arrowheads, G) compared to the continuous tight junctions between ependymal cells in tPA−/− mice (arrows, G). Quantification of ependymal GLUT1 and ZO1 staining (between the dashed lines in E,G) by pixel intensity from six (GLUT1) and four (ZO1) maximum intensity confocal Z-stacks per animal revealed an significant increase of both GLUT1 (F) and ZO1 (H) staining in the ventricular wall of tPA−/− mice compared to WT littermate controls. The data shown are representative quantifications of two to six maximum intensity confocal Z-stacks per animal from two independent staining experiments. The images display (A) stitched tiling of epifluorescent images (taken with 10× objective) and (D,E,G) maximum intensity projections generated from confocal Z-stacks (22 μm). Each picture is a representative from different individuals. Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo) or CD31. Data presented as (C) box-and-whiskers plot for distribution with min to max variance and F,H) mean ± SEM. Statistical significance was determined by student’s unpaired t-test and *P < 0.05; **P < 0.01 relative to control. Scale bars (A–B) 1 mm and (D,E,G) 20 μm. LV, Lateral ventricles. Arbitrary units; A.U.
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Figure 6: Lateral ventricular defects and abnormal ependymal lining in the brains of tPA−/− mice. Lateral ventricular abnormalities (arrowheads) and hypoplastic septum separating the lateral ventricles (LV; arrows) were seen in tPA−/− mice in (A)ex vivo sections visualized with DAPI and with (B)in vivo magnetic resonance imaging (MRI) of WT and tPA−/− adult mice. (B) Montage from MR scans of an adult WT female mouse (upper panel) and two tPA−/− mice (middle panels, images from a male adult tPA−/− mouse and lower panel, images from a female adult tPA−/− mouse). (C) Quantification of the ratio between the smallest to the largest ventricular size from the MRI scans confirmed that tPA−/− mice (n = 13) displayed a significant asymmetry in lateral ventricular size as compared to WT littermate controls (n = 10). The quantifications in (C) were made using Image J to trace the entire area of the right and left ventricle respectively. (D–H) Abnormal ependymal lining of the LV in tPA−/− mice were revealed through immunofluorescent stainings and confocal microscopy on brain sections from WT (n = 5) and tPA−/− (n = 5) mice. Analysis of the ependymal cell lining of the lateral wall of the LV was conducted using antibodies against S100B and Podocalyxin (Podo) (D), GLUT1 (E–F) and ZO1 (G–H). Note the thicker ependymal lining in tPA−/− mice (arrows, D) compared to WT littermate controls (arrowheads, D) and the punctate pattern of ZO1 staining in WT (arrowheads, G) compared to the continuous tight junctions between ependymal cells in tPA−/− mice (arrows, G). Quantification of ependymal GLUT1 and ZO1 staining (between the dashed lines in E,G) by pixel intensity from six (GLUT1) and four (ZO1) maximum intensity confocal Z-stacks per animal revealed an significant increase of both GLUT1 (F) and ZO1 (H) staining in the ventricular wall of tPA−/− mice compared to WT littermate controls. The data shown are representative quantifications of two to six maximum intensity confocal Z-stacks per animal from two independent staining experiments. The images display (A) stitched tiling of epifluorescent images (taken with 10× objective) and (D,E,G) maximum intensity projections generated from confocal Z-stacks (22 μm). Each picture is a representative from different individuals. Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo) or CD31. Data presented as (C) box-and-whiskers plot for distribution with min to max variance and F,H) mean ± SEM. Statistical significance was determined by student’s unpaired t-test and *P < 0.05; **P < 0.01 relative to control. Scale bars (A–B) 1 mm and (D,E,G) 20 μm. LV, Lateral ventricles. Arbitrary units; A.U.

Mentions: During the characterization of the tPA−/− mice we noted that these mice presented with asymmetric LV (Figure 6) similar to what we recently reported for Pdgfc−/− mice on C57BL/6 background (Fredriksson et al., 2012). Analysis of brain sections from tPA−/− mice (n = 5) by DAPI staining revealed that the abnormal LV (arrowheads) coincided with a hypoplastic septum, a defect that was not noted in any WT animals (n = 5; arrow, Figure 6A). To ensure that the ventricular abnormalities were not artifacts of tissue preparation, in vivo MRI in live mice was employed. Figure 6B shows the montage from MRI scans of one adult WT mouse (upper panel) and two tPA−/− mice (middle and lower panels). These MR scans illustrate displacement of the septum towards the side of the smaller ventricle in one of the tPA−/− brains (arrowhead) and a hypoplastic septum in the other tPA−/− mouse (arrow, Figure 6B). This displacement of the septum in the tPA−/− mice made the smaller ventricle appear compressed. Comparing the total area of the smallest to the largest ventricle from the MR scans revealed significant asymmetry of the LV (P < 0.05) in the tPA−/− mice (70 ± 5%, n = 13) as compared to WT littermate controls (86 ± 4%, n = 10; Figure 6C), thus confirming the observation from the DAPI analysis. The ventricular abnormalities seen in the tPA−/− mice appeared to be milder when compared to the asymmetry of the LV reported in Pdgfc−/− mice (56 ± 6%; Fredriksson et al., 2012).


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)

Lateral ventricular defects and abnormal ependymal lining in the brains of tPA−/− mice. Lateral ventricular abnormalities (arrowheads) and hypoplastic septum separating the lateral ventricles (LV; arrows) were seen in tPA−/− mice in (A)ex vivo sections visualized with DAPI and with (B)in vivo magnetic resonance imaging (MRI) of WT and tPA−/− adult mice. (B) Montage from MR scans of an adult WT female mouse (upper panel) and two tPA−/− mice (middle panels, images from a male adult tPA−/− mouse and lower panel, images from a female adult tPA−/− mouse). (C) Quantification of the ratio between the smallest to the largest ventricular size from the MRI scans confirmed that tPA−/− mice (n = 13) displayed a significant asymmetry in lateral ventricular size as compared to WT littermate controls (n = 10). The quantifications in (C) were made using Image J to trace the entire area of the right and left ventricle respectively. (D–H) Abnormal ependymal lining of the LV in tPA−/− mice were revealed through immunofluorescent stainings and confocal microscopy on brain sections from WT (n = 5) and tPA−/− (n = 5) mice. Analysis of the ependymal cell lining of the lateral wall of the LV was conducted using antibodies against S100B and Podocalyxin (Podo) (D), GLUT1 (E–F) and ZO1 (G–H). Note the thicker ependymal lining in tPA−/− mice (arrows, D) compared to WT littermate controls (arrowheads, D) and the punctate pattern of ZO1 staining in WT (arrowheads, G) compared to the continuous tight junctions between ependymal cells in tPA−/− mice (arrows, G). Quantification of ependymal GLUT1 and ZO1 staining (between the dashed lines in E,G) by pixel intensity from six (GLUT1) and four (ZO1) maximum intensity confocal Z-stacks per animal revealed an significant increase of both GLUT1 (F) and ZO1 (H) staining in the ventricular wall of tPA−/− mice compared to WT littermate controls. The data shown are representative quantifications of two to six maximum intensity confocal Z-stacks per animal from two independent staining experiments. The images display (A) stitched tiling of epifluorescent images (taken with 10× objective) and (D,E,G) maximum intensity projections generated from confocal Z-stacks (22 μm). Each picture is a representative from different individuals. Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo) or CD31. Data presented as (C) box-and-whiskers plot for distribution with min to max variance and F,H) mean ± SEM. Statistical significance was determined by student’s unpaired t-test and *P < 0.05; **P < 0.01 relative to control. Scale bars (A–B) 1 mm and (D,E,G) 20 μm. LV, Lateral ventricles. Arbitrary units; A.U.
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Figure 6: Lateral ventricular defects and abnormal ependymal lining in the brains of tPA−/− mice. Lateral ventricular abnormalities (arrowheads) and hypoplastic septum separating the lateral ventricles (LV; arrows) were seen in tPA−/− mice in (A)ex vivo sections visualized with DAPI and with (B)in vivo magnetic resonance imaging (MRI) of WT and tPA−/− adult mice. (B) Montage from MR scans of an adult WT female mouse (upper panel) and two tPA−/− mice (middle panels, images from a male adult tPA−/− mouse and lower panel, images from a female adult tPA−/− mouse). (C) Quantification of the ratio between the smallest to the largest ventricular size from the MRI scans confirmed that tPA−/− mice (n = 13) displayed a significant asymmetry in lateral ventricular size as compared to WT littermate controls (n = 10). The quantifications in (C) were made using Image J to trace the entire area of the right and left ventricle respectively. (D–H) Abnormal ependymal lining of the LV in tPA−/− mice were revealed through immunofluorescent stainings and confocal microscopy on brain sections from WT (n = 5) and tPA−/− (n = 5) mice. Analysis of the ependymal cell lining of the lateral wall of the LV was conducted using antibodies against S100B and Podocalyxin (Podo) (D), GLUT1 (E–F) and ZO1 (G–H). Note the thicker ependymal lining in tPA−/− mice (arrows, D) compared to WT littermate controls (arrowheads, D) and the punctate pattern of ZO1 staining in WT (arrowheads, G) compared to the continuous tight junctions between ependymal cells in tPA−/− mice (arrows, G). Quantification of ependymal GLUT1 and ZO1 staining (between the dashed lines in E,G) by pixel intensity from six (GLUT1) and four (ZO1) maximum intensity confocal Z-stacks per animal revealed an significant increase of both GLUT1 (F) and ZO1 (H) staining in the ventricular wall of tPA−/− mice compared to WT littermate controls. The data shown are representative quantifications of two to six maximum intensity confocal Z-stacks per animal from two independent staining experiments. The images display (A) stitched tiling of epifluorescent images (taken with 10× objective) and (D,E,G) maximum intensity projections generated from confocal Z-stacks (22 μm). Each picture is a representative from different individuals. Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo) or CD31. Data presented as (C) box-and-whiskers plot for distribution with min to max variance and F,H) mean ± SEM. Statistical significance was determined by student’s unpaired t-test and *P < 0.05; **P < 0.01 relative to control. Scale bars (A–B) 1 mm and (D,E,G) 20 μm. LV, Lateral ventricles. Arbitrary units; A.U.
Mentions: During the characterization of the tPA−/− mice we noted that these mice presented with asymmetric LV (Figure 6) similar to what we recently reported for Pdgfc−/− mice on C57BL/6 background (Fredriksson et al., 2012). Analysis of brain sections from tPA−/− mice (n = 5) by DAPI staining revealed that the abnormal LV (arrowheads) coincided with a hypoplastic septum, a defect that was not noted in any WT animals (n = 5; arrow, Figure 6A). To ensure that the ventricular abnormalities were not artifacts of tissue preparation, in vivo MRI in live mice was employed. Figure 6B shows the montage from MRI scans of one adult WT mouse (upper panel) and two tPA−/− mice (middle and lower panels). These MR scans illustrate displacement of the septum towards the side of the smaller ventricle in one of the tPA−/− brains (arrowhead) and a hypoplastic septum in the other tPA−/− mouse (arrow, Figure 6B). This displacement of the septum in the tPA−/− mice made the smaller ventricle appear compressed. Comparing the total area of the smallest to the largest ventricle from the MR scans revealed significant asymmetry of the LV (P < 0.05) in the tPA−/− mice (70 ± 5%, n = 13) as compared to WT littermate controls (86 ± 4%, n = 10; Figure 6C), thus confirming the observation from the DAPI analysis. The ventricular abnormalities seen in the tPA−/− mice appeared to be milder when compared to the asymmetry of the LV reported in Pdgfc−/− mice (56 ± 6%; Fredriksson et al., 2012).

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