Limits...
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

Decreased number of large diameter vessels in tPA−/− brains. Immunofluorescent staining of murine brain sections from tPA−/− deficient mice n = 5) and wild-type (WT) littermate controls (WT, n = 5) with (A–D) the endothelial cell marker CD31 and (E–F) podocalyxin (Podo) showed fewer large diameter vessels (arrowheads) and increased number of small diameter vessels (arrows) in tPA−/− mice compared to WT littermate controls. Quantification of the CD31 staining from four confocal Z-stacks per animal revealed that the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (B) and that there was a significant redistribution toward smaller diameter vessels in the cerebrovasculature of tPA−/− mice (C). The number of vessels analyzed is displayed on the respective bars. There was no significant difference in the overall amount of CD31 positive staining as quantified by pixel intensity (D). Staining with podocalyxin confirmed the results seen with staining for CD31 (E–F). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from four independent staining experiments with CD31 and Podocalyxin, respectively. The images display (A,F) maximum intensity projections generated from confocal Z-stacks (22 μm) and (E) stitched tiling of epifluorescent images (taken with 10× objective). Cell nuclei were visualized with DAPI. Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test. *P < 0.05; **P < 0.01; ns = non significant relative to control. Scale bars (A,F) left panels, 50 μm; right panels, 20 μm, (E) 1 mm. Arbitrary units, A.U.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663266&req=5

Figure 1: Decreased number of large diameter vessels in tPA−/− brains. Immunofluorescent staining of murine brain sections from tPA−/− deficient mice n = 5) and wild-type (WT) littermate controls (WT, n = 5) with (A–D) the endothelial cell marker CD31 and (E–F) podocalyxin (Podo) showed fewer large diameter vessels (arrowheads) and increased number of small diameter vessels (arrows) in tPA−/− mice compared to WT littermate controls. Quantification of the CD31 staining from four confocal Z-stacks per animal revealed that the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (B) and that there was a significant redistribution toward smaller diameter vessels in the cerebrovasculature of tPA−/− mice (C). The number of vessels analyzed is displayed on the respective bars. There was no significant difference in the overall amount of CD31 positive staining as quantified by pixel intensity (D). Staining with podocalyxin confirmed the results seen with staining for CD31 (E–F). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from four independent staining experiments with CD31 and Podocalyxin, respectively. The images display (A,F) maximum intensity projections generated from confocal Z-stacks (22 μm) and (E) stitched tiling of epifluorescent images (taken with 10× objective). Cell nuclei were visualized with DAPI. Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test. *P < 0.05; **P < 0.01; ns = non significant relative to control. Scale bars (A,F) left panels, 50 μm; right panels, 20 μm, (E) 1 mm. Arbitrary units, A.U.

Mentions: A growing body of evidence is showing that tPA is both necessary and sufficient to regulate cerebrovascular permeability (Yepes et al., 2003; Su et al., 2008; Fredriksson et al., 2015). In order to gain a better understanding of the role of tPA in controlling cerebrovascular events we performed a thorough analysis of the vascular bed in brain sections from tPA deficient (tPA−/−) mice. Immunofluorescent stainings using CD31 antibodies, a marker for vascular endothelial cells, revealed an abnormal vascularization in the brains of adult tPA−/− mice (n = 5) compared to littermate WT controls (n = 5; Figure 1A). In tPA−/− brains the vascular bed showed an apparent decrease in large diameter vessels (arrowheads) and increase in small diameter vessels (arrows) relative to WT mice (Figure 1A). This was confirmed by quantification of the CD31 stainings, showing a significant decrease (P < 0.01) in vessel diameter in tPA−/− mice (4.75 ± 0.2 μm) as compared to WT littermate controls (6.03 ± 0.3 μm; Figure 1B). This decrease in average vessel size in tPA−/− mice was due to a significant reorganization of the vascular bed in tPA−/− brains relative to WT littermate controls, with increased number of small diameter vessels (<5 μm; WT = 54 ± 6% vs. tPA−/− = 67 ± 4%, P < 0.05) and fewer large diameter vessels (>10 μm; WT = 10 ± 1% vs. tPA−/− = 1 ± 0.8%, P < 0.01; Figure 1C). This was accompanied with an overall, but non-significant (P = 0.31), reduction in the total amount of CD31 staining in tPA−/− brains (80 ± 12% of WT) compared to WT littermate controls (Figure 1D). Staining with podocalyxin antibodies, another marker of vascular endothelial cells, confirmed the loss of large diameter vessels (arrowheads) and increase in small diameter vessels (arrows) in tPA−/− brains relative to WT littermate controls (Figures 1E,F).


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)

Decreased number of large diameter vessels in tPA−/− brains. Immunofluorescent staining of murine brain sections from tPA−/− deficient mice n = 5) and wild-type (WT) littermate controls (WT, n = 5) with (A–D) the endothelial cell marker CD31 and (E–F) podocalyxin (Podo) showed fewer large diameter vessels (arrowheads) and increased number of small diameter vessels (arrows) in tPA−/− mice compared to WT littermate controls. Quantification of the CD31 staining from four confocal Z-stacks per animal revealed that the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (B) and that there was a significant redistribution toward smaller diameter vessels in the cerebrovasculature of tPA−/− mice (C). The number of vessels analyzed is displayed on the respective bars. There was no significant difference in the overall amount of CD31 positive staining as quantified by pixel intensity (D). Staining with podocalyxin confirmed the results seen with staining for CD31 (E–F). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from four independent staining experiments with CD31 and Podocalyxin, respectively. The images display (A,F) maximum intensity projections generated from confocal Z-stacks (22 μm) and (E) stitched tiling of epifluorescent images (taken with 10× objective). Cell nuclei were visualized with DAPI. Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test. *P < 0.05; **P < 0.01; ns = non significant relative to control. Scale bars (A,F) left panels, 50 μm; right panels, 20 μm, (E) 1 mm. Arbitrary units, A.U.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663266&req=5

Figure 1: Decreased number of large diameter vessels in tPA−/− brains. Immunofluorescent staining of murine brain sections from tPA−/− deficient mice n = 5) and wild-type (WT) littermate controls (WT, n = 5) with (A–D) the endothelial cell marker CD31 and (E–F) podocalyxin (Podo) showed fewer large diameter vessels (arrowheads) and increased number of small diameter vessels (arrows) in tPA−/− mice compared to WT littermate controls. Quantification of the CD31 staining from four confocal Z-stacks per animal revealed that the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (B) and that there was a significant redistribution toward smaller diameter vessels in the cerebrovasculature of tPA−/− mice (C). The number of vessels analyzed is displayed on the respective bars. There was no significant difference in the overall amount of CD31 positive staining as quantified by pixel intensity (D). Staining with podocalyxin confirmed the results seen with staining for CD31 (E–F). The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from four independent staining experiments with CD31 and Podocalyxin, respectively. The images display (A,F) maximum intensity projections generated from confocal Z-stacks (22 μm) and (E) stitched tiling of epifluorescent images (taken with 10× objective). Cell nuclei were visualized with DAPI. Data presented as mean ± SEM. Statistical significance was determined by student’s unpaired t-test. *P < 0.05; **P < 0.01; ns = non significant relative to control. Scale bars (A,F) left panels, 50 μm; right panels, 20 μm, (E) 1 mm. Arbitrary units, A.U.
Mentions: A growing body of evidence is showing that tPA is both necessary and sufficient to regulate cerebrovascular permeability (Yepes et al., 2003; Su et al., 2008; Fredriksson et al., 2015). In order to gain a better understanding of the role of tPA in controlling cerebrovascular events we performed a thorough analysis of the vascular bed in brain sections from tPA deficient (tPA−/−) mice. Immunofluorescent stainings using CD31 antibodies, a marker for vascular endothelial cells, revealed an abnormal vascularization in the brains of adult tPA−/− mice (n = 5) compared to littermate WT controls (n = 5; Figure 1A). In tPA−/− brains the vascular bed showed an apparent decrease in large diameter vessels (arrowheads) and increase in small diameter vessels (arrows) relative to WT mice (Figure 1A). This was confirmed by quantification of the CD31 stainings, showing a significant decrease (P < 0.01) in vessel diameter in tPA−/− mice (4.75 ± 0.2 μm) as compared to WT littermate controls (6.03 ± 0.3 μm; Figure 1B). This decrease in average vessel size in tPA−/− mice was due to a significant reorganization of the vascular bed in tPA−/− brains relative to WT littermate controls, with increased number of small diameter vessels (<5 μm; WT = 54 ± 6% vs. tPA−/− = 67 ± 4%, P < 0.05) and fewer large diameter vessels (>10 μm; WT = 10 ± 1% vs. tPA−/− = 1 ± 0.8%, P < 0.01; Figure 1C). This was accompanied with an overall, but non-significant (P = 0.31), reduction in the total amount of CD31 staining in tPA−/− brains (80 ± 12% of WT) compared to WT littermate controls (Figure 1D). Staining with podocalyxin antibodies, another marker of vascular endothelial cells, confirmed the loss of large diameter vessels (arrowheads) and increase in small diameter vessels (arrows) in tPA−/− brains relative to WT littermate controls (Figures 1E,F).

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