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 ASMA+ vessels in tPA−/− brains. (A) Immunofluorescent staining of murine brain sections from mice tPA−/− (n = 5) and WT littermates (n = 5) with the vascular smooth muscle cell (vSMC) marker for alpha-smooth muscle actin (ASMA) displayed a redistribution of ASMA-positive (ASMA+) vessels in tPA−/− mice compared to WT littermate controls. There appeared to be more ASMA+ vessels with smaller diameter (arrows) in tPA−/− brains and fewer large ASMA+ diameter vessels (arrowheads) commonly seen in WT brains. (B–D) Quantification of the ASMA staining from eleven confocal images per animal revealed that there was no significant difference in the overall amount of ASMA staining as quantified by pixel intensity (B), although the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (C). In addition, there was a significant redistribution toward smaller diameter ASMA+ vessels in the cerebrovasculature of tPA−/− mice compared to WT littermate controls (D). The number of vessels analyzed is displayed on the respective bars. The data shown are representative quantifications of four to eleven confocal images per animal from three independent staining experiments. (E) Staining with the vascular mural cell marker CD13, which stains pericytes, revealed that large diameter vessels positive for CD13, normally present in WT control brains (arrowheads), was essentially lost in tPA−/− brains. However, the pericyte coverage of capillaries appeared normal (arrows). (F) The loss of large diameter CD13+ vessels resulted in a significant reduction in CD13 staining in tPA−/− brains (n = 5) compared to WT littermate controls (n = 5). Quantification of CD13 pixel intensity was made from nine confocal Z-stacks per animal. The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from three independent staining experiments. The images display (A,E) maximum intensity projections generated from confocal Z-stacks (22 μm). Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo). 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,E) left and middle panels, 50 μm; right panels, 20 μm. Arbitrary units, A.U.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Decreased number of large diameter ASMA+ vessels in tPA−/− brains. (A) Immunofluorescent staining of murine brain sections from mice tPA−/− (n = 5) and WT littermates (n = 5) with the vascular smooth muscle cell (vSMC) marker for alpha-smooth muscle actin (ASMA) displayed a redistribution of ASMA-positive (ASMA+) vessels in tPA−/− mice compared to WT littermate controls. There appeared to be more ASMA+ vessels with smaller diameter (arrows) in tPA−/− brains and fewer large ASMA+ diameter vessels (arrowheads) commonly seen in WT brains. (B–D) Quantification of the ASMA staining from eleven confocal images per animal revealed that there was no significant difference in the overall amount of ASMA staining as quantified by pixel intensity (B), although the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (C). In addition, there was a significant redistribution toward smaller diameter ASMA+ vessels in the cerebrovasculature of tPA−/− mice compared to WT littermate controls (D). The number of vessels analyzed is displayed on the respective bars. The data shown are representative quantifications of four to eleven confocal images per animal from three independent staining experiments. (E) Staining with the vascular mural cell marker CD13, which stains pericytes, revealed that large diameter vessels positive for CD13, normally present in WT control brains (arrowheads), was essentially lost in tPA−/− brains. However, the pericyte coverage of capillaries appeared normal (arrows). (F) The loss of large diameter CD13+ vessels resulted in a significant reduction in CD13 staining in tPA−/− brains (n = 5) compared to WT littermate controls (n = 5). Quantification of CD13 pixel intensity was made from nine confocal Z-stacks per animal. The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from three independent staining experiments. The images display (A,E) maximum intensity projections generated from confocal Z-stacks (22 μm). Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo). 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,E) left and middle panels, 50 μm; right panels, 20 μm. Arbitrary units, A.U.

Mentions: Mural cells on the cerebral vascular tree include vascular smooth muscle cells (vSMCs) and capillary pericytes. These cells are known to play important roles in cerebrovascular events, including maintenance of the blood-brain barrier (BBB; Armulik et al., 2010; Daneman et al., 2010) and in regulation of vessel diameter and blood flow (Kornfield and Newman, 2014; Hill et al., 2015). To assess the distribution of vascular mural cell coverage on cerebral vessels in tPA−/− mice we performed immunofluorescent stainings using antibodies against ASMA, a marker for vSMCs, and CD13, a marker for pericytes. The stainings showed an apparent shift in the size of ASMA+ vessels, with more ASMA+ small diameter vessels (arrows) and fewer ASMA+ large diameter vessels (arrowheads) in tPA−/− brains (n = 5) compared to WT littermate controls (n = 5; Figure 3A). There was no significant difference in the overall amount of ASMA+ staining (P = 0.46; Figure 3B) but the average diameter of ASMA+ vessels was significantly smaller (P < 0.01) in tPA−/− mice (15.4 ± 0.7 μm) as compared to WT littermate controls (22.9 ± 1.3 μm; Figure 3C). tPA−/− mice displayed significantly increased numbers of ASMA+ small diameter vessels (<15 μm; WT = 17 ± 8% vs. tPA−/− = 61 ± 6%, P < 0.01) and fewer large diameter vessels (>30 μm; WT = 19 ± 3% vs. tPA−/− = 5 ± 1%, P < 0.01; Figure 3D). The microvascular capillaries in the adult murine brain of tPA−/− mice appeared to have normal coverage of pericytes as visualized by immunostainings with CD13 antibodies (arrows, Figure 3E). However, CD13 antibodies also stain vSMC in larger vessels (arrowheads, Figure 3E) and these vessels are essentially lost in tPA−/− mice, resulting in an overall reduction in CD13 staining relative to WT littermate controls (P < 0.05; Figure 3F).


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 ASMA+ vessels in tPA−/− brains. (A) Immunofluorescent staining of murine brain sections from mice tPA−/− (n = 5) and WT littermates (n = 5) with the vascular smooth muscle cell (vSMC) marker for alpha-smooth muscle actin (ASMA) displayed a redistribution of ASMA-positive (ASMA+) vessels in tPA−/− mice compared to WT littermate controls. There appeared to be more ASMA+ vessels with smaller diameter (arrows) in tPA−/− brains and fewer large ASMA+ diameter vessels (arrowheads) commonly seen in WT brains. (B–D) Quantification of the ASMA staining from eleven confocal images per animal revealed that there was no significant difference in the overall amount of ASMA staining as quantified by pixel intensity (B), although the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (C). In addition, there was a significant redistribution toward smaller diameter ASMA+ vessels in the cerebrovasculature of tPA−/− mice compared to WT littermate controls (D). The number of vessels analyzed is displayed on the respective bars. The data shown are representative quantifications of four to eleven confocal images per animal from three independent staining experiments. (E) Staining with the vascular mural cell marker CD13, which stains pericytes, revealed that large diameter vessels positive for CD13, normally present in WT control brains (arrowheads), was essentially lost in tPA−/− brains. However, the pericyte coverage of capillaries appeared normal (arrows). (F) The loss of large diameter CD13+ vessels resulted in a significant reduction in CD13 staining in tPA−/− brains (n = 5) compared to WT littermate controls (n = 5). Quantification of CD13 pixel intensity was made from nine confocal Z-stacks per animal. The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from three independent staining experiments. The images display (A,E) maximum intensity projections generated from confocal Z-stacks (22 μm). Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo). 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,E) left and middle panels, 50 μm; right panels, 20 μm. Arbitrary units, A.U.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Decreased number of large diameter ASMA+ vessels in tPA−/− brains. (A) Immunofluorescent staining of murine brain sections from mice tPA−/− (n = 5) and WT littermates (n = 5) with the vascular smooth muscle cell (vSMC) marker for alpha-smooth muscle actin (ASMA) displayed a redistribution of ASMA-positive (ASMA+) vessels in tPA−/− mice compared to WT littermate controls. There appeared to be more ASMA+ vessels with smaller diameter (arrows) in tPA−/− brains and fewer large ASMA+ diameter vessels (arrowheads) commonly seen in WT brains. (B–D) Quantification of the ASMA staining from eleven confocal images per animal revealed that there was no significant difference in the overall amount of ASMA staining as quantified by pixel intensity (B), although the average vessel diameter was significantly lower in tPA−/− brains as compared to WT littermate controls (C). In addition, there was a significant redistribution toward smaller diameter ASMA+ vessels in the cerebrovasculature of tPA−/− mice compared to WT littermate controls (D). The number of vessels analyzed is displayed on the respective bars. The data shown are representative quantifications of four to eleven confocal images per animal from three independent staining experiments. (E) Staining with the vascular mural cell marker CD13, which stains pericytes, revealed that large diameter vessels positive for CD13, normally present in WT control brains (arrowheads), was essentially lost in tPA−/− brains. However, the pericyte coverage of capillaries appeared normal (arrows). (F) The loss of large diameter CD13+ vessels resulted in a significant reduction in CD13 staining in tPA−/− brains (n = 5) compared to WT littermate controls (n = 5). Quantification of CD13 pixel intensity was made from nine confocal Z-stacks per animal. The data shown are representative quantifications of four to nine maximum intensity confocal Z-stacks per animal from three independent staining experiments. The images display (A,E) maximum intensity projections generated from confocal Z-stacks (22 μm). Cell nuclei were visualized with DAPI and vessels with podocalyxin (Podo). 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,E) left and middle panels, 50 μm; right panels, 20 μm. Arbitrary units, A.U.
Mentions: Mural cells on the cerebral vascular tree include vascular smooth muscle cells (vSMCs) and capillary pericytes. These cells are known to play important roles in cerebrovascular events, including maintenance of the blood-brain barrier (BBB; Armulik et al., 2010; Daneman et al., 2010) and in regulation of vessel diameter and blood flow (Kornfield and Newman, 2014; Hill et al., 2015). To assess the distribution of vascular mural cell coverage on cerebral vessels in tPA−/− mice we performed immunofluorescent stainings using antibodies against ASMA, a marker for vSMCs, and CD13, a marker for pericytes. The stainings showed an apparent shift in the size of ASMA+ vessels, with more ASMA+ small diameter vessels (arrows) and fewer ASMA+ large diameter vessels (arrowheads) in tPA−/− brains (n = 5) compared to WT littermate controls (n = 5; Figure 3A). There was no significant difference in the overall amount of ASMA+ staining (P = 0.46; Figure 3B) but the average diameter of ASMA+ vessels was significantly smaller (P < 0.01) in tPA−/− mice (15.4 ± 0.7 μm) as compared to WT littermate controls (22.9 ± 1.3 μm; Figure 3C). tPA−/− mice displayed significantly increased numbers of ASMA+ small diameter vessels (<15 μm; WT = 17 ± 8% vs. tPA−/− = 61 ± 6%, P < 0.01) and fewer large diameter vessels (>30 μm; WT = 19 ± 3% vs. tPA−/− = 5 ± 1%, P < 0.01; Figure 3D). The microvascular capillaries in the adult murine brain of tPA−/− mice appeared to have normal coverage of pericytes as visualized by immunostainings with CD13 antibodies (arrows, Figure 3E). However, CD13 antibodies also stain vSMC in larger vessels (arrowheads, Figure 3E) and these vessels are essentially lost in tPA−/− mice, resulting in an overall reduction in CD13 staining relative to WT littermate controls (P < 0.05; Figure 3F).

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