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Identification of pro-angiogenic markers in blood vessels from stroked-affected brain tissue using laser-capture microdissection.

Slevin M, Krupinski J, Rovira N, Turu M, Luque A, Baldellou M, Sanfeliu C, de Vera N, Badimon L - BMC Genomics (2009)

Bottom Line: Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery.Areas were compared for pro- and anti-angiogenic gene expression using targeted TaqMan microfluidity cards containing 46 genes and real-time PCR.In this work we have identified concurrent activation of key angiogenic molecules associated with endothelial cell migration, differentiation and tube-formation, vessel stabilization and stem cell homing mechanisms in areas of revascularization.

View Article: PubMed Central - HTML - PubMed

Affiliation: SBCHS, Manchester Metropolitan University, Manchester, UK. m.a.slevin@mmu.ac.uk

ABSTRACT

Background: Angiogenesis correlates with patient survival following acute ischaemic stroke, and survival of neurons is greatest in tissue undergoing angiogenesis. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery.

Results: Here, we have isolated active (CD105/Flt-1 positive) and inactive (CD105/Flt-1 minus (n=5) micro-vessel rich-regions from stroke-affected and contralateral tissue of patients using laser-capture micro-dissection. Areas were compared for pro- and anti-angiogenic gene expression using targeted TaqMan microfluidity cards containing 46 genes and real-time PCR. Further analysis of key gene de-regulation was performed by immunohistochemistry to define localization and expression patterns of identified markers and de novo synthesis by human brain microvessel endothelial cells (HBMEC) was examined following oxygen-glucose deprivation (OGD). Our data revealed that seven pro-angiogenic genes were notably up-regulated in CD105 positive microvessel rich regions. These were, beta-catenin, neural cell adhesion molecule (NRCAM), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hepatocyte growth factor-alpha (HGF-alpha), monocyte chemottractant protein-1 (MCP-1) and and Tie-2 as well as c-kit. Immunohistochemistry demonstrated strong staining of MMP-2, HGF-alpha, MCP-1 and Tie-2 in stroke-associated regions of active remodeling in association with CD105 positive staining. In vitro, OGD stimulated production of Tie-2, MCP-1 and MMP-2 in HBMEC, demonstrated a de novo response to hypoxia.

Conclusion: In this work we have identified concurrent activation of key angiogenic molecules associated with endothelial cell migration, differentiation and tube-formation, vessel stabilization and stem cell homing mechanisms in areas of revascularization. Therapeutic stimulation of these processes in all areas of damaged tissue might improve morbidity and mortality from stroke.

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A, tissue from the normal looking contralateral hemisphere showed no observable staining of Tie-2 (arrows; × 40). B, stroked brain tissue showing many microvessels strongly staining positive for Tie-2 (i; × 40) and (ii and iii) at higher magnification (× 100; arrows). C, small microvessels from the peri-infarcted zone positive for Tie-2 (× 100). Di-iii, double immunoflourescence showing co-localization of Tie-2 (red) and CD105 (green) in peri-infarcted stroke tissue (× 100; sections from I06232 were used).
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Figure 5: A, tissue from the normal looking contralateral hemisphere showed no observable staining of Tie-2 (arrows; × 40). B, stroked brain tissue showing many microvessels strongly staining positive for Tie-2 (i; × 40) and (ii and iii) at higher magnification (× 100; arrows). C, small microvessels from the peri-infarcted zone positive for Tie-2 (× 100). Di-iii, double immunoflourescence showing co-localization of Tie-2 (red) and CD105 (green) in peri-infarcted stroke tissue (× 100; sections from I06232 were used).

Mentions: HGF-α was expressed strongly in microvessels of varying size from all active stroke regions after stroke (Figure 4A–C; Bi–iii). Old infarcted regions with dead cells were not stained. In contrast, there was no observable expression of HGF-α in normal looking blood vessels from the contralateral region. Glia and neurons were-unstained however, occasional inflammatory cells were positively stained in stroked regions. Many of the HGF-α-positive vessels, particularly those with malformed/impatent morphology stained CD105 positive suggesting cellular activation or angiogenesis was occurring in these areas (Figure 3D). Tie-2 showed a similar pattern with strong staining in small and medium sized, blood vessels from peri-infarcted and infarcted regions (Figure 5A–D; Bii shows higher powered micrographs; × 100). Again, there with no observable staining in sections from normal looking contralateral tissue (A; arrows). D; Tie-2 also co-localised in CD105-positive immature neo-vessels suggesting an association with angiogenesis. Tie-2 expression was specific for blood vessels with no staining of glia or inflammatory cells; however occasional dying neurons from the infarcted core had weak cytoplamic staining. MMP-2 was strongly expressed in both relative mature and immature microvessels from stroked regions, particularly in penumbral regions undergoing active remodeling (Figure 6Bi–iii and 6E, showing co-localization with CD105) and also in cells with the morphological appearance of astrocytes (Figure 6C) and neurons (Figure 6D). Normal looking (contralateral tissue was unstained (Figure 6A, arrows). MCP-1 stained primarily CD105-positive microvessels in peri-infarcted regions with some positive vessels also in the infarcted zones (Figure 7Bi–iii and 7C–E). Normal looking tissue did not stain for MCP-1 (A; arrows point to blood vessels). Some glia and infiltrating inflammatory cells were positively stained for MCP-1. In all cases, old infarcted regions stained negatively for all the antibodies tested and the majority of staining was seen in active regions of remodeling and revascularization. A summary of the findings is given in Table 1.


Identification of pro-angiogenic markers in blood vessels from stroked-affected brain tissue using laser-capture microdissection.

Slevin M, Krupinski J, Rovira N, Turu M, Luque A, Baldellou M, Sanfeliu C, de Vera N, Badimon L - BMC Genomics (2009)

A, tissue from the normal looking contralateral hemisphere showed no observable staining of Tie-2 (arrows; × 40). B, stroked brain tissue showing many microvessels strongly staining positive for Tie-2 (i; × 40) and (ii and iii) at higher magnification (× 100; arrows). C, small microvessels from the peri-infarcted zone positive for Tie-2 (× 100). Di-iii, double immunoflourescence showing co-localization of Tie-2 (red) and CD105 (green) in peri-infarcted stroke tissue (× 100; sections from I06232 were used).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664824&req=5

Figure 5: A, tissue from the normal looking contralateral hemisphere showed no observable staining of Tie-2 (arrows; × 40). B, stroked brain tissue showing many microvessels strongly staining positive for Tie-2 (i; × 40) and (ii and iii) at higher magnification (× 100; arrows). C, small microvessels from the peri-infarcted zone positive for Tie-2 (× 100). Di-iii, double immunoflourescence showing co-localization of Tie-2 (red) and CD105 (green) in peri-infarcted stroke tissue (× 100; sections from I06232 were used).
Mentions: HGF-α was expressed strongly in microvessels of varying size from all active stroke regions after stroke (Figure 4A–C; Bi–iii). Old infarcted regions with dead cells were not stained. In contrast, there was no observable expression of HGF-α in normal looking blood vessels from the contralateral region. Glia and neurons were-unstained however, occasional inflammatory cells were positively stained in stroked regions. Many of the HGF-α-positive vessels, particularly those with malformed/impatent morphology stained CD105 positive suggesting cellular activation or angiogenesis was occurring in these areas (Figure 3D). Tie-2 showed a similar pattern with strong staining in small and medium sized, blood vessels from peri-infarcted and infarcted regions (Figure 5A–D; Bii shows higher powered micrographs; × 100). Again, there with no observable staining in sections from normal looking contralateral tissue (A; arrows). D; Tie-2 also co-localised in CD105-positive immature neo-vessels suggesting an association with angiogenesis. Tie-2 expression was specific for blood vessels with no staining of glia or inflammatory cells; however occasional dying neurons from the infarcted core had weak cytoplamic staining. MMP-2 was strongly expressed in both relative mature and immature microvessels from stroked regions, particularly in penumbral regions undergoing active remodeling (Figure 6Bi–iii and 6E, showing co-localization with CD105) and also in cells with the morphological appearance of astrocytes (Figure 6C) and neurons (Figure 6D). Normal looking (contralateral tissue was unstained (Figure 6A, arrows). MCP-1 stained primarily CD105-positive microvessels in peri-infarcted regions with some positive vessels also in the infarcted zones (Figure 7Bi–iii and 7C–E). Normal looking tissue did not stain for MCP-1 (A; arrows point to blood vessels). Some glia and infiltrating inflammatory cells were positively stained for MCP-1. In all cases, old infarcted regions stained negatively for all the antibodies tested and the majority of staining was seen in active regions of remodeling and revascularization. A summary of the findings is given in Table 1.

Bottom Line: Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery.Areas were compared for pro- and anti-angiogenic gene expression using targeted TaqMan microfluidity cards containing 46 genes and real-time PCR.In this work we have identified concurrent activation of key angiogenic molecules associated with endothelial cell migration, differentiation and tube-formation, vessel stabilization and stem cell homing mechanisms in areas of revascularization.

View Article: PubMed Central - HTML - PubMed

Affiliation: SBCHS, Manchester Metropolitan University, Manchester, UK. m.a.slevin@mmu.ac.uk

ABSTRACT

Background: Angiogenesis correlates with patient survival following acute ischaemic stroke, and survival of neurons is greatest in tissue undergoing angiogenesis. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion, enhanced neuronal survival and improved recovery.

Results: Here, we have isolated active (CD105/Flt-1 positive) and inactive (CD105/Flt-1 minus (n=5) micro-vessel rich-regions from stroke-affected and contralateral tissue of patients using laser-capture micro-dissection. Areas were compared for pro- and anti-angiogenic gene expression using targeted TaqMan microfluidity cards containing 46 genes and real-time PCR. Further analysis of key gene de-regulation was performed by immunohistochemistry to define localization and expression patterns of identified markers and de novo synthesis by human brain microvessel endothelial cells (HBMEC) was examined following oxygen-glucose deprivation (OGD). Our data revealed that seven pro-angiogenic genes were notably up-regulated in CD105 positive microvessel rich regions. These were, beta-catenin, neural cell adhesion molecule (NRCAM), matrix metalloproteinase-2 (MMP-2), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), hepatocyte growth factor-alpha (HGF-alpha), monocyte chemottractant protein-1 (MCP-1) and and Tie-2 as well as c-kit. Immunohistochemistry demonstrated strong staining of MMP-2, HGF-alpha, MCP-1 and Tie-2 in stroke-associated regions of active remodeling in association with CD105 positive staining. In vitro, OGD stimulated production of Tie-2, MCP-1 and MMP-2 in HBMEC, demonstrated a de novo response to hypoxia.

Conclusion: In this work we have identified concurrent activation of key angiogenic molecules associated with endothelial cell migration, differentiation and tube-formation, vessel stabilization and stem cell homing mechanisms in areas of revascularization. Therapeutic stimulation of these processes in all areas of damaged tissue might improve morbidity and mortality from stroke.

Show MeSH
Related in: MedlinePlus