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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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Related in: MedlinePlus

A, (i) Area with discreet region of CD105-positive (red; circle) and CD105-negative (blue: circle) vessels captured with the laser. (ii) RNA extraction from multiple combined laser cut sections showing good RIN and concentration. B, (i-ii) Shows non-significant differences in ΔCts between pre-amplified genes indicating no bias in gene amplification. (iii) Significant increases in gene expression of CD105 and Flt-1 in the positively selected samples indicated the sampling/IHC was accurate. (iv) Significant correlation between CD105 and Flt-1 expression in all the tested samples indicated reliability of the markers to discriminate between active and inactive vessels.
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Figure 2: A, (i) Area with discreet region of CD105-positive (red; circle) and CD105-negative (blue: circle) vessels captured with the laser. (ii) RNA extraction from multiple combined laser cut sections showing good RIN and concentration. B, (i-ii) Shows non-significant differences in ΔCts between pre-amplified genes indicating no bias in gene amplification. (iii) Significant increases in gene expression of CD105 and Flt-1 in the positively selected samples indicated the sampling/IHC was accurate. (iv) Significant correlation between CD105 and Flt-1 expression in all the tested samples indicated reliability of the markers to discriminate between active and inactive vessels.

Mentions: Areas rich in CD105-positive or CD31-positive/CD105-negative vessels were chosen for laser-capture as shown in (Figure 1). The cDNA obtained from 1 ng of total RNA was pre-amplified using the TaqMan Applied Biosystems PreAmp Master Mix Kit (Figure 2Ai–ii).


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, (i) Area with discreet region of CD105-positive (red; circle) and CD105-negative (blue: circle) vessels captured with the laser. (ii) RNA extraction from multiple combined laser cut sections showing good RIN and concentration. B, (i-ii) Shows non-significant differences in ΔCts between pre-amplified genes indicating no bias in gene amplification. (iii) Significant increases in gene expression of CD105 and Flt-1 in the positively selected samples indicated the sampling/IHC was accurate. (iv) Significant correlation between CD105 and Flt-1 expression in all the tested samples indicated reliability of the markers to discriminate between active and inactive vessels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: A, (i) Area with discreet region of CD105-positive (red; circle) and CD105-negative (blue: circle) vessels captured with the laser. (ii) RNA extraction from multiple combined laser cut sections showing good RIN and concentration. B, (i-ii) Shows non-significant differences in ΔCts between pre-amplified genes indicating no bias in gene amplification. (iii) Significant increases in gene expression of CD105 and Flt-1 in the positively selected samples indicated the sampling/IHC was accurate. (iv) Significant correlation between CD105 and Flt-1 expression in all the tested samples indicated reliability of the markers to discriminate between active and inactive vessels.
Mentions: Areas rich in CD105-positive or CD31-positive/CD105-negative vessels were chosen for laser-capture as shown in (Figure 1). The cDNA obtained from 1 ng of total RNA was pre-amplified using the TaqMan Applied Biosystems PreAmp Master Mix Kit (Figure 2Ai–ii).

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