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BMC Genomics. 2009; 10: 113.
Published online Mar 17, 2009. doi:  10.1186/1471-2164-10-113
PMCID: PMC2664824
Identification of pro-angiogenic markers in blood vessels from stroked-affected brain tissue using laser-capture microdissection
Mark Slevin,corresponding author1,2 Jerzy Krupinski,3 Norma Rovira,2 Marta Turu,2 Ana Luque,2 Maribel Baldellou,2 Coral Sanfeliu,4 Nuria de Vera,4 and Lina Badimon2
1SBCHS, Manchester Metropolitan University, Manchester, UK
2Centro de Investigación Cardiovascular, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
3Hospital Universitari Mútua de Terrassa, Department of Neurology, Cerebrovascular Diseases Unit, Pl. Dr. Robert, 5, 08221 Terrassa, Barcelona, Spain
4Dept of Brain Ischemia and Neurodegeneration, IIBB, CSIC-IDIBAPS, Barcelona
corresponding authorCorresponding author.
Mark Slevin: m.a.slevin/at/mmu.ac.uk; Jerzy Krupinski: krupinski/at/csub.scs.es; Norma Rovira: nrovira/at/csic-iccc.org; Marta Turu: mturu/at/csic-iccc.org; Ana Luque: aluque/at/csic-iccc.org; Maribel Baldellou: mbaldellou/at/csic-iccc.org; Coral Sanfeliu: cspfat/at/iibb.csic.es; Nuria de Vera: nvcfat/at/iibb.csic.es; Lina Badimon: lbadimon/at/csic-iccc.org
Received November 28, 2008; Accepted March 17, 2009.
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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