title: Identification of pro-angiogenic markers in blood vessels from stroked-affected brain tissue using laser-capture microdissection.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: SBCHS, Manchester Metropolitan University, Manchester, UK. m.a.slevin@mmu.ac.uk
authors.forename: Mark
authors.initials: M
authors.lastname: Slevin
authors.forename: Jerzy
authors.initials: J
authors.lastname: Krupinski
authors.forename: Norma
authors.initials: N
authors.lastname: Rovira
authors.forename: Marta
authors.initials: M
authors.lastname: Turu
authors.forename: Ana
authors.initials: A
authors.lastname: Luque
authors.forename: Maribel
authors.initials: M
authors.lastname: Baldellou
authors.forename: Coral
authors.initials: C
authors.lastname: Sanfeliu
authors.forename: Nuria
authors.initials: N
authors.lastname: de Vera
authors.forename: Lina
authors.initials: L
authors.lastname: Badimon
mesh_terms: D042501:Angiogenic Proteins / Q000378:metabolism*; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000097:blood; D001808:Blood Vessels / Q000737:chemistry / Q000378:metabolism; D001921:Brain / Q000098:blood supply* / Q000378:metabolism*; D018932:Chemokine CCL2 / Q000378:metabolism; D000071063:Endoglin; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D042282:Microdissection / Q000379:methods; D009389:Neovascularization, Pathologic / Q000378:metabolism; D042787:Receptor, TIE-2 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D020521:Stroke / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism
chemical_list: D042501:Angiogenic Proteins; D015703:Antigens, CD; D015415:Biomarkers; C111835:CCL2 protein, human; D018932:Chemokine CCL2; C064206:ENG protein, human; D000071063:Endoglin; D011956:Receptors, Cell Surface; C501162:FLT1 protein, human; D042787:Receptor, TIE-2; D040281:Vascular Endothelial Growth Factor Receptor-1; D020778:Matrix Metalloproteinase 2