Abstract
BackgroundAngiogenesis 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.ResultsHere, 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.ConclusionIn 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.
Highlights
Angiogenesis correlates with patient survival following acute ischaemic stroke, and survival of neurons is greatest in tissue undergoing angiogenesis
Pre-amplification of RNA demonstrated equality of multiplication of key genes Pre-amplification analysis demonstrated that Flt-1, CD105 and CD31 amplified to a similar extent and within acceptable limits with ΔΔCt lower than the cut off point of 1.5 and no significant differences between the Cts of any of the post-amplified genes (Figure 2Ci and 2Cii)
Selected immuno-positive CD105-positive tissue samples were enriched with CD105 and Flt-1 gene expression Confirmation was made that the samples were CD31/ CD105/Flt-1 enriched using TaqMan real-time PCR
Summary
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. Neuronal survival in peri-infarcted regions determines the extent of patient recovery [1]. Restoration of cerebral microvascular circulation following angiogenesis/ revascularization in peri-infarcted regions may salvage tissue, enhance neuronal survival and enhance functional recovery after stroke [3]. Conventional technology have employed microarrays to identify general changes in gene and protein regulation in biopsies from normal and abnormal regions of atherosclerotic plaques [5,3,6] and stroke tissue [7,8], but have failed to discover cell-specific changes, and in particular, those associated with angiogenesis. When combined with the latest RNA microscale extraction and analysis technology, this provides a powerful and sensitive tool for identification of genetic changes associated with blood vessel activation
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