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

SBCHS, Manchester Metropolitan University, Manchester, UK.
BMC Genomics (Impact Factor: 4.04). 02/2009; 10:113. DOI: 10.1186/1471-2164-10-113
Source: PubMed

ABSTRACT 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.
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.
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 abstract] [Hide abstract]
    ABSTRACT: Background: Matrix metalloproteinases (MMPs) are key players in proteolytic blood-brain barrier (BBB) disruption during ischemic stroke, leading to vascular edema, hemorrhagic transformation and infiltration by leukocytes. Their effect is dampened by the endogenous tissue inhibitors of metalloproteinases (TIMPs). The respective cellular source of specific MMPs and TIMPs during BBB breakdown is still under investigation. Methods: We analyzed the MMP and TIMP release of human brain microvascular endothelial cells (BMECs) under oxygen glucose deprivation (OGD). Cultured human BMECs (the hCMEC/D3 cell line) were subjected to OGD (6, 12, 18 and 24 h). Gene expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were serially measured by quantitative real time-PCR and compared to ELISA-detected cell culture medium levels. Results: OGD induced a significant and long-lasting increase in MMP-2 gene expression, reaching a plateau after 12 h. Medium protein levels of MMP-2 were correspondingly elevated at 12 h of OGD. The MMP-9 synthesis rate was detectable at very low levels and remained unaffected by OGD. TIMP-1 gene expression and secretion declined under OGD, whereas both expression and secretion of TIMP-2 remained stable. Contrary to the respective gene expression rate, medium levels of MMP-2, TIMP-1 and TIMP-2 started a simultaneous decline after 12 h of OGD. This is most likely due to an impaired synthesis and enhanced consumption rate under OGD. Conclusions: The objective of our study was to determine the contribution of human BMECs to the MMP metabolism under in vitro OGD conditions simulating ischemic stroke. Our results suggest that human BMECs switch to a proinflammatory state by means of an enhanced production of MMP-2, attenuated release of TIMP-1, and unaffected production of TIMP-2. Thus, human BMECs might participate in the MMP-mediated BBB breakdown during ischemic stroke. However, our data does not support human BMECs to be a source of MMP-9.
    Cerebrovascular Diseases 06/2013; 35(6):514-520. DOI:10.1159/000350731 · 3.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In this article, I discuss the hallmarks of hypoxia in vitro and in vivo and review work showing that many types of stem cell proliferate more robustly in lowered oxygen. I then discuss recent studies showing that alterations in the levels and the types of cell and substrate adhesion molecules are a notable response to reduced O(2) levels in both cultured primary neural stem cells and brain tissues in response to hypoxia in vivo. The ability of O(2) levels to regulate adhesion molecule expression is linked to the Wnt signaling pathway, which can control and be controlled by adhesion events. The ability of O(2) levels to influence cell adhesion also has far-reaching implications for development, ischemic trauma and neural regeneration, as well as for cancer and other diseases. Finally I discuss the possibility that the fluctuations in O(2) levels known to have occurred over evolutionary time could, by influencing adhesion systems, have contributed to early symbiotic events in unicellular organisms and to the emergence of multicellularity. It is not my intention to be exhaustive in these domains, which are far from my own field of study. Rather this article is meant to provoke and stimulate thinking about molecular evolution involving O(2) sensing and signaling during eras of geologic and atmospheric change that might inform modern studies on development and disease.
    Cell adhesion & migration 01/2012; 6(1):49-58. DOI:10.4161/cam.19582 · 3.40 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Chemokines act mainly in guiding leukocyte migration along the endothelium. Apart from angiogenesis or neuronal survival, chemokines are involved in damage and repair of brain tissue after ischemic stroke. We studied the presence of chemokines directly in neurons and brain blood vessels that were obtained by means of laser microdissection from human ischemic brains. Using multiple ELISA Searchlight® array we evaluated nine chemokines (CCL1−5, CCL11, CCL17, CCL22, and CXCL8) in microdissected samples. We found higher levels of CCL1 and CCL2 in neurons than in vessels; CCL5 and CCL22 were decreased in the infarcted areas.
    06/2014; 3. DOI:10.1016/j.trprot.2014.03.001

Full-text (3 Sources)

Available from
May 21, 2014