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

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.

Stem cells from human cardiac adipose tissue depots show different gene expression and functional capacities.

BACKGROUND: The composition and function of the adipose tissue covering the heart are poorly known. In this study, we have investigated the epicardial adipose tissue (EAT) covering the cardiac ventricular muscle and the EAT covering the left anterior descending artery (LAD) on the human heart, to identify their resident stem cell functional activity. METHODS: EAT covering the cardiac ventricular muscle was isolated from the apex (avoiding areas irrigated by major vessels) of the heart (ventricular myocardium adipose tissue (VMAT)) and from the area covering the epicardial arterial sulcus of the LAD (PVAT) in human hearts excised during heart transplant surgery. Adipose stem cells (ASCs) from both adipose tissue depots were immediately isolated and phenotypically characterized by flow cytometry. The different behavior of these ASCs and their released secretome microvesicles (MVs) were investigated by molecular and cellular analysis. RESULTS: ASCs from both VMAT (mASCs) and the PVAT (pASCs) were characterized by the expression of CD105, CD44, CD29, CD90, and CD73. The angiogenic-related genes VEGFA, COL18A1, and TF, as well as the miRNA126-3p and miRNA145-5p, were analyzed in both ASC types. Both ASCs were functionally able to form tube-like structures in three-dimensional basement membrane substrates. Interestingly, pASCs showed a higher level of expression of VEGFA and reduced level of COL18A1 than mASCs. Furthermore, MVs released by mASCs significantly induced human microvascular endothelial cell migration. CONCLUSION: Our study indicates for the first time that the resident ASCs in human epicardial adipose tissue display a depot-specific angiogenic function. Additionally, we have demonstrated that resident stem cells are able to regulate microvascular endothelial cell function by the release of MVs.

Novel methods for accurate identification, isolation, and genomic analysis of symptomatic microenvironments in atherosclerotic arteries.

A challenge facing surgeons is identification and selection of patients for carotid endarterectomy or coronary artery bypass/surgical intervention. While some patients with atherosclerosis develop unstable plaques liable to undergo thrombosis, others form more stable plaques and are asymptomatic. Identification of the cellular signaling mechanisms associated with production of the inflammatory, hemorrhagic lesions of mature heterogenic plaques will help significantly in our understanding of the differences in microenvironment associated with development of regions susceptible to rupture and thrombosis and may help to predict the risk of plaque rupture and guide surgical intervention to patients who will most benefit. Here, we demonstrate detailed and novel methodologies for successful and, more importantly, accurate and reproducible extraction, sampling, and analysis of micro-regions in stable and unstable coronary/carotid arteries. This information can be applied to samples from other origins and so should be useful for scientists working with micro-isolation techniques in all fields of biomedical science.