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Interactions between peripheral blood mononuclear cells (PBMC) and the endothelium critically determine vascular repair and reparative angiogenesis, but also pathological processes, such as atherosclerosis. Current methodology to study these interactions mostly regards PBMC as a homogenous population or it restricts its focus on individual cell types and therefore does not appreciate the differential behavior of individual PBMC subpopulations, which synergize or antagonize each other to obtain the overall effect. We therefore developed a flow cytometry-based in vitro assay to assess multiple parameters of interaction between several individual populations of PBMC and an endothelial monolayer. Freshly isolated, unlabelled human PBMC were left to adhere to or transmigrate through a monolayer of fluorescence-labeled human aortic endothelial cells grown to confluence on the filter membrane of sterile transwell migration inserts. Monocyte chemoattractant protein-1 (MCP-1) was applied as a chemoattractant to the lower compartment of the migration chamber. After 6 h, transmigrating PBMC were harvested from the lower compartment, while nonadherent and adhering cell populations were harvested from the upper compartment by sequential washing/detachment. All three cell fractions were then individually stained with fluorescence-labeled monoclonal antibodies and analyzed by flow cytometry. Quantification was achieved by the usage of counting beads. Endothelial cells were separated from PBMC during the analysis by a multiparametric gating strategy. Using the newly established assay, we observed distinct migration patterns for inflammatory CD14(hi) CD16(neg) and resident CD16(pos) monocytes. These cell types differed in their basal adhesion and transmigration patterns as well as their responses to the CCR2 ligand MCP-1. This assay allows for the parallel study of interactions between multiple individual leukocyte populations and an endothelial layer. Several readouts can be derived from the same experiment, like the composition of adhering and transmigrating cell fractions or the individual adhesion/migration behavior of several distinct cell types., (Copyright © 2011 International Society for Advancement of Cytometry.)

Abstract: Objectives: This study sought to examine the effects and underlying mechanisms of systemic VEGF inhibition in experimental atherosclerosis and aortic endothelial cells. Background: Pharmacological inhibition of vascular endothelial growth factor (VEGF), a major mediator of angiogenesis, has become a widely applied treatment of certain cancers and multiple ocular diseases including age-related macular degeneration. However, recent clinical trials raise concern for systemic vascular adverse effects, prompting the Food and Drug Administration to revoke the approval of bevacizumab for metastatic breast cancer. Methods: Eight-week old apolipoprotein E knockout mice received a high-cholesterol diet (1.25% cholesterol) for 24weeks and were exposed to a systemic pan-VEGF receptor inhibitor (PTK787/ZK222584, 50mg/kg/d) or placebo (gavage) for the last 10weeks. Atherosclerotic lesions were characterized in thoraco-abdominal aortae and aortic arches. Mechanistic analyses were performed in cultured human aortic endothelial cells. Results: Systemic VEGF inhibition increased atherosclerotic lesions by 33% whereas features of plaque vulnerability (i.e. necrotic core size, fibrous cap thickness) remained unchanged compared with controls. Aortic eNOS expression was decreased (trend). In human endothelial cells VEGF inhibition induced a dose-dependent increase in mitochondrial superoxide generation with an uncoupling of eNOS, resulting in reduced NO availability and decreased proliferation. Conclusion: Systemic VEGF inhibition disrupts endothelial homeostasis and accelerates atherogenesis, suggesting that these events contribute to the clinical cardiovascular adverse events of VEGF-inhibiting therapies. Cardiovascular safety profiles of currently applied anti-angiogenic regimens should be determined to improve patient selection for therapy and allow close monitoring of patients at increased cardiovascular risk. [Copyright &y& Elsevier]

Summary: Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDLCKD), a population with high cardiovascular risk, we have demonstrated that HDLCKD in contrast to HDLHealthy promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDLCKD that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDLCKD reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension. [ABSTRACT FROM AUTHOR]