Introduction Hypoxia drives angiogenesis in a range of pathologies. Mutations in von hippel lindau protein (vhl) lead to excessive angiogenesis via upregulation of hypoxic signalling, due to impaired HIF-1α degradation. Physical forces exerted by blood flow have been shown to contribute to vascular remodelling. We therefore used vhl mutant zebrafish to observe the interplay between hypoxic signalling, haemodynamic flow and vascular development. Since NO has been shown to be both pro-angiogenic and released in response to haemodynamic force, we assessed whether NO contributed to angiogenesis in this model. Methods Vhl mutant zebrafish were crossed with a fli1; GFP transgenic that expresses Green Fluorescent Protein (GFP) in the endothelium. Embryonic vascular development was observed in mutants and wild type siblings by confocal microscopy. To determine the role of blood flow in the angiogenic response, cardiac troponin t2 was knocked down by morpholino antisense injection. To assess the contribution of nitric oxide, embryos were treated with either L-NAME (nitric oxide synthase inhibitor) (1mM) or sodium nitroprusside (NO donor) (100μM) from 24-h post fertilisation (hpf) until imaging at 4dpf. Results Imaging of the developing trunk vasculature revealed that vhl mutant embryos display excessive and aberrant angiogenesis from 3dpf (Abstract 72 figure 1A, B). Cardiac troponin T2 knockdown prevented any cardiac contraction, but embryos develop normally due to passive oxygen diffusion. Loss of blood flow did not alter normal intersegmental vessel patterning in either controls (Abstract 72 figure 1C) or vhl mutants (Abstract 72 figure 1D). However, loss of blood flow completely prevented excessive angiogenesis in vhl mutants (Abstract 72 figures 1D and 2), implying that both blood flow and hypoxic signalling are required for “pathological” angiogenesis but not developmental angiogenesis (vasculogenesis). NO synthase inhibition with L-NAME had no effect, suggesting that the contribution of flow to excessive angiogenesis in response to upregulated hypoxic signalling is NO independent. Conclusion Angiogenesis in response to hypoxic signalling is critically dependent upon haemodynamic force, compared with developmental vasculogenesis that can proceed in the absence of any blood flow. This indicates a different mechanism of development for hypoxia driven angiogenesis and vasculogenesis which may have important therapeutic implications.

In-vitro studies of tenocytes aim to understand the relationship between the cells and their surrounding matrix [1], and how this interaction is affected by external stimuli placed upon them such as mechanical forces. For this reason it is important to understand whether cell proliferation is altered by mechanical or other stimuli.Currently the most commonly used methods of measuring cell proliferation rely on the use of BrdU (5-bromo-2'-deoxyuridine) or PCNA (proliferating cell nuclear antigen), which are visualised using antibody staining, therefore, relying on the permeabilisation of the cell membrane to detect what stage of the cell cycle the cells are in. EdU (5-ethyny1-2'-deoxyuridine) is a novel alternative to the BrdU assay for the measurement of active DNA synthesis. EdU is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis [2]. Detection of EdU is based on a "click" reaction, which is a copper (I) catalyzed reaction between an azide and an alkyne [3].EdU was added to media at 50mM and incubated with the cells for 6 - 22 hours. Cells were washed and fixed with ethanol and stained for 30 min with Alexa Fluor 488. Cells grown in monolayer were then imaged using a fluorescence microscope or explanted tendon fascicles were imaged using mulitphoton microscopy. Total number of cells present was calculated by staining with DAPI.Cells incorporating EdU brightly fluoresced and were easily identifiable from non-proliferating cells. Preliminary studies have found that 30-40% of tenocytes in monolayer were actively synthesizing DNA after a 6 hour labeling period. In contrast, when the cells were stained in-situ in intact tendon fascicles for 22 hours, only 2-5% of the cells were found to be synthesizing DNA.This method enables the simple visualization of proliferating tenocytes within intact tendons facilitating the investigation of biomechanical effects on proliferation both in vivo and ex vivo.

Progressive saphenous vein graft (SVG) narrowing and occlusion remains a major limitation of coronary artery bypass grafting and is an important target for gene therapy. Ex vivo adenoviral gene transfer of tissue inhibitor of metalloproteinase 3 (TIMP-3) reduces adverse SVG remodelling postarterialization, but concerns remain over the use of viral vectors in patients. Ultrasound exposure (USE) in the presence of echocontrast microbubbles (ECM) substantially enhances nonviral gene delivery. We investigated the effects of ultrasound-enhanced gene delivery (UEGD) of TIMP-3 plasmid on vascular remodelling in porcine SVG. Maximal luciferase activity (3000-fold versus naked plasmid alone) and TIMP-3 transgene expression in porcine vascular smooth muscle cells in vitro was achieved using USE at 1 MHz, 1.8 mechanical index (MI), 6% duty cycle (DC) in the presence of 50% (v/v) BR14 ECM (Bracco). These conditions were therefore utilized for subsequent studies in vivo. Yorkshire White pigs received carotid interposition SVG that were untransfected or had undergone ex vivo UEGD of lacZ (control) or TIMP-3 plasmids. At 28 d postgrafting, lumen and total vessel area were significantly greater in the TIMP-3 group (10.1+/-1.2 and 25.5+/-2.2 mm2, respectively) compared to untransfected (6.34+/-0.5 and 20.8+/-1.9 mm2) or lacZ-transfected (6.1+/-0.7 and 19.7+/-1.2 mm2) controls (P<0.01). These data indicate that nonviral TIMP-3 plasmid delivery by USE achieves significant biological effects in a clinically relevant model of SV grafting, and is the first study to demonstrate the potential for therapeutic UEGD to prevent SVG failure.