Objectives The purpose of this study was to examine the role of heparanase in controlling thrombosis following vascular injury or endovascular stenting. Background The use of endovascular stents are a common clinical intervention for the treatment of arteries occluded due to vascular disease. Both heparin and heparan sulfate are known to be potent inhibitors of thrombosis. Heparanase is the major enzyme that degrades heparan sulfate in mammalian cells. This study examined the role of heparanase in controlling thrombosis following vascular injury and stent-induced flow disturbance. Methods This study used mice overexpressing human heparanase and examined the time to thrombosis using a laser-induced arterial thrombosis model in combination with vascular injury. An ex vivo system was used to examine the formation of thrombus to stent-induced flow disturbance. Results In the absence of vascular injury, wild type and heparanase overexpressing (HPA Tg) mice had similar times to thrombosis in a laser-induced arterial thrombosis model. However, in the presence of vascular injury, the time to thrombosis was dramatically reduced in HPA Tg mice. An ex vivo system was used to flow blood from wild type and HPA Tg mice over stents and stented arterial segments from both animal types. These studies demonstrate markedly increased thromboses on stents with blood isolated from HPA Tg mice in comparison to blood from wild type animals. We found that blood from HPA Tg animals had markedly increased thrombosis when applied to stented arterial segments from either wild type or HPA Tg mice. Conclusions Taken together, this study’s results indicate that heparanase is a powerful mediator of thrombosis in the context of vascular injury and stent-induced flow disturbance.

β-Lap showed NQO1-dependent efficacy against two triple-negative breast cancer (TNBC) xenografts. NQO1 expression variations in human breast cancer patient samples were noted, where ~60% cancers over-expressed NQO1, with little or no expression in associated normal tissue. Differential DNA damage and lethality were noted in NQO1(+) versus NQO1-deficient (NQO1(-)) TNBC cells and xenografts after β-lap treatment. β-Lap-treated NQO1(+) cells died by programmed necrosis, whereas co-cultured NQO1(-) TNBC cells exhibited DNA damage and caspase-dependent apoptosis. NQO1 inhibition (dicoumarol) or H2O2 scavenging (catalase [CAT]) blocked all responses. Only NQO1(-) cells neighboring NQO1(+) TNBC cells responded to β-lap in vitro, and bystander effects correlated well with H2O2 diffusion. Bystander effects in NQO1(-) cells in vivo within mixed 50:50 co-cultured xenografts were dramatic and depended on NQO1(+) cells. However, normal human cells in vitro or in vivo did not show bystander effects, due to elevated endogenous CAT levels. Innovation and Conclusions: NQO1-dependent bystander effects elicited by NQO1 bioactivatable drugs (β-lap or deoxynyboquinone [DNQ]) likely contribute to their efficacies, killing NQO1(+) solid cancer cells and eliminating surrounding heterogeneous NQO1(low) cancer cells. Normal cells/tissue are protected by low NQO1:CAT ratios.

The shear stresses derived from blood flow regulate many aspects of vascular and immunobiology. In vitro studies on the shear stress-mediated mechanobiology of endothelial cells have been carried out using systems analogous to the cone-and-plate viscometer in which a rotating, low-angle cone applies fluid shear stress to cells grown on an underlying, flat culture surface. We recently developed a device that could perform high-throughput studies on shear-mediated mechanobiology through the rotation of cone-tipped shafts in a standard 96-well culture plate. Here, we present a model of the three-dimensional flow within the culture wells with a rotating, cone-tipped shaft. Using this model we examined the effects of modifying the design parameters of the system to allow the device to create a variety of flow profiles. We first examined the case of steady-state flow with the shaft rotating at constant angular velocity. By varying the angular velocity and distance of the cone from the underlying plate we were able to create flow profiles with controlled shear stress gradients in the radial direction within the plate. These findings indicate that both linear and non-linear spatial distributions in shear stress can be created across the bottom of the culture plate. In the transition and “parallel shaft” regions of the system, the angular velocities needed to provide high levels of physiological shear stress (5 Pa) created intermediate Reynolds number Taylor-Couette flow. In some cases, this led to the development of a flow regime in which stable helical vortices were created within the well. We also examined the system under oscillatory and pulsatile motion of the shaft and demonstrated minimal time lag between the rotation of the cone and the shear stress on the cell culture surface. Biotechnol. Bioeng. 2013;110: 1782-1793.

The metastatic spread of cancer is a major barrier to effective and curative therapies for cancer. During metastasis, tumor cells intravasate into the vascular system, survive in the shear forces and immunological environment of the circulation, and then extravasate into secondary tumor sites. Biophysical forces are potent regulators of cancer biology and are key in many of the steps of metastasis. In particular, the adhesion of circulating cells is highly dependent upon competing forces between cell adhesion receptors and the shear stresses due to fluid flow. Conventional in vitro assays for drug development and the mechanistic study of metastasis are often carried out in the absence of fluidic forces and, consequently, are poorly representative of the true biology of metastasis. Here, we present a novel high-throughput approach to studying cell adhesion under flow that uses a multi-well, mechanofluidic flow system to interrogate adhesion of cancer cell to endothelial cells, extracellular matrix and platelets under physiological shear stresses. We use this system to identify pathways and compounds that can potentially be used to inhibit cancer adhesion under flow by screening anti-inflammatory compounds, integrin inhibitors and a kinase inhibitor library. In particular, we identify several small molecule inhibitors of FLT-3 and AKT that are potent inhibitors of cancer cell adhesion to endothelial cells and platelets under flow. In addition, we found that many kinase inhibitors lead to increased adhesion of cancer cells in flow-based but not static assays. This finding suggests that even compounds that reduce cell proliferation might also enhance cancer cell adhesion during metastasis. Overall, our results validate a novel platform for investigating the mechanisms of cell adhesion under biophysical flow conditions and identify several potential inhibitors of cancer cell adhesion during metastasis.